Biotechnology Ebook

MICROBIAL METABOLISM AND BIOTECHNOLOGY
Horst W.DoeIIe, DSc, DSc [hc]

Deputy-Director, MIRCEN-Biotechnology Brisbane and Pacific Regional Network
Past Chairman, nternational Organisation of Biotechnology and Bioengineering
[ Parts of this book have been published in the Unesco sponsored Encyclopedia for Life Support Systems
http://www.eolss.net]
Content:

Chapter 1- Scope, Resources and AppIications

Chapter 2 - Nature's Concept of CIean Environment and SustainabiIity - CycIes of
Matter, Interactions with Microorganisms, PIants and AnimaIs.
1. Introduction
2. CycIes of Matter
2.1 Carbon CycIe
2.2 Nitrogen CycIe
2.2.1 Nitrogen fixation
2.2.2 Symbiotic nitrogen fixation
2.2.3 Ammonification
2.2.4 Nitrification
2.2.5 Denitrification
2.3 SuIfur CycIe
2.3.1 Oxidative suIphur transformation
2.3.2 Reductive suIphur transformation
2.4 Phosphorous CycIe
2.5 Iron CycIe
3. InterreIations between the CycIing of IndividuaI EIements
3.1 Interactions amongst microorganisms
3.2 Microorganism-PIant Interactions
3.2.1 Rhizosphere
3.3.2 Mycorrhiza
3.2.3 DetrimentaI Interactions
3.3 Microorganism-AnimaI Interactions
4. BibIiography

Chapter 3 - BiotechnoIogy - OId and Modern Concepts
1. HistoricaI DeveIopment
2. Present DeveIopment
2.1 FundamentaIs in BiotechnoIogy
2.2 AgricuIturaI BiotechnoIogy
2.3 MedicaI BiotechnoIogy
2.4 IndustriaI BiotechnoIogy
2.5 EnvironmentaI BiotechnoIogy
2.6 SociaI Aspects of BiotechnoIogy
3. Trends for Future DeveIopment in BiotechnoIogy
4. BibIiography

Chapter 4 - BiotechnoIogy and Human DeveIopment
1. Introduction
2. The DeveIopment of RuraI and Urban Societies
2.1 BiotechnoIogy and the Corporate WorId
2.2 HeaIth and SurvivaI
2.3 DNA TechnoIogy
2.4 ReIigion and Ethics
3. SociaI Aspects of BiotechnoIogy
3.1 HeaIth
3.2 Poverty
3.3 Starvation
3.4 Waste Management and RecycIing: CIean and Green TechnoIogies
4. The RoIe of MicrobioIogy
5. Future Perspectives for Life and Human DeveIopment
6. BibIiography

Chapter 5 - The Importance of Basic MicrobioIogicaI KnowIedge for a better Life,
SeIf-Efficiency and SustainabiIity
1. Introduction
2. The MicrobiaI Biochemistry Concept
2.1 IsoIation, Identification and InitiaI SeIection of MicrobiaI Strains
2.1.1 CuIture Preservation
2.1.2 Stock CuIture Maintenance
2.1.3 Storage of CuIture
2.1.4 CuItue CoIIection Resources and Services
2.2 Modification of the genetic structure to increase Product Formation
2.3 Nutrition, OptimaI NutritionaI and PhysicaI Requirements for Growth
2.3.1 MicrobiaI Nutrition
2.3.2 Growth Measurements
2.3.3 Growth Curve
2.3.4 Optimisation of NutritionaI and PhysicochemicaI Factors
2.4 Process Strategy
2.4.1 Primary MetaboIites
2.4.2 Secondary MetaboIites
2.4.3 Bioconversions
3. The BiochemicaI Engineering Concept
3.1 Identification of Main Products and Substrates
3.2 Stoichiometry of the Process
3.3 Kinetic and Process Rate
3.4 Reactor Design
3.5 Product Recovery
3.6 Waste Treatment
4. BibIiography

Chapter 6 - MicrobiaI CeII Type and Structure
1. Introduction
2. Prokaryotes and Eukaryotes
2.1 Prokaryotes
2.1.1 CeII Structure and Function
2.1.2 Gram-negative aerobic chemoheterotrophic rods and cocci
2.1.3 Gram-negative aerobic chemoIithotrophic rods and cocci
2.1.4 FacuItative Anaerobic Gram-negative Rods
2.1.5 Anaerobic Gram-negative Rods
2.1.6 Anaerobic and microaerophiIic Gram-negative Bacteria
2.1.7 Other Gram-negative Bacteria
2.1.8 Gram-positive Bacteria
2.1.9 Non-sporing Gram-positive Rods
2.1.10 Anoxygenic photosynthetic Bacteria
2.1.11 Oxygenic photosynthetic Bacteria
2.1.12 Archaebacteria
2.1.13 Actinomycetes
2.2 Eukaryotes
2.2.1 CeII Structure and Function
2.2.2 AIgae
2.2.3 Yeast
2.2.4 Fungi or MouIds
3. OsmoreguIation
4. Structure of the Chromosome
5. Viruses
6. BibIiography

Chapter 7 - MicrobiaI CeII CuItivation Systems
1. Introduction
2. Btach CuItivation System
3. Continuous Growth CuItivation System
4. Fed-batch CuItivation System
5. RecycIing CuItivation System
6. InocuIum Cascading System
7. SoIid-State and SoIid-Substrate CuItivation System
7.1 PrincipIes
7.2 GeneraI Features
7.3 MicrobiaI Basis of Processes
7.4 Importance of InocuIum
7.5 Bioreactor Design
7.6 AppIication of SSC
8. ImmobiIised CeIIs and/or Enzyme Systems
8.1 AIginate
8.2 Carrageenan
8.3 Ion Exchange Resin
8.4 PoIyurethan Foam
8.5 CeII Aggregation/FIoccuIation
8.6 CovaIent CoupIing
8.7 Passive ImmobiIisation
8.8 ImmobiIised Bioreactor Design
8.9 Biosensors
9. BibIiography

Chapter 8 - Thermodynamics, SoIute Transport and Enzyme CataIysis
1. Concept of Thermodynamics of BioIogicaI Systems
1.1 Modes of Energy Production
1.2 Modes of Energy Conservation
1.2.1 Proton-transIocating eIectron tansport chain
1.2.2 Proton-transIocating ATPase compIex
2. Membrane and SoIute Transport
2.1 Passive Diffusion
2.2 FaciIitated Diffusion
2.3 Active Transport
2.4 Group TransIocation
3. Concepts of MetaboIism
3.1 Photosynthasis
3.2 Aerobic Respiration
3.3 Anaerobic Respiration
3.4 Fermentation
4. Concept of Enzyme CataIysis
5. BibIiography

Chapter 9 - Basic Strategies of Energy MetaboIism under Aerobic Conditions
[Respiration]
1. Introduction
2. RenewabIe Substrates
2.1 PoIymer HydroIysis
2.1.1 Starch HydroIysis to GIucose
2.1.2 CeIIuIose HydroIysis to GIucose
2.1.3 Protein HydroIysis to Amino Acids
2.1.4 Fat HydroIysis to Fatty Acids
2.2 Monomer UtiIisation
2.2.1 Carbohydrate UtiIisation
2.2.2 Amino Acid UtiIisation
2.2.3 Fatty Acid UtiIisation
3. Non-renewabIe Substrates
3.1 Hydrocarbon UtiIisation
3.2 SingIe Carbon Compound UtiIisation
4. BibIiography

Chapter 10 - Basic Strategies for Energy MetaboIism under Anaerobic Conditions
[Fermentation]
1. Introduction
2. Carbohydrate Fermentation
2.1 EthanoI Formation
2.2 Acetone-ButanoI Formation
2.3 Organic Acid Formation
2.3.1 Propionic and Succinic Acid Formation
2.3.2 MaIo-Iactic Acid Fermentation
2.3.3 Formation of DiacetyI, Acetoin and ButanedioI
3. Protein and Amino Acid Fermentation
3.1 SingIe Amino Acid Fermentation
3.2 Fermentation of Pairs of Amino Acids
3.3 Fermentation of SingIe Amino Acids in Combination with a Keto
Acid
4. Fatty Acid Fermentation
5. BibIiography

Chapter 11 - Basic Strategies for Biosynthesis [AnaboIism] of CeIIuIar Components
and MetaboIic ReguIation
1. Introduction
2. Autotrophic Carbon AssimiIation
3. Heterotrophic Carbon Biosynthesis
3.1 Formation of Protein
3.2 Formation of RNA and DNA
3.3 Formation of Lipids
3.4 CeII WaII Formation
4. Biosynthesis of the Enzyme Protein CataIyst
4.1 Transcription
4.2 TransIation
4.3 Activation
4.4 Initiation
4.5 EIongation
4.6 Termination-ReIease
4.7 PoIypeptide FoIding and Formation of FunctionaI Protein
5. MetaboIic ReguIation
5.1 Enzyme Activity ReguIation
5.2 Enzyme Synthesis ReguIation
5.3 CataboIite Repression
6. BibIiography

Chapter 12 - MicrobiaI BiotechnoIogy in Industry - Enzyme Production
1. Introduction
2. ChemicaI Nature and CIassification
2.1 Oxidoreductases
2.2 Transferases
2.3 HydroIases
2.4 Lyases
2.5 Isomerases
2.6 Ligases
3. Enzyme Assays
4. Production of Enzymes
4.1 Screening of Enzyme Producers
4.2 Strain SeIection
4.3 Strain DeveIopment
4.4 Strain Maintenance
4.5 GeneraI Fermentation Process
4.6 Purification
5. Enzyme ImmobiIisation
5.1 Methods of ImmobiIisation
5.2 Properties of ImmobiIised Enzymes
6. Biosensors
7. BibIiography

Chapter 13 - MicrobiaI BiotechnoIogy in Industry - IndustriaI AppIications of
Enzymes
1. Pectinases
1.1 Pectin MethyIesterases
1.2 Pectin DepoIymerases
2. Lipases
2.1 Pancreatic Lipases
2.2 MicrobiaI Lipases
3. Proteases
3.1 Serine Proteases
3.2 MetaIIoproteases
3.3 Acid Proteinases
4. GIucose Oxidase
5. CataIase
6. GIucose Isomerase
7. IndustriaI Use of ProteoIytic Enzymes
8. Enzymes used in the Detergent Industry
9. Enzymes used in the Leather Industry
10. Enzymatic Synthesis of Aspartame
11. Enzymes used in the Meat Industry
12. Enzymes used in the Dairy Industry
12.1 Enzymes from Rennet and Rennt Substitutes
12.2 Beta-1,4-gaIactosidases
12.3 Lysozyme
12.4 SuIfhydryI Oxidase
12.5 Production of Aroma and Texture
13. Enzymes in the Starch Processing and Baking Industry
13.1 Syrup and Sweetener
13.2 FueI AIcohoI
13.3 Baking
13.4 GIucose Isomerisation
14. AnaIysis
15. BibIiography

Chapter 14 - MicrobiaI BiotechnoIogy in Industry - Production of MicrobiaI Biomass
for Food, Feed and FertiIiser
1. Introduction
2. Microorganisms
2.1 Bacteria
2.2 Yeast
2.3 Fungi
2.4 AIgae
3, Production of MicrobiaI Biomass as a NutritionaI Protein Source
3.1 Pruteen Process
3.2 Baker's Yeast Production
3.3 Fodder Yeast Production
3.4 PekiIo Process
3.5 Mushroom Production
3.6 AIgaI Biomass Production
4. Production of MicrobiaI Biomass as a Protein-enrichment for AnimaI Feed
4.1 Protein-enriched Starch
4.2 Protein-enriched Whey
4.3 Conversion of LignoceIIuIose into feed using white-rot Fungi
5. SiIage
5.1 EnsiIing Process
5.2 SiIage MicrofIora
5.3 SiIage Additives
5.4 SiIage QuaIity
5.5 SiIage in TropicaI Areas
5.6 SiIage from Crop Residues and By-Products
6. Composting
6.1 PhysicaI Factors
6.2 ChemicaI Factors
6.3 MicrobioIogy
6.4 HeaIth Risks from Pathogens
6.5 Odour Sources
6.6 ConcIusions
7. BibIiography

Chapter 15 - MicrobiaI BiotechnoIogy in Industry - Bioenergy Production
1. Introduction
2. BiofueIs from SoIids as EIectricity and Heat
2.1 Pretreatment of Biomass
2.2 Direct Combustion
2.3 Co-Firing
2.4 Gasification
2.5 SmaII ModuIar Systems
3. BiofueI in the Form of Gas
3.1 Hydrogen
3.2 Methane [Biogas]
4. BiofueI in the Form of a Liquid
4.1 EthanoI
4.2 DieseI
5. BiofueI from PhytopIankton
6. BibIiography

Chapter 16 - MicrobiaI BiotechnoIogy in Industry - Production of Bio-ChemicaIs
1. Introduction
2. Primary Product Formation
2.1 Acetic Acid
2.2 Citric Acid
2.3 Lactic Acid
2.4 Amino Acids
3. Secondary Product Formation
3.1 PoIysaccharides
3.1.1 Dextran
3.1.2 Xanthan Gum
3.1.3 AIginate
3.1.4 Approaches to Improvement of MicrobiaI PoIysaccharide
Production
3.1.5 PoIy-beta-Hydroxybutyrate [PHB]
3.2 Antibiotics
3.2.1 Mode of Action
3.2.2 Production
4. Bio-Insecticides
4.1 PrincipIes
4.2 Stages in the Investigation
4.3 PresentIy used Candidates for BioIogicaI ControI Agents
4.4 Production of BioIogicaI Insecticides
4.4.1 Submerged Fermentation
4.4.2 Surface CuIture
4.4.3 ,Q YLYR CuIture
4.5 Bioassays
4.6 FormuIation and Use of Bio-Insecticides
4.7 Safety Testing of Bio-Insecticides
4.8 Future
5. BibIiography

Chapter 17 - TraditionaI MicrobiaI Production of Food
1. Introduction
2. Fermented Food and CuIture
3. Southeast Asian Region
3.1 Ang-Kak
3.2 Bagoong
3.3 Puto
3.4 Doza and IdIi
3.5 Fish Sauce
3.6 Miso
3.7 Natto
3.8 Oncom [ontjom]
3.9 Soy Sauce
3.10 Tempeh
4. African Region
4.1 Gari
4.2 Ogi
4.3 OIive Fermentation
5. European Region
5.1 Bread
5.2 Cheese
5.3 Yoghurt
5.4 ButtermiIk
6. BibIiography

Chapter 18 - Socio-Economic Strategies for RuraI Farming and Agro-IndustriaI
Processing Industries
1. Introduction
2. Impact of Fermentation TechnoIogy and the IndustriaI RevoIution on
CuIture and Society in the now DeveIoped Countries
3. Present Situation in DeveIoping Countries
3.1 TechnoIogy Transfer
3.2 New Trends in MicrobiaI BiotechnoIogy
4. Impact of Integrated RuraI Fermentation TechnoIogy on CuIture and
Society in DeveIoping Countries
4.1 TropicaI Wet Zone
4.2 TropicaI Arid Zone

5. Joint Venture CapitaI Investment for CIean TechnoIogies
5.1 CIean TechnoIogies and Eco-Efficiency
5.2 Infrastructure
5.3 Existing Joint Venture ProbIems
6. Socio-ecoIogicaI Strategies for Future SustainabiIity
6.1 Information TechnoIogy Coordinators for Education and
Discussions on SustainabIe BiotechnoIogy
6.2 HistoricaI DeveIopment of Internet Conferences on Boi-Integrated
Systems
6.3 Scope and Purpose of Internet Conferencing
6.4 IndustriaI Eco-Systems
6.6 RuraI Ecosystems
6.6 ConcIusions
7. BibIiography

Chapter 19 - Concept of a Bio-Refinery: I. Processing of LignoceIIuIosic Biomass,
Human and AnimaI Waste with ControI of Pathogens
1. Introduction
2. Community InvoIvement and Joint Venture CapitaI
3. Bio-Refinery Concept
4. Products from LignoceIIuIosic Biomass
4.1 Energy [eIectricity and/or heat]
4.1.1 Direct Combustion
4.1.2 Cofiring
4.1.3 Cogeneration
4.1.4 Gasification
4.2 Mushrooms
4.3 FertiIiser through composting
4.4 AnimaI Feed through siIage
5. Products from Human, AnimaI and ResiduaI AgricuIturaI Biomass
5.1 Energy [biogas] and FertiIiser
5.2 Food [fish and aIgae through aquacuIture]
6. BibIiography

Chapter 20 - Concept of a Bio-Refinery. II. Processing of SimpIe PoIymer Biomass
[starch, sugar, oiI, protein] from various agricuIturaI crops
1. Introduction
2. Enzyme Production
3. Starch Crops
3.1 Introduction
3.2 Grain
3.3 Cassava and potato
3.4 SagopaIm
4. Sugar Crop - Sugarcane
5. Fatty Acid and OiI containing Crops - OiI PaIm
6. Fish Processing Industries
7. BibIiography

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Deputy-Director MIRCEN-Biotechnology Brisbane and Pacific Regional Network;

Chapter 1
Scope, Resources and AppIications
n 1975, Unesco established the first Microbiological Resources Centres [MIRCENs] for
the preservation of our microbial gene pool. The World Data Centre in Brisbane, Australia,
under the Directorship of Professor V.B.D.Skerman was the Centre of the gene pool
preservation campaign. Since that time, more than 35 MIRCEN centres were created,
forming the global network of MRCENs today [MRCEN 2001]. This worldwide network of
MRCENs has the objectives:
a) to provide a global infrastructure, which would incorporate national, regional, and inter-
regional cooperating laboratories geared to the management, distribution and utilisation of
the microbial gene pools;

b) reinforce the conservation of microorganisms with emphasis on Rhizobium gene pools
in developing countries with an agrarian base;

c) to foster the development of new inexpensive technologies native to the the specific
regions;

d) to promote the economic and environmental applications of microbiology;

e) to serve as focal centres in the network for the training of manpower.
As a member of the Department of Microbiology at the University of Queensland, which
housed the World Data Centre [WDC], since 1964, had the fortune of being able to help
developing countries in addition to Australians to understand the intricacies of the
microbial world in nature and our total reliance on nature and the microbial world for our
food and livelihood. On retirement of the Director of the WDC in 1986, the World Data
Centre moved to Japan, where it is still located at present with its Director Dr.H.Sugawara
and a new MRCEN-Biotechnology was formed in Brisbane responsible for the Regional
Pacific Region. had the honour of being Director of this MRCEN from 1987 - 2001.
Most MRCENs are affiliated with the World Federation of Culture Collections [WFCC], and
a significant number of MRCENs joint over the years the nternational Organisation for
Biotechnology and Bioengineering [OBB] in order to participate nin the technological
application of our microbial gene pool for industrial and/or commercial purposes.
Therefore, MIRCENs are one of the cornerstones of biotechnoIogy.
n numerous Unesco, CRO [nternational Cell Research Organisation], UNEP [UN
Environmental Program], OBB, MRCENs, UNDO [UN industrial Development
Organisation], ADAB and other supported training courses, in which the author
participated, the foundation was laid for the training and development of microbial
biotechnology in developing countries of SEAsia, Africa as well as Central America (Table
1). Over the past few years the author has received many requests from participants in
these countries to make lectures as well as presentations available to all as a basis for
their own teaching. n considering these

UNEP/Unesco/ICRO/WFCC Workshop on 'Preservation of genetic pools
and establishment of regional culture collection centres
Brisbane, 7-22 1uly 1975

UNEP/Unesco/ICRO/WFCC Workshop on 'The preservation of genetic
pools and the establishment of regional culture collections of
microorganisms in developing countries.
Brisbane, Australia 1977

UNEP/Unesco/ICRO/WFCC Training course on 'Techniques of Microbial
Gene Pool Preservations and their use by MIRCENs in
Environmental Management' Brisbane, Australia, 4-18. 1uly 1977

UNEP/Unesco/ICRO Regional Training Course on 'Fermentation of Solid Substrates' Mexico
City, Mexico, 7.-1.1.1978

UNEP/Unesco/ICRO/IOBB Advanced Training Course on 'Biochemical
and Microbiological Technology'
Lagos, Nigeria, 19.-3.1.1978

Unesco/UNEP/WFCC/ICRO Training Course on 'Culture Collection
Techniques and Identification Procedures and their use by
MIRCENs in Environmental Management.
Brisbane , Australia, 4.-18.1uly 198
Unesco/UNEP International Workshop on 'Biotechnology in Waste
Management' Waterloo, Canada, 27. 3. 1uly 198

ASEAN/UNEP/Unesco/Niftal/Government of Thailand Training Course
on 'Identification Techniques of Microorganisms in Culture
Collections' Bangkok, 1hailand, 15-28. November 1981
Unesco/MIRCEN Symposium on 'Microbial & Engineering Technology
In Waste Treatment.
Hong Kong, 3- December 199
Unesco/ICRO/MIRCEN Regional Training Course on 'The importance of
microbiological biotechnology for community and economic
development
Motupore Island, Papua New Cuinea, 2-7 September 1991
Unesco Regional Workshop on 'Molecular genetics of lactic acid
bacteria and its role in traditional fermented foods
Bangkok, 1hailand, 24th October 2nd November 1992
Unesco/ICRO/MIRCEN Regional Training Course on 'Fermentation
Technology for the conservation of the environment'
Shanghai, Republic of China, 2-13 November 1992
Unesco International Symposium: 20th Anniversary of International
Post-Graduate University Training Course in Microbiology
Osaka, 1apan, 2-22 September 1993

Unesco Professorship in Biotechnology at Food Industrial Research
Institute FIRI] in Hanoi
Hanoi, Jietnam, 2.1. 1.12.1993
Unesco/ICRO/French Foreign Affairs Training Course on 'Microbial
Process Development and the Ecological Environment in
relation to the development of Fermentation Industries
Hanoi, Jietnam, 24th October 2nd November 1994
ICRO/MIRCEN/Unesco/Biotec Training Course on 'Treatment and
Utilization of agro-industrial waste for a cleaner environment and
sustainability.
Hat Yai, 1hailand, 4-1 August 1997
ASM/Unesco/MIRCEN Workshop Series on 'General Aspects of
Available biotechnological systems for a sustainable
development of the Pacific Island Nations'
Suva, Fiji, 25-28 February 1997
Nuku'alofa, 1onga, 13 March 1997
ASM/Unesco/MIRCEN Workshop on 'The role of biotechnology in
health, food and energy supply for a sustainable development of
the Pacific Island Nations
Apia, Western Samoa, 4-7 March 1997
Unesco/ICRO/IOBB Training Course on 'Current Trends in Microbial
Technology for a sustainable environment: Exploring microbial
biodiversity for novel processes
Kuala Lumpur, Malaysia, 12-24 October 1998

TabIe 1: Unesco sponsored Training Courses and Workshops participation and/or
organisation between 1975 - 1999.

request and the task to coordinate and at the same time keep the material presented up-
to-date, it was decided to include material from various lecture and training courses, which
were sponsored by the respective governments [Table 2] together with a number of invited
articles written for various scientific journals and conference papers presented at
nternational Conferences.
GIAM V, Global Impact of Applied Microbiology, UNEP, Unesco/ICRO
Panel
Kuala Lumpur, Malaysia 1978
Visiting Professorship in Biochemical Engineering at Department of
Chemical Engineering , University of Lagos
Lagos, Nigeria, th 1anuary to 31st August 1978
Fuel Ethanol, Research and Development Workshop
Canberra, Australia, 198
National Conference on Fuels from Crops
Melbourne, Australia, 28-29 September 1981
Seminar on 'Appropriate Biotechnology for the Development of Mexico'
Mexico City, Mexico, 13th February to 3rd March 1982
ADAB/UQ Training course for developing countries on 'Microbial
Culture Collection
Brisbane, Australia, 1th 1anuary to 12th February 1983
Intensive Short Training Course on 'Biotechnology Principles,
Practice and Economics
Brisbane, Australia, 4-9 1uly 1983
ADAB/ASEAN Workshop on 'Solid Substrate Fermentation'
Cebu City, Philippines, 3.-9.October 1983
ADAB Lecture tour to Prince of Songkla University in Hat Yai
Thailand], Kasetsart and Chulalongkorn University in
Bangkok Thailand] and University of Kelaniya in Colombo
Sri Lanka]
21st 1anuary to 17th February 1984
Training Course on 'Fermentation Technology of Recombinant
Organisms.
Brisbane, Australia, 3-4 September 1984
International Workshop on 'Molecular Bioscience and Biotechnology'
Southern Petrochemical Company SPIC]
Madras, India, 21-24. August 1985
ADAB/Sri Lanka Government Training Course on 'Fermentation
Technology'
Colombo, Sri Lanka, 25.-31.August 1985
Emerging Biotechnologies for Agriculture Symposium on
'Biotechnology and the Sugar Industry
Canberra, Australia, November 1985
GIAM VIII Recent Advances in Biotechnology and Applied Biology
Hong Kong, 1-5 August 1988
Beijing International Conference on Biotechnology
Beijing, 8-1 1uly 1989
GIAM IX Global Impact of Applied Microbiology.
Malta, 15.-21.September 1991
IPARD, Report and Recommendations on the Introduction of
Biotechnology for Estate Crop Development.
1akarta, Indonesia, February 1992
International Workshop on 'Fermentation Technology'
Awka & Enugu, 7-19 September 1992
CONACYT Fellowship at Institut of Ecology
Xalapa, Jer., Mexico, 13th 1anuary to 3th 1une 1994

Course of Lectures on 'Microbial Process Development'
University of Jeracruz, Orizawa, Mexico, March 1994
Louis Pasteur International Symposium on 'Microbes, Environment,
Biotechnologies.
Papeete, 1ahiti, 8-12 May 1995

GIAM X International Conference on Global Impacts in Applied
Microbiology.
Elsinor, Denmark, -12 August 1995
10th International Biotechnology Symposium
Sydney, Australia, 25-3 August 199
3rd Asia-Pacific Biotechnology Congress
Manila, 22-24 May 199
IUMS Congress 8th International Congress of Bacterial and Applied
Microbiology Division
1erusalem, Israel, 18-23 August 199
10th National Biotechnology Seminar on 'Biotechnology towards the
next millenium'
SIRIM, Malaysia, 27-28 October 1998
InFoRM 2000 workshop on 'Integrated Food Production and Resource
Management.
Brisbane, Australia , 9-1 November 2
A complete Course on 'Systematic Waste Management'
Kuching, Sarawak/Malaysia, 1-18 September 22

TabIe 2: Lecture courses, seminars and conferences attended
Furthermore, this comprehensive lecture course on microbial metabolism and
biotechnology will have links to many up-to-date webpages and articles, so that all
participants will be able to keep abreast with new development.
t is also the aim of the author to keep the material up-to-date as much as possible,
although time may be a limiting factor and thus an invitation is sent out for help to keep this
document alive for the training of new scientists interested in our environment and those
who would like to improve nature's work for a sustainable future.

Deputy-Director, MIRCEN-Biotechnology and Pacific Regional Network;
Past-Chairman, nternational Organisation of Biotechnology and Bioengineering
CHAPTER 2
Nature's Concept of a CIean Environment and
SustainabiIity - CycIes of Matter, Interactions with
Microorganisms, PIants and AnimaIs
Content:
1. Introduction
2. CycIes of Matter
2.1 Carbon CycIe
2.2 Nitrogen CycIe
2.2.2 Symbiotic Nitrogen fixation
2.2.4 Nitrification
2.2.6 Nitrate ammonification
2.3 SuIfur CycIe
2.3.1 Oxidative suIfur transformation
2.3.2 Reductive suIfur transformation
2.4 Phosphporous CycIe
2.5 Iron CycIe
3. InterreIations between the CycIing of IndividuaI EIements
3.1 Interactions amongst microorganisms
3.1.1 CommensaIism
3.1.2 Synergism
3.1.3 MutuaIism or symbiosis
3.1.4 AmmensaIism
3.2 Microorganism-PIant Interactions
3.2.1 BeneficiaI Interactions
3.3 Microorganism-AnimaI Interactions
4. References
1. Introduction
The ecosphere or biosphere, which constitutes the totality of living organisms on earth and
the abiotic surroundings they inhabit can be divided into atmo-, hydro-, and litho-
ecospheres (Atlas & Bartha 1987). These divisions respectively describe the portions of
the global expanse inhabited by living things in air, water, and soil environments. Each of
the major divisions of the ecosphere contains numerous habitats, whereby a habitat
represents the physical location where an organism, plant and animal can be found.

The naturaI habitats of microorganisms are exceedingly diverse. Any habitat suitable for
the growth of higher organisms will also permit microbial growth (Brock et al. 1984), but in
addition, there are many habitats unfavourable to higher organisms, where
microorganisms exist and even flourish. Because microorganisms are usually invisible,
their existence in an environment is often unsuspected, yet microbial action is usually of
considerable importance of the ecosystem.
Within a habitat, some microorganisms are said to be autochthonous or indigenous to
that habitat. These autochthonous microorganisms, which are capable of survival,. Occupy
the environmental niches available to the microbial populations in a given ecosystem.
Autochthonous microorganisms generally exhibit features that make them physiologically
compatible with their physical and chemical environment. n contrast, some
microorganisms may be foreign and are referred to as allochthonous. These
microorganisms are transient members of their habitat, do not occupy the functional niches
of that ecosystem, and exhibit great variation in the lengths of time that they can survive in
foreign ecosystems.
Microbial populations in natural environments vary widely and will depend on the activity of
the individual cell and what kind of processes they carry out. As a general rule, 10
6
cells/g
of soil of ml of water can have an appreciative effect.
The atmosphere is not known to support autochthonous microbial populations, but it
serves as a medium for the rapid and global dispersal of many types of micro-organisms.
There certainly exist important transfers of microorganisms and gaseous metabolites
among atmosphere, hydrosphere and lithosphere. n contrast to the atmosphere, both,
hydro- and lithosphere contain large microbial populations, which generally have
physiological adaptations that allow them to survive and carry out metabolic activities that
provide for energy flow through the ecosystem and materials cycles within the system.
Microorganisms are the principal producers as well as decomposers in aquatic
ecosystems. n soils, microorganisms play a subordinate role to plants as primary
producers, but have a critical role in organic matter decomposition and mineral cycling.
2. CycIes of Matter
Biogeochemical cycling describes the movement and conversion of materials by
biochemical activities within the ecosphere through which elements circulate in
characteristic paths or cycles between the biotic and abiotic portions of the ecosphere.
This cycling occurs on a global scale, producing profound effects on the geology and
present environment of our planet. Biogeochemical cycles include physical
transformations, such as dissolution, precipitation, volatilisation, and fixation. They also
include chemical transformations such as biosynthesis, biodegradation, oxido-reductive
biotransformations, as well as various combinations of physical and chemical changes.
Biogeochemical cycling is driven directly or indirectly by the radiant energy of the sun.
Energy is absorbed, converted, temporarily stored and eventually dissipated, which means
that energy flows through the ecosystems. Whereas energy flows through the ecosystem,
materials undergo cyclic conversion that tend to retain materials within the ecosystem.

The intensity rate of biogeochemical cycling for each element roughly correlates to the
amount of the element in the chemical composition of biomass. The major elemental
components of living organisms (C,H,O,N,P and S) are cycled most intensely.

Microbiologically mediated portions of biogeochemical cycles are essential for growth and
survival of plant and animal populations. Some of the critical metabolic activities of
microorganisms that directly influence plant and animal populations are well known to
microbiologists. t is important to recognise that the biogeochemical cycling activities of
microorganisms determine, in large part, the potential productivity that can be supported
within a habitat. Alterations in the biogeochemical cycling activities of microbial
populations caused by human activities - by pollution etc - can result in changes in
transfer rates of elements between reservoirs and the size of reservoirs of elements
in particular chemical forms within habitats. This change alters the biochemical
characteristics of a habitat and the populations that can be supported, both in
quantitative and qualitative terms
The turnover of the elements that compose living organisms constitutes what we refer to
as the cycIes of matter. All organisms participate in various steps of these cyclic
conversions, but the contribution of microorganisms is particularly important, both
quantitatively and qualitatively.
Let us have a look at the major cycles and apply our knowledge in the basic fundamentals
of microbiology to the maintenance and sustainability of nature and thus mankind.
2.1 Carbon CycIe
When examining the cycles of an individual element it is useful to consider first the global
reservoirs of this element, the size and whether or not these reservoirs are being actively
cycled.

The most actively cycled reservoir of carbon is atmospheric CO
2
(0.03% of the
atmosphere. The dissolved inorganic forms of carbon (CO
2
, H
2
CO
3
, HCO
3
-
and
CO
3
<SUP2-< sup>) in surface water are in direct equilibrium with the atmospheric CO
2
.
The living biomass in terrestrial and aquatic environments contains slightly less carbon
than the atmosphere.
The natural rate of carbon cycling in oceans and on land are close to a steady state, that
is, the rates of movement of carbon between the atmosphere and trees or between algae
and the dissolved inorganic carbon of the oceans do not change measurably from year to
year and tend to balance each other (Hobbie & Melillo 1984). However, human activities
have recently introduced changes in the carbon cycle that are large enough to be
measured. For example, the flux of carbon from algae into dissolved organic carbon in the
open ocean is at steady state because human activities are not as yet great enough to
perturb the rate. n contrast, the reservoir of carbon (as CO
2
) in the atmosphere is no
longer in a steady state and is growing from year to year. Thus the global carbon cycle is
out of balance. Atmospheric CO
2
, because it is a relatively small carbon pool, has been
measurable affected by industrial CO
2
release (Bolin et al. 1979). The increase in
atmospheric carbon dioxide is largely due to the burning of fossil fuels, with additional
largely as CO
2
contributed from forest biomass and soil humus in the course of forest
clearing for agricultural land.
The concentration of CO
2
is largely set by the competing processes of photosynthesis and
respiration [Figure 1]. Under favourable environmental conditions of light intensity and
temperature, the rate of photosynthesis and therefore the rate of plant growth is limited by
the concentration of CO
2
available to the plant.

Figure 1: Generalised Carbon Cycle in Nature

When CO
2
is dissolved in slightly alkaline water, bicarbonate (HCO
3
-
) and carbonate
(CO
3
2-
) ions are formed as mentioned earlier

Therefore, bicarbonate serves as the reservoir of carbon for photosynthesis in aquatic
environments. The bicarbonate concentration of ocean waters acts as reservoir for CO
2
for
the atmosphere, the ocean trap a large fraction of the CO
2
produced on land, keeping its
atmospheric concentration at a relatively low and constant level.
The carbonate ions in the oceans combine with dissolved calcium ions and become
precipitated as calcium carbonate. The latter is also deposited biologically in the shells of
protozoan, corals, and molluscs. This is the geological origin of the calcareous rock or
limestone that is an important constituent of the surface of the continents. The formation
and solubilisation of calcium carbonate are brought about primarily by changes in
hydrogen ion concentration, and microorganisms contribute indirectly to both processes as
a consequence of pH changes that they produce in natural environments. For example,
such microbial processes as sulfate reduction and denitrification [see later] cause an
increase in alkalinity of the environment, which favours the deposition of calcium
carbonate in the ocean and other bodies of water.
Microorganisms also play an important role in the solubilisation by production of acid
during nitrification, sulfur oxidation and fermentation.
As a general principle, anaerobic environments tend to serve as sinks in which organic
materials accumulate because fewer organic materials can be metabolised anaerobically
than aerobically. But methanogenesis provides a major route by which organic material
can escape from an anaerobic environment to an aerobic one, where it can be
metabolised further to CO
2
and water. Sulfate reducing bacteria also play an important role
in oxidising products of fermentation.

Figure 2: Carbon Redox CycIe (Brock et al. 1984)

The degradation and recycling of organic matter in most habitats is accomplished by
heterotrophic macro- and microorganisms. Microbial activities are crucial not only in terms
of quantity but also of quality of their contribution. Under aerobic conditions, macro- and
microorganisms share the ability to biodegrade simple organic nutrients and some
biopolymers such as starch, pectines and protein etc., but microorganisms are unique in
their capacity to carry out anaerobic (fermentative) degradation of organic matter. They are
responsible for the recycling of most of the very abundant but difficult to digest
biopolymers lignin and cellulose. The greatest range of carbon transformation occurs
under aerobic conditions (see chapter 9). On the other hand, certain carbon
transformations, such as methanogenesis, occur exclusively under anaerobic conditions
(see chapter 10). This leads to a biogeochemical zonation of habitats. Respiratory
metabolism yields more energy to cells than fermentative metabolism (see chapter 8).
Fermentation therefore requires a greater consumption of organic matter to support the
same biomass as respiration. Complete respiration results in the production of carbon
dioxide, whereas fermentation results in the accumulation of low molecular weight
alcohols, organic acids, carbon dioxide and hydrogen.

Since the industrial revolution, human exploitation of the stored deposits of organic carbon
in the earth's crust has resulted in their rapid mineralisation. A consequence of this very
rapid burning of fossil fuels has been an increase in the rate of production of CO
2
over the
rate at which it is utilised in biological fixations. Over the past 100 years the net increase of
CO
2
in the atmosphere has been about 15%. Although this increase is relatively small, if it
continues, its impact could be profound because atmospheric CO
2
tends to prevent the
loss of radiant energy from the earth, thus causing its average temperature to increase,
perhaps to dangerous levels (greenhouse effect). This danger could be counteracted by
increasing the photosynthesis by about 1% and a balance could be restored.

The rapid increase in the total size and local density of human population that have
occurred over the past century contributed also to modifications in the environment and
thus modifying not only the carbon cycle (see figure 2), but also all other cycles as we will
see later. Within the past century these factors have led to local environmental changes
comparable in scale to those produced by major geological upheavals in the past history of
the earth. The spread of agriculture, the denudation of forests, the mining and burning of
fossil fuels, and the pollution of the environment with human and industrial wastes have
profoundly affected the distribution and growth of other forms of life.

As a result of the concentration of the human population in large cities, the disposal of
organic wastes, both domestic and industrial, has become a major ecological problem. In
order to get our pIanet back into an ecoIogicaI baIance, it is necessary to investigate
microorganisms and their biochemicaI potentiaI and use these for the removaI of
the effects human popuIation has exerted on the naturaI cycIes of matter.
2.2 Nitrogen CycIe
Plants, animals and most microorganisms require combined forms of nitrogen for
incorporation into cellular biomass since the element N is a key constituent of protoplasm
(Brown & Johnson 1977). Nitrogen, which has stable valency states ranging from -3 [NH
3
]
to +5 [NO
3
2-
], occurs in numerous oxidation states. Thermodynamically however, nitrogen
gas [N
2
] is the most stable form of nitrogen, and it is this form that nitrogen will react to
under equilibrium conditions. This explains the fact that a major reservoir for nitrogen on
earth is the atmosphere contrasting carbon as a minor component of the atmosphere. The
high energy necessary to break the N=N bond of molecular nitrogen means that the
utilisation of N
2
is an energy-demanding process. Only a relatively small number of
microorganisms are able to utilise N
2
(nitrogen fixation), thus the recycling of nitrogen on
earth involves to a great extent the more easily available forms, ammonia and nitrate (as
chemical fertilisers). Since N
2
, however, constitutes by far the greatest reservoir of
nitrogen available to living organisms, the ability to utilise N
2
is of great ecological
importance. n many environments, productivity is limited by the short supply of combined
nitrogen compounds, putting a premium on biological nitrogen fixation. While many
habitats depend on plants for a supply of organic carbon, that can be used as source of
energy, aII habitats depend either on the bacterial fixation of atmospheric nitrogen or on
human invention chemical fertilisers.

Figure 3: Simplified Diagram of the Nitrogen Cycle in Nature

The nitrogen cycle (Fig. 3, Fig. 4 , and Fig. 5) is the conversion of nitrogen between the
different forms mentioned earlier. Nitrogen gas constitutes 80% of the earth's atmosphere,
is chemically inert and not a suitable source for most living forms. Access to an adequate
supply of nitrogen in some form is a prerequisite for all forms of life. n a simplified form,
the nitrogen cycle could be drawn as exhibited in Figure 3.

Figure 4: Nitrogen Cycle in Nature

Figure 5: Nitrogen Redox Cycle (Brock et al. 1984)

The fixation of nitrogen or the conversion of nitrogen gas into ammonia is carried pout in
nature in two classical ways:

1. free living microorganisms, which include the blue-green algae
2. symbiotic nitrogen fixing microorganisms, which belong mainly to the genus Rhizobium
infecting the roots of legumes, Frankia, Klebsiella, Beijerinckia.

The most important agents of non-symbiotic nitrogen fixation are heterocyst-forming blue-
green bacteria such as Anabaena and Nostoc (table 1). A wide variety of other bacteria
are also capable of fixing nitrogen, which include both aerobic bacteria (eg Azotobacter
group, Azospirillum and Bacillus polymyxa) and anaerobic bacteria (eg photosynthetic
bacteria, Clostridium sp.).

Bacterial nitrogen fixation is mediated in part by free-living bacteria, but the symbiotic
fixers are quantitatively more important (table 2).

The most thoroughly studied of the symbiotic fixers are representatives of the genus
Rhizobium, because they form associations with agronomically important leguminous
crops. Because of the critical agronomic importance of fixed nitrogen, the current world
food crisis, and the fact that manufacture of nitrogen fertilisers by the Haber process
requires large expenditures of energy, biological nitrogen fixation has become an intensive
subject of investigation (Balatti & Freire 1996).

Biochemically, nitrogen fixation is the reduction of the inert N
2
to ammonia by the unique
enzyme nitrogenase (Smith 1982). The nitrogenase enzyme system has two major
component proteins, one containing molybdenum plus iron and the other only iron-sulfur.
Nitrogenase is extremely sensitive to oxygen, requiring low oxygen tensions for activity.
The fixation of nitrogen needs not only nitrogenase, but also ATP and reduced ferredoxin.
Ammonia is formed as the first detectable product:

The highly positive indicates that the reaction requires a high energy input.
The electrons for nitrogen reduction are transferred to the enzyme via ferredoxin, a low
redoxpotential carrier. The ATP requirement for nitrogen fixation is very high, about 4-5
ATP for each 2 e
-
transferred. ATP is apparently required to lower the redox-potential of
the system to -0.4 V at which level the enzyme combines with ATP and transfers the
electrons to ferredoxin. From ferredoxin the electrons travel via the two iron-sulfur proteins
and reduce N
2
.
Nitrogenase is not specific for N
2
, but will also reduce cyanide (CN
-
), acetylene (CH=CH)
and several other compounds. The reduction of acetylene is only a two-electron process
and ethylene (CH
2
=CH
2
) is produced. t is being used to measure the activity of nitrogen-
fixing systems
2.2.2 Symbiotic Nitrogen Fixation
One of the most interesting and important symbiotic relationships is that between
leguminous plants and bacteria of the genus Rhizobium. Legumes are a large group that
includes important plants such as soybean, clover, alfalfa, string beans and peas.
Under normal conditions, neither legume nor Rhizobium alone is able to fix nitrogen as
only the interaction between the two leads to the development of nitrogen-fixing ability.
The infection of the roots of one of the legumes with the appropriate strain of Rhizobium
leads to the formation of root nodules. n the nodule, precise oxygen levels are controlled
by the O
2
-binding protein leghemoglobin, which is a red, hemoglobin-like protein, which is
always found in healthy N
2
-fixing nodules. To colonise the root and produce nodules, the
rhizobia bacteria must migrate to the root surface. This occurs via a chemotaxis movement
along a concentration gradient of a chemical and electrotaxis movement along electric
currents flowing into actively growing parts of the root. Plant root exudates stimulate
growth and movement and switches on the rhizobial genes for nodulation (nod). The
bacteria multiply rapidly within the root cell and it is here where nitrogen fixation occurs
after the nif gene has been turned on.
About 90% of all leguminous species are capable of becoming nodulated. There is a
marked specificity between species of legume and strains of Rhizobium. The effectiveness
is determined by genes in the bacterium that can be lost by mutation or gained by genetic
transformation.

n recent decades there has been a great deal of interest in enhancing biological nitrogen-
fixation during the production of forages and other legumes. Biological nitrogen fixation
provides a form of nitrogen that is less expensive and more sustainable than conventional
nitrogen fertilisers. t was further recognised that incorporating more biological nitrogen
fixation into agriculture might help reduce the dependence on synthetic nitrogen fertilisers
and thus lower the energy inputs associated with nitrogen fertilisation of crops. Since the
early 1970s considerable research has been conducted in order to help producers utilise
biological nitrogen fixation more often and more effectively in food and forage production.
Unesco has recognised this urgent demand and three (3) of the presently thirty (30)
Microbiological Resources Centers [MRCENs] are in fact Rhizobium-MRCENs in Brazil,
Kenya and Hawaii. Some of the benefits expected are:
1. a greater use of biological nitrogen fixation will reduce society's current dependence on
synthetic nitrogen fertilisers. The production of widely used synthetic nitrogen fertilisers
such as anhydrous ammonia requires the use of relatively large amounts of energy from
non-renewable energy sources such as natural gas. Distribution and application of these
fertilisers also requires relatively large amounts of non-renewable energy sources such as
petrol and diesel fuels.

2. a greater use of biological nitrogen fixation can help enhance environmental quality by
reducing problems with air and water pollution. The over-application of synthetic nitrogen
fertilisers has been linked to excessive nitrate (NO
3
-
) levels in groundwater in a number of
locations around the world. Excessive nitrate concentrations may have detrimental effects
on human health. Both the manufacture and application of synthetic nitrogen fertilisers
involves burning non-renewable fuels such as natural gas, diesel fuel, and petrol, which
have been shown to contribute to air pollution.

3. a greater use of biological nitrogen fixation can help lower production costs and thus
increase profit margins for producers. The use of crops that fix nitrogen in crop rotation
can significantly reduce nitrogen fertiliser needs for crops in rotation.

4. a greater use of biological nitrogen fixation can help enhance sustainable food
production by improving soil fertility and tilth. Some producers have found that the use of
so-called green manure crops is a more sustainable fertiliser alternative than purchased
synthetic fertiliser. Green manure crops are crops grown specifically to be incorporated
into the soil rather than for harvest. Using green manure crops that fix atmospheric N
2
may
potentially increase soil nitrogen levels and organic matter content over time. Additional
organic matter in soils generally improves the tilth of a soil, where tilth refers to desirable
physical properties of soil such as proper drainage, water holding capacity, aeration and
structure (Forage nformation System 1998). Good examples exist in Southern China,
where rice paddocks are allowed to generate blue-green algae to fix nitrogen before the
soil is tilted using water buffaloes

Many plants, animals, and microorganisms are capable of ammonification, a process in
which organic nitrogen is converted to ammonia. Nitrogen in living and dead organic
matter occurs predominantly in the reduced amino form. Under anaerobic conditions,
ammonia is stable, and it is in this form that nitrogen predominates in anaerobic
sediments. n soils, much of the ammonia released by aerobic decomposition is rapidly
recycled and converted into amino acids in plants. Because ammonia is volatile, some loss
can occur from soils (eg high alkaline soils) by vaporisation, and major losses occur in
areas of dense animal population (eg feedlots). n acidic and neutral aqueous
environments, ammonia exists as ammonium ions. The release of ammonia from a simple
nitrogenous organic compound such as urea can be described as:

The importance of organic nitrogen mineralisation for continued ecosystem productivity
has been emphasised (Blackburn 1982).
The initial incorporation of ammonia into living organic matter is often accomplished either
by glutamine synthetase/glutamate synthase reactions or by a direct amination of an -
ketocarboxylic acid to form an amino acid (Doelle 1975; Gottschalk 1979).

2.2.4 Nitrification
The conversion of ammonia to nitrate via nitrite is referred to as nitrification. This process
of nitrification is limited to a restricted number of autotrophic bacteria (Doelle 1975; Fochl &
Verstraete 1977). The two steps of nitrification are carried out by different microbial
populations. Normally, these two processes are closely coupled and an accumulation of
nitrite does not occur. Both processes are energy-yielding processes. This is possible,
because the nitrifying bacteria are chemolithotrophs and utilise the energy derived from
nitrification to assimilate CO
2>
. The bacteria in the soil, which carry out these processes
belong to the genera with the prefix nitroso- [ammonia nitrite] and nitro- [nitrite - nitrate].
The most common genera are therefore Nitrosomonas and Nitrobacter . Other ammonia
oxidisers are Nitrosospira, Nitrosococcus and Nitrosolobus, whereas other nitrite oxidisers
belong to the genera Nitrospina and Nitrococcus.
Nitrosomonas and Nitrobacter have wider growth ranges than the others and are much
more numerous in soils and water. The others tend to inhabit specialised habitats. The
growth rates are often slow - generation times of 20-40 hours are common in culture and
are undoubtedly slower in the natural environment.
They are 'inhibited' by high organic carbon concentrations (eg they do not grow well on
agar plates) probably because any organic carbon in the environment causes metabolism
and therefore loss of nitrate or nitrite and also changes the microbial environment. They do
not seem directly inhibited by the presence of organic carbon. They gain energy from the
inorganic nitrogen reactions and gain their carbon from carbon dioxide, that is one of the
reasons why they belong to the chemoautotrophs.
Ammonia is possibly oxidised to nitrite via the following intermediates:

Hydroxylamine is the major intermediate. The oxidation of hydroxylamine to nitrite is
connected with the cytochrome system and thus an exergonic energy step. This reaction
also yields hydrogen ions and lowers the pH of the environment in which it occurs.

Although oxygen dependent, the second step of nitrification obtains the oxygen for the
formation of nitrate from a water molecule; the molecular oxygen serves only as an
electron acceptor:

This whole process is the removal of electrons from a hydrated nitrite ion.

The reactions of Nitrobacter are inhibited by small quantities of ammonia gas (1.4 mg/l
ammonia inhibits 99%). The result of this is that ammonia in soils leads to the
accumulation of nitrite since only Nitrobacter is inhibited. Nitrite is continually formed by
Nitrosomonas , but is not utilised by Nitrobacter , when it is inhibited. This nitrite can
accumulate to levels toxic to plants.
The process of nitrification is especially important in soils, because the transformation of
ammonium ions to nitrite and nitrate ions results in a change of charge from positive to
negative. Positively charged ions tend to be bound by negatively charged clay particles in
soil, while negatively charged ions freely migrate in the soil water. The process of
nitrification therefore must be viewed as a nitrogen mobilisation process within soil
habitats. Ammonia in soil is normally oxidised very rapidly by nitrifying bacteria. Plants
readily take up nitrate ions into their roots for assimilation into organic compounds. Nitrate
and nitrite ions, however, can also be readily leached from the soil column into the ground
water. This is an undesirable process, since it represents a loss of fixed forms of nitrogen
from the soil.
The appearance of nitrite in ground water is a serious concern, since nitrite can react
chemically with amino compounds to form nitrosamines, which are highly carcinogenic.
Nitrate and nitrite in ground water is a problem in agricultural areas receiving heavy
concentrations of synthetic nitrogen fertiliser.
Denitrification is the reversal of nitrification

n contrast to nitrification, which is an aerobic process, denitrification is an anaerobic
process, whereby nitrate serves as the final electron acceptor (see chapter 8 ), a process
often also referred to thermodynamically as anaerobic respiration. Many facultative
microorganisms can use nitrate in place of oxygen as final electron acceptor. The most
common organisms are Pseudomonas spp., Achromobacter spp., Paracoccus spp.,
Moraxella spp., Bacillus spp., Alcaligenes spp., and Gluconobacter spp. All are relatively
common soil bacteria.
Thus, whenever organic matter is decomposed in soil or water an oxygen is exhausted as
a result of aerobic microbial respiration, certain species of these aerobes will continue to
respire the organic matter if nitrate is present. By this process, combined nitrogen is
removed from the soil and water, releasing nitrogen gas into the atmosphere.
Denitrification is a process of major ecological importance. t depletes the soil of an
essential nutrient for plants, thereby decreasing agricultural productivity (Fig. 6) . Such
losses are particularly important from fertilised soils.The detailed biochemical process of
denitrification is catalysed by the three enzymes nitrate reductase, nitrite reductase and
hyponitrite reductase. Typically, nitrous oxide will be produced early in the reaction and
nitrogen will be produced later. At high concentrations of nitrate, higher amounts of nitrous
oxide are formed. Nevertheless, not all the consequences of denitrification are detrimental.
Denitrification is vital to the continued availability of combined nitrogen on the land masses
of the earth. The highly soluble nitrate ion is constantly leached from soil and carried to the
oceans. Without denitrification, the earth's supply of nitrogen, including the dinitrogen of
the atmosphere, would eventually accumulate in the oceans, precluding life on the land
masses except for a fringe near the oceans. Denitrification also maintains the potability of
fresh waters, because high concentrations of nitrate ions may be toxic.
Figure 6: Process of Denitrification
The nitrogen cycle starting with nitrogen gas fixation to ammonia, nitrification to nitrate is
closed by employing denitrification. The nitrogen cycle is important for our life, to grow
plants and make the nitrogen for the atmosphere available as nitrogen fertiliser. Any
interference could lead to acidic soils (nitrite accumulation), polluted waterways and
oceans (nitrate accumulation) or cessation of nitrogen fixation (oversupply of ammonia).
The leaching of nitrogen causes eutrophication along lakes and bays causing anaerobic
conditions.

2.2.6 Nitrate ammonification
Nitrate ammonification plays an important role in stagnant water, sewage plants, and some
sediments (Koike & Hattori 1978). Unlike assimilatory nitrate reduction, dissimilatory nitrate
reductase is not inhibited by ammonia and can be excreted in relatively high
concentrations. As compared to denitrification, nitrate ammonification is an environment-
tally less significant process for the reductive removal of nitrate and nitrite ions.
Simultaneously with denitrification, organic matter is oxidised. The utilisation of glucose
through nitrate reduction by Pseudomonas denitrificans

involves the dissimilatory nitrate and nitrite reductase systems. Denitrification is more
common in standing waters than in running rivers.
2.3 SuIfur CycIe
Sulfur is a reactive element with stable valency states from -2 (S
2-
) to +6 (SO
4
2-
) and is
among the ten most abundant elements in the crust of the earth. At an average
concentration of 520 ppm, it rarely becomes a limiting nutrient.

Figure 7: Sulfur Cycle in Nature
Plants, algae, and many heterotrophic microorganisms assimilate sulfur in the form of
sulfate (Figure 7). For incorporation into cysteine, methionine, and coenzymes in the form
of sulfhydryl (SH
-
) groups, sulfate needs to be reduced to the sulfide level by assimilatory
sulfate reduction. A direct uptake as sulfide is not feasible for most microorganisms
because of the very high toxicity of H
2
S. n assimilatory sulfate reduction, toxicity is
avoided by immediately reacting the reduced sulfur with an acceptor, eg serine, to yield
cysteine.

2.3.1 Oxidative suIfur transformation
n the presence of oxygen, reduced sulfur compounds are capable of supporting chemo-
lithotrophic microbial metabolism. Beggiatoa, Thiovolum, Thiothrix, and more recently
described thermophilic Thermothrix are filamentous, microaerophilic bacteria capable of
oxidising H
2
S

Sulfur globules are deposited within the cells. n the absence of H
2
S, these sulfur globules
are slowly further oxidised to sulfate. These typical gradient organisms position
themselves on the interface of an anaerobic environment, the sediment, and the partially
oxygenated water in contact with the sediment.
Some species of Thiobacillus (Thiobacillus thioparus, Thiobacillus novellus) also oxidise
H
2
S and other reduced sulfur compounds and, because they have a low acid tolerance,
deposit elemental sulfur rather than generate sulfuric acid by further oxidation. The
filamentous sulfur bacteria and these Thiobacillus species are facultatively
chemolithotrophic. Other members of the genus Thiobacillus produce sulfate from the
oxidation of elemental sulfur and other inorganic sulfur compounds:

TheseThiobacillus species are acidophilic, grow well at pH 2-3, and are obligate
chemolithotrophs, obtaining their energy exclusivel y from the oxidation of inorganic sulfur
and their carbon from the reduction of CO
2
. Most Thiobacillus species are obligate
aerobes requiring molecular oxygen for the oxidation of the inorganic sulfur compounds.
Thiobacillus denitrificans, however, can utilise nitrate ions as terminal electron acceptor in
the oxidation of inorganic sulfur compounds:

This organism is not capable of assimilatory nitrogen reduction and requires ammonium as
a nitrogen source. Members of the genus Sulfolobus oxidise elemental sulfur in hot acidic
habitats to generate their required energy.
Chemoautotrophic sulfur-oxidising bacteria are widely distributed and they are very active
in soils and aquatic habitats. A variety of other heterotrophic microorganisms oxidise
inorganic sulfur to sulfate or thiosulfate, but do not appear to derive energy from this
transformation.

Hydrogen sulfide is also subject to phototrophic oxidation in anaerobic environments.
Photosynthetic sulfur bacteria, the Chromaticeae and Chlorobiaceae, are capable of
photoreducing CO
2
while oxidising H
2
S to S
0
, in striking analogy to the photosynthesis of
eukaryotes:

Most Chromataceae store sulfur globules intracellularly, whereas Chlorobiaceae excrete
sulfur globules. Both have only a marginal capacity to oxidise sulfur further to sulfate and
they may contribute towards biological sulfur deposition.
Microbial oxidation of reduced sulfur is essential for continued availability of this element in
nontoxic form, but the chemoautotrophic fixation of carbon dioxide connected with this
activity contributes only minimally to the carbon cycling in most ecosystems.

2.3.2 Reductive SuIfur Transformation
The analogy between H
2
O and H
2
S in oxygenic and anaerobic phototrophy goes even
further. According to that analogy, elemental sulfur should assume a role similar to oxygen
in respiratory processes. Desulfotomaculum acetoxidans grows for example on acetate,
anaerobically reducing stoichiometric amounts of S
0
to H
2
S:

Although the free energy is very low, no other sources of carbon and energy are required
for growth. Desulfuromonas is unable to reduce sulfate or live by fermentative metabolism
and uses the substrate acetate that is not metabolised by most sulfate reducers.
Desulfuromonas occurs in anaerobic sediments rich in sulfide and elemental sulfur. t also
lives syntrophically with the phototrophic green sulfur bacteria (Chlorobiaceae) that
photooxidise H
2
S to S
0
and excrete elemental sulfur extracellularly. Desulfuromonas
regenerates H
2
S by sulfur respiration, using at least in part, organic matter leaked by
Chlorobium cells.
The use of sulfate as terminal electron acceptor in anaerobic respiration has been known
since the time of Beijerinck. When obligately anaerobic bacteria carry out dissimilatory
sulfate reduction, they are referred to as sulfate reducers. The traditional sulfate-
reducing genera Desulfovibrio and Desulfotomaculum were recently joined by several
newly described types, Desulfobacter, Desulfobulbus, Desulfococcus, Desulfonema and
Desulfosarcina. The reduction of sulfate results in the production of hydrogen sulfide:

n addition to anaerobic sulfate reducing bacteria, some species of Bacillus,
Pseudomonas, and Saccharomyces have been found to liberate hydrogen sulfide from
sulfate, but these additional genera do not appear to play a major role in the dissimilatory
reduction of sulfate. Sulfate reduction can occur over a wide range of pH, pressure,
temperature, and salinity conditions. Only relatively few compounds can serve as electron
donors for sulfate reduction, the most common of which are pyruvate, lactate, and
molecular hydrogen. Sulfate reduction is inhibited by the presence of oxygen, nitrate, or
ferric ions. The rate of sulfate reduction is often limited by carbon availability. The addition
of organic compounds to marine sediments can result in greatly accelerated rates of
dissimilatory sulfate reduction.
The production of even small amounts of hydrogen sulfide by sulfate reducers can have a
marked effect on populations within a habitat. Hydrogen sulfide is extremely toxic to
aerobic organisms because it reacts with the heavy metal groups of the cytochrome
systems. Hydrogen sulfide also has antimicrobial activity and can adversely affect
microbial populations in soil.
n contrast to the specialised dissimilatory sulfate reducers, many organisms are capable
of assimilatory sulfate reduction. Assimilatory sulfate reduction produces low
concentrations of hydrogen sulfide, which are immediately incorporated into organic
compounds. Many microorganisms and plants can utilise sulfate ions as the source of
sulfur required for incorporation into proteins and other sulfur-containing biochemicals.
Plant roots readily take up sulfate from soils, incorporating it into organic matter.
The assimilation of inorganic sulfate involves a series of transfer reactions initiated by the
reaction of sulfate with ATP to form adenosine-5'-phosphosulfate (APS) and
pyrophosphate (PP). A second reaction between ATP and APS produces 3-phospho-
adenosine-5-phosphosulfate (PAPS) and ADP:

The active sulfate of PAPS is subsequently reduced to yield sulfite and adenosine 3'5-
diphosphate (PAP). A second reduction step yields sulfide that is immediately incorporated
into an amino acid:

The biochemical mechanism involved in dissimilatory sulfate reduction is similar to the one
described, but the generated hydrogen sulfide is released to the environment. n sulfate
rich marine environments, most of the hydrogen sulfide originates from dissimilatory
sulfate reductions, but in rich organic sediments, hydrogen sulfide may accumulate also
from the decomposition of organosulfur compounds.
Sulfate reduction contributes to the atmospheric cycling of sulfur. n the atmosphere,
hydrogen sulfide is rapidly photooxidised to SO
2
, SO
3
and H
2
SO
4
. Hydrogen sulfide and
other reduced volatile sulfur compounds are rapidly oxidised to sulfate in aerobic soils and
sediments.
Sulfur oxidation produces substantial amounts of strong mineral acid. Within soils, this can
lead to solubilisation and mobilisation of phosphorous and other mineral nutrients with a
generally beneficial effect on both microorganisms and plants. The activity of Thiobacillus
thiooxidans may be used for adjusting soilpH . Thiobacillus thiooxidans and Thiobacillus
ferrooxidans are used in microbial mining operations. When mining activities , especially
strip mining, uncover large amounts of reduced sulfide rock, the activities of the same
thiobacilli give rise to acid mine drainage, a destructive pollution phenomenon.

Fossil fuels, especially coal and some heating oils, contain substantial amounts of sulfur.
Much of this sulfur is present as pyrit (FeS
2
) and originates from hydrogen sulfide
produced by sulfate reducers. When burned, most of the sulfur is converted to SO
2
, which
combines with atmospheric moisture to form sulfuric acid (H
2
SO
4
). Atmospheric inversions
in urban areas during the winter heating season can lead to the formation of highly
irritating and unhealthy acid smog. This conditions differs from the urban smog that arises
in warmer weather predominantly from automobile exhaust, in which ozone and nitrogen
oxides are the main irritants.
On the larger scale, the burning of fossil fuels gives rise to the formation of acid rain.
Rainwater, which normally has a pH just below neutrality due to the weak acidity of
carbonic acid, becomes quite acidic (pH 3.5-4.0) from sulfurous acid. Acid rain corrodes
buildings and mnuments, especially those made of limestone and marble. t may also
damage plant leaves. Because of the acid rain problem, air pollution standards limit sulfur
dioxide emissions, and there is pressure to further tighten these standards. The option to
burn clean fuels, like natural gas and low sulfur oil, is becoming increasingly expensive.

Microorganisms show some potential for reducing the acid rain problem by removing sulfur
from fossil fuels prior to burning. An important practical implementation of the sulfur cycle
is the anaerobic corrosion of steel and iron structures set in sulfur-containing soils and
sediments. This type of corrosion can severely damage or destroy pipes and pilings and
has posed unexpected engineering problems.

2.4 Phosphorous CycIe
Phosphorous is an essential element in all living systems. Within biological systems, the
most abundant forms are phosphate esters, eg nucleic acid. Phosphate also forms the
essential portion of the ATP molecule and phospholipids are essential components of cell
membranes.

The microbial cycling of phosphorous for the most part does not alter the oxidation state of
phosphorous and can be viewed as transfers of inorganic phosphate to organic phosphate
or as a transfer of insoluble forms of phosphorous to soluble compounds. Phosphate can
also serve as a terminal electron acceptor in the absence of sulfate, nitrate and oxygen
with the final reduction phosphine (PH3). Phosphines are highly volatile and
spontaneously ignite on contact with oxygen and methane producing a green glow.
Soluble forms of inorganic phosphorous can be readily taken up by plants and
microorganisms and assimilated to organic phosphates.
The biggest problems with soluble phosphorous occurs in lakes as a result of over-
fertilisation causing so-called eutrofication. A eutrophic lake is one with large amounts of
algal nutrients so that development of undesirable large amounts of algal biomass occurs.
Analytical data on algal populations and measurement of nutrient uptake led to the
equation

which indicates a molar ratio of C:N:P of 106:16:1 in algal protoplasm. This formula was
proven to exist in many aquatic systems, both marine and freshwater. The data also
showed that phosphorous is the most likely limiting nutrient for algal productivity. This
eutrophication has been of greatest concern in lakes because of the importance of
freshwater to human welfare. There appears to be an excellent correlation between P and
chlorophyll content. t is therefore of great importance to limit phosphorous content in
aquatic systems to prevent algal growth to occur.
2.5 Iron CycIe
ron is one of the most abundant elements in the earth's crust, but is a relatively minor
component in aquatic systems because of its relative insolubility in water. Only a small
portion of the iron is available for biogeochemical cycling (Ehrlich 1981, Nelson 1983). The
cycling of iron consists largely of oxidation-reduction reactions that reduce ferric iron to
ferrous iron and oxidises ferrous iron back to ferric iron. These oxidation-reduction
reactions are important in both organic iron-containing compounds and in inorganic iron
compounds.

The bacterial reduction of ferric iron to the ferrous state is a major means by which iron is
solubilised in nature. Many of these microorganisms also reduce nitrate, and since they
are facultative anaerobes, they can also use oxygen. The overall reaction of ferric iron
reduction can be expressed as:

n addition to the bacterially catalysed reduction, if H
2
S is present, as it is in many
anaerobic environments, ferric iron is also reduced chemically to FeS. This type of ferric
iron reduction is very common in water-logged soils and anaerobic lake sediments. The
ferrous iron can leach out of the soil and can result in the transport of considerable
amounts of iron. Once this iron-laden water reaches aerobic regions, the ferrous iron is
quickly oxidised and ferric compounds precipitates. Such deposits can cause serious
problems in drinking water pipes:

Although the initial oxidation of ferrous iron consumes hydrogen ions and thus leads to a
rise in pH, the hydrolysis of Fe
3+
and formation of Fe(OH)
3
consumes hydroxyl ions and
leads to an acidification of the medium.
3. InterreIations between the cycIing of individuaI eIements
Although we have dealt with four of the major cycles in nature, it needs to be emphasized
that in reality the cycle of each element is dependent on, or at least influenced by, the
cycling of other elements. This is not only true for C, H, and O, that are cycled by the same
two processes of photosynthesis and respiration, but also for the other cycles that are
driven by different biochemical processes and performed by distinct microorganisms.

The reductive portions of the N,S, and Fe cycles are driven by chemical energy fixed in
organic substances during photosynthesis. The chemolithotrophic reoxidation of N,S, and
Fe are, in turn, linked to the conversion of carbon dioxide into cell material, again involving
the cycling of C,H, and O. Acids from nitrification and sulfur oxidation help to mobilise
phosphorous, whereas photosynthesis and respiration are required for its uptake and
conversion into high energy phosphates.
From the pool of potential electron acceptors, the microbial community selects the one that
maximises energy yield from the available substrate. This seemingly intelligent decision is
in part due to metabolic regulation within a single population, and in part due to the
inevitable outcome of competition between populations with diverse metabolic capabilities.
Through various regulatory mechanisms, facultatively anaerobic microorganisms shut off
their less efficient fermentative or dissimilatory nitrate reduction pathways in the presence
of oxygen. When competing for a common substrate (eg hydrogen), methanogens have a
lower utilisation efficiency and a higher threshold for hydrogen uptake as compared to
sulfate reducers. Consequently, methanogens cannot effectively compete with sulfate
reducers until all or most of the sulfate is deleted.
The classical sequence of electron acceptor utilisation is further illuminated but not altered
by recently recognised patterns of interspecies hydrogen transfer. Hydrogenoges, that
is, hydrogen producing fermentative microorganisms, are at a thermodynamical
disadvantage if hydrogen accumulates. They live syntrophically with hydrogentrophs,
hydrogen consumers, such as sulfate reducers, methanogens, and acetogens. The
syntrophic relationship allows the fermentation to proceed and at the same time supplies
the sulfate reducers or methanogens with the hydrogen necessary for sulfate or carbon
dioxide reduction, respectively. Recent measurements on anaerobic sewage sludge and
lake sediments showed that most of the hydrogen-dependent methanogenesis in these
ecosystems occurs via interspecies hydrogen transfer rather than by utilisation of
hydrogen dissolved in water.
In summary about the cycles of matter one should therefore realise that the major
elements nitrogen and sulfur are cycled in a complex, oxidoreductive fashion. Their
reduced forms support chemolithotrophic metabolism. Their oxidised forms are
used as electron sinks in anaerobic environments. Denitrification is balanced by the
agriculturally important process of dinitrogen fixation.
3.1 Interactions amongst Microorganisms
Microbial species rarely exist alone in nature. When two or more kinds of organisms are
present in a limited space, possibilities exist for interactions that can be beneficial or
harmful to one or more of them. When two organisms live together, the relationship is
called symbiotic. When both organisms are benefited by the association, the relationship
is called mutualistic, and when one organism is benefited and the other harmed, the
relationship is parasitic. Occasionally, one of the organisms is benefited and the other is
unaffected, in which case one refers to commensalistic. Finally, if two populations living
together have no effect on each other, the term neutralistic is being used.
Beneficial relationships in the microbial world vary widely, but two classes can be formed:
(1) relationships between two or more microorganisms; and (2) relationships between
microorganisms and higher order organisms such as plants and animals.

There is a growing literature describing many categories of interacting assemblages of
microorganisms (Bushell & Slater 1981), although one of the present difficulties is to know
exactly how significant some of these associations may be. However, it is possible to
suggest a simple classification of the various microbial communities which have been
isolated (Slater & Lovatt 1981).
1. Structure due to the provision of specific nutrients between different members of the
community
2. Structure due to the removal of metabolic products which may be inhibitory to the
producing member of the community, including hydrogen transfer communities
3. Structure and stability due to interactions which may result in the modification of
individual population growth parameters resulting i n a more competitive or efficient
community
4. Structure due to the effect of a concerted, combined metabolic capability, not expressed
by the individual populations acting alone;
5. Structure due to a cometabolic stage;
6. Structure due to the transfer of hydrogen ions;
7. Structure is the result of the presence of more than one primary substrate utiliser - in
many cases the nature of the interactions are unknown.

3.1.1 CommensaIism
There are a number of physical and chemical bases for relationship of commensalism.
This type of interaction often occurs, when the unaffected population in the course of its
normal growth and metabolism, modifies the habitat in such a way that another population
benefits because the modified habitat is more suitable to its needs. For example, the
production of growth factors forms the basis of many commensal relationships between
microbial populations. This is of particular importance in soil habitats, where some strains
produce and excrete vitamins which are required by other genera as growth factors. This
allows rather fastidious microbial populations to develop in natural habitats.
Another very common basis for commensalism occurs between fungi and bacteria. Some
fungi are capable of producing and excreting enzymes, which convert complex natural
biomass such as lignocellulose, cellulose, and starch into their monomeric forms glucose,
which can be readily used by a large microbial population. Such a system may exist in
oriental food fermentations used in many Asian countries.
3.1.2 Synergism
Synergism is a relationship whereby two populations may benefit from each other. Both
populations are capable of surviving in their natural environment on their own, but brought
together are capable of producing products which neither population could produce alone.

Synergistic relationships are very common in global geochemical cycling. Azotobacter
populations in soil are able to fix nitrogen if they have a sufficient supply of organic carbon.
Other soil bacteria are able to utilise the fixed forms of nitrogen supplying the Azotobacter
population with needed organc compounds.
3.1.3 MutuaIism or Symbiosis
Mutualism or symbiosis can be considered as an extended synergism and is an obligatory
relationship between two populations that benefits both. The best examples of such
relationship are the Iichens, a relationship or association of an algae or cyanobacterium
with fungi. This relationship usually leads to the formation of a plant-like structure called a
thallus, which can be seen by the naked eye. These lichens are widespread in nature and
found often to develop on bare rocks, tree trunks and house roofs. Lichens are essential
food for the reindeers in the northern part of Scandinavia.
The mutualistic association in a lichen is very delicately balanced and can be disrupted by
environmental changes (Gilbert 1969). Although they occupy some hostile habitats, they
are particularly sensitive to industrial air pollution and have been disappearing from
industrialised areas. t appears that the sulfur dioxide inhibits their development.

3.1.4 AmmensaIism
When one microbial population produces a substance inhibitory to other populations, the
relationship is called amensalism. Amensalism lead to the preemptory colonisation of a
habitat. The production of lactic acid or similar low molecular weight fatty acids is inhibitory
to many populations (Wolin 1969). Conditions that favour antibiotic production are not
normally found in natural habitats, as antibiotics are secondary metabolites. Therefore it
appears that antibiotics do not play a major and widespread role within soil and aquatic
habitats (Waksman 1961), but can be destructable if introduced by man.
3.2 Microorganism-PIant interactions
Microorganisms not only exhibit relationships of neutralism, commensalism, synergism,
mutualism etc. among microbial populations, the same types of interactions als occur
between microorganisms and plants (Fitter 1985). Some of these interactions are
beneficial to both the plant and the microbial populations, whereas others are detrimental
to either of the two.

3.2.1 Rhizosphere [BeneficiaI Interactions]
Symbiotic nitrogen fixation. One of the most important mutualistic relationship between
microorganisms and plants involves the invasion of the roots of suitable host plants by
nitrogen fixing bacteria, eg Rhizobium, resulting in the formation of a nodule within which
the bacteria are able to fix atmospheric nitrogen (see nitrogen fixation). This symbiotic
fixation of nitrogen is of extreme importance for the maintenance of soil fertiliyt and, in
agricultural practices, to increase crop yields.

Plant roots provide suitable habitats for the growth of microorganisms. nteractions
between soil microorganisms and plant roots satisfy important nutritional requirements for
both the plant and associated microorganisms (Katznelson 1965; Rovira 1965). The
density of microorganisms can also be high in the rhizosphere soils, whereby the size of
the rhizosphere depends on the particular plant root structure, which contributes to the
establishment of the rhizosphere microbial population. Within the rhizosphere, plant roots
have a direct influence on the composition and density of microbial communities. This
interrelationship is based largely on interactive modification of the soil environment by
processes such as water uptake by the plants, release of organic chemicals to the soil by
the plants, microbial production of plant growth factors and microbially mediated
availability of mineral nutrients.

Plant root excretion of organic matter is influenced by the surrounding microbes in the
rhizosphere. Roots surrounded by microorganisms excrete many more carbohydrates than
sterile roots (Rovira 1969).
Just as plant roots have a direct effect on the surrounding microbial populations,
microorganisms in the rhizosphere have a marked influence on the growth of plants.
Microbial populations in the rhizosphere benefit the plant through increased recycling and
solubilisation of mineral nutrients, synthesis of vitamins, amino acids, auxins, and
gibberellins. They also may stay in antagonism with potential plant pathogens through
competition and development of an amensal relationship based on antibiotic production.

3.2.2 Mycorrhiza. Some fungi enter into a mutualistic relationship with plant roots,
whereby the fungi become integrated into the physical structure of the roots. The fungus
derives nutritional benefits from the plant roots, contributes to plant nutrition, and does not
cause plant disease. Mycorrhizal associations differ from other rhizosphere associations
between plants and microorganisms by the greater specificity and organisation of the
plant-fungus relationship.

There exist two basic types of mycorrhizal associatoin. n ectomycorrhizae, the fungus
(ascomycetes or basidiomycetes) forms an external sheath more than 40 m thick and
constituting up to 40% of the dry weight of the combined root-fungus structure. The
morphology of the root is altered, forming a shorter, dichotomously branching cluster, with
a reduced meristematic region. n contrast to the predominantly exogenous
ectomycorrhiza, endomycorrhizae invade the living cells of the root, which become filled
with mycelial clusters.

Microbial diseases of plants are also of ecologic and economic importance (Campbell
1985). Plant pathology is a very extensive field and can only be mentioned here. n a
broad sense, microbial diseases of plants cause malfunctions which result in the reduced
capability of the plant to survive and maintain its ecological niche. One of the best
examples is the potato blight in reland in 1845 causing starvation in that country. Since
obligately plant pathogenic microorganisms have a limited period of viability outside the
host plant tissues, it is possible to control plant pathogens of agricultural crops by
appropriate management procedures. These include the planting of resistant species and
using crop rotation. Plants also have natural resistance mechanisms and such resistance
can be genetically selected. Genetic breeding of crop plants allows for the selection of
resistant varieties. A good example of such a resistance breeding concerns the viral Fijian
disease of sugarcane, which was eliminated by Fijian researchers using the protoplasm
fusion method of genetical resistance breeding.

3.3 Microorganism - AnimaI Interactions

Microorganisms are important contributors to the nutrition of some animals. Many animals
lack the enzymes necessary to digest some or all of the food resources available to them,
and thus require the assistance of microbial populations. Of particular importance is the
ability of various microbial populations to produce the extracellular enzymes that degrade
complex plant polymers such as cellulose, which otherwise would be unavailable to the
animal. The activities of microbial populations in the gastrointestinal tract of mammals and
humans thus contribute to food digestion, supply vitamins and provide protection against
pathogenic invaders.
The best known and studied contribution of microbial population to food digestion occurs
within ruminant animals (Hungate 1975). The rumen harbors a great diversity of
microorganisms including species of Bacteroides, Ruminococcus, Succinimonas,
Methanobacterium, Butyrivibrio, Selenomonas, Succinivibrio, Streptococcus, Eubacterium
and Lactobacillus. The high diversity of microbial populations in the rumen allows the
microbial community to respond to changes in the ruminant diet.

The spread of disease among animal populations is an ecological process that is
dependent on the biological properties of the causative organism, the biological properties
of the host organism and abiotic and biotic factors that affect the transmission of the
pathogen between hosts. The relationship between pathogenic microbial populations and
host animal populations is important in determining the size and distribution of each
population. Resistance to microbial diseases is a measure of fitness that acts through
natural selection to determine the success of particular animal populations.

4. References
AtIas,R.M. & R.Bartha 1987 - Microbial Ecology. 2nd ed. The Benjamin/Cummings Publ.
Comp., California
BaIatti,A.P. & J.R.J.Freire 1996 - Legume inoculants. Selection and characterisation
ofstrains. Production, use and management. Kingraf, LaPlata, Argentina
BIackburn,T.H. 1983 - The microbial nitrogen cycle. n 'Microbial Geochemistry'
C.W.E.Krumbein, ed.), pp. 63-89; Blackwell Scientific Publications, Oxford
BoIin,B., E.T.Degens, P.Duvigneaud & S.Kempe 1979 - The global biogeochemical
carbon cycle. n The Global Carbon Cycle (B.Bolin, E.T.Degen, S.Kempe & P.Ketner,
eds), John Wiley and Sons, New York
Brock,T.D., D.W.Smith & M.T.Madigan 1984 - Biology of Microorganisms.
Prentice-Hall nc.
Brown,C.M. & B.Johnson 1977 - norganic nitrogen assimilation in aquatic
microorganisms. Advances Aquatic Microbiology 1,49-114
BusheII,M.E. & J.H.SIater 1981 - Mixed Culture Fermentations. Academic Press nc.,
London
CampbeII,R. 1985 - Plant Microbiology. E.Arnold Publisher, London
DoeIIe,H.W. 1975 - Bacterial Metabolism. 2nd ed., Academic Press nc, New York
EhrIich,H.L. 1981 - Geomicrobiology. Marcel Dekker, New York
Fitter,A.H. 1985 - Ecological Interactions in the soil environment: plant, microbes and
animals. Blackwell Scientific Publ., Oxford
Focht,D.D. and W.Verstraete 1977 - Biochemical ecology of nitrification and
denitrification. Adv. Microbial Ecology 1,135-214
Forage Information System 1998 - Biological Nitrogen Fixation. 2. Describe the benefits
of BNF in economic and environmental terms.
Http://web.css.orst.edu/Classes/NFC/Topics/BNF/2/Body.html
GiIbert,O.L. 1969 - The effect of SO2 on lichens and bryophytes around Newcastle upon
Tyne. n European Symp. On Influences of Air Pollution on Plants and Animals. Center for
Agricultural Publ. & Documentation, Wageningen
GottschaIk,G. 1979 - Bacterial Metabolism. Springer-Verlag, Heidelberg
Hobbie,J.E. & J.M.MeIiIIo 1984 Comparative carbon and energy flow in ecosystems. n
Current Perspectives in Microbial Ecology (M.J.Klug, C.A.Reddy, eds.) American Society
for Microbiology, Washington, DC, pp 389-393
Hungate,R.E. 1975 - The rumen microbial ecosystem. Ann.Rev.Microbiology 29,39-66
KatzneIson,H. 1965 - Nature and importance of the rhizospehere. n Ecology of soil-borne
plant pathogens (K.F.Baker & W.C.Snyder, eds.), pp 187-207, Univ. California Press
Koike,I. & A.Hattori 1978 - Denitrification and ammonia formation in anaerobic coastal
sediments. Applied & Environmental Microbiol. 35,278-282
NeaIson,K.H. 1983 - The microbial iron cycle. n Microbial Geochemistry (W.E.Krumbein,
ed.), Blackwell Scientific Publ., Oxford
Rovira,A.D. 1969 - Plant root exudates. Botanical Revs. 35,35-57
SIater,J.H. & D.Lovatt 1981 - Biodegradation and the significance of microbial
communities. n Biochemistry of microbial degradation (D.T.Gibson, ed.), Marcel Dekker,
New York
Waksman,S.A. 1961 - The role of antibiotics in nature. Perspectives of Biological
Medicine 9,271-278
WoIin,M.J. 1969 - Volatile fatty acids and the inhibition of Escherichia coli growth by
rumen fluid. Applied Microbiology 17,83-87

Horst W. DoeIIe, DSc, DSc [hc]
Deputy-Director, MIRCEN-Biotechnology Brisbane and Pacific Regional Network;

Chapter 3
BIOTECHNOLOGY - oId and modern concepts
[This chapter has been adapted from a publication, which appeared as: Biotechnology. n:'Biotechnology'
[eds. H.W.Doelle and E.DaSilva], Encyclopedia for Life Support Systems (EOLSS), Oxford Univ Press]
Content:
2.6 SociaI Aspects of BiotechnoIogy
4. BibIiography
Biotechnology is a technology using biological systems and parts thereof. Since the basic
unit of any biological system is the cell, any biotechnological approach will and does
involve living and/or resting cells or their enzymes of many kinds, such as bacteria
(prokaryotes), yeast, fungi, plants and animals (eukaryotes) including man. Depending on
the specific purposes and needs, wild-type cells, natural mutants or genetically modified
cells are employed. n most cases, the cells are not grown under natural conditions but are
cultivated under more or less strict control in semi-artificial or artificial environments.
Biotechnology has its roots in fermentation (see chapter 10), a process requiring a ferment
to convert complex molecules into different chemical compounds. Fermentation itself has
been practised for many centuries. Bread, cheese, pickled cabbage together with beer,
mead and wine making are believed to have occurred under Egyptians, Romans, Greeks
and Germans around 5000 BC. Algae of the genus Spirulina were harvested for food from
alkaline ponds by the Aztecs in Mexico. The origins of a variety of indigenous 'fermented'
foods and sauces in Africa and Asia using surface culture methods go back thousands of
years.
The earliest report of the observation of microorganisms is that of Antonie van
Leeuwenhoek in 1680 describing them as 'animalculus' (Prescott et al. 1993). t was in
1856, nearly two hundred years later, that Louis Pasteur concluded that yeast cells
(Leeuwenhok's animalculus) are the ferment converting sugars into ethanol and carbon
dioxide that the connection between the living cell and the products were established
(Pasteur 1861). A further milestone in this development came in 1881, when the medical
doctor Robert Koch pioneered the development of pure culture techniques and in 1897
when Buchner discovered the enzymes in yeast cells (Buchner 1897).
The establishment of pure culture techniques together with the now ever increasing
biochemical knowledge of unicellular functions [bacteria, yeast] led to the commercial
exploitation of these 'organic catalysts' in an industry, coining the name 'ndustrial
Microbiology' (Prescott & Dunn 1982; Rehm 1980).
The first successful culture of plant cells was achieved in the mid-1930s, but had to wait
until the 1950s and 1960s before the development of cultivation techniques allowed further
strain development (Petersen & Alfermann 1993; Taylor 2003). n a very similar
development, the classical mammalian cell culture techniques started only in 1910, when
Harrison and Carrel were able to establish complex media for growth. t was not until the
late 1950s that many of the nutritional requirements of cultured mammalian cells were fully
understood.(Marquis 2003)
As soon as it was realised that not only microorganisms, but also plants and mammalian
cells could be cultivated and used as organic catalysts in product formation, and that cells
of either source could be manipulated by transferring genes from one to the other, that the
term ndustrial Microbiology had to be widened and became, around 1980, Biotechnology.
t is of interest to realise that the word 'biotechnology' itself has gone through an
evolutionary development since it first was introduced in 1919 by the Hungarian
agricultural economist Karl Ereky. He coined his new word to cover the interaction of
biology with technology. The first use of the word in the English language appeared in the
journal Nature in 1933. The most important definition of biotechnology, however, was
published in 1938, when Julian Huxley stated that biotechnology will in the long run be
more important than mechanical and chemical engineering (Kennedy 1991). n 1962, the
'Journal of Microbiological Technology and Engineering' changed its name to
'Biotechnology and Bioengineering', whereby its editor, Elmer Gaden, used the word
biotechnology representing 'all aspects of the exploitation and control of biological
systems'. t was only in the late 1970s and early 1980s that the word became more
associated with genetic engineering.
t is therefore not surprising to realise why the first commercial applications of technology
of biological systems occurred with microbial cells around 1900. t was at that time that the
engineering profession got involved in microbial process development joining forces with
the microbiologists, and thus the subject areas of industrial microbiology [or applied
microbiology] and biochemical engineering evolved. Processes such as bread making,
cheese manufacture, brewing and distilling developed to meet modern commercial
requirements for large-scale production, high and consistent quality, competitive cost and
product variety (Prescott & Dunn 1982). During World War (1914-1918) in Germany,
baker's yeast grown on sugarbeet molasses was produced as a protein supplement for
human consumption [SCP] and during World War (1939-1945) it was Candida utilis
grown on sulfite waste liquor from pulp and paper manufacture. Commercial production of
lactic acid using Lactobacillus delbrueckii began in 1881 and citric acid around 1923. The
latter began as a surface culture method [= solid substrate fermentation](Doelle et al.
1994) with the submerged Aspergillus niger process being introduced after World War .
t is often forgotten that the first cars ever built used ethanol as fuel, produced chemically
as well as microbiologically. ndustrial alcohol and acetone-butanol fermentations
dominated until the 1950s, when oil became cheap and ethylene was introduced into the
market.
This enormous development in the chemical and microbial fermentation industry led to the
evolvement of the 'chemical engineer' and 'biochemical engineer', creating the term
'Biochemical Engineering' in the engineering sciences and 'Fermentation technology' in the
biological sciences. This was particularly pronounced with the start of the antibiotic
industries. Although Louis Pasteur suggested that the antagonistic effects of
microorganisms might have therapeutic potential, it was Alexander Fleming in 1928,
exactly 72 years later, who observed that the fungus Penicillium notatum is able to kill the
pathogenic bacterium Staphylococcus aureus. He was able to demonstrate that the
product of the fungus, which he called penicillin, displayed inhibition toward many
pathogenic bacteria (Fleming 1929). t took, however, until 1940 and the enormous
casualties in World War for the first commercial plant to be established in the USA,
starting the ever increasing search and production of antibiotics to combate infectious
diseases and thus improving health care.
n the context of the antibiotic industry development it should also be of interest to realise
that the ability to confer resistance to disease by vaccination was first described in 1798
and the first mass application of the first cholera vaccine was administered in 1885 (Koch
1892/93). Mass vaccinations against diphtheria started in the 1930s and further programs
and the development of new vaccines happened in the 1950s. This resulted in the
explosive development of the commercial production of antibiotics and vaccines around
the same time, only 50-60 years ago.
The elucidation of the double helix structure of DNA in 1953 by James Watson and Francis
Crick (1953) opened the floodgates for the development of gene technology in the field of
genetics, which was given the name 'genetic engineering' (Phler 1993). Since, by this
time, cultivation techniques for biological cells of microbial, plant and animal/mammalian
origin, had already been developed, the new gene technology soon spread to all biological
systems. t involves the formation of new combinations of heritable material by insertion of
foreign genes, produced outside the cell, into a host organism in which they do not
naturally occur. The first experiments in which DNA fragments were joined in vitro and the
recombinant molecule reintroduced into living cells were performed in 1973. Only two
years later, the basic technology for the production of monoclonal antibodies from a single
ancestor or clone was established. The basic information obtained in these experiments
together with new findings in all fields of the biosciences as well as in the chemical,
physical and computer sciences have led to what is now the development of modern
genetic engineering. This new powerful methodology can be regarded as a set of
biological, genetic, biochemical, chemical and physical procedures that enable the
localisation, isolation, characterisation, modification, synthesis and transfer of genetic
material (Phler 1993).
The application of gene technology or genetic engineering as a research tool has
undoubtedly changed nearly all areas of the biosciences and has dramatically increased
the rate at which data can be obtained.
The enormous and rapid development in plant and animal cell cultivation together with the
new gene technology applicable to all biological systems (Panyim & Angsuthanasombut
2003), led bioscientists and biochemical engineers to realise in the early 1980s, that the
existing terminologies did not encompass this development and it was decided to combine
fermentation technology, plant cell technology, animal cell technology, and gene
technology under the one name Biotechnology. Therefore, over the past 20 years, all
technological applications based on biological systems, whether small or large, rural or
urban, medical or non-medical, natural or engineered, fall under this one name
Biotechnology. What the inorganic catalyst represents for the chemical industries, finds its
counterpart in the organic or biological catalyst of the biotechnology industries.
Often misunderstood, biotechnology is best considered as the integration of the natural
and engineering sciences in order to achieve the application of organisms, cells, parts
thereof, and molecular analogues for products and services. Biotechnology generically
embraces a variety of bioprocesses in which the traditional and novel practices co-exist
and reinforce one another with accompanying socio-economic impact in the industrial,
environmental, agricultural, medical, pharmaceutical, energy, and food sectors of our
everyday life. Genetically engineered strains have been designed to produce potent
biopharmaceuticals, agrobiochemicals (such as biofertilisers), biopesticides, and
bioremediation agents (Kleinkauf & Dohren 1997). Biotechnology has even been used to
counteract deterioration of valuable objects of cultural heritage such as monuments,
ancient books and valuable paintings as well as outside strategic metals through improved
microorganism-mediated bioleaching. Different components of the microbial world
contribute through interlocking biogeochemical cycles to the recycling of nutrients in food
chains in aquatic and terrestrial niches, and to the bioconversion of waste residues into
useful products, reflecting the various kinds of physiological activities in the maintenance
and management of the planet's biological fabric (Rehm & Reed 1993).
There is no doubt that the application of gene technology or genetic engineering of plant
cells has made a tremendous impact on traditional agriculture (Sasson 1990; Thomson
2003; Karanya & Kahindi 2003)). The implantation of genes for pest resistance should
reduce the application of pesticides (Freman 2003; Moazami 2003). Other genes have led
to higher yields and to crops containing higher nutritional supplements. This field of plant
genetical engineering will largely replace and speed up the natural breeding, mutation and
adaptation processes. This field, plus the successes in animal cell cloning to produce
transgenic plants (Hefferon 2003) and animals (Wang & Yang 2003) has been revered for
its potential to be the Holy Grail solution to any of the world's problems. This thought has
also sent shockwaves through various communities, because it is feared that biological
tinkering is both dangerous and uncontrollable, and perhaps even immoral.
With its transformative capacity in fostering and driving the world's economy,
biotechnology has set in motion changing technical and market profiles for the health,
pharmaceutical, agricultural, food and environmental sectors. Coupled to this development
come the revolutionary networking information technologies, bioinformatics and its
applications in novel fields such as telemedicine, tissue engineering, edible vaccines,
transgenic food market, DNA-chip computers and bioprobes for use in space exploration
(Editor 2000). n fact, biotechnology will be a key and integral component of the network
household of the future, which is already on the horizon. Such advances and benefits will
be accompanied by concerns and problems that focus on biosafety, proprietary and
intellectual property rights and ethics. Cloning and the mimicking of life-sustaining
processes and their interaction with the financial markets will be some of the issues that
will continue to make the headlines.
Despite the ever increasing new direction of biotechnology over the last two decades, it
should not be forgotten that the practice of basic biotechnology from several hundred
years ago is still with us (Prescott & Dunn 1982; Rose 1982; Frazier 1967; Barzena &
Sineriz 2003). As outlined earlier, microbes have unwittingly been used by humans in
various fields. The cultural heritage of virtually every civilisation provides indelible
testimony of the domestication of microbial fermentations. Traditional foods of strong
cultural and national identity, such as kefir (Bulgaria), chichi (Venezuela), taettemjolk
(Scandinavia), dahi (ndia), mopwo (Kenya), leben (Egypt and Syria), tofu, tempeh
(ndonesia), soysauce (SEAsia)and many others are produced from fermentation
processes going back several thousands of years. Such traditional knowledge reinforced
by important research advances in the field of nutrition and food preservation has
contributed to the production of quality controlled fermented foods that are consumed by
of the world's population. Today, the preparation of fermented foods is widespread,
especially in Southeast Asia. Food products derived through the fermentative action of
microorganisms on seed, milk, meat, fish and vegetables are delicacies that supply
protein, and that fortify predominantly starchy staple diets with vitamins and other nutrients
badly needed by millions of people subsiding on marginal incomes. Fermented foods such
as tempeh, ontjom and tofu are today commercially processed in industrialised societies
as vegetable protein meat substitutes and as sources of meat-like flavours and sauces.
The start of the third millennium has therefore been marked by a paradox in the life
sciences. Modern genetic engineering techniques and sophisticated culture skills honed
into a fine art over the last two decades has given rise to a new agriculture. Crops have
been genetically altered to increase yields for food and energy production, or for
resistance to disease and pests, which in turn has led to a steady loss of the wild strains
and of biological diversity. How will this affect our natural cycles of matter and the
environment? Have we asked ourselves that question? How will genetic engineering affect
traditional food production and will pests overcome the resistance of pest resistant plants
as the bacteria developed resistant strains against the antibiotics produced?
During the past two decades, biotechnology has further expanded into socio-economics
and the field of sustainability. David del Porto established in 1999 the coefficient of
sustainability as

This field of socio-economic biotechnology moves away from the purely commercial
application into the rural and urban development level. Topics such as 'bio- integration',
'multi-product development', 'urban- and farm-level applications' have emerged which may
help to secure farm development and reduce the trend towards urbanization (Doelle 1998).
The introduction of biotechnology in the global economy has been marked by extensive
public debates on the role of science and technology in global society. Policy makers
around the world are beginning to understand the revolutionary implications of
biotechnology for economic transformation.

Our planet world has gone from the traditional heritage through the ndustrial Revolution,
the Green Revolution to the Biotechnology Revolution, which is claimed will be followed by
the Socio-economic and Socio-ecological Revolution (Doelle 1989). t can be argued that
every step in our historical development has improved health and living standard of man to
a certain extent. Have we, however, asked at what cost to people, the environment and
the natural cycles of matter vital for the existence of all biological systems have these
benefits come? (Doelle 2001). Can the biotechnology revolution rectify our earlier mistakes
and improve and sustain Nature's existence?
Over the past two decades, biotechnology has provided good evidence that the joined
forces of all disciplines in the sciences and engineering field are capable to work in this
direction. t would be wrong, however, to fail to take into account the concerns of the non-
scientific community in the future directions of our scientific programs. One of the key
problems is, of course, that we still have a major division in the world, between the
developed countries (mostly situated in the more temperate climatic zones) and the still
developing countries situated mainly in the tropical regions with 80 percent of the world's
population. There are indications that the economic and technological gap is widening
instead of closing (see chapter 4). What are the reasons for this and why does it happen
despite the Green and Biotechnology Revolutions?
There is no doubt today that the biotechnologists are facing a tremendous task to feed an
ever increasing world population, lifting their health standard through waste management
and disease control and at the same time making sure that the natural cycles of matter
and the ecological environment are sustained.
There is also no doubt that the biotechnology revolution started to make some inroads into
these tasks, although the road will be hard and rocky as the achievements will have to be
aligned to social structure, ethic and moral standards (Macer 2000) as well as
conservation of cultural heritage and biodiversity.
The basic and fundamental unit of biological systems is the individual cell. Whereas
microorganisms like bacteria and yeast are predominantly unicellular, septated fungi,
plants and animals are predominantly multicellular. n order to be able to use these cells
for biotechnological applications, it is important:
1. to find the cell and keep the cell alive, in other words to cultivate the cell;
2. to determine the optimal nutritional requirements for growth;
3. to determine the optimal and most economical requirements for product formation;
4. to find preservation techniques; and
5. to be able to modify the genetic structure of the cell to achieve the required product
formation with enhanced yields.
All five aspects not only require a thorough knowledge in growing and cultivating the cell
(chapter 7), but also in its thermodynamics (chapter 9, 10).
n sharp contrast to the usual requirements for academic research, organism isolation and
initial selection for an industrial process is dependent on a range of criteria that are
relevant to the optimization of the particular process. Their features may be morphological,
physiological, genetical, immunological and the sum of all these features of a
microorganism is referred to as its phenotype. A phenotype therefore represents any
visible and/or measurable characteristic or distinctive trait possessed by an organism. n
contrast, the genotype represents all genes possessed by a cell or organism. This
genotype can therefore be explored via phenotypic expression (chapter 5).
The new and fast developing area of gene technology, which has its basis in the
improvements of recombinant DNA technologies, allows us to expand the phenotypic
expression of a cell through rearrangements of its genotype.
Every manipulation of cells requires, however, certain precautionary measures, which fall
under the biosafety requirements (Simon and Frommer 1993). Any cell within biological
systems are potentially dangerous under certain conditions, in particular when cultivated in
artificial and thus un-natural conditions (Lonsane et al. 1994).
The development of agriculture over the past 50 years was given first priority in the 1950s
with the introduction of the Green Revolution. New mechanised farm management policies
did indeed triple the average yield per hectare of corn and many other crops. The
development of higher yielding varieties allied to the greater use of irrigation [water
resources], chemical fertilisers, pesticides and herbicides have played a large part in
ensuring that the world's food shortages are not even more acute Johnston & Sasson
1986; Sasson 1990). Although this development had been an enormous success in food
production, the adverse effects on the environment started to show very clearly about 30
years later. t has been estimated that today 40 percent of our soils are becoming infertile
and the crop yields are dropping again, tendencies towards large farms to justify
mechanisation drove farmers into the cities increasing urbanisation even more than
before, and the use of pesticides and insecticides reducing biodiversity significantly.
With the help of the new gene technology it was possible to transfer genes expressing
resistance to many pests into crop plants significantly reducing the use of pesticides,
herbicides and insecticides, and making them hopefully redundant in the not too far future.
Plant gene technology together with traditional plant breeding methods, micropropagation,
germplasm exchange and protoplast fusion have played a major role in this area and has
developed into a multi-million dollar industry. Today, approximately 80 percent of all corn
grown in the US is of the genetically modified pest resistant type. Cotton crops are another
success story in this field [see FAO].
Whether or not the pests will adapt and mutate to become again a pest to the crop
remains to be seen. Further possible disadvantages may arise that the pest resistance
may be transferred through pollination to weeds, which then also become resistant and
interfere with crop harvesting. Further disputable areas of concern are whether other
insects or useful microorganisms might be killed by these transgenic plants or whether the
foreign genes in the plant affect the consistency of the crop itself (Collard et al.2003;
Johnson & Hope 2003; Braun 2003). The biotechnologist working in this area has
therefore to be aware of the enormous concern by the communities in regard to the so-
called 'GMO food'. Can such changes have a negative effect on human health, or will the
effect be similar to simple natural mutation?
One solution to the concerns of cross-pollination suggested incorporating a so-called
'terminator gene', which would prevent the germination of the crop seed. Such a solution,
of course, raised enormous concern among farmers, who fear that the global agriculture
can now be dominated and ruled by a few seed producing companies in developed
countries and thus this idea had to be stalled. This is a typical example for the possible
consequences of a perfect solution to a problem.
The controversies in these areas are of a social and of ethical nature. Has the consumer
the right to choose its product to be used either as food in the house or as seed on the
farm. What about the neighbouring farmer of a transgenic plant user, who opposes
transgenic plant usage and fears that cross-pollination, may affect his crop.
The biotechnologist has also taken up the steadily reducing yield problem. Using gene
technology, new varieties producing higher yields have been developed with the most
spectacular success with transgenic rice being just recently released. Will the companies
release their secrets to make this new variety available to everybody?
Many other crops have been nutritionally improved through gene technology. However, the
questions raised are also of ethical and social nature (Macer 2003). Has the consumer the
right to refuse the product and will the producer clearly label its product so the consumer
will be able to choose its preference?
The new biotechnology area of gene technology has clearly proven it can help the farmer
in regard of pesticide control and yield, a tremendous success story in such a short time.
The debate over the potential environmental impacts of genetically modified organisms
has taken on significant dimensions and, if not resolved appropriately, threatens to pose a
serious impediment to the diffusion and hence development of agricultural biotechnology
[Brodnig 1999; Ashiya 1999]. These concerns are currently being addressed by the
Convention on Biological Diversity with 177 member countries as part of the negotiations
for a Protocol on Biosafety. The main objectives of the Convention are
a) conservation of biodiversity;
b) sustainable use of the components of biodiversity; and
c) fair and equitable sharing of the benefits arising from the use of genetic resources.
One of the main environmental concerns is the possible flow of genes from GM crops to
their wild relatives, which would be serious in cases, where the genes code for traits such
as herbicide resistance. Although the introduction of insect-resistant crops is considered
as an option for reducing the use of pesticides, there is also the risk that natural selection
could favour the proliferation of pests that are resistant to the natural insecticide. Such
potential environmental impacts cannot be neglected.

Regulatory approaches to the environmental aspects of biotechnology vary between the
major regions of the World. Whereas the US seeks to base its approaches on evolving
science along with a risk-benefit analysis, Europe has generally been in favour of the
precautionary principle that suggests a wait-and-see approach that requires further
elaboration and exploration of the possible risks of the new technology. Developing
countries tend to follow the Convention on Biological Diversity, as they are concerned with
some of the broader socio-economic ramifications of the environmental risks of
biotechnology. This is not surprising if one realises that the income gap between the fifth of
the world's people living in developed countries and the fifth in the developing countries
has increased from 30:1 in 1960 to 74:1 in 1974 and only 1/10 of the total spending on
R&D in biotechnology was spent in developing countries with 80 percent of the world
population (see MRCENs [Microbiological Resource Centres]. As was mentioned earlier,
the ecosystem has been disturbed by many farming operations, when fixed nitrogen is not
returned to the soil. This is even more so with successive harvesting and modern intensive
production techniques. An alternative to chemical fertiliser addition to supply the soil with
the nitrogen required is the use of biological nitrogen fixation (chapter 2) using the soil
bacteria Rhizobium and Bradyrhizobium. The development of gene technology has helped
to elucidate the enormously complicated nitrogen fixing process [nitrogenase complex] and
the improvement resulting from the incorporation of these bacteria in leguminous and other
plants. This provides the possibility of using leguminous plants to obtain free nitrogen from
the air to make the combined nitrogen available to the plant and other soil microorganisms.
The enormous benefits of such inoculants is slowly being realised in developing countries
such as Brazil and Kenya. However, the enormous potential of this nitrogen restoration
benefit has unfortunately so far been poorly exploited despite the low productivity in
agriculture
The field of gene technology has also shown a great impact on biopesticide usage in
agriculture. Although the former USSR used Bacillus thuringiensis in aerial sprays over
wide areas in the 1950s, it was gene technology together with the fields of protein
engineering and recombinant protein production, which firstly elucidated the Bt-toxin
protein, altered the protein and finally producing this biopesticide [Biotechnology MRCEN
Tehran, Moazami 2003]. Bt-toxin, together with other biopesticides, has been successfully
used against pests such as caterpillar in cabbage and other vegetable fields (Unesco-
MRCEN Training Course 1994).
t is unknown, however, whether the pest will become resistant. There have been reports
claiming that such a resistance has occurred. However, protein engineering is presently
used to overcome this problem. t is feared, however, that Bt-toxin as a microbial product
and agent may follow the line of antibiotics and here again only gene technology is able to
stay ahead.
Furthermore it has been observed that plants sprayed with Bt-toxin actively exudate this
compound through their roots into the rhizosphere. The question now posed is whether the
toxin will affect the microbial population in the rhizosphere, which is so important for plant
growth.
These few examples demonstrate the enormous success of the new biotechnological
development in agriculture, but the potential negative effects on the environment and
ecological management need careful monitoring. One of the most pertinent question is, of
course, how these improvements affect health [GMO food], re-use of biomass waste, and
the natural biogeochemical cycles of matter.
t should not be forgotten, however, that the traditional agriculture has also benefited
enormously through the achievements in plant and animal cell cultivation. These
cultivation techniques improved and reduced the time for new variety breeding
dramatically. Techniques such as micropropagation, germplasm exchange, protoplast
fusion etc were instrumental in producing the first pesticide resistant varieties in various
crops. For example, the devastating Fijian Virus disease in sugarcane was combated
using protoplast fusion techniques. Many plant breeders therefore believe that traditional
plant breeding techniques are a more natural way for pesticide resistant crop production
and do not believe that gene technology with its recombinant DNA techniques are
necessary.
t is therefore important to realise that our biotechnological progress and development has
not only created a new field of genetic manipulation, but has also benefited significantly
the improvement of the traditional techniques used in agriculture.
Antibiotic production started a new era for the pharmaceutical industry. t was an era
where everybody thought the 'magic bullet' was found for disease control and elimination,
securing an ultimate health and survival standard on this globe. Antibiotics made a
tremendous impact on the health of people and almost eradicated certain infectious
diseases from the developed world.
However, antibiotic use and misuse has soared since penicillin was first introduced on the
market and now includes many non-medicinal applications. n 1954, approximately 1
million kg were produced in the US, the figure exceeds 30 million kg now. Human
treatment accounts for roughly half the antibiotics consumed, whereas the other half is
being exploited in animal husbandry and even in aerosols to combat bacterial infections in
fruit tress. This over- and misuse has created serious problems not foreseen at the time of
the introduction of these drugs. Thus we are facing now the challenge of antibiotic
resistance. Worldwide, many strains of Staphylococcus aureus, one of the most deadly
bacterium, are already resistant to all antibiotics except vancomycin. We have again
reached the stage of having unstoppable killer bacteria and the death rate for some
communicable diseases have started to rise again, after having declined significantly in the
industrialised nations. t is time that people realise that although antibiotics are needed to
control bacterial infections, they can have broad, undesirable effects on microbial ecology.
The compounds should only be used when they are truly needed.
The new gene technology enabled us to find the course of this resistance build-up. Only
when we understand the reasons for resistance development, can we work or search for a
cure. t was found that the two main forces are the prevalence of resistant genes and the
extent of antibiotic use. f the bacterial biota in a community has no genes conferring
resistance to a given antibiotic, the latter will successfully eliminate infection. On the other
hand, if the biota possesses resistance genes and the community uses the drug
persistently, bacteria are able to defy eradication.
Through the new gene technology we were able to learn that bacteria can acquire
resistance genes through a few routes. Many inherit the genes from their forerunners and
at other times genetic mutation, which occurs readily in bacteria, will spontaneously
produce new resistant traits. t is also common, that bacteria will gain a defence against an
antibiotic by taking up resistance genes from other bacteria. Such a horizontal exchange of
genes is very common in the bacterial systems. Resistance genes are carried on plasmids
or occur occasionally on the bacterial chromosome. Viruses, that occasionally extract a
gene from one bacterial cell and inject it into a different one, can also transfer resistance
genes.
The extensive worldwide exploitation of antibiotics in medicine, animal husbandry and
agriculture constantly selects for antibiotic resistance bacteria. f the drugs are to retain
their power against pathogens, they have to be used more responsibly. Society, in
particular public health officials, physicians, veterinarians and farmers have to start to think
about the administration of these drugs to avoid further resistance build-up and reduce
their dangerous environmental impacts.
As well as combating the above mentioned infectious diseases, biotechnology and in
particular gene technology has been on the forefront in helping to cure human disorders,
which result from imbalances or defects in the body's chemistry (Reeves 2000). n the
case of hormones, genetic engineers turned their attention to polypeptide hormones, such
as insulin, growth hormones and nerve growth factors. The intense interest in insulin was
twofold:
(a) can genetically engineered microorganisms be efficiently incorporated into the
pharmaceutical industry?; and
(b) will the gene transferred from an animal cell into a microbial cell produce a safer
and cheaper product?.
n both cases gene technology triumphed and is able to help millions of diabetics today.
Further success stories can be seen in the use of growth hormones and steroid hormones.
Enormous potential benefits are hoped for in gene disorders, cancer treatment and
monoclonal antibodies. The fields of organ transplants and cardiovascular diseases are
under investigation and show very promising results.

The field of medical biotechnology may be the area of greatest success for gene
technology for improving health and increasing the life span of humans (Hattori et al.
2000).
Despite these enormous achievements of the new biotechnology in the medical and
pharmaceutical fields, infectious diseases are still on the rampage in developing countries
with 80 percent of the world population (see chapter 4). Since most of the spectacular
progress has been in areas of developed [industrialised] world health problems, do we
need some re-thinking in the area of medical biotechnology development ? The most
recent action by one drug company to decrease the price of its drug by 85 percent for its
use in Africa is certainly an encouraging sign
Highly significant developments in the area of medical biotechnology have undoubtedly
occurred in the diagnostic field. DNA finger printing for forensic purposes and also for the
early detection of genetic diseases in unborn children together with enzymatic analyses
and lab-on-chip technologies help not only to clarify which suspects were at crime scenes,
but help in the diagnosis of particular diseases at an early stage of development. The
combination of computer and enzyme technologies has revolutionized our pathology
laboratories.
The rapid pace of advances in current times has only been achieved on the basis of
understanding the life processes involved. Enormous advances in the chemical
engineering field, eg process fermenters and control, instrumentation, process
optimisation, upstream and downstream processing together with the developments of
appropriate physical and chemical methodologies all owed the development of a multi-
billion dollar biotechnology industry. These developments benefited many of the 'old
industrial microbiology' and made possible mass production of many new products. The
close cooperation between the biological scientists and the chemical engineering
profession made this proliferation possible. The range of commercial products is directed
towards medical health, pollution control and waste management. However, the majority, if
not all process industries, still remain a mono- or single product industry causing more or
less severe waste and environmental problems, albeit with steady improvement through
stricter regulations.
One of the most spectacular improvements in production processes occurred undoubtedly
in the ethanol industry. The re-discovery that ethanol can be used as motor fuel [replacing
gasoline or petrol] let the production rise from approximately 650 million litres per year in
1991 to a worldwide production volume of 33 x 10
9
L per year in 1997 and is still
increasing. The largest market for fuel ethanol can be found in Brazil and in the USA. The
reasons for this increase in production were both, control of pollutants emitted through the
exhaust pipes of internal combustion engines of gasoline- and diesel-powered vehicles,
and foreign exchange savings in the case of Brazil. n the US, this industry created a
significant boost to the US corn farmer being able to sell their corn and thus secure their
income. The residual product from the corn-ethanol industry is being used as animal feed
supplement, using the microorganisms as a beneficiary protein supplier. n areas where
cars are driven by 100 percent ethanol as fuel, air pollution through carbon monoxide (CO)
has been reduced significantly, improving health standards of man. The same has
occurred in many cities of the US where ethanol was used as a blend with gasoline. The
enzymatic production of high fructose corn syrup during the period of sugar shortage in the
USA was yet another success.
Apart from improving old technologies, enormous progress has been made in the
production of biodegradable polymers, bioplastics and biosurfactants, which are all
biodegradable and thus friendly to the environment. These polymers, such as dextrans,
xanthans, etc., are all produced by microorganisms under certain cultivation conditions
and find use mainly in our food industries, medicine and in households. A large field for
gene technology applications has been opened for the search for new biopolymers of any
kind.
The extensive use of petrochemicals by our industries has led to significant environmental
problems over the past decades. Chemicals from these industries include the earlier
mentioned pesticides and insecticides, but also oil from spills and inorganics from mining
industries. To combat the deleterious effects of these chemicals and secure a clean water
supply, a whole new area concerned with bioremediation has developed (Klein and Winter
2000). Since most of these chemicals from the chemical industries are of the synthetic
type, the natural biological systems are not sufficiently prepared to degrade effectively and
completely these toxic compounds and it is again gene technology, which can help to
overcome these shortcomings of the wild types. Decontaminating our ecosystem requires
gene technology to obtain microorganisms capable of degrading the so-called xenobiotic
compounds and other artificial chemicals seeping through our soils into our water supply
(Klein 2000).
The improvements in the process industry has also made it possible to produce new
pharmaceuticals connected with recombinant DNA, such as recombinant protein
production, the production of monoclonal antibodies from mammalian cells and secondary
products from plant cells (Kleinkauf and Dohren 1997). These enormous achievements in
commercialisation of products from mammalian and plant cells, required and were due to
the success in the development of new methodologies such as biosensors and many other
analytical instruments.
The improvements in the process industry has also made it possible to produce new
pharmaceuticals and nutraceuticals with or without recombinant DNA. Examples for the
former are recombinant protein, enzyme, antibiotic, biopolymer and bioplastic as well as
biosurfactant productions and the already mentioned biopesticide production. On the other
hand, improvements in cultivation techniques make it possible to obtain nutraceuticals
from mushrooms and pharmaceuticals from plants and algae.
The recent strong development in all areas of biotechnology should therefore be visualised
as occurring with and without the use of gene technology.
The basic natural cycles of matter originally maintained our environment, when all
biological systems were in balance (chapter 2). An increasing population over the past
century, and their increasing demands for food and other products led to increases in
animal breeding, farmland use, as well as the establishment of many chemical industries.
As a consequence, the organic content of domestic, agricultural and industrial wastes is
causing large pollution problems, overloading our environment with utilisable carbon, and
also with organic compounds foreign to natural degradation cycles. This has led to the
contamination of our soils and water resources with soluble and/or insoluble organic and
inorganic material, which can only be purified and kept clean by a combination of
mechanical, chemical and/or biological purification procedures.
The treatment of all kinds of human wastes has a long history and reaches back to
Egyptian and Roman times with procedures depending on the lifestyle of the population
and legislation. Unfortunately, this aspect of waste treatment has not changed, since major
waste problems of this kind still exist for 80 percent of the world population, causing
illnesses and severe health problems (chapter 4). n developed countries, the first
treatment systems were established around 1910 with the construction of public sewer
systems and have improved over the years giving these countries a high living and health
standard.
Wastewater was perceived not only as a waste, but also as a raw material for enhanced
agricultural or aquatic production. Apart from the fertilising effect of sewage sludge, the
wastewater itself could also serve as fertiliser for agricultural soil.
One of the major 'biotechnological' discoveries was, however, the realisation that
anaerobic treatment of these wastes can produce methane or 'biogas', a valuable energy
resource for cooking and other purposes (Hobson & Wheatlet 1993). Although the first
anaerobic digesters were developed in ndia, China was, and still is, on the forefront of
making the so-called biogas available for cooking, and appropriate generators were
developed to use the biogas for electricity and power generation in the Philippines. This
new biotechnological development is now spreading from these countries around the
globe as one of the many possibilities of renewable energy or alternative energy resource.
Over the past decades, this type of waste treatment has been further explored scientifically
and now includes all types of animal and agricultural wastes or biomass.
Since the Green Revolution, pesticides, insecticides, xenobiotic compounds and
inorganics have started to seriously damage our environment. New biotechnological
techniques such as genetically modified organisms with enzyme mutants were found
enabling the degradation of these artificial and non-natural compounds.
The debate over the potential environmental impacts of genetically modified organisms
has recently taken on significant dimensions. The concerns are largely based on the
assumption that new products may have harmful effects and need to be introduced in a
precautionary manner. One of the main issues of concern is the possible flow of genes
from GM crops to their wild relatives, which would have disastrous consequences in the
case of genes coding for traits such as pesticide or insecticide resistance and also for the
disturbance and reduction in biodiversity.
Environmental biotechnology is a complex subject (Winter 1999; Klein 2000; Klein &
Winter 2000). One expects that the familiarity with nature might mean rural communities
are much more knowledgable about the ecological cycles than urban dwellers, which have
in general a much higher per capita income compared to the farmer. t is therefore
alarming to realise that the effect of technology on the rural depopulation and dramatic
decrease in the number of farms, which in turn has a dramatic effect on culture, families
and employment for the unskilled.
n order to make further progress, biotechnology has to develop the area of sustainable
agriculture (Pretty 1998), which essentially means to utilise and regenerate local or internal
resources more effectively and make better use of available biophysical and human
resources. There are an increasing number of proven resources - conserving and
regenerative technologies for pest, nutrient, soil, water and energy management. Many of
these have arisen on farms where steps have already been taken to reduce both costs
and the adverse environmental effects. Wastes from one sector of the farm are becoming
inputs to another sector. n this way farms remain productive and negative impacts on the
environment are reduced. Such a development is often referred to as 'old' or 'low
technology'. This is one of the most fundamental misconceptions amongst scientists.
Sustainable agriculture and environment quality mai ntenance implies an incorporation of
recent innovations that may originate with biotechnology development, with farmers, or
better yet with both (Doelle 1998).
2.6 SociaI aspects of BiotechnoIogy
Biotechnology has gone through a tremendous development over the past 20-30 years,
mainly due to the discovery of new instruments, computers, process equipment and gene
technology. This rapid development is one of the reasons why biotechnological products,
especially GM foods have entered international trade at a rapid rate (Brodnig 1999). This
rapid entrance into the international trade has, of course, caused considerable concern,
especially with GM foods as was mentioned earlier. General concerns about the overall
impact of globalisation is not only based on environmental and human health grounds, but
also on the overall trading framework. Biological resources and their use have been a key
element of international diplomacy, mainly in regard to the biological resources on the one
hand and advances in molecular biology on the other. A host of multilateral organisations
are involved in policy-making, harmonisation of national regulations and the administration
of a wide range of legal instruments (Ashiya 1999). One of the most significant
developments associated with the 'new biotechnology' has been the strengthening of
intellectual property protection for biological inventions. ntellectual property rights arise
from the information generated from genetic resources. They are the legal instruments,
that confer protection on processes or products of research and development efforts and
formally assure the allocation of benefits to the inventor in return for full disclosure to
society. Access to a particular process is therefore likely to be dictated by the degree to
which corporations are willing to license key technologies to smaller corporation and
developing countries and the cost at which they are willing to do so. This is of particular
concern to developing countries, which may view biotechnology as a potentially fruitful
economic sector and yet may not be able to achieve this objective due to high licensing
costs.
n this context, bioprospecting (Sagar & Daemmrich 1999) is another area of concern at
present. Bioprospecting refers to the systematic identification and development of new
sources of chemical compounds, genes, organisms and other economically valuable
products found in nature. t therefore has relevance to biodiversity conservation as well as
to the biotechnology industry. The anti-cancer drug Taxol from the bark of the Pacific yew
tree is a typical example. The attractiveness of bioprospecting agreements for developing
countries is based on expected benefits from the use of resources from their territories.
Whereas bioprospecting offers a potentially interesting approach for linking biodiversity
conservation with the biotechnology sector, critics of bioprospecting have in turn charged
that these practices could result in 'biopiracy', the use of intellectual property laws to gain
exclusive monopoly control over genetic resources without just compensation to the
source country, let alone to local farmers.

"Biotechnology industries question the scientific basis of the consumer protests
and have accused these consumers of slowing down scientific development.
However, this remains an inadequate response as biotechnology industries are not
addressing consumer preferences and the basis of consumers' decisions, be this
scientific, political, ethical or religious reasons or just a matter of price and taste.
The rift between private industries and consumers indicates once again that science
and technology cannot and should not be developed in isolation...Farmers are
important actors in the supply and demand of biotechnology. Despite the strategic
role of farmers, research and development in biotechnology, be it private or public
sector, has often ignored farmers needs in developing countries.'[Editorial,
Biotechnology and Development Monitor 41,2000]

The development of biotechnology, in particular the rapid growth of biotechnology
industries in developed countries has also impacted international politics in the various
regions. At the present time the social, political and educational aspects of biotechnology
are becoming more and more important. t is therefore not surprising to realise that the
survival of mankind requires a different approach from the present, commercially driven,
biotechnology, which means a different approach to what is called economical in
developed countries. The opposition to many of the products of biotechnology, in particular
GM foods, indicates that the individual society or communities in general are not
consulted, involved and/or educated in the vast development. The realisation amongst
leaders of organisations and governments that processes which are economical for one
nation may not be economical for another (irrespective of whether these countries are
developed or less developed), lead to the development of a socio-economic approach for
a sustainable agriculture. n the future, the new gene technology approach as well as
newly developed process technologies, may find that they can only succeed in joining
forces with these new strategies. t means that all technological developments should be
aimed at improving the quality of life of a community of people and that developing
countries are looking for programmes reducing the risk to health and achieving
sustainable, economic growth conducive to a higher per capita income of the community. t
has become very clear over the past two decades that the major beneficiaries of the new
biotechnology revolution are of high socio-economic status. This can easily be shown by
WHO (2001) figures that the death rate amongst children is still on the increase and that
around 90 percent of these fatalities occur in developing countries.
Socio-economics assumes therefore that economics is embedded in society, politics and
culture, and is not a self-contained system. Sustainability and sustainable development
means 'the development that meets the needs of the present without compromising the
ability of future generations to meet their own needs'. There are an increasing number of
process resource-conserving and regenerative technologies for pest, nutrient, soil, water
and energy management. Many of these have arisen from farms and should be taken up
by biotechnologists for further improvements. n this way farms remain productive as well
as reducing negative impacts on the environment. The important issue is to conserve
existing farm resources and introduce new elements into the farming system that add to
the stocks of these resources.
n this aspect of sustainability it should not be forgotten that we have apart from the rural
community also an urban community. Urban agriculture is concerned with growing food in
urban spaces, especially from urban waste. This represents an important paradigm shift in
food production as both intensive food production techniques and improved urban waste
treatment technologies develop in parallel. t means growing food where it is needed to
reduce transport costs and enhance freshness. The most successful urban agriculture
integrates agriculture, horticulture, aquaculture, agro forestry and mini-livestock husbandry
with sound water management and waste management, plus helping urban planning and
regulation.
These socio-economic and/or integrated biosystems are becoming extremely popular in
the densely populated areas of the developing countries, at present mainly in rural areas
to stop or at least minimise the trend into the cities. These systems are exploiting the
natural resources for food, feed and fertiliser production incorporating a good waste
management for the production of energy, food, biofertiliser and water recycling. t
combines the food production and removal of soil nutrients with a replenishment of the soil
to secure soil fertility and farming stability (Doelle & Foo 2000).

Where will the biotechnology revolution lead us in the future? There is no doubt that gene
technology will continue to be of great importance and become even more vital in the
future. The most exciting area will undoubtedly be medical biotechnology. Establishing the
human genome sequence will help combating hereditary defects and reduce our disabled
population. Recombinant proteins and monoclonal antibodies will help us in cancer
therapy and related illnesses. However, securing general health and food production can
only progress if gene technology and the socio-economic movement join forces. One
should not forget that GM foods are absolutely useless, if the soils are infertile. Natural and
GM crops require a healthy soil, which can only be maintained by sound waste
management and an integration of food production with sound waste and soil
management.
The areas of bioremediation, marine biotechnology, biotransformation (Kelly 2000; Kelly &
Peters 2000) are areas open for immense development.
The biotechnologist has to become aware, however, that clean technologies are required,
which foster the environment and secure our food supplies. Furthermore, the
biotechnologist has to be aware that in the not too distant future, all presently used non-
renewable process industries have to be replaced by renewable process industries. This
enormous challenge requires massive input by a joined force of biologists, engineers and
other disciplines. t will be the farmer producing the renewable resources, who will be at
the centre of this development and thus no product will succeed without the involvement of
the individual societies at large in the future development of old and new biotechnological
systems.
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Chapter 4
Biotechnology and Human Development.
Part of this article has been published in the Electronic Journal of Biotechnology [see http://www.ejb.org]
Content

1. Introduction
2. The DeveIopment of RuraI and Urban Societies
2.3 DNA TechnoIogy
2.4 ReIigions and Ethics
3.1 HeaIth
3.2 Poverty
3.3 Starvation
3.4 Waste Management and RecycIing CIean and Green TechnoIogies
6. References
1. Introduction
Of all the life forms occurring in nature within the biodiversity there should be no doubt to
realise that microorganisms are the most powerful creatures in existence. They
determine life and death on this planet. They can kill merciless humans, animals and
plants, but at the same time they can be harnessed to sustain life (Doelle 1997; Doelle et
al. 1987; DaSilva et al. 1987; Sasson 1988; DaSilva & Sasson 1989). Nature has provided
us with a perfect balance in Carbon, Nitrogen and Phosphorous cycles (see chapter 2) to
sustain microbial, plant, animal and human life. Any interference in these cycles can swing
the pendulum very quickly into the direction of killing or sustaining mankind. It is the
microorganism, which determines the growth and existence of pIants, animaIs and
humans on this pIanet. t is therefore of utmost importance that we give microbiology a
first priority, as we have to isolate and investigate the biochemistry and behaviour of the
microorganism in order to understand how nature works. Only when we obtain this
information will we be able to sustain and improve life in our community. The
microorganism is much more flexible and adaptable to environmental changes than plants,
animals and humans.
Biological resources constitute an asset with a great deal of immediate as well as potential
benefit for the quality of life. These resources evolved through evolutionary processes of
life on earth involving genetic differentiation and genomic adaptation to a
microenvironmental variation of habitats. As a result these biological resources consisting
of a tremendous diversity of species of microorganisms, fungi, pIants and animaIs
occur in a variety of ecological settings throughout the world, from the richest areas in
tropical to the poorest in the temperate and cold regions of the planet earth. Such a variety
of life forms is collectively known as biological diversity or biodiversity. Biodiversity
therefore encompasses all levels of organisation of life from genes to populations, species,
ecological communities and ecosystems. They are essential resources for human survival
(Baimai & Brockelman 1998; Braun & Ammann 2003). The human species is therefore an
integrated part of this biodiversity.
2. The DeveIopment of RuraI and Urban societies
Since his appearance, man has always lived in an uncertain, sometimes precarious,
symbiosis with nature, obtaining his nourishment needed from plants and animals
personally accessible to him. n accordance with the climatic and environmental
conditions, the search for food (life as Nomades) soon developed into actively growing,
storing and preparing the food (life as Settlers and Farmers). Water, sun availability and
soil conditions determined the type of original food for the particular society.
t was the farmer, who was responsible for the growth and harvesting of the crops,
whereas the families used their own recipes for the conversion of the agricultural products
into edible and palatable products for themselves, their animals and later the market place.
This practice is still followed today in many societies in SEAsia, Africa and Latin America.
The culture of biotechnology originated therefore in the rural areas, where people
experimented with the regeneration of soil fertility, breeding of new crop varieties and
fermentation for a palatable and well digestible food. Whereas cold and temperate zones
used mainly grain for their food and fermentation, tropical zones of SEAsia and Africa
produced numerous foods from rice, soybean, cassava [manihot] and other plants ( see
chapter 17 ).
A second type of fermentation technology was accidentally introduced very soon in form of
beverages such as wine, met [honey beer], and beer and in different types of food such as
bread, milk products such as cheese, butter and yoghurt. Whereas the societies of cooler
climates preferred beer and wine from barley and grapes, respectively, it was pulque from
the sweet juice of the Mexican Agave in Aztec countries of Latin America, the saki from
rice in SEAsia and the palm wine from palms in some societies of Africa. Other societies in
Africa did not encourage this second type of fermentation out of personal and/or religious
beliefs.
Since food preparation and fermentation was carried out according to 'local society
tradition', handed down from generation to generation, these complex preparations were
much more an art than a science. Nevertheless, the traditionally fermented protein-rich
foods are highly acceptable to millions of people until today, because they are easily made
and are generally more attractive to the consumers than the cooked original substrates.
The organoleptic characteristics of the substrate are improved by the fermentation
process. These fermentations also increase the nutritional value of the substrate, since the
amount of vitamins are significantly increased as well as the digestibility. f properly
fermented, these foods are not hazardous to health since the microorganisms responsible
for these processes are not toxin producers (Wang & Hesseltine 1982).
t was not before A. van Leeuwenhoek in the 18th century was able to construct the first
microscope and Louis Pasteur in the 19th century found the reasons for the fermentations
to occur (Doelle & DaSilva 2003), was the complexity of the traditional food fermentation
realised (see also chapter 17).

Figure 1: DeveIopment of societies

The impetus of the industrial revolution during the 18th and 19th centuries transformed the
very nature of society in many parts of the world, which are now referred to as the
'developed countries '. These societies were now not only using renewable resources, but
also consuming vast amounts of non-renewable resources. The industrial society
developed by the accumulation of scientific knowledge, the spread of technological
innovations, and the exploitation of enormous natural resources. Traditional vegetable and
animal fibres were increasingly replaced by synthetics manufactured by an ever increasing
chemical industry from coal and petrochemicals. This development in the 20th century
fundamentally altered the pattern of consumption, land use and international trade, and the
distribution of wealth (King & Cleveland 1980). Longer life expectancy, higher survival
rates, and dramatic population increases followed through better housing and sanitation,
the production of antibiotics and vaccines. The quality of life was improved by the
introduction of petrol and the motor industry among others. The impact on society was
dramatic and on culture devastating as a large proportion of the traditional way of life was
lost through this development owing to an ever increasing urbanisation.
The majority of societies on this planet, however, were bypassed by the grey revolution
and chemical industry development. These countries are in general referred to as
'developing ' or 'less developed ' countries and are situated in two climatic zones, the
tropical wet and the tropical arid zones. ronically, the largest reservoirs of non-renewable
resources required for the development of the industrial revolution in the 'developed'
countries were found in countries of the tropical arid zone. The exploitation of these non-
renewable energy sources situated in predominantly dry dessert areas, where agricultural
food production has always been minimal, led to large increases in population which
became totally dependent on food imports. The resulting starvation, malnutrition together
with an unavailability of antibiotics for the fight against diseases kept living standards and
survival rates at a relatively low level.
The green revolution (Sasson 1990) was introduced to secure food for the ever increasing
population in all parts of the world.
The question therefore arises, can scientific knowledge and technology improve quality of
life, life expectancy and thus increase the survival rate for all societies without affecting
culture and society in such disastrous ways as occurred in the developed countries ?
The reasons for such an impact on culture and society are manifested in the principles of
the 'industrial systems' organisation'(Fernandez & Ocampo 1980),which dominates today's
society in the developed countries. This organisation is based on short-term profit, with a
production to sell attitude, with preference given to the production of luxury consumer
goods over goods required for basic needs, particularly at the level of the large energy
systems, such as coal, hydrocarbons, and nuclear energy. Such a production is an
obstacle to the total realisation of individuals and society.
The principal energy sources of antiquity were all derived directly from the sun: human and
animal muscle power, wood, flowing water and wind. About 300 years ago, the industrial
revolution began with stationary wind-powered and water-powered technologies, which
were essentially replaced by fossil hydrocarbons: coal in the nineteenth century, oil since
the twentieth century, and now, increasingly, natural gas(Hall et al. 2003; Chow et al.
2003). The global use of hydrocarbons for fuel by humans has increased nearly 800-fold
since 1750 and about 12-fold in the twentieth century.
Hydrocarbon-based energy is important for the three main areas of human development:
economic, social and environmental. Energy prices have an important effect on almost
every major aspect of macroeconomic performance, because energy is used directly and
indirectly in the production of all goods and services. Both theoretical models and empirical
analyses of economic growth suggest that a decrease in the rate of increase in energy
availability will have serious impacts (Smulders & de Nooij, 2003).
The results of these chemical and manufacturing industries are accompanied by ever
increasing amounts of effluents of both heat and toxic substances, many of which are non-
biodegradable. Modern agriculture is now strongly based on the application of chemical
fertilisers and ever-increasing amounts of organic pesticides, mainly as a consequence of
an enormous and rapid expansion in world population demanding an ever greater quantity
with increasing quality of food and goods of all kinds. This, in turn, encourages the use of
still further quantities of non-renewable resources and energy. This development led in the
1970s to a turning point in the perception of man's relation to his natural environment, the
biosphere, as well as a shift in man's relationship to the man-made environment, the
technosphere (King & Cleveland 1980). The question was raised whether the earth and its
atmosphere can provide an infinite sink and absorb the waste products of industry,
agriculture and urban living as they become more and more prevalent. The processes of
physical planning are now challenged and well established procedures are under severe
scrutiny.
The green revolution was introduced to secure the food for the ever increasing population
in all parts of the world as a consequence of the industrial development (Sasson 1990).
Thus, the 'industrial system organisation' was introduced into agriculture in the hope that
an equivalent development could be achieved. The introduction of farm mechanisation and
monoculture systems initially brought the promised results of higher yields, but drove the
small farmer into an already overflowing urbanising area and produced a wonderful
breeding ground for insect and other crop pests (Oerke et al. 1994). Farm mechanisation
also lead to a destruction of the natural soil structure and severe nutrient deficiencies. As a
consequence, chemical fertilisers and pesticides were developed and introduced to
combat these deficiencies. After the initial boost in productivity it was soon realised that
the indiscriminate use of fertilisers killed and changed the microflora in the soil and around
the plant causing ever increasing soil infertility and salination (Vierling & Kimpel 1992). The
pesticides in form of chemicals foreign to nature's soil population and thus undegradable,
destroyed not only mixed populations of microorganisms necessary for the ripening of the
crops and subsequent fermentations, but also killed many insects which served as food for
many birds. Furthermore, both types of chemicals often applied in excess appeared in
waterways endangering human and animal life. t is becoming more and more clear that
many changes in the human life [eg immune system] could be due to the application of
these man-made chemicals.
The industrial and agricultural development over the past century has, unfortunately,
changed our society from a nature caring and observing society into one full of obsession,
greed and disrespect, a so-called 'throw-away society, which does not care anymore about
its own environment and the beauty of nature (Chantalakhana 1998). These phenomena
pose a serious threat to sustainable development and species diversity may well be our
planet's most important and irreplaceable resource (Mahidol & Puchirawat 1998; Schell &
Mayer 1987). ndustries and urbanised societies started to use Nature as a bottomless
sink, into which one can throw anything not wanted. Our planet earth is therefore in an
existential crisis, which is of an ecological as well as an economic nature (Wohlmeyer
1987).
Throughout the past century, humankind has made a tremendous effort to understand the
biological intricacies of nature. t started with the traditional fermentation of food to the
commercial exploitation of all types of biological cells. The most incredible advances
occurred since the mid 1940s with the discovery of the life saving antibiotics to the present
rapid progress in understanding the genetic basis of the living cells. The latter progress
has led to the development of new products and processes useful in human and animal
health, food and agriculture, and the environment (ADB 2001). t appears, however, that
these enormous discoveries could not be integrated into the natural cycles of matter. As a
consequence, prevention is being replaced by curing continuously occurring medical and
agricultural ailments. This can easily be visualised by the enormous over- and misuse of
antibiotics causing a lowering of the immune systems and an ever increasing resistance
against these drugs among microorganisms, which in turn requires the never ending
search for new antibiotics and vaccines (Ananthaswamy 2003). This is becoming even
more difficult as it was found recently that the non-stop battle against parasites appears to
be altering the human genome more radically than could ever be imagined (Weatherall
2003), which could explain the reasons why some cultural societies show higher infectious
disease rates than others.
The intensification of agriculture during the green revolution with its reliance on antibiotics
and hormones in feeding animals in so-called 'animal factories' [eg chicken, pigs, cows] or
breeding animals for fast growth (D'Silva 2003) as well as on irrigation and chemical inputs
in crop fields has led also to serious health and environmental problems (ADB 2001).
The reason for this unfortunate development must be sought in the fact that research and
development (Doelle 2001), personnel and finance are concentrated in rich countries, led
by global corporations and following the global market demand dominated by high-income
consumers (UNDP Report 2001). As a result, research has neglected opportunities to
develop technologies for poor people in developing countries representing approximately
80% of the world population. According to the UNDP report on Human Development, in
1998 global spending on health research was US$ 70 billion, but just US$ 300 million was
dedicated for vaccines for HV/ADS and about US$ 100 million to malaria research. Of
special concern is the fact that new drugs and vaccines are being developed to export for
profit rather than to sell cheaply to local people (Bloom & Track 2001). Patent, license and
royalty have become a tool to create wealth for the developed countries.
Although people are the real wealth of nations, it has so far not been possible to create an
environment in which people can develop their full potential and lead productive lives in
accordance with their needs and interests. s it therefore really surprising to see that some
societies rebel against the further development in science and technology ?
2.3 DNA TechnoIogy
The third millennium has therefore been marked by a paradox in the life sciences. Modern
genetic engineering techniques and sophisticated culture skills honed into a fine art over
the last two decades has given rise to new approaches in the medical and agricultural
sciences (DaSilva 2001).
n the medical sciences, these new approaches led to significant improvements in the
fight against human diseases increasing life expectancies (Lehtolainen et al. 2003). There
is no doubt, that the DNA technology approach in the medical sciences could improve
significantly health and living standards of mankind providing the products will be readily
available and affordable to all societies. Stem cell research will hopefully correct body
malfunctions and cancerous diseases in human life. However, the use of stem cells and
cloning techniques requiring human embryos are at present disputed areas of research a
both techniques involve the manipulation of human cells. How far should the scientist be
allowed to go (Editorial 2003). Many societies and religious groups are opposed to the
manipulation of human cells and this field of biotechnology is becoming a big ethical issue
(Braun 2003).
n the agricultural sciences, plant cells have been genetically altered for resistance to
diseases and pests as well as to increase yields and nutrition for food and energy. This
development saves undoubtedly the use of naturally foreign chemicals, but has led to a
steady loss of wild strains and of biological diversity (Johnson & Hope 2003). How different
is genetical manipulation to natural selection or old fashioned breeding by farmers, which
also alters biological diversity ? (Davies 2003; Garcia-Espinosa 2003) How will this rapid
selection affect our natural cycles of matter and the environment ? s genetic manipulation
more favourable to the environment than changing our farm management system, which
fosters pest breeding ? How will genetic engineering affect traditional food fermentation
and will pests overcome the resistance as the bacteria developed resistant strains against
antibiotics ? How will the culture and society react to the change of dominance from the
people of rural communities to corporate bodies ? s it really a wonder that society is
asking questions and is divided on the issue of GM crops (Masood 2003);

'Public debates about the safety of new products introduced in the market go back
centuries and were often based less on science than on politics of the time. Similarly
today, much of the debate about agricultural biotechnology is steered by myths and
misinformation and not by science. The scientific community, with stronger support from
governments, must do more to openly address science and technology issues with their
publics' [Juma 2003].

s this statement therefore really true considering that the culture of biotechnology is to be
found in food and its various fermentations developed over the centuries by society and
not corporate bodies or politicians. Should positive and negative developments in our
societies not be recognised together with the rather overpowering development and
secrecy of corporate bodies ? Every debate and opposition has a reason and it is often
forgotten to look for and investigate these reasons and rush into conclusions which offend
large parts of our society, which can lead in the extreme to war, whereas science and
technology should rather foster peace.
Thomas Sinclair (2003) asked What are realistic options for increasing resource
availability ? He believes that the solutions are in researching basic questions leading to
understanding, improving and managing soils and crops, even though these do not seem
to be currently politically attractive to research funders. Thus, genetic engineering need not
be yield-increasing technology to have an impact, but should simply need to be yield
'permitting' technology (Parrott 2003). Such an immediate application to stop current yield
losses to pests and abiotic stresses should be enough to stop food shortages in many
developing countries, in particular in dessert areas. Parrott (2003) indicated that existing
transgenic technology to protect crops against many diseases is well established and
highly effective, whereas more should be done in the field of abiotic stresses. One should
distinguish between wholesale degradation of soils, such as the reduction of a productive
forest slope to bedrock or fertile soils to salinity and reduction in biodiversity per se through
loss of wild organisms (Jenkins 2003). Latest GM crop trials in the Uk clearly indicate that
one should not generalise and declare all GM is good or all GM is bad (Pincock 2003) and
that some GM crops such as oilseed rape and sugar beet fare badly whereas maize
appeared to have beneficial results for the environment (Hunter 2003). Since herbicide-
tolerant crops can also be generated without genetic manipulation, they may have the
same effect on biodiversity than the GM crops (Walgate 2003). t is very encouraging to
see the successes of soil improvement techniques in some developing countries (Bamire
& Manyong 2003; Bttner & Hauser 2003; Wijuhoud et al. 2003) where improved nutrient
management has started to show success in higher crop yields. Poor soils, on the other
hand, lead to further yield reduction despite the use of GM crops (Masood 2003).
The development of knowledge and the application with respect to genetic engineering are
socio-technical processes, embedded in a social, economic, cultural and political context
(Haribabu 2003). Modern technologies should be blended in with traditional technologies
(McHughen 2003, 2000). Furthermore, the future of biotechnology depends very much on
quality science education (Alberts & Labov 2003).
2.4 ReIigion and ethics
A further critical point in the scientific and biotechnological development is, of course the
question about the relationship between science, religion and ethics, which certainly
affects the impact of science and technology on society (Dickson 2003; Park 2003).
Whereas their appeared to be conflicts in some cases, Watts (2003) outlined that the
relationship between science and religion needs to be based on mutual respect. t should
be realised that science is not as culturally neutral and value free as is sometimes
supposed as it is dominated at present by America and other Western countries, in other
words Christian countries. However, the most recent First Arab Human Development
Reports (AHDR 2002; 2003) lead the way to a better mutual understanding. These
conflicts become even greater, spreading through all religions and societies if cloning is
expanded to humans in the medical and health areas (Trounson 2003). n regard to ethics,
it has been claimed that ethics is on the corporate payroll (Cohen 2002) and subsequently
developed countries have been warned on imposing ethics onto developing countries
(Richards 2002). On the other hand, stem cell research shows great promises in the battle
against human diseases. There is no doubt that the scope of research ethics has to be
broaden (Benatar 2002) in order to achieve a mutual agreement and or respect between
the scientist and the community. One area is undoubtedly science education together with
the realisation by the corporate bodies that it can and should not dictate the way of life of
societies. Despite these controversies one should never forget that the seeds of
biotechnology are rooted deep in the past, but the fruits of its growth should benefit all
societies in one way or the other (Unesco 1991)

n order to establish the real need for what type of biotechnology is required for developing
countries, one has first to realise that there exist three major climatic zones, namely
a) the temperate zones of the developed world;
b) the tropical zones of developing countries; and
c) the arid zones of developing countries

Moreover, there is no escaping the fact that over 90% of biotechnological research and
development is occurring in the temperate zones of our world.
Secondly, the most serious problems in the developing countries concern:
1. HeaIth. t has been estimated by UNDP (UNDP 2001) that 2.4 billion people are
without access to basic sanitation and 11 million children under five are dying
annually from preventable causes.
2. Poverty. Approximately 1.2 billion people live on less than US$ 1 a day and 2.8
billion on less that US$ 2 a day.
3. Starvation. The developing world has still 826 million undernourished people, living
predominantly in the arid zone areas of Africa and Asia. For example, Sub-Saharan
Africa has an infant mortality rate of more than 100 and an under-five mortality rate
of more than 170 per 1,000 live births.

There is absolutely no doubt in my mind, that our present biotechnological knowledge is
able to abolish the health and poverty problems, with the reduction or even eradication of
starvation in arid zones well on the way using modern genetically modified agriculture. t
should therefore be possible to create an environment in which people can develop their
full potential and lead productive and creative lives in accordance with their needs and
interests. Since most of the biotechnology research and development is concentrated in
the temperate climatic zones, a closer cooperation has to occur to facilitate an adaptation
of the old and newly developed technologies to the appropriate climatic zone, the
particular society and the local environment.
What are the most urgent biotechnoIogicaI issues in deveIoping countries for the
improvement of human deveIopment ?
n considering all the available technologies together with those under development, the
first and basic priority thinking has to be the fact that 'technologies in general do not
transfer from developed to developing countries. Rather they need to be built up in situ
using local knowledge and innovative ability after which, if successful, they are being
adopted' (Doouthwaite & Ortiz 2001). Social aspects of psychology, religion and gender
are of paramount importance ( Warner 2000).
3.1 HeaIth
t is very hard to understand why our nternational Agencies have failed to eliminate the
health problems in developing countries. Biotechnologists and in particular microbial
technologists must fail to comprehend why the numerous existing technologies have
neither been supported nor implemented. Basic sanitation should be made available as a
first priority in human development. t is well known that the handling of human and animal
excreta or manure depends on and varies with the social and religious background of a
particular society, but the technologies available today caters for all aspects of human
society.
The basis for socio-economical integrated biosystems, recently referred to also as
'ecological sanitation' (Esrey 2001) has been with us for the last century in form of
a) composting toilets wet or dry
b) composting together with household waste and other biomass (see also chapter 14)
c) anaerobic digestion or biogas digesters.(see also chapters 15 & 19)
Neither of these systems requires any handling of excreta or manure, as these can be
channelled through pipelines to a cesspit, compost or anaerobic digester. Whereas
composting and anaerobic digestion takes care of all pathogens, the cesspit requires the
addition of lime or ash to help desiccate the manure and raise the pH, which effectively
kills off all pathogens within several months (Esrey 2001). This pathogen-free manure can
directly be returned to the soil or, in order to be safe, be added to compost heaps before
improving the fertility of soils.
Composting reaches a thermophilic range of 50-80
0
C and anaerobic digestion reduces
the redoxpotential to such an extent that pathogens are killed.
n the case of cesspits, compost and anaerobic digestion, the residue can be safely used
for soil improvement, replacing chemical fertilisation. Whether this organic fertilisation is
used on fields of flower or food crops is dependent on the local society (Kasule 1998) and
their religious belief. Technologies are also available to treat and recycle the water
[greywater] (Guenther 2001) effectively for reuse.
The best ecological as well as economic sanitation system is undoubtedly the use of
anaerobic or biogas digesters. n this case, the families will be able to use the biogas
formed for cooking and electricity. These systems are becoming very popular in
Bangladesh, Vietnam, Cambodia and China. Biodigesters are also the best socio-
economic systems with establishment costs becoming very cheap as has been
demonstrated in Vietnam (An et al 1997), Bangladesh (Shakti 2000) and Cambodia using
polyethylene tubing as construction material . Sizes of these digesters in use at present
range from 6 - 20 m
3
, whereas digesters up to 2000 m
3
require concrete or steel
construction.
t is very distressing to realise that many international organisations, eg FAO, do not
condemn the use of raw manure for soil improvement, and do not emphasize the absolute
need for treatment before use as was shown in one of the latest electronic conferences on
'Area wide integration of crops and livestock production' (FAO 2001).
f these old and improved biotechnological techniques are fully supported and properly
funded in a way the introduction of GMO crops are funded by UNDP, UNEP, UNDO, FAO,
WHO etc, health in developing countries could be quickly raised to the level of developed
countries.
n addition to these disease prevention technologies, nternational Agencies must also be
forced or force corporations to make biotechnological products available to the 2 billion
people [one third of the world population], who still have no access to low-cost essential
medicines such as penicillin, a technological process developed in the late 1940s (UNDP
2001). Such access would eliminate outbreaks of measles, cholera, meningitis and
haemorrhagic fever (WHO 2001).
3.2 Poverty
The term poverty is very often misinterpreted with starvation. Whereas poverty is
flourishing in most developing countries, this is certainly not the case with lack of food
causing starvation. However, poverty may cause starvation as the people are not able to
buy the available food. Poverty is mainly caused through strong increases in urbanisation,
as low income rural farmers stream into the cities to find work and a better income. This
depletes very efficient and productive rural agriculture, and reduces the possible maximal
agricultural food or crop production. Whereas the green revolution technologies resulted in
increased food production in favourable and irrigated environments, they had little impact
on the millions of smallholders living in rainfed and marginal areas, where poverty is
concentrated in Asia [ADB 2001]. The reasons for this trend are manyfold, but can mainly
be traced to changes in farm management [single crop production] as well as farm
mechanisation [big farms] resulting in a severe reduction of small farm holders, and a
severe reduction of funding and investment into agriculture. n order to alleviate poverty
and make certain that we are able to continue with feeding an ever increasing population,
we need a socio-economic biotechnology revolution (Doelle & Prasertsan 2002) realising
that we have to learn from the mistakes of the green revolution and secure a proper
income to the farmer. One major problem of the green revolution was that the farmers
were made to believe in the production for markets, forgetting their own consumption. t is
evident that farmers in some developing countries grow crops solely under contract for
supplying processing factories, while they have to buy the food for their own consumption.
Traditional food was demoted, whereas canned and bottled food was promoted. Local
(traditional) wisdom and knowledge in food preservation and medicine were treated as an
'uncivilized way of life' and is disappearing, so the younger generation is not practising it
anymore.
Such a new biotechnology revolution has to take into consideration
1. that farming in developing countries is profoundly different from developed countries
with crops like cassava, rice, soybean, sago etc;
2. that farms are small and should stay small with minimal mechanisation but more
intensive and integrated farming;
3. that single crop production must make way to a multi-product farming, including
livestocks on the farm;
4. that we use existing biotechnological techniques to develop biotechnological industries
using locally grown biomass such as sago palm and cassava;
5. combine the biomass waste, excessive amounts of agro-industrial wastes with human
and animal waste treatments for novel product and renewable energy production.
Such a sustainable socio-economic biotechnology revolution can best be established in
form of so-called 'biorefineries', whereby all the biomass is used to improve the living
standard of the people [Doelle 2002]. Such biorefineries require a host of different
biotechnological and physical techniques ranging from anaerobic digestion of wastes to
surplus biomass conversion to renewable energy, food, feed and commodity product
formation. All biotechnological and physical techniques are readily available and can
immediately be implemented.
The additional incorporation of aspects of modern biotechnological techniques (DaSilva
2001) will come as soon as society has learned the advantages of the existing
technologies. Some biotechnological issues such as pest-resistant plants and organic
fertilisation will involve some GMO plants. However, one should always be aware and not
forget, that local breeding experience may be a better way to go initially than GMO
introduction.
t is very surprising, for example, that agricultural biotechnology development sofar has
totally ignored plants such as cassava and the sago palm as a starch resource, as they
are hardly known in developed countries. Cassava can yield up to 65 t/ha with a 65%
starch content in marginal soils and the sagopalm can easily produce 25 t of starch/ha in
swampy areas unsuitable for any other crop (Doelle 1998) . Since the average intake of
food for human is, in general, about 250 kg of grain per year, one hectare of sago
plantation can feed 100 people and a 1000 ha sago plantation can subsequently save
100,000 humans from hunger, a clear example of the potential of sago as a major starch
crop of the world (shizaki 1997). Both crops are ideal for obtaining a variety of bioproducts
ranging from biofuel, bioplastics, biodetergents, biolubricants to bio-pharmaceuticals
[Bujang and Ahmad 2000; Bujang et al. 2001].
The establishment of biorefineries (see chapter 19 & 20) will diversify rural farming, keep
people employed in the rural areas and will also increase the income of the individual
farmer, helping in the alleviation of poverty.
3.3 Starvation
The elimination of starvation in the arid zones of the world is the biggest challenge to
agricultural biotechnology. Much more effort should be put into the breeding or genetic
modification of crops for drought resistance [Dodds et al. 2001]. mprovement of soil
condition using treated human and animal manure should go hand-in-glove with the
introduction of drought resistant or at least drought tolerant crop varieties. The most
beneficial aspect of GMO crops in these areas would immediately improve livestock and
food production. However, one has to be aware that different regions have different types
of crop demands. Genetic modification for drought resistance should occur with local
plants and crops used by the local societies. We should stop introducing GMO plants from
temperate zones in developed countries and respect the local food demand and varieties.
Such a project would cause much less opposition than genetically modified foreign crops.
The elimination of starvation in arid zones could become an ideal place for combining 'old'
biotechnological concepts of soil fertility improvement with 'new or modern'
biotechnological concepts of increasing crop and livestock production. Soil fertility
improvement should also go hand in hand with a reduction or elimination of rain and other
forest clearings, which in turn would stop the expansion of the arid zone areas. Such a
combined concept would create more small holding farms with a proper income, helping to
stop the development of further poverty.
What are realistic options for increasing resource availability ? The solutions are in
researching basic questions leading to understand, improve and manage soils and crops
(Sinclair 2003). Modern gene technology can certainly take care of crop destruction and
spoilage, but what about the soil ? Here we have to remember that Nature has given us
the perfect answer in the cycles of matter (see chapter 2), which have worked well for
centuries until we used the soil and the environment as bottomless sinks. How do these
cycles work and what can we do to regenerate and adapt these cycles to modern need ?
3.4 Waste management and RecycIing: CIean and Green TechnoIogies
During the past decades, production systems have been based on the assumption that
wastes are an unavoidable part of our daily lives but that ecological and environmental
destruction could be avoided because of the planet's vast natural resources. However,
ecosystems and renewable resources are being destroyed at an increasingly rapid rate
and the problems of pollution and waste disposal are growing. t is thus becoming
increasingly evident that new production methods must be devised to fulfil society's basic
needs. n order to maintain the vitality of the rural sector and preserve the environment, a
system must be devised in which energy, food, animal feed, fuel and fertiliser
requirements can all be met from renewable resources used at a sustainable level (Doelle
1989; Raymond & Larvor 1986; Szmant 1986; White & Plaskett 1981; Moo-Young &
Gregory 1986; Sasson 1990).
n regard to waste management, environmentally friendly waste incineration plants
(CADDET 2003; Kathirvale et al. 2003) are becoming very popular in developed as well as
developing countries. t varies from the largest waste to energy plant in Denmark [772,000
t of waste annually] to the municipal solid waste to energy conversion plant in Kuala
Lumpur [Malaysia] operating on 1,500 t/day [= 540,000 t/year] producing 640 kW/day. The
Danish Plant has been calculated to save the emission of 80,500 t of CO
2
/year.
Ecological engineering was used for wastewater treatment using aquaculture principles,
which not only reduced carbon, nitrogen and phosphorus, but the additional anaerobic
treatment reduced also metals by 48-72% (Guterstam 1996).
These examples demonstrate that municipal solid wastes can be incinerated or in
connection with industrial wastewater anaerobically digested for recycling energy, fertiliser
and water in an environmentally friendly way (Riggle 2000).
The new socio-economic concept is based on the requirement for full exploitation of a
harvested renewable resource and the replacement of monoculture/monoproduct farming
with a multiproduct system (Doelle & Prasertsan 2003). Because it produces a variety of
products, this system will hopefully enjoy a constant and reliable market demand and will
be able to secure income for the rural sector as well as for joint venture industries. t also
will help to reduce the vast multiplication rates of pests in mono-culture system operations
(see chapter 20).
The vast majority of the populations in developing countries live in villages and the
importance of rural technology development in those countries cannot be overstated
(DeBruin & DeBoer 1986). n the context of technological development it is generally
considered imperative to see that such developments are of benefit to their lives as well.
Corporate bodies have to realise that they should not and cannot dominate rural
technologies. t certainly is recognised that rural life is affected by many factors, such as
political and social institutions, rural economic structures, communication, education and
technology. An initial step is therefore that building up the rural technology capacity is one
of the tools for development. Another step is the recognition that the technology employed
or developed should suit locally available resources and skills and be in harmony with local
culture (DeBruin & DeBoer 1986; Doelle et al. 1987).
n order to develop an appropriate biotechnology, resources available together with the
social structure of the population are of vital importance. t is often the need and not the
economics of the process, which is of importance. Here seems to lie one of the cardinal
differences between the thoughts of appropriate technology in developed, from those in
developing countries. n this context, a new concept has been developed specifically for
rural communities: the so-called integrated rural biotechnology systems. The
development of these systems depends primarily on the climatic conditions of the regions.
Two of the premises of sustainable development are that economic growth has to be in
harmony with the environment and that a rational and sustainable use of natural resources
has to be implemented (Olguin 2000). n congruence with such premises, industrial
development has to change from the degradative to the sustainable style, which requires
the adoption of cleaner production systems.
The United Nations Environment Program (UNEP) defines the cleaner production concept
as 'the continuous application of an integrated preventive environmental strategy to
processes, products and services to increase eco-efficiency and reduce risks to humans
and the environment' (UNEP 1996). One of its distinctive features is that reduction of the
quantity and toxicity of all emissions and wastes is made before they leave the process
stream. n the case of services, environmental concerns should be incorporated into
design and delivery.
Eco-efficiency, on the other hand, involves 'the delivery of competitively priced goods and
services that satisfy human needs and bring quality of life while progressively reducing
ecological impacts and resource intensity, throughout the life cycle, to a level at least in
line with the Earth's estimated capacity' (UNEP 1994).
t is becoming very clear that adoption of clean production systems by industries calls for
fundamental changes, not only at the technological level but also at the legislative level
(Olguin 2000). Cleaner bioprocesses are under intensive research and development
following the general guidelines for cleaner production.
Since microorganisms are responsible for diseases and/or sustaining life, we have to
start educating people and the whole society on the role of microorganisms in their
life. This is a very difficult task as we can not see them in nature [the good ones], but only
feel them [the bad ones, if sickness occurs]. We have to educate them that it is the Society
itself which has and still is causing not only the destruction of the environment, but also the
life and existence of the Society. It is not onIy the fauIt of Governments if infectious
diseases occur, as we have no one else to blame than ourselves.
With the heIp of the Society, microbioIogy and its appIication, microbiaI technoIogy,
can Iead very quickIy to meeting and soIving many, if not aII, of above probIems.
This cooperation can bring back a cIean and sustainabIe environment without
stopping or hoIding back further deveIopment. If one interprets 'sustainabiIity' as
the recognition of Iong-term management and use of naturaI resources together
with the protection of the environment it becomes cIear that we have to continue
our 'sustainabIe deveIopment' for higher Iiving standards in tune with Nature.
This can be achieved using microbial and fermentation technologies involving man,
biomass and industry emphasizing the utilisation of renewable resources with a low
environmental impact and a high regeneration capacity (see chapters 18-20). Microbial
technology must and can provide for
Environmental Management through the bioconversion of domestic and animal wastes
into two non-polluting fuels such as biogas, ethanol and value-added products such as
algae, fresh water fish etc.
Bioconversion of agro-industrial residues and products into value-added products such
as mushroom, biofertiliser through composting, silaging, protein-enriched feed etc.
Enhancement of soil fertility and stability through the direct application of anaerobically
digested sludge material or microbial fertilisers [e.g. sound and responsible farm
management, rhizobia and algae etc.]
Public health programme by eliminating enteric parasites and microorganisms through
anaerobic digestion processes or cleaner ecological environment management [e.g.
composting, garbage collection etc.]
Waste water treatment and waste utilisation through microbial-based systems

Concentration and leaching of valuable minerals and removal of heavy metals from
rivers and estuaries from mining waters, low grade ores as well as contaminated rivers

Substitution of toxic chemicals in using biopesticides [e.g. Bacillus thuringiensis,
antagonistic microorganisms]
Food through improvement of indigenous fermentations, e.g. puto, nata de coco, suka,
tempeh, bagoong etc.
Very soon we have to reIy totaIIy on microorganisms for our suppIy on food, feed,
fertiIiser, energy and disease controI. This we can do provided we harnass and
protect the biodiversity of microorganisms, pIants and animaIs. The roIe of
microorganisms in heaIth, food, energy and agricuIture is unIimited, but we need
urgentIy microbioIogists, not for biomedicaI areas, but for agro-business and food
fermentation areas. Here we do not need geneticaI engineering, but a sound
knowIedge in microbioIogy, biochemistry and some aspects of chemicaI
engineering. (see chapter 5).
The importance of microbial technology for the future of mankind and thus each society
can be seen in the establishment of an international network by Unesco, UNEP and CRO.
At present there exist 29 Microbiological Resources Centres (MRCENs) worldwide with a
World Data Centre in Japan. These institutions together with OBB [nternational
Organisation for Biotechnology and Bioengineering] try to help the exchange, training and
research of manpower as well as in an advising capacity.
n the field of microbial biotechnology, the world activities at present center very much
around agricultural biotechnologies and waste utilisation. Agriculturally based
biotechnology is mainly concerned with renewable biomass resource production, which led
not only to plant disease resistance and higher yields, but also to an increasing self-
efficiency in some developing countries. This development gives the microbial
biotechnologist the task to:
1. secure that the farmer is able to stay on his farm by changing mono-products to
value-added multiproducts;
2. produce products which can be used domestically taking the pressure from a
depressed or fluctuating export market;
3. introduce flexibility and convert a one-product farming system into a multi-product
farming system.
4. Stabilising the farming community is the only safeguard for the continuous
availability of farm products and thus food for the future.
The biotechnology issues for developing countries in future requires a change from the
presently commercially driven to a more human development, combining 'old' and 'modern'
biotechnological techniques for the improvements in the health and living conditions of
80% of our world population. t is very encouraging to observe the establishment of new
organisations such as the Program for Research and Documentation for a Sustainable
Society (ProSus), ecological sanitation (Ecosan) and many others together with the
information distributors Livestock Research and Rural Development (LRRD), Centre for
the Analysis and Dissemination of Demonstrated Energy Technologies (CADDET),
Ecosan Newsletters as well as discussion groups such as ntegrated Biosystems (BS)
emphasizing the need for such a development (see also Doelle and Foo 2000). t is a
great opportunity for the nternational Organisations [UNDP, UNEP, UNDO, FAO, WHO
etc] to take up the challenge and realising that the so-called 'modern biotechnology' alone
cannot solve the problems. As long as the present biotechnology development is driven
only by commercial enterprise, human development in developing countries will lag
increasingly behind and cannot progress as it would with the application of a total
biotechnology concept, such as a socio-economic sustainable bio-integrated system.
Sustainable development and human development should not and can not go separate
directions.
f one combines all these efforts reported into an socio-economic strategy (Doelle et al.
1998; 2000), farms and/or farm cooperatives as well as agro-industries are able to
combine natural renewable resource production with bioenergy, food, feed, biofuel and
fertiliser production (see chapter 18). Such a system must be and can be flexible and
should be adaptable to local conditions. The new term Bio-Refinery [see chapter 19] has
been given to these systems. n all our consideration one should never forget the natural
cycles of matter and the microbial soil population, both so important for the production of
our renewable resources, maintenance, environment, and improvement of living
standards.
Sustainability can be obtained together with higher health and living standards, if
appropriate technologies are applied according to the climatic region and local society
(Doelle 2002a). n order to determine what part of biotechnology can be used in rural
farming as well as in the biorefineries, we have to analyse what type of renewable
resources are available, the climatic zone of the region, the amount of waste produced
from humans and animals, and the cultural and societal structure of the local village and
regional communities. The demands of communities in developed countries is different
from those in developing countries. All over the world, however, rural communities are
characterised by certain common factors (Doelle 2002b).
n using disease resistant seeds obtained through conventional breeding or DNA-
recombinant technology together with proper soil enrichment fertilisation should secure a
safe income to the rural farmer as well as secure all the food required for the urban
societies (Peczon et al. 2002; Dharmsthiti & Flegel 2002).
We need a balance between man and the natural world which surrounds him. Nature
tends to conserve the information network of the environment, which sustains us as living
creatures. Our only strategy can be to respect it, if we do not want to carelessly disappear.
The utilisation of hidden 'biochemical pathways of nature' can help us in this major change
of direction. We always should remember the first principles of the United Nations
Conference on the Human Environment of June 1972 (Wohlmeyer 1987), which says:
Man has a fundamentaI right to freedom and suitabIe conditions of Iife in an
environment that is so constructed that a Iife of dignity and weII-being is possibIe.
He has the soIemn duty to preserve and improve the environment for this
generation and for future generations.

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Chapter 5
The Importance of Basic MicrobioIogicaI KnowIedge for
a better Life, SeIf-efficiency and SustainabiIity
[Large parts of this chapter are based on chapters 2-4 of the book 'Microbial Process Development' , two of
which are contributions by Dr.L.Sly, Director of MRCEN-Biotechnology Brisbane]
Content:
1. Introduction
2.1 IsoIation, Identification and InitiaI SeIection of MicrobiaI Strains
2.1.1 CuIture Preservation
2.1.2 Stock CuIture Maintenance
2.1.3 Storage of cuIture
2.1.4 CuIture CoIIection Resources and Services
2.2 Modification of the Genetic Structure to Increase Product Formation
2.3 Nutrition, OptimaI NutritionaI and PhysicaI Requirements for Growth
2.3.1 MicrobiaI Nutrition
2.3.2 Growth Measurements
2.3.3 Growth Curve
2.3.4 Optimisation of NutritionaI and PhysicochemicaI Factors
2.4 Process strategy
2.4.1 Primary MetaboIites
2.4.2 Secondary MetaboIites
2.4.3 Bioconversions
3.1 Identification of main products
3.2 Stoichiometry of the Process
3.3 Kinetic and process rates
3.4 Reactor Design
3.5 Product Recovery
3.6 Waste Treatment
4. BibIiography

1. Introduction
The use of any of the available microbial types for the industrial processing of materials to
provide mankind with desirable products, re-establish the natural cycles of matter (chapter
2) and to re-establish a clean ecological environment, is called microbial technology,
which incorporates the well known area of fermentation technology.
The essence of this microbial technology is its interdisciplinary nature. t is a technology
that brings microbiology, in particular the physiological and biochemical side, together
with biochemistry and chemical engineering and demands intimate collaboration
between scientists in these disciplines. n addition, the scientist working in this area needs
to be conscious of diverse economic, sociological and legal constraints that impinge on the
success or failure of the operation under consideration (Doelle 1997).
The development of a microbial process for the formation of biomass or products is aimed
at maximising three factors:
1. the yieId of product per gram of substrate;
2. the concentration of the product;
3. the rate of product formation.
n order to achieve this, the following main features of a microbial process development
have to be observed:
a) isolation, identification and initial selection of microorganisms;
b) modification of the genetic structure of the organism to increase product
formation;
c) nutrition, optimal nutritional and physical factor requirements;
d) strategy of product formation

All four aspects are basically concerned with the adjustment of metabolic regulation in the
organism, whereby metabolism means that all of the available carbon is converted into
biomass and the endproduct(s) of energy metabolism. Microbial process development can
therefore be regarded as the ideal example for basic scientific research with an applied
goal. The knowledge gained in such process development can then be translated into
microbial process technology, which in turn can be classified into high, intermediate and
low or village technology. Over the past decade, biotechnology has emphasised the
development of technologies for organisms preserved in culture collections, which have
never been investigated along the lines mentioned above. f one wants to develop a
technology of a process, one has to know the catalyst first. The latter, of course, is the
appropriate microorganism in question and the suitability for a process falls under
microbial process development (Doelle 1994).
n terms of total biomass of our planet, microorganisms are equal to the animal kingdom
(including humans), together taking about half and higher plants the other half. The
question was thus raised whether mankind has taken or is taking full advantage of this
almost untapped natural resource (DaSilva 1980). Microorganisms are still most frequently
referred to as the cause of disease in human beings, animals and plants, and only slowly
do we recognise that many more types are beneficial than harmful to higher forms of life.
The reasons for this increasing awareness over the last decade is the realisation that
biological systems may be utilised for many new purposes in addition to food production. It
is the bioIogicaI sciences which are expected to provide important potentiaIities for
deveIopment in the forthcoming miIIennium and thus twenty-first century.
2.1 IsoIation, identification and initiaI seIection of microbiaI strains
A great number of culture collections, together with the recently established MRCENs
(Microbiological Resources Centers), contain large lists of microbial strains of more or less
known characteristics. f one looks for a particular strain, the World Data Centre on
microorganisms is available to locate the strain in the particular affiliated culture collection.
The majority of these available strains, however, have neither been isolated nor explored
with an aim for process development. t is therefore necessary to search for new, more
suitable cultures (Hunter-Cevera et al. 1986), which possess the properties for producing
the desired product in high yield, or reinvestigate the existing strains from culture
collections with the same aim, and at the same time economically utilise the available
substrate. New cultures may be found by chance observations (eg Fleming's culture of
Penicillium notatum) or more likely by a systematic search.
A systematic search for new cultures may depend on two major approaches:

1. the pure scientific approach;
2. the process development oriented search.

Whichever direction is chosen, it is absolutely necessary to be well acquainted with the
microorganisms, that is, one must be able to place them correctly into the system of living
entities (Sly 1994). Every isolation is connected with an evaluation of various features of
microorganisms. The initial features in microbial process development would undoubtedly
be related to resource utilisation and/or product formation. n sharp contrast to the usual
requirements of academic research, organism isolation and initial selection for an industrial
process is dependent on a range of criteria that are relevant to the optimisation of the
particular process. Their features may be morphological, physiological, genetical,
immunological etc. and the sum of all these features of a microorganism is referred to as
its phenotype. A phenotype therefore represents any measurable characteristic or
distinctive trait possessed by an organism. n contrast, the genotype represents all genes
possessed by an organism. The genotype can be explored via the phenotypic
expressions. The isolation, identification and initial selection of microorganisms for
microbial process development depends therefore on the phenotypic expression of the
organism. Despite the selective aim, one should not forget that every microbial culture
must possess certain general attributes:

1. the strain should be a pure culture;
2. the strain must be genetically stable;
3. the strain must produce readily many vegetative cells, spores or other reproductive
units;
4. the strain should grow vigorously and rapidly after inoculation;
5. the strain should produce the required product within a short period of time;
6. if possible, the strain should be able to protect itself against contamination;
7. the strain should produce the desired product, which should be easily separable
from all others; and
8. the strain should be amenable to change by certain mutagenic agents.

n most cases it is useful to isolate a culture from a natural resource or decomposing or
organic materials. Rapid screening techniques for testing the phenotypic expression
normally combines isolation and selection simultaneously. The techniques used for these
tests are numerous and depend, of course, on the expected phenotypic expression. Any
isolated culture should immediately be deposited with a culture collection for maintenance
and preservation.
The isolation and identification of an organism of a new culture on phenotypic expression
also gives some indication on the metabolism of the organism. t is of utmost importance,
however, to investigate in details the basic metabolic processes of the organism as part of
the selection programme. Traditionally, screening procedures are based on agar plate
techniques or enrichment cultures (Demain & Solomon 1986). t should be realised that
both techniques can be very restrictive if one aims at certain microbial process
developments. The agar plate techniques are very important for extracellular enzyme- and
antibiotica-producing strains. They give excellent results for polymer degradation (eg
starch, cellulose) by exoenzymes or antibiotic production, that is, phenotypic expression
related to products excreted out of the cell. They also could be indicators for acidic or
alkaline product formation. However, these procedures are very labour intensive and time
consuming. Enrichment cultures, on the other hand, are carried out under substrate
excess conditions and thus select microorganisms on the basis of maximum specific
growth rate [see chapter 7]. This characteristic may not be the key criterion for the process
under development. t also must be realised that in batch enrichment the time of sampling
is important for the selection of the most desirable organism, since the growth conditions
change as a function of time. t could therefore be possible to miss the particular stage
when the particular organism is present in sufficient numbers to guarantee its isolation.
An attractive alternative has been developed during the last decade, which involves a
continuous flow enrichment technique. This technique allows the selection and
isolation of organisms on the basis of their substrate affinity (using a chemostat),
maximum specific growth rate (using a turbidostat), resistance to toxic material etc.
Different screening techniques (White et al. 1986) select therefore different types of
organisms and it is in the experimenter's hand to choose which one of these techniques
would lead to the isolation and selection of the microorganism wanted for the envisaged
process development.
t was mentioned earlier that sound knowledge in microbial biochemistry, that is the basic
metabolic processes, is an absolute requirement for a successful and speedy isolation and
selection programme. Aerobic, facultative anaerobic and anaerobic organisms can be
isolated separately for their substrate specificity, growth rate or product formed.

2.1.1 CuIture Preservation.
f a suitable microorganism has been found for the required industrial purpose, it is of great
importance to preserve the organism in a way that its genetic make-up remains
unchanged and that the organism is constantly available (Chang & Elander 1986). Such a
preservation of the properties of a microorganism must be assured not only for the
constant product formation, but more importantly when that microorganism is pertinent to a
patent application. The subject cuIture has to be deposited in a culture collection
recognised by the Patent Office of the country in which the patent application has been
filed.
Most industrial microbiological establishments have a collection of all those
microorganisms employed in the manufacturing process. This stock of microorganisms is
maintained in a low metabolic state in which replication of the cells is kept to a minimum or
even entirely restricted. ndustrially important microorganisms are often mutants, and the
condition of low metabolism in which they are kept limits their tendency to revert to their
low yielding ancestors. A stock of microorganisms or culture collection therefore serves as
a source of regular supply of microorganisms to be used in industrial fermentations.
Organisms maintained in a low metabolic state in a culture collection are called ' primary
stock cuIture'. n some circumstances organisms are maintained in a more active state in
which they are virtually ready for use in fermentation preculture experiments or research.
These cultures are referred to as 'maintenance cuIture'. From the maintenance culture,
the organisms are then transferred into an active state, called ' working stock ', from
which the inoculum or precultures are prepared. t must be borne in mind that the
maintenance, working stock as well as the precultures stand the chance of contamination
and/or mutation, two serious problems in industrial fermentation. There exist various kinds
of culture collections around the world. A list of culture collections can be found in the
WorId Directory of CuItures of Microorganisms.

2.1.2 Stock CuIture Maintenance.
Easy access to actively growing cultures is a requirement of most microbiological
laboratories. Cultures are routinely required generally on a day to day basis for quality
control, comparative testing, inocula for bioassays and for various other reasons. New
isolates or accessions are usually maintained as actively growing stocks until the strain
can be confidently preserved by one or more of the long-term methods available (Sly
1991). There are some cultures for which no long-term preservation methods are available
and these cultures must be continually maintained in active culture.
Periodic transfer or subculture is the traditional method used by microbiologists to maintain
isolates in the laboratory. However, to minimise genetic change and for reasons of
financial and labour savings it is advisable to engage in as little subculturing as possible
and to have back-up stocks of well preserved cultures.
Periodic transfer is not recommended for long term preservation. Genetic change through
selection of variants is likely to occur, the chances of contamination and mislabelling are
high and the risk of culture loss is greater than in other methods. Nevertheless, periodic
subculture is an essential technique at various stages of culture propagation in
microbiology, but its limitation should be taken into account.
Subculturing involves the inoculation of cultures onto fresh medium in bottles, tubes or
plates and the growth of the subculture under suitable incubation conditions. After growth,
the new cultures are stored under suitable conditions, usually in a refrigerator. The period
between subcultures depends on the microorganism and may range from one or two days
for delicate cultures [eg Haemophilus] to months or even years [eg Escherichia coli,
Staphylococcus aureus, fungi]. The longevity of such cultures is influenced by the nature
of the organism itself, the composition and pH of the medium, the degree of aeration and
hydration, and the temperature of storage.
Various techniques such as overlayering with sterile paraffin oil have been employed to
reduce the culture's metabolic rate and reduce dehydration, and so extend the period
between subculture. Low storage temperatures usually around 40
0
C are preferred but
there are exceptions with some strains preferring storage at room temperature.
The growth medium is also a matter of consideration. For reasons of economy most
laboratories try to keep the number of media in use to a minimum. Calcium carbonate is
usually added to the medium of acid producing strains [eg Lactobacillus spp, Acetobacter
spp] so that excess acid does not kill off the culture.
A two culture technique of first and second stock cultures is usually used to minimise
contamination. The first stock is only used to prepare a new first or seed stock. The
second stock is used as a working stock culture for routine inoculations.
The objective of preservation is to maintain the viability and genetic stability of the culture
by reducing the organism's metabolic rate thereby extending the period between
subcultures. t is important to realise that there is no universal method of preservation
that is successful for all microorganisms. Taxonomic groups of microorganisms respond
differently to different preservation methods. The preservation methods used reflect
therefore the different biological properties of the various groups of microorganisms such
as bacteria, viruses, fungi, yeasts, algae and protozoa, and their responses to changes in
their environment. An evaluation of the application of various methods of preservation to
the different groups of microorganisms is shown in tables 1 and 2:

TabIe 1: Preservation methods for microorganisms [Sly 1991]

TabIe 2: Comparison of methods of preservation of microbial cultures.

All preservation methods follow an essentially similar protocol. The process begins with
a final check on the purity and authenticity of the culture and the preparation of the
ampoules which are the usual vessels in which cultures are preserved. The type of
ampoule, its size, composition and shape often depends on the supplies available. Or the
nature of the equipment in use. The ampoules for drying methods are usually made of
soda-glass or borosilicate glass and for freezing methods are usually borosilicate glass or
propylene.

2.1.3 Storage of cuItures
The culture is grown on its usual maintenance medium and suspended in a preservation
medium as soon as it has reached the early stationary phase of growth, at which stage
cultures are generally most resistant to damage (see chapter 7. A dense suspension of
around 10
8
- 10
10
viable cells per ml is usually used as some loss of viability occurs during
the various stages in the preservation process. The suspended cells are dispensed into
the ampoules in 0.1-0.5 ml quantities depending on the preservation method and
preservation is carried out. The ampoules are then stored at an appropriate temperature
and accurate records of ampoule stocks kept.
Quality control is an important part of preservation. A representative of each ampoule
batch should be checked for purity, viability and authenticity. t is essential that the
important characteristics, for which it is considered necessary to preserve the culture, are
retained.

Preservation suspending media. Most preservation methods rely on suspending media
to protect the living organisms from damage during the preservation process. Their
functions include stabilisation of protein, prevention of freezing damage and protection
against overdrying. The choice of the preservative therefore depends on the preservation
process and the nature of the organism. The preservative must ensure a minimum loss of
viability during preservation, maintain the microorganism in a viable state during storage
and allow easy recovery from the preserved state.

Agar storage. Organisms stored on suitable agar at normal growth temperatures attain
the stationary phase and begin to die because of the release of toxic material and the
exhaustion of nutrients. These cultures are therefore transferred refrigerated as soon as
adequate growth is attained. Agar dishes are often sealed with a flexible tape to avoid
water evaporation.
Aerobic organisms are stored in so-called agar slants, and anaerobic organisms in agar
stabs. The shortcoming of this method is that the organisms are still metabolising and
have to be transferred in certain intervals. Every transfer increases risks of mutant
selection and contamination.
The nature of the medium on which microorganisms are stored is of great importance. A
medium prepared from natural rather than chemically defined medium is preferable, since
a defined medium may, because of lack of a component present in the natural
environment, select another organism specifically capable of growing on this medium. t
should also be not rich on carbohydrates, as acids are formed from these carbon sources.

Distilled water storage. Many organisms die in distilled water because of water
absorption by osmosis. However, some have been known to survive for long periods of
time, if such storage is carried out in a refrigerator.

Freezing without dehydration. The physiological basis for storage by freezing is the
extremely attenuated growth of the organism. n this technique, a broth culture of the
organism is sealed in a tube, or preferably put in a small screw-cap bottle and frozen in
liquid nitrogen, whose temperature can be as low as -196
0
C. Organisms to be frozen
should preferably be in the stationary phase of growth, at which organisms have been
shown to be most resistant to freezing and thawing. Storage at low temperatures may
result in damage to the organism, but such damage may be minimised by the introduction
of cryoprotective substances such as glycerol.
The use of liquid nitrogen has become expensive. Nevertheless it has the advantage that
organisms preserved in this way can be used as working as well as primary stock since
frozen cultures can rapidly be thawed and brought into use without any further preparation.

Storage in dry state of solid substrate. The most commonly used form of storage in a
dry state is the use of dry sterile soil. n this method, sterile dry soil is inoculated with a
broth or agar culture of the organism. Subsequently the culture can be refrigerated. This
method has been widely and successfully used to spore sporulating organisms especially
clostridia and fungi.
Another method of solid dry storage is the use of silica gel. Dry crystals of the gel are
placed in close proximity to the organism in a tube and the cultures consequently become
dry. Fungi including yeasts have been preserved for several years using this method.
A third method is the storage on filter paper. Sterile filter paper is soaked in the broth
culture of the organism to be dried, placed into a tube, which is then evacuated and
sealed.

Drying with refrigeration. Freeze-drying or lyophilisation is widely used. The principle
of the method is that the organism is first frozen. Subsequently water is removed by direct
evaporation of the ice with the introduction of a vacuum. Because the suspension is not in
a liquid state, distortion of shape and consequently cell damage is minimised. At the end of
the drying period, the ampoule containing the organism is sealed and stored under
refrigeration.
The suspending medium must be carefully chosen, because of differences in the
cryoprotection properties of different substrates. Lyophilisation is preferred for the
preservation of most microorganisms because of its success with a large number of
organisms. However, organisms with delicate cellular membranes, eg algae, can not be
lyophilised, but are better stored under liquid nitrogen.

2.1.4 CuIture CoIIection resources and services.
Culture collections occupy a fundamental and central position in microbiology and
biotechnology. The overall management of the collection is under the control of the curator
who is responsible for implementing culture collection policy, and the collection's
programmes. Curators of culture collections hold a very responsible position. They should
be well qualified and need a sound knowledge of systematics and systematics procedures
The curator also overseas the acquisition of new cultures and should be familiar with
literature on new microorganisms and should seek to obtain those which may have an
immediate or future use in the research or service programmes of the collection. The
management of such a culture collection involves an organisational system which may be
divided into a number of distinct areas of operating which are conveniently summarised in
a flow diagram (Fig.1).

Figure 1: Culture Collection Management[Sly 1994]

Apart from their basic role of preserving cultures of past, present or future interest, culture
collection offer a number of other services by virtue of the expertise developed in their own
infrastructure. Culture collections are important centres of information and offer advice on
the availability of cultures, maintenance and preservation, identification, classification and
nomenclature, postal and quarantine regulations and patent culture regulations.
The international culture collection community primarily fostered by the World Federation
for Culture Collections, Unesco, UNEP and the Microbiological Resources Centres global
network [MRCENs] has been actively working towards the documentation of the world's
culture collection resources through the World Data Centre for Microorganisms. The
concept of the Microbiological Resource Centre has led to the formation of an active and
productive network of laboratory centers throughout the world. An international community
of microbiologists and biotechnologists has resulted from this innovation of the United
Nations that was initiated in 1975 with the formation of the World Data Center , when
centers in developing regions of the world were established to preserve and use microbial
resources. n 1984, pilot centers were established in a few countries and today more than
30 centres in 25 countries (MRCEN 1996), a significant testimony to the worthiness of the
concept and the practicality of the mission of the MRCENs.
The microbial gene pool conserved in culture collections is a world resource, the
significance of which may only be recognised in the light of future scientific discoveries and
technological developments.
2.2 Modification of the genetic structure to increase product formation
The production of intermediates of the degradative pathways or any compound connected
with the biosynthesis of macromolecules, which also includes the secondary products,
require the use of mutation techniques (Baltz 1986).
n order to understand fully the nature of mutagenesis it is necessary to have a general
idea of the manner in which genetic information is encoded and deciphered into gene
products. A mutation is any heritable change in the base pair sequence of an organism's
DNA and is defined by any detectable, heritable change in an organism's phenotype.
Changes within a coding region, for example, can alter the amino acid sequence of an
enzyme and thereby affect its activity. This could lead to so-called auxotrophic mutants.
With the removal of one enzyme from the biosynthetic sequence, the intermediate or
substrate for this particular enzyme accumulates as a product. n order to ensure the
continuation of growth and thus product formation, the would-be product of this enzyme or
any of the subsequent intermediates must be fed into the fermentation vessel. The slight
alteration of a promoter sequence could also increase the probability that RNA polymerase
will bind the promoter and thus enhance the rate of transcription. This is very important in
the case of enzyme production. Mutations in operator regions or in a regulatory gene can
prevent the binding of a represser and thereby greatly increase transcription. These so-
called reguIatory mutants are frequently used in amino acid, vitamin etc production, as
they remove feedback regulation.
There exist several ways to bring about such changes or induce mutagenesis. Chemical
mutagens can induce genetic changes by direct induction of base mispairing, whereas
physical agents damage DNA and thus can also cause gene mutations. Mutagens hit
genes at random, which makes it impossible to cause a particular gene to mutate
preferentially. To improve a strain by mutation one has therefore to rely on sensitive tests
that make it possible to recognise and select the rare mutants which have the desired
characteristics.
Auxotrophic and regulatory mutants together with parents strains are still the most
commonly used microorganisms in industrial applications. Microbial process development
is mainly concerned with selecting first the type of organism necessary to either develop a
new or improve existing processes.
Strain improvement can also be achieved by hybridisation, which is any crossing of
genetically different individuals leading to an offspring with a genotype different from that
of either parent. Hybridisation is therefore a recombination of genetic material. Here
one has to distinguish between the sexual hybridisation of eukaryotes and the parasexual
hybridisation of prokaryotes, imperfect eukaryotes and eukaryotic tissue cultures.
Heterogenic incompatibility, however, restricts the use of this method and one should be
aware that one cannot expect that fresh genetic information taken from nature by
screening techniques will be suitable for the purpose of strain improvement by
hybridisation. n the case of parasexual processes, mechanisms known as conjugation,
transduction, transformation and mitotic recombination have been used frequently to bring
the genetic material together. The barriers of homogenic and heterogenic incompatibility
have been overcome to a large extent using the techniques of protopIast fusion and
protopIast infection with DNA, which then lead to a whole new field of gene technoIogy,
genetic engineering, recombinant DNA technoIogy and recombinant protein
technoIogy (Phler 1993)
.
Mutation alters a microorganism's genes, whereas recombination rearranges genes
or part of genes and brings together in an individual organism genetic information
from two or more organisms

The new and fast developing area of gene technology has its basis in the improvement of
recombinant DNA techniques (Watson 1965). The first process was that of transferring
plasmids. Plasmids are small circular molecules of extrachromosomal DNA found in
bacteria and yeast, that are capable of autonomous replication within the cell and are
inherited by daughter cells. These plasmids often carry genes that give certain bacteria
specialised properties. They can be transferred from one bacterial strain to an unrelated
strain and sometimes to a different species, to introduce totally different new genetic
properties to that bacterium. This ability to isolate plasmid DNA from a culture and induce
another culture to take it up is the basis of most recombinant DNA manipulation.
The improvement of recombinant DNA techniques over the past decades has let to the
use of plasmids as gene carrier and has opened the area for what is now known as
genetic engineering. A gene or genes taken from an unrelated organism or an artificially
synthesised gene can now be spliced into a plasmid and the plasmid introduced into a new
microbial cell. The plasmid serves therefore as a vector for genes that have no counterpart
in the recipient organism and could not be stably inherited in it through other recombinant
techniques such as hybridisation. The enormous development of gene technology or
genetic engineering was made possible mainly because of the discovery of those enzymes
which cause the above mentioned restrictions in gene modification, protecting the species
against foreign genetic information. These so-called restriction endonucleases are now
used

a) to cleave a given DNA into characteristic fragments, and
b) to split foreign DNA into a vector molecule such as the plasmid.

There exists a special system of nomenclature for the large number of individual restriction
endonucleases. The second discovery was the ligating enzymes, which are cable of
rejoining DNA fragments. This gene technology was soon extended from the prokaryotes
to eukaryotic systems, and the use of plasmid vectors further to viruses as vectors,
particularly in the case of animal and plant cells.
Recombinant DNA techniques can be applied in various ways for a number of different
industrial purposes. The most widely known objective is the production by a
microorganism of a protein it does not normally synthesise, such as an enzyme or a
hormone. The idea is to transfer an individual gene coding for the desired product into a
host microorganism and grow this microorganism in large volume to yield the product. A
different approach or objective of gene technology is the genetic improvement of an
existing strain. nstead of introducing a brand-new genetic capability one can improve the
efficiency of an existing strain by modifying its genetic information. Finally, the technology
will make it possible to improve the precision of a more traditional approach by bringing
about the mutation of specific sites in particular genes, overcoming the random nature of
normal mutagenesis.
The generalised scheme in Figure 10 outlines the approach taken to date in obtaining
cultures for microbial process development of a certain specified product. This does not
mean, however, that large scale processing can now commence. The modified strains
using the various types of genetic modification have again to be submitted to optimisation
studies. It is completely wrong to assume that the modified strain would behave in
the established medium in exactly the same way as the parent strain. The isolation
and growth optimisation may have been carried out in terms of product formation. Both
aspects have now to be brought under one and the same optimisation conditions.

Figure 2: General Flowsheet for Microbial Process Development

2.3 Nutrition, optimaI nutritionaI and physicaI requirements for growth
Despite their constant genotype, microorganisms are amazingly flexible in their ability to
alter their composition and metabolism in response to environmental changes. By virtue of
metabolic regulatory mechanisms, microbial cells do not generally oversynthesise
metabolites despite environmental variations. Both, microbial growth and product
formation therefore occur in response to the environment. t is essential to understand the
relationship between the chemical and physical environment and regulation of microbial
metabolism. The next step with our new culture is thus concerned with the establishment
of a medium economically useable on a process scale. This goal automatically excludes
solid media in favour of liquid cultures, because the liquid media and cultures are
amenable to standard chemical engineering techniques and equipment although solid
substrate cultivation techniques are improving and are on the increase even on process
scale [eg mushroom production, enzymes etc]. The requisite conditions for growth of
biomass in a culture are:
1. a viable culture,
2. an energy source,
3. nutrients to provide the essential material from which the cell is synthesised
4. the absence of inhibitors,
5. suitable physicochemical conditions.

2.3.1 MicrobiaI nutrition.
All substances in the environment, which can be used by the cell for catabolism and
biosynthesis are called nutrients. A culture medium must therefore contain, in quantities
appropriate to the specific requirements of the microorganisms for which it is designed, all
necessary nutrients. However, microorganisms are extraordinarily diverse in their specific
physiological properties, and correspondingly in their nutrient requirements. Literally
thousands of different media have been proposed for their cultivation, and in the
description of these media the reasons for the presence of the various components are
often not clearly stated. Nevertheless, the design of a culture medium can and should be
based on scientific principles, the principles of nutrition. The chemical composition of the
cell, broadly constant throughout the living world indicates the major material requirements
for growth. Water accounts for some 80-90% of the total weight of cells and is always
therefore the major essential nutrient in quantitative terms. The solid matter of cells
contains, in addition to hydrogen and oxygen, carbon, nitrogen, phosphorous and sulfur, in
order of decreasing abundance. These six elements account for about 95% of the cellular
dry weight.
The nutrients can be divided into two major classes:

1. necessary nutrients, without which the cell can not grow,
2. useful, but dispensable nutrients, which are used if present, but are not essential.

Some nutrients are the building blocks from which the cell makes macromolecules and
other structures, while other nutrients serve only as energy source without being
incorporated directly into the cellular material and sometimes a nutrient can play both
roles.
When a substrate or nutrient is only incorporated into the biomass, the amount of biomass
formed can be estimated from the stoichiometry. f, however, a substrate is used to
provide energy for metabolism, the efficiency with which the microorganism utilises the
energy becomes the factor determining the amount of growth. The yields of biomass
and/or other microbial products have significant implications on several aspects of
industrial microbiology.
The required substances can therefore be divided into two groups, macronutrients and
micronutrients, depending upon whether they are required in large or small amounts.
The carbon source is obviously one of the most important nutrients in the growth of
microorganisms. The element carbon is the most abundant element and represents
approximately 50% of the biomass. f it is a limiting factor, the total biomass X is
proportional to the initial concentration of the organic source of carbon, which gives the
yield constant for the substrate and organism:

t is therefore possible to calculate the minimum quantity of a carbon substrate to obtain a
specific yield of biomass [see chapter 7].
Nitrogen, which is needed for amino acids, purine and pyrimidine biosynthesis, can be
obtained by microorganisms from either inorganic or organic forms. The most inorganic
nitrogen sources are nitrate and ammonia [see chapter 2]. Although ammonia has the
same oxidation state as an amino group, its assimilation into amino acids still requires
expenditure of energy. The most common way is the direct introduction of an amino group
in exchange for a keto group:

Once the amino acid has been incorporated into glutamate, transaminases transfer the
amino group into the appropriate carbon skeleton.
When nitrate is used as a nitrogen source, it has to be reduced to ammonia first, a process
called assimilatory nitrate reduction [see chapter 2], which involves the enzymes nitrate
reductase and nitrite reductase. A third possibility is the utilisation of nitrogen gas (N
2
) as a
source of nitrogen, a process called nitrogen fixation. This process is a property of only
certain bacteria and cyanobacteria. n the fixation process, dinitrogen s reduced to
ammonia, which then can be used as mentioned earlier.

norganic sulphate is absolutely necessary for growth to synthesise the sulfur-containing
amino acids cysteine and methionine as well as the vitamins thiamine, biotin and lipoic
acid. The assimilation of sulphate first involves its activation by a reaction with ATP in two
steps to form phosphoadenosine phosphosulfate (PAPS). Subsequently, the sulphate
radical attached to PAPS is reduced to sulphite (SO
3
2-
), which is further reduced to
hydrogen sulphide (H
2
S). The incorporation of the sulfur into organic sulfur compounds
always occurs via serine [see chapter 2].

Phosphorous occurs in nature in the form of organic and inorganic phosphates and is
utilised by microorganisms primarily to synthesise phospholipids and nucleic acids. Thus
all microorganisms utilise inorganic phosphate for growth. Organic phosphate compounds
in nature are utilised as phosphate sources through the action of phosphatases, which are
enzymes hydrolysing the organic phosphate ester.
A variety of other minerals are required for growth, with potassium, magnesium, calcium
and in some cases silicon as macronutrients. Of those, magnesium is an essential
nutrient as it functions to stabilise ribosomes, cell membranes and nucleic acids.
Magnesium is also required for the activity of many enzymes, especially those involving
phosphate transfer. Gram-positive bacteria require about 10-times more magnesium than
do gram-negative bacteria. Without magnesium, there is no growth possible.
Calcium ions play a key role in the heat stability of bacterial spores and may also be
involved in the stability of the cell wall. Calcium, however, can not replace magnesium.
Potassium is universally required for the activation of some enzymes involved in protein
biosynthesis.
Sodium requirement reflects only the environment. Sea water, for example, has a high
sodium content and thus marine microorganisms generally require sodium for growth.
Tracer elements , or micronutrients, requirement are difficult to determine, since most
macronutrients contain enough tracer elements to satisfy the demand. The tracer elements
commonly required by most microorganisms are zinc, copper, manganese and
molybdenum. These metals function in enzymes or coenzymes.

Iron is a rather special case, as it requires in fairly significant amounts, although not at a
level of macronutrients. Since iron is normally present in the environment in a very
insoluble form, organisms must have special mechanisms for obtaining iron from their
habitats. ron has to oxidation states, ferrous (Fe
2+
) and ferric (Fe
3+
), with the ferrous
compounds generally more soluble. ron forms complexes with a wide variety of organic
compounds, specifically with metals which are called chelators. These chelators play a
special role in iron transport. Many microorganisms produce specific iron-binding organic
compounds called ironophores, which solubilise ferric ions and transport it into the cell.
Some of these ironophores are also referred to as siderochromes or ferrichromes.
Mostly, these organic compounds are derivatives of hydroxamic acid or phenolic acids. n
culture media, iron is rendered available by providing it in chelated form with a synthetic
chelating agent EDTA (ethylenediaminotriacetic acid) or NTAA (nitrolotriacetic acid). A
concentration of 10 g/ml would be in excess for virtually any microbial culture.
n addition to these nutrient requirements, some organisms and in particular mutants often
require so-called growth factors. Growth factors are specific organic compounds that are
required in very small amounts and can not be synthesised by the cell. Substances
frequently serving as growth factors are vitamins, amino acids, purines and pyrimidines. n
practice, any deficiency in biosynthesis or requirement for growth is compensated by the
addition of yeast extract or peptone.
These growth factor requirements have been widely employed for the examination of
foods, pharmaceuticals and other preparations. Such ' microbiological assays ' have the
virtues of specificity, sensitivity and simplicity. To perform the assay, a culture medium is
used in which all substances needed by the microorganism for growth are supplied, with
the exception of the substance to be assayed. The growth factor is then added to the
medium at some low concentration. Under these conditions, the amount of growth
obtained after incubation is proportional to the concentration of the limiting growth factor
(Figure 3):

Figure 3: Microbiological Assay

Growth factor requirements are greatest under anaerobic growth conditions and the least
under aerobic conditions.
n constructing a Culture Medium for any microorganism, the primary goal is to provide a
balanced mixture of the required nutrients at concentrations that will permit good growth. t
might seem at first sight reasonable to make the medium as rich as possible by providing
all nutrients in great excess. However, this approach is not a wise one. n the first place,
many nutrients become growth inhibitory or toxic as the concentration is raised, This is
true of many organic substrates, such as salts of fatty acids and even of sugars. Some
inorganic constituents may also become inhibitory if supplied in excess. Second, even if
growth can occur in a concentrated medium, the metabolic activities of the growing
microbial population will eventually change the nature of the environment to the point
where it becomes highly unfavourable and the population becomes physiologically
abnormal or dies.
The rational point of departure for the preparation of media is to compound a mineral base,
which provides all these nutrients that can be supplied to any organism in inorganic form.
This base can then be supplemented as required with a carbon source, an energy source,
a nitrogen source, and any required growth factor.
A medium composed entirely of chemically defined nutrients is termed a synthetic
medium. One that contains ingredients of unknown chemical composition is termed a
compIex medium.
n microbiology, every medium is finally sterilised before inoculation with the one specific
microbial strain under investigation. t is not wise to sterilise the mineral base medium
containing the carbon source, particularly if sugars are involved. Sugars do caramelise in
the presence of inorganic salts and thus only become partly available for microbial
utilisation. Carbon sources and mineral base solutions should be sterilised separately and
mixed aseptically prior to inoculation.
n order to encompass the variety of nutritional pattern known to exist amongst bacteria,
the following nutritional terminology has been introduced:
Autotroph: a microorganism that is able to use carbon dioxide as sole carbon source for
growth (cell carbon);
Heterotroph: a microorganism that requires carbon sources more reduced than carbon
dioxide for growth (cell carbon);
Photolithotroph: a microorganism that derives its energy from light and uses inorganic
compounds as electron donor (mostly photoautotrophs);
Photoorganotroph: a microorganism that derives its energy from light and uses organic
compounds as electron donor (mostly photoheterotrophs);
Chemolithotrophs: a microorganism that derives its energy from biochemical reactions
and uses inorganic compounds as electron donor (mostly chemo-autotrophs);
Chemoorganotrophs: a microorganism that derives its energy from Biochemical
reactions and uses organic compounds as electron donor (mostly chemoheterotrophs).
n order to take into account the requirement for growth factors an additional pair of terms,
prototrophy and auxotrophy, are sometimes employed. A prototroph can derive all
carbon requirement from the principal carbon source, whereas an auxotroph requires in
addition to the principal carbon source one or more organic nutrients.

2.3.2 Growth measurements.
n order to follow the course of growth, it is necessary to make quantitative measurements
Posten & Cooney 1993; Prescott et al. 1993; Doelle 1994). As a matter of convenience,
the properties measured are usually cell mass or cell number.
Dry weight. The only direct way to measure cell mass is to determine the dry weight of
cell material in a fixed volume of culture by removing the cells from the medium, washing
them in water or buffer solutions, drying them typically at 80
0
C for 24 hrs or at 110
0
C for 8
hrs, and then weighing them to constancy. Such determinations are time consuming and
relatively insensitive. Furthermore, it is difficult to weigh with an accuracy of less than 1
mg, the dry weight of which may still represent as many as 5 billion bacteria.
Optimal measurement of microbial biomass. The determination of the amount of light
scattered by a suspension of cells is a technique based on the fact that small particles
scatter light proportionally, within certain limits, to their concentration. When a beam of
light is passed through a suspension of bacteria, the reduction in the amount of light
transmitted as a consequence of scattering is thus a measure of the bacterial mass
present. Such measurements are usually made in a photometer or spectrophotometer
using appropriate wavelengths.
Absorbency is defined as the logarithm of the ratio of light striking the suspension (o) to
that transmitted by the suspension ():

Since scattering is inversely proportional to the fourth power of the wavelength of light
being scattered, the sensitivity of the measurements increases sharply if light of shorter
wavelength is used. n general, however, the lower limit of sensitivity of the method is
reached with bacterial suspensions that contain about 10 million bacteria/ml. Thus the
proportionality between absorbency and dry weight is linear only at low values of
absorbance (eg up to approx. 0.5 absorbency units).
Total cell count. The number of unicellular organisms in a suspension can be determined
microscopically by counting the individual cells in an accurately determined very small
volume. Such counting is usually done with special microscope slides known as counting
chamber or haemocytometer. These are ruled with squares of known area and are so
constructed that a film of known depth can be introduced between the slide and the cover
slip. Consequently, the volume of fluid overlaying each square is accurately known. Only
suspensions that contain 10 million or more cells/ml can be counted with any degree of
accuracy by this technique. The resulting data indicate the total number of cells, but do not
quantitate the number of viable cells unless a viable stain such as methylene blue is
used. This stain is oxidised to a colourless form by cells capable of respiring, a trait usually
associated with viability. Dead or non-respiring cells are stained blue.
Cell counts can also be carried out using an electronic equipment, the Coulter Counter. n
this technique, a portion of the suspension is passed through a fine orifice (30 m) into a
small glass tube. The orifice serves to complete an electric circuit through the suspending
medium between electrodes on the interior and exterior of the tube. Detection depends on
the difference in conductivity between the bacterium and the suspending liquid. Each time
a bacterium or particle passes through the orifice, the conductivity drops. The suspending
liquid must therefore be scrupulously free of inanimate particles (eg dust) since smaller
ones will be counted as cells and larger ones will plug the orifice.

Viable counts. Since single viable cells separated from one another in space by
dispersion on or in agar medium give rise through growth to separate, macroscopically
visible colonies, the enumeration of unicellular organisms can also be made by plate
count. Hence, by preparing appropriate dilutions of a bacterial population and using them
to seed an appropriate medium, one can ascertain the number of viable cells in the initial
population by counting the number of colonies that develop after incubation of the plates,
and multiplying this figure by the dilution factor. n contrast to direct microscopic
enumeration and electronic counting or dry weight determination, this technique measures
only those cells that are capable of growth on the plating medium used.
The viable count is by far the most sensitive method of estimating bacterial number, since
even a single viable cell in a suspension can be detected. ts accuracy depends on
observing certain precautions. Rapid chilling prior to or during dilution can cause death of
a significant portion of the population in certain cases, a phenomenon known as cold
shock.
A combination of total cell and viable count can be used to determine the fraction of viable
cells in the population.

Cell constituent measurement. Sometimes, because of growth patterns (eg cells may
grow as filaments or form clumps) or of complexity of the medium, it is impractical to
measure cell mass or numbers. n these cases growth can be measured by determining
the amount of a particular cell constituent, eg protein, peptidoglycan, RNA, DNA or ATP in
the medium. Such measurements are often the most practical way to determine microbial
biomass and growth in a natural environment.

2.3.3 Growth Curve
f we follow bacterial growth by cell mass or cell number measurements and plot the
absorbance or number against time, we obtain the so-called growth curve of
microorganisms. We will find that the cell population passes through four (4) main phases
(Fig. 4): lag, logarithmic (log), stationary and death phase:

Figure 4: A typical microbial growth curve

The lag phase is the adaptation period required and depends largely on the preculture
medium from where the inoculum is obtained. f the organism has been grown in the same
medium as the experiment is carried out, all enzymes should be fully adapted and
functional and the lag phase should be shortest or not even in existence. f, however, the
preculture was grown under different conditions, the organism requires an adaptation
period for carrying out the necessary metabolic changes.
Once the organism has adapted itself, balanced growth occurs and the population
multiplies, whereby a straight line relationship exists between the logarithm of cell mass or
number and time. This particular phase is referred to as the exponential or logarithmic
phase of growth.
Microbial populations seldom maintain exponential growth at high rates for long and it is
normally limited either by exhaustion of available nutrients or by the accumulation of toxic
products of metabolism. As a consequence, the rate of growth declines and can either
continue for a while arithmetically or goes straight into the stationary phase. This transition
involves a period of unbalanced growth during which the cellular components are
synthesised at unequal rates. Consequently, cells in the stationary phase have a chemical
composition that is different from that of cells in the exponential phase. They are also more
resistant to adverse physical and chemical agents.
From these measurements it follows that, when microbial cells are inoculated into a
nutrient broth and incubated at a suitable temperature, a sequence of changes occur. n
order to be able to characterise the behaviour of various microorganisms under a variety of
cultivation conditions and in order to find optimal growth conditions, it is necessary to use
some form of mathematical description and a survey of the different types of cultivation
techniques available (see chapter7).

2.3.4 Optimisation of nutritionaI and physicochemicaI factors
All microorganisms used for microbial process development require organic compounds
both as source of carbon and energy. The element carbon is the most abundant element
and represents approximately 50% of the biomass. n the case of algae and
photosynthetic bacteria, the energy source is light and the carbon source is carbon
dioxide, chemoautotrophic bacteria can utilise inorganic compounds as energy source and
carbon dioxide as carbon source, whereas chemoheterotrophic organisms require organic
compounds for both. For any microbial process development, the carbon source is
therefore the largest ingredient. f it is a limiting factor, the total biomass X is proportional
to the initial concentration of the organic source of carbon, which gives the yield constant
as was outlined earlier:

This yield constant comes from the original definition developed by Monod

t is the carbon source which therefore is predominant and is selected, of course, from the
substrate available. The intimate relationship between the substrate as carbon and energy
source can be found in cases when the energy yield [ATP] is known. n this situation there
exists a proportionality between the number of moles ATP formed and the biomass
produced (see chapter 8). n anaerobic cultures this yield factor is around 10. Under
aerobic conditions, however, this yield factor varies greatly and is manyfold higher, since
much larger proportions of carbon substrates are converted into biomass. t is possible to
calculate the minimum quantity of a carbon substrate to obtain a specific yield of biomass.
f one assumes a 50% biomass carbon requirement and required 50 g of bacterial cells on
a dry weight basis per litre, one would require:

The nutrients that are amenable to measurement and to cell growth are, apart from the
carbon source, nitrogen, oxygen, mineral salts and some specific growth factors, eg amino
acids or vitamins. The reliability of the method depends on the ratio of cell mass produced
per unit nutrient consumed (cell yield), the accuracy of the analytical methods and the
presence of substances which interfere with the analysis. Furthermore, if the substrate is
also used for product formation and the ratio of product to cell mass is large and/or
variable, a substantial error will result unless another independent measurement for
product concentration is available to correct the measurement of substrate used for cell
biosynthesis.
A very significant difference in approach for nutrient optimisation depends on the kind of
process envisaged: anaerobic or aerobic. n the latter case, oxygen is a vital nutrient,
whereas redoxpotential replaces oxygen in anaerobic cultures. Optimisation of
anaerobic cultures requires a carbon balance between carbon substrate, biomass
and product, whereas in aerobic cultures it is mainly a balance between carbon
substrate and biomass.
Microbial growth is, however, also a function of temperature, which has been described by
the Arrhenius equation:

whereby A is the Arrhenius constant, Ea is the activation energy, R is the gas constant and
T is the absolute temperature. This means, of course, that temperature also affects the
efficiency of the carbon energy substrate conversions to cell mass and thus a variety of
metabolic processes in the cell. The most important factor in the optimisation of a microbial
process is therefore the design of the growth and production medium. From above outlined
considerations, a first approximation of the minimum requirement could be achieved using
the stoichiometry for growth and product formation. n contrast to academic research,
several economical and technical constraints should be considered or built in for microbial
process development. These constraints include cost, availability of raw materials,
requirement for specific carbon and nitrogen sources, recovery and pollution control. t
should always be remembered that the ultimate goal or objective function is the
development of a process with minimum cost per unit product.
When the microbial cell is faced with more than one utilisable substrate, it has to make a
choice. f it would produce all the enzymes necessary for the utilisation of all the substrates
present, this would be less economic than producing enzymes for the utilisation of one
substrate after the other. The cell thus produces enzymes to utilise the best substrate
present first and only after the exhaustion of this primary substrate are the enzymes
formed for the next substrate. This phenomenon is called catabolic repression and referred
to as diauxie[see chapter 11].
n order to grow and remain alive, microorganisms must carry out a tremendous number of
enzyme-catalysed reactions. f a cell is placed in an environment containing a polymer
such as starch plus ammonia and salts, it must first hydrolyse the starch to glucose, bring
the glucose into the cell and then degrade the hexose to three- and two-carbon
compounds, and feed these smaller molecules into the tricarboxylic acid cycle to provide
energy and intermediates. The intermediates formed by this and other reaction sequences
must be converted to building blocks, such as 20 amino acids, 4 ribonucleotides, 10 or so
vitamins, fatty acids, sugars, sugar acids, and hexoseamine. These building blocks must
then be converted into about 2000 proteins, DNA, three types of RNA, mucopeptides,
polysaccharides, coenzymes and lipids. These molecules are then used to form cell
structures such as nuclei, ribosomes, flagella, cell walls, membranes and in eukaryotes
also mitochondria and other inclusion bodies.
The DNA of the microbial cell dictates the detailed synthesis of the enzymatic machinery.
Despite their constant genotype, microorganisms are amazingly flexible in their ability to
alter their chemical composition and metabolism in response to environmental changes.
The environment does not change the genetic make-up of the cell but markedly affects the
phenotypic expression of the genes. By virtue of metabolic regulatory mechanisms,
microbial cells do not generally oversynthesise metabolites despite profound
environmental variations.
Whereas degradation enzymes are usually regulated by induction and catabolic regulation,
the biosynthetic enzymes that convert metabolic intermediates to the building blocks of
macromolecules are mainly controlled by feedback inhibition (see chapter 11). n addition
to metabolic regulation, cells possess another selective mechanism essential to their
economy - permeability control. The main permeability barrier is the cytoplasmic
membrane. Whereas metabolic regulation prevents oversynthesis of metabolites and
macromolecules essential to the life of the cell, the permeability barrier allows cells to
retain concentrated solutions of these same molecules and so selectively bring into the cell
required nutrients. Environmental conditions exercise a strong control over permeability.
Permeability can be increased or decreased by mutations and by changes in the
environment. The different types of transport are of great importance.
These mechanisms control the degradation of substrates at the cell membrane level and
thus are also responsible for the uptake of the substrate. Since the substrate taken up by
the cell is used by the organism to provide energy for growth and biosynthesis of
molecular compounds such as enzymes, a proviso has to be made for a possible
overproduction of energy, which would lead to heat dissipation and thus overheating of the
cell. This type of energy regulation is most prevalent in aerobic metabolism and has been
quantified as

This formula not only regulates the activities of the catabolic enzymes but also the
biosynthetic enzymes that utilise ATP. The existence of such a control again indicates the
coordination of control between catabolism or substrate utilisation and growth or
biosynthesis of enzymes and other growth-requiring compounds.
2.4 Process Strategy
Microbial cells have two main commercial applications. The first is a source of protein,
primarily for animal feed. Since growth of a microorganism can be followed relatively
easily, optimisation methods have been devised along the lines mentioned earlier. A great
number of industrial processes have been developed over the last decade. The second
commercial application is, however, the more difficult one, as it uses the microbial cell to
carry out biological conversions and thus leads to organic chemicals or enzyme
production. These biological conversions or microbial transformations can be
accomplished with growing cells, non-growing cells, immobilised cells, spores or even
dried cells. There are signs visible already for replacing certain chemical industries
because of their heavy energy input and/or pollution/waste management demands.
Biological conversions have many advantages over chemical conversions. Besides a
strong energy input, chemical conversions require generally solvents and inorganic
catalysts, both of which could be strong pollutants. n contrast, biological conversions are
carried out with water as solvent and at moderate biological temperatures. The commercial
important products from microorganisms can be categorised into three major classes:

1. the large molecules such as enzymes;
2. the primary metabolic products (compounds essential for growth);
3. the secondary metabolic products (compounds not required for growth).

2.4.1 Primary metaboIites.

Primary metabolites include end products of low molecular weight that are formed during
the utilisation of the carbon source and include all intermediates of catabolic and
biosynthetic pathways. Amino acids, purine, pyrimidine nucleotides, vitamins, organic
acids, alcohols, solvents are all considered to be primary products or metabolites.
Overproduction, however, of primary metabolites is avoided by microorganisms since it is
a wasteful process that decreases survival ability in nature. Some microorganisms survive
with aberrations in their regulatory mechanisms, and these overproducers are the cultures
chosen in screening programmes designed to isolate potential fermentation cultures.
These cultures are then subjected to intensive development programmes in which
environmental and genetic modifications are used to decrease regulation and increase
overproduction. An increase in permeability of the bacterial membrane is another method
and is responsible for the overproduction of glutamic acid, the most important commercial
amino acid in Southeast Asia. Two common characteristics of the microorganisms
involved represent the biochemical keys to their glutamate overproduction: a deficiency of
2-ketoglutarate dehydrogenase and a nutritional requirement for biotin (necessary for cell
wall biosynthesis). Ketoglutarate being a TCA [tricarboxylic acid cycle] intermediate is
converted to glutamate via a reductive amination catalysed by glutamate dehydrogenase.
Without a modification of the permeability by biotin limitation, feedback inhibition would
restrict very severely glutamate production.

2.4.2 Secondary metaboIites.
Secondary metabolites are molecules synthesised by certain microorganisms, usually late
in the growth cycle. Although not required for growth, these metabolites may have survival
value for the organism producing them. The best known secondary metabolites are the
antibiotics, mycotoxins and pigments.

2.4.3 Bioconversions.
Bioconversions are processes in which microorganisms convert a compound to a
structurally related product. Such conversions are often called microbial transformations.
The processes comprise only one or a small number of enzymatic reactions, as opposed
to the multireaction sequences of fermentation pathways.
One of the earliest known bioconversions involves the manufacture of vinegar from
ethanol by acetic acid bacteria. Organisms catalysing bioconversions act a stereospecific
catalysts. The ultimate in specificity are the steroid bioconversions so useful in providing
new intermediates for chemical conversion into improved steroidal drugs. The main types
of steroid modifications include monohydroxylation, dihydroxylation, epoxidation,
dehydrogenation and hydrogenation. High yields are usually obtained in bioconversions.

The application of chemical engineering principles is required for the analysis of design
and operation of bioreactors, recovery of products and waste treatment, if necessary
(Atkinson & Mavituna 1983; Bailey & Ollis 1986; Stephanopoulos 1993). Classical
approaches to the analyses, however, are limited by a number of constrains:

1. the reactant mixture is relatively complex. Microbial biomass increases with the
biochemical transformation and the catalyst is synthesised as the reaction proceeds;
2. the bulk densities of suspended microbial cells and substrate particles generally
approach those of their liquid environment so that relative flow between the dispersed
and continuous phase is normally low;
3. the size of microbial cells are very small compared to chemical particles: coupled with
the above constraints it is generally difficult to promote high velocities and turbulent
flow conditions;

4. polymeric substrates or metabolites and mycelial growth often produce very viscous
reaction mixtures which are generally pseudoplastic non-Newtonian, again these
conditions tend to limit desirably high flow dynamics in the bioreactors;
5. many multicellular microbial growths, especially fungal ones, generally form
relatively large cell aggregates such as clumps or pellets. Compared to catalyst
particles, intraparticle diffusional resistance are often serious in these systems, eg
leading to anaerobiosis;
6. bioreactors frequently require critically close control of solute concentrations, pH,
temperature and local pressures in order to avoid damage or destruction of live or
labile compounds which are essential to the process;
7. very low concentrations of reactants and/or products in aqueous media are normally
involved in bioreactors, so concentration forces for mass transfer are often severely
limited;
8. microbial growth rates are substantially lower than chemical reaction rates so hat
relatively large reactor volumes and residence times are required.

n order to combat these problems, at least two specialisations took place within the
chemical engineers. Efficiency and reproducibility are major concerns of reaction
engineering, whereas the applications of chemical engineering principles and practices
in microbiological processes constitutes the main activities of fermentation engineering.

Figure 5: General diagram of the Chemical Engineering Concept

n the development of an industrial process, good engineering means the following tasks
must be undertaken in a logical manner:

3.1 Identification of main products and substrates and, if possibIe, aIso main
intermediates.
n the case of a fermentation process, such information is usually supplied by the
microbiologist and biochemist working with laboratory microbial cultures.
3.2 Stoichiometry of the process.
> For a fermentation, an exact material balance of the process is not always possible.
Nevertheless, as much information as possible should be gathered of details regarding the
ratios among products, substrates (eg carbon source, nitrogen sources, oxygen supply),
and known intermediates and regarding the variations of these ratios responding to
environmental changes. n some fermentation processes, such as those producing
microbial biomass protein (MBP) from hydrocarbon, energy balance is of great
importance..
3.3 Kinetic and process rate.
Often, problems (3.1) and (3.2) cannot be fully answered without the consideration of a
time scale. n batch processes, the accumulated changes in products, intermediates, and
substrates are of great concern. However, the time span and the manner by which such
changes take place, and thus the kinetics and rate of the involved processes, are also
necessary information. n the case of continuous fermentation, which is still gradually
gaining in its popularity, the design and analysis of the reactors are usually based upon the
rates of change of these quantities and dilution rate.
3.4 Reactor design.
nformation on (3.1), (3.2) and (3.3) is a prerequisite for the ultimate objective of microbial
reaction engineering, which deals with the design and analysis of fermenters. Even though
stirred tanks still represent the most popular geometry of fermenters, an increasing
number of other physically shaped vessels, such as tower fermenter, do appear in
industry. Even with the same stirred tank, considerations regarding a proper choice from
different modes of operation, including batchwise, fed-batchwise, continuous and others,
are also part of the reaction engineering activity.
Complete, accurate and detailed record keeping is essential in order to be able to
understand and describe the dynamic behaviour of fermentation processes in terms of
kinetic models. With this understanding it is then possible to expand the control of
fermentation processes beyond independent closed-loop feedback control of culture
conditions, such as temperature, pH and dissolved oxygen, into the more sophisticated
strategies of adaptive or interactive control.
3.5 Product recovery.
Let us now assume that we have designed a plant and it operates. The next task of the
chemical engineer comes in regard to recovery and isolation of the products produced by
the fermentation. The basic problems that confront the separation technologists, whether
they are chemists or engineers, are the complexity in the starting material or feed to the
isolation process. Furthermore, it is of extreme importance to know:
a) what is the value of the product ?

Figure 6: Biotechnological Process Flowsheet

b) what is an acceptable product quality for the proposed end-use ?
c) where is the product in the complex mixture ?
d) where are the impurities in the complex mixture ?
e) what are the physical and chemical properties of the product and the impurities ?
f) what are the economics of various isolation possibilities ?

Careful consideration of these questions will provide answers that will enable the
isolation technologist to teach the goals of adequate product quality and high recovery
with minimum effort. The last two questions (e) and (f) obviously affect the final cost of
the product appreciably.
An extremely important factor before commercialisation progresses is the need for an
extensive economic evaluation. Unique characteristics of fermentation processes,
including the use of agricultural commodities, high energy use per unit of product, and
need for aseptic conditions require the involvement of chemical engineers. t is
assumed that a totally new facility is to be built, site allocation is an early requirement.
f one looks at the generalised flowsheet in figure 14, there are certain unique
requirements which should be considered in fermentation plant design.

3.6 Waste treatment
. Finally, there is the waste treatment design which may or may not be required, depending
upon the effluent from the plant. The most economic consideration would be, of course, if
a waste utilisation design could be involved for additional product formation. Microbial and
fermentation technology today involves man, biomass, and industry in emphasising the
utilisation of renewable resources with a Iow environmentaI impact and a high
regenerative capacity. Microbial technology must therefore provide for
Environmental Management through the bioconversion of domestic wastes into non-
polluting fuels, such as methane, ethanol and methanol;
Bioconversion of Agroindustrial Residues and products into value- added products;
Enhancement of Soil Fertility and Stability through the direct application of sludge
material or microbial fertilisers;
Public Health Program by elimination of enteric parasites through the anaerobic digestion
process or cleaner ecological environment;
Waste Water Treatment and Waste Utilisation through microbial- based systems;
Concentration and leaching of valuable minerals from mining waters and low grade
ores;

Substitution of Toxic Chemical Products and pesticides by microbial preparations.
n terms of available resources, the choice of technology to be used depends upon a
variety of factors:
High-Capital Technology involves relatively heavy financial investments, sophisticated
operation procedures, large-scale plants with complex equipment and high maintenance
costs. This type of technology has been of benefit mainly to industrialised countries.
Intermediate Capital Technology involves moderate financial investments, medium scale
plants with appropriate equipment and maintenance costs and less complex operating
procedures. The growth of such a technology can be linked to a re-evaluation of the
management of energy and other natural resources, and to environmental considerations
necessitating the utilisation of wastes that 'are resources out of place'.
Low-Capital Technology is featured by the low financial investment, small-scale
operating procedures, use of simple, indigenous equipment. Aimed at the eradication of
rural and village poverty, conservation of the environment and use for social action-
oriented programmes, such technology is best exemplified in biogas production,
mushroom cultivation, fermented food preparation and photosynthetic oxidation pond
systems [algal technology].
Microbial technology s a very challenging area and the following lectures are meant to help
fostering further development in this are and stress the importance of microbiology for the
survival of mankind.
4. References
Atkinson,B. and F.Mavituna 1983 Biochemical Engineering and Biotechnology
Handbook. MacMillan Publishers, London

BaiIey,J.E. and D.F.OIIis 1986 Biochemical Engineering Fundamentals. McGraw-Hill
Book Comp., New York

BaIts, R.H.(ed.) Strain mprovement. n 'Manual of Industrial Microbiology and
Biotechnology ' [A.L.Demain & N.A.Solomon, eds.], pp 154-215. American Society for
Microbiology, Washington

Chang,L.T. and R.P.EIander 1986 Long-term Preservation of ndustrially mportant
Microorganisms. n 'Manual of Industrial Microbiology and Biotechnology' [A.L.Demain &
N.A.Solomon, eds.], pp 49-56. American Society for Microbiology. Washington

DaSiIva,E.J. 1980 Trends in microbial technology for developing countries. n 'Renewable
Resources A systematic approach. (E.Campos-Lopez, ed.), pp. 329-368. Academic Press,
London

Demain,A.L. and N.A.SoIomon [eds.] 1986 Manual of ndustrial Microbiology and
Biotechnology. American Society for Microbiology, Washington

DoeIIe,H.W. 1994 Microbial Process Development. World Scientific Publishers, Singapore

Hunter-Cevera,J.C., Fonda,M.E. and A.BeIt 1986 solation of Cultures. n 'Manual of
Industrial Microbiology and Biotechnology (A.L.Demain & N.A.Solomon, eds.], pp 3-23.
American Society for Microbiology, Washington

MIRCENs 1997 Microbiological Resource Centers: MRCENs. A Resource for Global
Cooperation. American Society for Microbiology, Washington

Posten,C.H. and C.L.Cooney 1993 Growth of Microorganisms. n 'Biological
Fundamentals ' (H.Sahm, ed.), pp 113-162. Biotechnology, VCH Weinheim, Germany

Prescott,L.M., HarIey,J.P. and D.A.KIein 1993 Microbiology. 2nd ed., Wm.C.Brown
Publishers, Oxford

PhIer,A. (ed.) 1993 Genetic Fundamentals and Genetic Engineering. Vol. 2 of
Biotechnology. VCH Weinheim, Germany

SIy,L.I. 1991 Culture Collection Technologies and the Conservation of Our Microbial
Heritage. Unesco-MRCEN Training Course on 'The Importance of Microbiological
Biotechnology for Community and Economic Development ', Motupore sland, Papua New
Guinea

SIy,L.I. 1994 solation, Characterization and dentification of Microorganisms. n 'Microbial
Process Development ' (H.W.Doelle, ed), pp. 21-42. World Scientific Publishers,
Singapore

SIy,L.I. 1994 Maintenance and Preservation of Microbial Cultures. n 'Microbial Process
Development ' (H.W.Doelle, ed.), pp. 55-74. World Scientific Publishers, Singapore

StephanopouIos,G. (ed.) 1993 Bioprocessing. Vol. 3 of Biotechnology, VCH Weinheim,
Germany

Watson, J.D. 1965 Molecular Biology of the Gene. W.A.,Benjamin nc., New York

White,R.J., Maiese,W.M. and M.Greenstein 1986 Screening for new products. n 'Manual
of Industrial Microbiology and Biotechnology' [A.L.Demain & N.A.Solomon, eds), pp. 24-
31. American Society for Microbiology, Washington

Chapter 6
MICROBIAL CELL TYPE AND STRUCTURE
[This chapter represents part of the lectures given during lecture courses given at the Prince of Singkla
University in Hat Yai, Thailand and the University of Veracruz at Orizaba, Mexico in the 1990s]

Content
1. Introduction
2.1 Prokaryotes
2.1.1 CeII structure and functions
2.1.2 Gram-negative aerobic chemoheterotrophic rods and cocci
2.1.3 Gram-negative aerobic chemoIithotrophic rods and cocci
2.1.4 FacuItative anaerobic gram-negative rods
2.1.5 Anaerobic gram-negative rods
2.1.6 Aerobic and microaerophiIic gram-negative bacteria
2.1.7 Other gram-negative bacteria
2.1.8 Gram-positive bacteria
2.1.9 Non-sporing gram-positive rods
2.1.10 Anoxygenic photosynthetic bacteria
2.1.11 Oxygenic photosynthetic bacteria
2.2 Eukaryotes
2.2.1 CeII structure and functions
2.2.2 AIgae
2.2.3 Yeast
2.2.4 Fungi or MouIds
3. OsmoreguIation
5. Viruses
6. BibIiography
1. Introduction
One of the great unifying theories of biology is, that the cell is the fundamental unit of all
living matter. A single cell is an entity, isolated from other cells by a cell wall or membrane
and containing within it a variety of materials and subcellular structures. Cellular organisms
share a common chemical composition, their most distinctive chemical attribute being the
presence of three classes of complex macromolecules: deoxyribonucleic acid (DNA),
ribonucleic acid (RNA) and proteins. DNA is the constituent that carries in coded form all
the genetic information necessary to determine the specific properties of the organism,
known collectively as phenotype. The genetic information is initially transcribed into
complementary RNA sequences, in the form of molecules of RNA known as messenger
RNA (mRNA). The mRNA molecules subsequently serve as templates for the synthesis of
all the specific protein molecules characteristic of the organism. The translation of the
transcribed genetic message is mediated by organelles known as ribosomes, composed of
protein subunits and a special class of RNA, ribosomal RNA (rRNA). A third class of RNA
molecule, the transfer RNAs (tRNAs) also participate in protein synthesis as carriers of the
amino acids that are assembled into linear sequences in the primary step of protein
synthesis [see Chapter 11]. The proteins of an organism includes the enzymes that
catalyze its activities, and the subunits from which many classes of proteinaceaous cellular
microstructures are assembled.
The chemical activities of an organism, catalysed by its specific array of enzymes, are
known collectively as metabolic activities [see Chapters 9 and 10]. They include (a) the
biosynthesis of the macromolecular constituents from much simpler chemical substances
referred to as nutrients derived from the external environment and (b) the reactions
necessary to generate the energy-rich substances that drive the process of biosynthesis
[see Chapter 8].
Most organisms share a common physical structure, being organised into microscopic
subunits termed cells. All cells are enclosed by a thin membrane, the cytoplasmic
membrane, which retains within its boundary the various molecules necessary for the
maintenance of biological function, and which at the same time regulates the passage of
solutes between the interior of the cell and its external environment [see chapter 8]. Cells
never arise de novo, but are always derived from pre-existing cells by the process of
growth and cell division [see chapter 7].
Viruses are not in general regarded as cell types, as they lack many attributes of a cell and
acquire the attributes of a living cell only when associated with a cell.
Around 1950 occurred the development of the electron microscope and associated
preparative techniques for biological materials, which made it possible to examine the
structure of cells with a degree of resolution many times greater than that previously
possible with the light microscope. This led to the recognition of a profoundly important
dichotomy among the various groups of organisms with respect to the internal architecture
of the cell. Depending on their detailed structures, two radically different kinds of cells were
found. The less complex prokaryotic cell is the unit of structure in two microbial groups:

1. the eubacteria [including the cyanobacteria, formerly known as the blue-green algae]
and
2. the archaebacteria, a heterogenous group of microorganisms with prokaryotic structure
but with a cell chemistry that is strikingly different from that of eubacteria.

The differences between the eubacteria and the archaebacteria are so profound that most
microbiologists now believe that this distinction reflects an evolutionary separation as
fundamental as that which divides the eukaryotes from either of the two groups.
The more complex eukaryotic cell is the unit of structure in plants, metazoan animals,
protozoa, fungi, yeast and all algae. Despite the extraordinary diversity of the eukaryotic
cell as a result of its evolutionary specialisation in these groups, as well as the
modifications that it can undergo during the differentiation of plants and animals, its basic
architecture always has many common denominators.
We can thus distinguish on the basis of cell structure and function three major groups of
cellular organisms (see table 1): the eubacteria, archaebacteria and the eukaryotes.

TabIe 1: Relative difference between Gram-positive and Gram-negative prokaryotes.
The eubacteria can be further subdivided into Gram-negative and Gram-positive
eubacteria on the basis of the structure of the cell wall. There exists a small group of
eubacteria that cannot be assigned to either of these groups because they lack a cell wall.
Our knowledge of the diversity of the archaebacteria is still too rudimentary to attempt a
systematic subdivision. The eukaryotes can be subdivided into the following groups: the
yeasts, the fungi, the algae, the plants, the animals, and the protists.
2.1 Prokaryotes
2.1.1 CeII structure und functions
As was mentioned earlier, prokaryotic cells are the simplest living cells in terms of
structure and are very small. They can be made visible under the normal light microscope
by various staining methods, of which the Gram stain is the most important one as it this
staining method indicates the fundamental differences in the cell wall structure.
Despite their small size, there is a wide variation in size among different organisms. Most
bacteria have distinctive cell shapes, which remain more or less constant, although shape
is more or less influenced by the environment. Bacteria shaped like spheres are called
cocci, whereas those shaped like cylinders are referred to as rods. f a rod is many times
longer than it is wide, it is usually called a filament. Some bacteria are shaped like spirals
and a long spiral has the shape like a helix The shape of the cell definitely affects its
behaviour and stability. Cocci, for example, being round , become less distorted upon
drying and thus can survive normally more severe desiccation than rods or spirals. Rods,
on the other hand, have more surface exposed per unit volume than cocci do and thus can
more readily take up more nutrients from dilute solutions.
The material (cytoplasm) of the prokaryotic cell is surrounded by a cytoplasmic membrane,
which controls passage of materials into and out of the cell (see Chapter 8). External to
and thus protecting the cytoplasmic membrane is a rigid cell wall made up of chemical
substances unique to prokaryotes. Some prokaryotes possess threadlike appendages
which originate in the cytoplasm and extend beyond the cell wall. These are called flagella
and are responsible for the microorganism's motility. Some cells have a slimy covering
around the rigid cell wall, which is referred to as capsule or slimy layer.
Within the cytoplasmic area of the prokaryotic cell, the following structures can be
observed:

Ribosomes: small particles consisting of proteins and ribonucleic acid, which are involved
in the synthesis of new proteins and enzymes;
GranuIes: deposits of various chemical substances which may serve as reserves or store
food
NucIear materiaIs: strands of desoxyribonucleic acid (DNA), the carrier of genetic
material and genetic information and ribonucleic acid (RNA);
PIasmids: small, circular, extrachromosomal genetic components
Mesosomes: folds or invaginations of the cytoplasmic membrane into the cytoplasm.

Prokaryotes have many shapes: round, rodlike, twisted, and branched. A coccus with a
diameter of 1.2 has a volume of 10
-2
cm
3
, the specific weight is 1.1 and the mass about
1.1x10
-12
g.
The prokaryotic ceII waII is chemically quite different from that of any eukaryotic cell. t
consists of peptidoglycan, a large polymer built around subunits of N-acetylglucosamine
and N-acetylmuramic acid. A four unit side chain consisting of the amino acids L-alanine,
D-glutamic acid, diaminopimelic acid and D-alanine crosslinks the peptidoglycan
molecules forming a tight mesh. The peptidoglycan content of the bacterial cells vary
greatly from more than 50% in the walls of Gram-positive to less than 10% in Gram-
negative cells. The relative differences between the two types are given in table 8.1. t is of
particular interest to note that Gram-positive cells are much less resistant to antibiotic
treatment compared with Gram-negative cells.
n Gram-negative cells, the inner layer of the cell wall is poor in peptidoglycan and the
outer layer is rich in lipoprotein and lipopolysaccharides, which are mainly responsible for
antigenic specificity.
The peptidoglycan structure is present only in prokaryotes, and is found virtually in all
species. The sugar N-acetylmuramic acid is never found in eukaryotes and the amino acid
diaminopimelic acid (DAP) is also never found in eukaryotic cell walls. However, not all
prokaryotic organisms have diaminopimelic acid in their peptidoglycan. This acid is present
in all Gram-negative bacteria and in some Gram-positive species, but most Gram-positive
cocci have lysine instead of DAP. Another unusual feature of the prokaryotic cell wall is the
presence of two amino acids that have the D-configuration, D-alanine and D-glutamic acid.
n proteins, amino acids are always in the L-configuration.
The formation of the peptide cross-links involves an unusual type of peptide bond
formation called transpeptidation, which is also important because it is inhibited by the
antibiotic penicillin. Cycloserine is another antibiotic which functions by blocking
transpeptidation. The inhibition of transpeptidation by penicillin leads to the formation of a
peptidoglycan which lacks strength. The further damage to the cell, resulting in lysis and
death occurs because there are enzymes in the cell, called autolysin, that are involved in
the opening up of the peptidoglycan structure as growth occurs. These enzymes continue
to act, but because new peptidoglycan cross-links cannot occur, the cell wall will become
increasingly weaker and osmotic lysis occurs. This lysis by penicillin can be prevented by
adding an osmotic stabiliser such as sucrose. Under such conditions, continued growth in
the presence of penicillin leads to the formation of protoplasts and spheroplasts.
Penicillin-induced lysis only occurs with growing cells.
n most Gram-negative bacteria, the outer waII Iayer exists as a true unit membrane.
However, the outer membrane layer is not constructed solely of phospholipid as is the
plasma membrane, but also contains additional lipid plus polysaccharide and protein. The
lipid and polysaccharide are intimately linked in the outer layer to form specific
lipopolysaccharides (LPS) structures. Although complex, the chemical structures of
some LPS layers are now well understood. Here the polysaccharide consists of two
portions, the core polysaccharide and the O-polysaccharide. n Salmonella, the core
polysaccharide consists of ketodeoxyoctonate, seven carbon sugars or heptoses, glucose,
galactose and N-acetylglucosamine. Connected to this core polysaccharide is the O-
polysaccharide, which usually contains galactose, glucose, rhamnose aqnd mannose as
well as one or more unusual dideoxysugars such as abequose, colitose, paratose or
tyvelose. The functional importance of the outer layer is that it serves as an outer barrier
through which materials must penetrate if they are to reach the cell. The outer layer is
permeable to small molecules, but not to enzymes or other large molecules. n fact, one of
the main functions of the outer layer may be its ability to keep certain enzymes, which are
present outside the peptidoglycan, from leaving the cell. These enzymes are present in the
area called periplasmic space. The outer layer in many Gram-negative bacteria possesses
toxic properties and is responsible for some of the symptoms of infection. A type of such
toxic substance called endotoxin is either part of or equivalent to the LPS.
Although Gram-positive bacteria do not have a lipopolysaccharide outer layer attached to
their cell wall, they generally have acidic polysaccharides called teichoic acid, which
contain repeating units of either glycerol or ribitol. These polyol units are connected by
phosphate esters and usually have other sugars and D-alanine attached. Because they
are negatively charged, teichoic acids are partly responsible for the negative charge of the
cell surface as the whole. Teichoic acids have been shown to regulate autolysin action to
keep it in balance with cell wall synthesis.
Certain glycerol-containing acids are bound to the membrane lipid of Gram-positive
bacteria. As these teichoic acids are intimately associated with lipid, they have been
referred to as lipoteichoic acids.
The area between the outer layer and the peptidoglycan is called the periplasmic space,
in which many enzymes are held. The outer layer can also possess toxic properties and
endotoxins are either part of or equivalent to lipopolysaccharides. Gram-positive cells have
no such outer layer, but have attached to their cell wall acidic polysaccharides , called
teichoic acid. Because of the thinner peptidoglycan content, Gram-negative cells can be
much easier broken or destroyed by mechanical means compared to Gram-positive cells.
The pIasma membrane is a thin structure and a critical barrier separating the inside of the
cell from its environment. The main components are phospholipids and proteins. The
phospholipid molecules disperse themselves in water in such a way that water-insoluble
(hydrophobic) groups associate together and the ionic (hydrophilic) groups associate
together, leading almost automatically to the formation of a double-layered membrane.
The structure of the plasma membrane is stabilised mainly by hydrogen and hydrophobic
bonding. However, cations such as Mg
2+
and Ca
2+
also combine with some of the negative
charges of the phospholipids and help stabilise the membrane structure. t becomes
therefore a tight barrier, so that the passive movement of solute molecules does not
readily occur. Such movement can only occur by means of specific transport systems (see
chapter 8). The reasons for this restricted movement are that ionised molecules such as
amino acids, inorganic salts etc are repelled by the electric charges on the surface of the
membrane.
n addition to the plasma membrane at the periphery of the cell, most prokaryotes possess
internal membranes, which can often been seen in electron micrographs. These internal
membranes may be simple extensions or invaginations of the cell membrane, or they may
be much more complicated. n photosynthetic prokaryotes, the photosynthetic membranes
often form an extensive internal membrane system, and the nitrifying as well as the
methane-oxidizing bacteria also frequently have elaborate internal membranes.

ProtopIast and SpheropIast. Most bacterial environments have solute concentrations
considerably lower than the solute concentration within the cell. Since water always
passes from lower to higher solute concentrations (osmosis effect), the cell would soon
burst were it not for strength of the cell wall. f the cell wall is damaged or treated with an
enzyme called lysozyme or zymolase, the cell bursts or lyses. These enzymes hydrolyse
the cell wall polysaccharide, thereby weakening the cell wall. f, however, the solute
concentration outside the cell is made equal to the inside of the cell, the action of the
enzyme lysozyme would destroy the cell wall, but the cell is held together by the plasma
membrane. Such a protoplast is therefore a structure completely devoid of the cell wall. f
parts of the cell wall are still present, the structure of the protoplast becomes spherical and
this structure is referred to as spheroplast. Both protoplast and spheroplast are osmotic
sensitive structures.

f solute concentration is higher in the medium than in the cell, water flows out, the cells
become dehydrated, and the protoplast collapses, a process called plasmolysis. This is
one reason why foods can be protected from bacterial spoilage by curing them with strong
salt or sugar solutions.

CeII division. Prokaryotic cells multiply asexually, almost always by the formation of a
septum or crosswall, after DNA has been replicated. From parent cell, two daughter cells
are formed. Lack of proper nutrients can often inhibit complete separation of the daughter
cells and as a result, chains or mycelium type structures appear.

MotiIity. Some bacteria are motile, others are not. The ability to move independently is
usually due to a special organelle, the flagellum. Flagella are arranged differently on
different bacteria. n polar flagellation the flagella are attached at one or both ends of the
cell. Occasionally, a tuft of flagella may arise at one end of the cell, an arrangement called
lophotrichous. n peritrichous flagellation , the flagella are not localised, but grow from
many places on the cell surface. The type of flagellation is often used as a taxonomic
characteristic.
Bacterial flagella are composed of protein subunits. The protein is referred to as flagellin.
The basal region of the flagellum is different in structure from the rest of the flagellum.
There is firstly a wider region at the base of the flagellum called the 'hook'. Attached to this
hook is the 'basal body', a complex structure involved in the connection of the flagellar
apparatus to the cell envelope.

Fimbriae and piIi are structures that are somewhat similar to flagella but are not involved
in motility. Fimbriae are considerably shorter than flagella and are more numerous. Not all
organisms have fimbriae and the ability to produce them is an inherited trait. The functions
are not exactly known, but it is assumed that they enable bacteria to stick to inert surfaces,
or to form pellicles on liquid surfaces.
Pili are similar structurally to fimbriae but are generally longer and only one or a few pili are
present on the surface. t is assumed that pili are very important in the mating process and
also in the attachment to human tissues by some pathogenic bacteria.

CapsuIes. Most prokaryotic organisms secrete on their surfaces slimy or gummy material
which consists of polysaccharide slimes (dextrans etc), which are referred to as capsules.
These capsules can be easily observed in ink preparations under the light microscope.

Gas vesicIes. A number of prokaryotic organisms that live a floating existence in lakes
and the sea produce gas vesicles, which confer buoyancy upon the cells. The most
dramatic instances of flotation due to gas vesicles can be seen in cyanobacteria (blue-
green algae) in lakes. Gas-vesiculated cells rise to the surface of the lake and are blown
by wind into dense masses.

Endospores. Endospores can be formed by many microorganisms. Such spore formation
makes the organism extremely resistant against sterilisation and other extraordinary
environmental conditions. Endospores are readily seen under the light microscope as
strongly refractile bodies. The structure of the spore is much more complex than that of the
vegetative cell in that it has many layers. t contains a chemical substance, dipicolinic acid,
which is specific for endospores. Spores are dormant and can remain stable for long
period of time. Under proper conditions, however, dormancy is rapidly broken and
germination occurs.

Chemotaxis. Chemotaxis is a behavioural phenomenon and suggests some kind of
nervous response. t is the movement of an organism toward or away from a chemical.
Positive chemotaxis refers to movement toward a chemical and is usually exhibited when
the chemical is of some benefit, eg nutrients. Negative chemotaxis is the movement away
from a chemical, usually one that is harmful. Chemicals that induce positive chemotaxis
are called 'attractants', and chemicals that induse a negative chemotaxis are called
'repellents'.
A type of chemotactic behaviour can be observed in the laboratory with the swarming
phenomenon on agar plates. As they metabolise a nutrient in their immediate environment,
they deplete the concentration of that substance and create their own chemical gradient.
They thus move out from the center of the plate following the gradient of nutrient in form of
a ring located always at the gradient.

2.1.2 Gram-negative Aerobic chemoheterotrophic Rods and Cocci
Pseudomonas.The genus Pseudomonas contains straight or slightly curved rods, that are
motile by one or several polar flagella. These chemoheterotrophs are aerobic and carry
out respiratory metabolism with oxygen and are also able to use nitrate as final electron
acceptor. All pseudomonads have a functional tricarboxylic acid cycle and can oxidise
substrates to carbon dioxide. Most hexoses are degraded via the Entner-Doudoroff
pathway. t is a very heterogeneous taxonomic group, which is subdivided according to
properties such as the presence of poly--hydroxybutyrate (PHB), the production of
fluorescent pigment, pathogenicity and glucose utilisation.
This group of microorganisms has a very important impact in several ways:
1. many of the species are able to degrade an exceptionally wide variety of organic
compounds and thus are very important in the mineralisation process in nature and in
sewage treatment. They are also important in regard to bioremediation.
2. Some pseudomonads are major plant and animal pathogens
3. Many species such as Ps.fluorescens are involved in the spoilage of food because of
their ability to grow at low temperatures.

Azotobacter and Rhizobium are our most important nitrogen fixers. Whereas Rhizobium
grows symbiotically within root nodule cells of legumes as nitrogen-fixing bacteroid,
Azotobacter is a free-living soil genus and fixes atmospheric nitrogen nonsymbiotically.
The genus Agrobacterium has been placed into the family of Rhizobiaceae as it also
invades plants. However, in contrast to Rhizobium, agrobacteria do not fix nitrogen but
invade the crown, roots, and stems of many plants forming proliferating tumor cells. The
best known species A.tumefaciens causes the crown gall disease.

Methylococcaceae consist of rods, vibrios and cocci which use methane and methanol as
their sole source of carbon and energy sources under aerobic or microaerophilic
conditions. They are referred to as methylotrophs. The family contains two genera:
Methylococcus and Methylomonas. Methylotrophic growth depends on the presence of
methane and related compounds, a major reason why these organisms can be found
above anaerobic habitats.

2.1.3 Aerobic Gram-negative chemoIithotrophic rods and cooci
Chemolithotrophic bacteria are those bacteria that derive their energy and electrons from
reduced inorganic compounds. Normally they employ carbon dioxide as their carbon
source [chemolitho-autotrophs] , but some can also use reduced organic carbon sources
[chemolitho-heterotrophs]. These bacteria are divided into groups based on the type of
inorganic compound they prefer to oxidise: nitrifiers, sulfur oxidisers, hydrogen oxidisers
and metal oxidisers.
Nine genera of nitrifying bacteria are presently recognised in the family Nitrobacteraceae.
They are all aerobic organisms without endospores and are able to oxidise either ammonia
[Nitrosomonas europaea, Nitrosococcus oceanus, Nitrosospira briensis, Nitrosolobus
multiformis] or nitrite [Nitrobacter winogradskyi, Nitrococcus mobilis]. As their names
suggest, nitrifiers may be rod-shaped, ellipsoidal, spherical, spirillar or lobate and they
possess either polar or peritrichous flagellation.
Nitrifying bacteria are very important ecologically and can be isolated from soil, sewage
disposal systems, freshwater and marine habitats. When twogenera such as Nitrobacter
and Nitrosomonas grow together, ammonia is converted to nitrate, a process called
nitrification (see chapter 2).
Like the nitrifiers, the suIfur oxidisers [ or colourless sulfur bacteria] are a very diverse
group. Of the genera Thiobacillus, Thiomicrospira, Thiobacterium, Thiospira and
Macromonas, the first two are the most investigated ones. Whereas Thiobacillus is a
gram-negative rod, Thiomicrospira is a long spiral cell. These sulfur oxidisers have a wide
distribution and are of great practical importance. Thiobacillus grows in soil and aquatic
habitats. Because of their great acid tolerance, these bacteria prosper in habitats they
have acidified through their own sulfuric acid production. The production of large amounts
of sulfuric acid and ferric ions by T.ferrooxidans corrodes concrete and iron pipe
structures. The beneficial importance of these organisms is in their capability to increase
soil fertility by releasing sulfate are extensively used in ore leaching because of their ability
to leach metals from ore.

2.1.4 FacuItative anaerobic Gram-negative rods
This category contains 27 genera, 20 of which fall amongst the three families
Enterobacteriaceae, Vibrionaceae and Pasteurellaceae.
The family Enterobacteriaceae is the largest of the three families and is categorised
according its metabolic properties. Members of the so-called enteric bacteria all degrade
sugars via the Embden-Meyerhof pathway. The majority carry out a mixed acid
fermentation and produce mainly lactate, acetate, formate and ethanol [Escherichia,
Proteus, Salmonella, Shigella], whereas Enterobacter, Serratia, Erwinia and Klebsiella are
butanediol fermenters. The differentiation of these bacteria occurs normally via the so-
called biochemical tests.
Escherichia coli is the most common bacterium in this group as it is a major inhabitant of
the colon of humans and other warmblooded animals. t is used as an indicator for fecal
contamination in our waterstreams. Some of the strains cause gastroenteritis or urinary
tract infections. Other genera such as such as Salmonella cause typhoid fever and
gastroenteritis, Shigella a bacillary dysentery, Klebsiella pneumonia and Yersinia the
plague. Members of the genus Erwinia are major plant pathogens.

Vibrionaceae consist of four genera: Vibrio, Photobacterium, Aeromonas and
Plesiomonas. Many of the vibrios are important pathogens, such as V. cholera causing
cholera disease and V. parahaemolyticus is the culprit causing gastroenteritis in human
following consumption of contaminated seafood. V. anguillarum is responsible for many
fish diseases.
Several members of the family are marine bacteria capable of bioluminescens because
of the enzyme luciferase. Some are free living bacteria and others live symbiotically in the
luminous organs of fish.

The family Pasteurellaceae contains three genera: Pasteurella, Haemophilus and
Actinobacillus. These members are best known for their diseases they cause in humans
and animals. Of these, H.influenzae is probably the most vicious , as it causes a variety of
diseases including meningitis in children.

2.1.5 Anaerobic Gram-negative rods
The family Bacteroidaceae and 13 genera are all obligately anaerobic, non-sporing rods
of various shape. They are chemoheterotrophs and usually produce a mixture of organic
acids as fermentation endproducts. About 30% of the bacteria isolated from human faeces
are members of the genus Bacteroides and may provide extra nutrition by degrading
cellulose , pectins and other complex carbohydrates. B.succinogenes and B.ruminicola are
major components of the rumen flora. This family is also involved in human diseases, in
particular of major organ systems ranging from the central nervous system to the skeletal
system.
The dissimilatory sulfate- or sulfur-reducing bacteria contain a morphologically diverse
group of seven genera. The best studied sulfate-reducing genus is Desulfovibrio and of
sulfur reduction Desulfuromonas. These bacteria are very important in the cycling of sulfur
within the ecosystem. Often sulfur and sulfate reduction is apparent from the smell of
hydrogen sulfide in marshes, the blackening of water and sediment by iron sulfide and
corroded iron.

2.1.6 Aerobic and microaerophiIic Gram-negative Bacteria
This group is very diverse ecologically and is found in soil, water and marine habitats.
Some species are associated with plant roots or grow in the intestinal tract, oral cavity and
reproductive organs of humans and animals.
The genera Spirillum, Aquaspirillum, Oceanospirillum and Azospirillum are widely
dispersed and readily isolated from various environments. Azospirillum is associated with
the roots of forage grass, crops such as sorghum, legumes and maize [corn] and fixes
nitrogen at low oxygen tensions.
Campylobacter contains both nonpathogens and pathogens. C.fetus causes reproductive
disease and abortions in cattle and sheep and is associated with a variety of conditions in
humans ranging from septicemia to enteritis.
Bdellovibrio preys on other Gram-negative bacteria and alternates between a nongrowing
predatory phase and an intracellular reproductive phase. After entry, Bdellovibrio takes
control of the host cell and grows in the space between the cell wall and plasma
membrane while the host cell loses its shape and rounds up. The life cycle resembles that
of bacteriophages.
Several small groups of nonmotile, Gram-negative exist that are curved to varying
degrees. They are widespread in soil, freshwater and marine habitats. Many of these
bacteria are placed into the family Spirosomaceae containing the three genera Spirosoma,
Runella and Flectobacillus. The remainder are assigned to four other genera, of which
Microcyclus is the best studied genus.
The genus Zymomonas has come to the forefront over the past decades as a prolific
ethanol producer. n particular the species Zymomonas mobilis can be found readily in
ripening and overripe fruit such as pineapple, pear and others. This genus is an unusual
Gram-negative rod as it uses in contrast to all the other Gram-negatives bacteria, the
Entner-Doudoroff pathway and has ethanol and carbon dioxide as its sole endproduct. t
has, however, a very limited substrate utilisation system because of its adaptation to the
environment and grows only on glucose and some subspecies may also utilise sucrose.

2.1.7 Other Gram-negative bacteria
The spirochetes are a group of gram-negative, chemoheterotrophic bacteria distinguished
by their structure and mechanism of motility. They are slender, long bacteria with a
flexible, helical shape. Spirochetes can be anaerobic, facultative anaerobic, or aerobic and
carbohydrates, amino acids, long-chain fatty acids may serve as carbon and energy
source. The group is exceptionally diverse ecologically and grow in habitats ranging from
mud to the human mouth. Some members of the genera Treponema, Borrelia and
Leptospira are important pathogens.
Rickettsiales and Chlamydiales are obligate intracellular parasites as they grow and
reproduce only within host cells. Although they assemble viruses in their intracellular
existence, they differ from viruses in having both DNA and RNA, a plasma membrane,
functioning ribosomes and other features. The order Rickettsiales contains many important
pathogens, since R.prowazekii and R.typhi are associated with typhus fever and Coxiella
burnetii with Q fever in humans. They are also important pathogens of domestic animals.

2.1.8 Gram-positive bacteria
The family Micrococcaceae contains the two most important genera Micrococcus and
Staphylococcus. The genus Micrococcus contains aerobic, catalase-positive cocci that
occur mainly in pairs, tetrads or irregular clusters. They are widespread in soil, water and
on mammalian skin. They do not seem to be particularly pathogenic. Members of the
genus Staphylococcus are facultatively anaerobic, nonmotile, gram-positive cocci that
usually form irregular clusters. Staphylococci are normally associated with the skin, skin
glands, and mucous membranes of warm-blooded animals. S.aureus is the most important
human staphylococcal pathogen and causes boil, abscesses, wound infections,
pneumonia, food poisoning and many other diseases. t produces the enzyme coagulase,
which causes blood plasma to clot.
The genus Streptococcus is an important member of a group of facultatively anaerobic or
microaerophilic, catalase-negative Gram-positive cocci. They are all chemoheterotrophs
that ferment sugars to lactic acid. The genus is large and diverse and is subdivided into
the three genera Streptococcus, Enterococcus and Lactococcus. The genus
Streptococcus still contains most of the important human pathogens.
The genus Leuconostoc contains facultative gram-positive cocci which lack catalase and
carry out heterolactic fermentation by converting glucose to D-lactate and ethanol. The
genus is mainly used in the wine industry, fermentation of vegetables, eg sauerkraut and
pickles, and in the manufacture of buttermilk, butter and cheese. L.mesenteroides
synthesises dextrans from sucrose. A variety of gram-positive bacteria produce lactic acid
as their major fermentation product and often are referred to as Iactic acid bacteria.
Streptococcus, Enterococcus, Lactococcus and Leuconostoc are all members of this
group. The largest group amongst these lactic acid bacteria is the genus Lactobacillus,
which carries out a homolactic fermentation using the Embden-Meyerhof Pathway or a
heterolactic fermentation with the pentose phosphate pathway. The genus is found on
plant surfaces and in dairy products, meat, water, sewage, beer, fruis and many other
materials. Lactobacilli are also part of the normal flora of the human body in the mouth,
intestinal tract and are usually not pathogenic. Lactobacillus is indispensable to the food
and dairy industry, but also occur as spoilage organisms in beer, milk and meat.
Another important genus causing severe food poisoning is Listeria. They are widely
distributed in nature, particularly in decaying matter. Listeria monocytogenes is a pathogen
of humans causing listeriosis.
There exist genera of endospore-forming bacteria. The two most important genera being
Bacillus and Clostridium. Both contain gram-positive, endospore-forming,
chemoheterotrophic rods that are either peritrichously flagellated or nonmotile. The genus
Clostridium is obligately anaerobic and lacks a complete electron transport chain, whereas
the genus Bacillus is aerobic or sometimes facultative aerobic.
Both genera are of considerable industrial importance as members of the genus Bacillus
produce antibiotics such as bacitracin, gramicidin and polymyxin. Other species such as
B.cereus cause some form of food poisoning and can infect humans and B.anthracis is the
causative agent of the disease anthrax, which can be devastating to animals and humans
alike. Other species, such as B.thuringiensis and B.sphaericus form a solid protein crystal
next to their spores during endospore formation. The B.thuringiensis crystal or parasporal
body contains a toxin that will kill over 100 species of moths by dissolving in the alkaline
guts of caterpillars and destroying their gut epithelium causing paralysis and death. One of
these toxin proteins has been isolated and successfully used for bioinsecticide
applications. The B.sphaericus crystal or parasporal body contains protein toxins against
mosquito larvae.
Members of the genus Clostridium also have a great practical impact. Since they are
anaerobic and form heat resistant endospores, they are responsible for many cases of
food spoilage. C.botulinum is the causative agent of botulism. Their general metabolism of
proteins under anaerobic conditions causes the production of unpleasant odours arising
during putrefaction, eg hydrogen sulfide, amines and others. Several clostridia produce
toxins and are major disease agents, eg C.tetanus causes tetanus and C.perfringens gas
gangrene and food poisoning. Apart from these 'proteolytic clostridia', there are also a
large group of 'saccharolytic clostridia', which use carbohydrates instead of proteins as
their major carbon and energy source. Many of these species are of great industrial
importance as for example C.acetobutylicum, which produces butanol and other species
manufacture the solvent acetone.
2.1.9 Nonsporing Gram-positive rods
The genus Arthrobacter contains aerobic rods , which change their morphology during
growth. Although they appear as irregular branched rods during exponential growth, in
stationary phase they change to a coccoid form. Their usual habitat is the soil and some
species can even degrade some herbicides and pesticides.
The genus Corynebacterium comprise aerobic slightly curved rods with a tapered end,
giving the organism a club-shaped form. Although some species are harmless soil and
water saprophytes, C.diphtheriae is the causative agent of diphtheria in humans.
Members of the genus Actinomyces are straight or slightly curved rods that vary in shape
and filaments. They require carbon dioxide for better growth and are normal inhabitants of
mucosal surfaces of humans . They are responsible for actinomycosis, ocular infections
and peridontal diseases.
Species of the genus Mycobacterium are free-living saprophytes and are best known as
animal pathogens. M.bovis causes tuberculosis in cattle and can also cause the same
disease in humans. One reason why milk has to be pasteurised to kill this pathogen.
Therefore it is M.tuberculosis which is the chief source of tuberculosis in humans. Another
species. M.leprae, is responsible for the disease leprosy.

2.1.10 Anoxygenic Photosynthetic Bacteria
There are two groups of photosynthetic prokaryotes performing an anoxygenic
photosynthesis: the purple bacteria and the green bacteria. Neither of these organisms
produces oxygen as they are unable to use water as their electron donor. They employ
inorganic molecules such as hydrogen sulfide, sulfur or hydrogen and in some cases
organic matter as their electron donor for the generation of the reducing power NADH+H
+

and NADPH+H
+
. They often produce sulfur granules either inside the cell [purple bacteria]
or outside the cell [green bacteria].
Amongst the green bacteria there are two groups, the green suIfur and the green non-
suIfur bacteria.
The green suIfur bacteria [Chlorobacteriaceae] are a small group of obligately anaerobic
photolithoautotrophs that use hydrogen sulfide, elemental sulfur and hydrogen as electron
sources. Their photosynthetic pigments are located in ellipsoidal vesicles called
chlorosomes or chlorobium vesicles which are attached to the plasma membrane but are
not continuous with it. They contain accessory bacteriochlorophyll pigments, but the
reaction center bacteriochlorophyll is located in the plasma membrane. They are very
diverse in their morphology and can appear as rods, cocci or vibrios. There main
representatives are Chlorobium, Chloropseudomonas, Clathrochloris, Cylindrogloea,
Prosthecochloris and Pelodictyon.
The green nonsuIfur bacteria [Athiorhodaceae] have as their main representative
Chloroflexus, which is a gliding, filamentous and thermophilic bacterium that is often
isolated from hot springs, very often in association with cyanobacteria. Chloroflexus can
carry out anoxygenic photosynthesis with organic compounds as carbon sources and may
also be able to grow aerobically as a chemoheterotroph.
The purpIe suIfur bacteria belong to the families Chromatiaceae and Ectothiorhodospira.
They are strict anaerobes and usually photolithoautotrophs. They oxidise hydrogen sulfide
to sulfur and deposit it internally as sulfur granules. Thiospirillum, Thiocapsa and
Chromatium are typical purple sulfur bacteria.
The purpIe nonsuIfur bacteria are exceptionally flexible with their energy source. They
normally grow anaeronbically as photoorganoheterotrophs, trap light energy and employ
organic molecules as both electron and energy sources. n the absence of light, these
bacteria can greow aerobically as chemoorganoheterotrophs, but some species carry out
fermentations. As oxygen inhibits the formation of bacteriochlorophyll and carotenoids,
these organisms appear colourless. Morphologically, this group is also very diverse and
appear as spirals [Rhodospirillum], rods [Rhodopseudomonas], circles [Rhodocyclus], or
buds [Rhodomicrobium]. They are most prevalent in mud and water of lakes and ponds
with abundant organic matter and low sulfide levels, causing severe eutrophication.

2.1.11 Oxygenic Photosynthetic Bacteria
Two groups of oxygenic photosynthesizers can be found amongst the bacteria,
cyanobacteria and Prochlorales. Of these, the cyanobacteria are the largest and most
diverse group. Although cyanobacteria are true prokaryotes, their photosynthetic system
closely resembles that of the eukaryotes because they possess chlorophyll a and a
photosystem , which produces oxygen from water. Although many cyanobacteria are
obligate photolithoautotrophs, some can grow slowly in the dark as chemoheterotrophs by
oxidising glucose. At present there exist five orders of cyanobacteria, Chroococcales,
Pleurocapsales, Oscillatoriales, Nostocales and Stigonematales. Amongst the
Chroococcales we find the genera Gloeobacter, Synechococcus and others, the
Pleurocapsales contain the genera Pleurocapsa and Dermocarpa, the better known
Oscillatoriales comprise the genera Oscillatoria, Spirulina, and Pseudoanabaena, the
order Nostocales the well known Anabaena, Cylindrospermum, Nostoc, Calthrix and the
Stigonematales the genera Fischerella, Stigonema and Geitleria.
Cyanobacteria are very tolerant of environmental extremes and are present in almost all
aquatic and terrestrial habitats. n nutrient-rich or eutrophic warm ponds and lakes,
Anacystis and Anabaena can produce rapidly and form blooms. Other cyanobacteria such
as Oscillatoria are so pollutant resistant and characteristic of fresh water with high organic
matter that they act as water pollution indicators.
The genus Spirulina is a well known nitrogen and phosphate scavenger containing
valuable vitamins and are often used for pollution control and as valuable fish feed.
Some cyanobacteria such as Anabaena have frequently been used in rice paddies
because of their nitrogen fixing ability.
Cyanobacteria are very successful in establishing symbiotic relationships with other
organisms, eg lichen, mosses and other species.

Archaebacteria are different from both eubacteria and eukaryotes and become a very
important group in environmental biotechnology (Bertoldo and Antranikian 2003).
As a group the archaebacteria are very diverse, both in morphology and physiology. They
can be aerobic, facultative aerobic or strictly anaerobic. Nutritionally they range from
chemolithoautotrophs to organotrophs, and some are mesophilic, while others are extreme
thermophiles.. They are often present in anaerobic, hypersaline, or high-temperature
environments.
The most distinctive feature of the archaebacteria is the nature of their membrane lipids.
They differ from both eubacteria and eukaryotes in having branched chain hydrocarbons
attached to glycerol by ether links rather than fatty acids. These lipids can be combined in
various ways to yield membranes of different rigidity and thickness. This is the cause of
their resistance to attack by lysozyme and lactam antibiotics such as penicillin.
Taxonomically, the archaebacteria are divided into five major groups: methanogenic
archaebacteria, archaebacterial sulfate reducers, extremely halophilic archaebacteria, cell
wall-less and extremely thermophilic S-metabolisers.
Methanogenic bacteria or methanogens are strict anaerobes and obtain energy by
converting carbon dioxide, hydrogen, formate, methanol, and acetate to either methane or
mostly to methane plus carbon dioxide. This is the largest group of archaebacteria with at
least three orders and 13 genera. Methanogens thrive in anaerobic environments rich in
organic matter: the rumen and intestinal systems of animals, freshwater and marine
sediments, swamps and marshes, and hot springs. Because of their capability to produce
methane gas, methanogens are being intensively used in anaerobic digesters, to reduce
pollution from human and animal manure and at the same time produce a valuable energy
source. This makes these methanogens of great ecological significance. The rate of
methane production can be so great that bubbles of methane will sometimes rise to the
surface of a lake or pond. A kilogram of organic matter can yield up to 600 litres of
methane, a clean burning gas and important source of pollution-free energy.
Methane is, of course, also an ecological problem. t absorbs infrared radiation and thus is
a greenhouse gas and contributes significantly to future global warming
ArchaebacteriaI suIfate reducers contains gram-negative cocci with walls consisting of
glycoprotein subunits. Sofar only one genus has been established, Archaeglobus. Which is
an extrem thermophile and is able to reduce sulfate, sulfite or thiosulfate to sulfide. t also
possesses the unusual methanogen coenzymes F420 and methanopterin.
The extreme haIophiIes consist of six genera within one family, Halobacteriaceae. They
are aerobic chemoheterotrophs and require complex nutients for their growth. The most
obvious disinguishing trait of this family is its absolute dependency on a high concentration
of NaCl (at least 1.5 M) and they will grow on salt concentrations up to 36% NaCl. The cell
wall of Halobacterium disintegrates as soon as the salt concentration drops below 1.5 M
NaCl.
Probably the best studied member of this group is Halobacterium salinarium, which can
actually trap light neergy photosynthetically without having any chlorophyll. They are able
to produce a modified cell membrane referred to as 'purple membrane', which contains the
protein bacteriorhodopsin, which produces ATP with a unique type of photosynthesis.
The extremeIy thermophiIic archaebacteria contains bacteria, many of which are
acidophiles and sulfur dependent. There are three order [Thermococcales,
Thermoproteales, Sulfolobales] and at least nine genera. The best studied genera are
Thermoproteus and Sulfolobus. Both are calssified as thermoacidophiles, because they
grow best at acid pH values and high temperatures. They are strict anaerobes and grow
mainly in hot springs.
This group of aerobic, gram-positive bacteria forms branching filaments or hyphae and
asexual spores. Actinomycetes have a considerable practical significance, as they are
primarily soil inhabitants and are widely distributed. They can degrade an enormous
number and variety of organic compounds and are extremely important in the
mineralisation of organic matter. Actinomycetes also produce most of the medically useful
natural antibiotics. Although most are free-living microorganisms, a few are pathogenic to
humans, animals and some plants. The actinomycetes are divided into seven groups,
primarily based on properties such as cell wall type, conidia arrangements, and the
presence and absence of sporangium [Nocardia, frankia and Dermotophilus,
Actinoplanetes, Streptomyces, Maduromycetes, Thermomonospora,
Thermoactinomycetes].
Of all these groups, the genus Streptomyces is undoubtedly the most important genus with
at present more than 300 species. Streptomycetes are very important, both ecologically as
well as medically. The natural habitat is the soil, where they may constitute between 1 and
20% of the culturable populaiton. The odour of most of the moist earth is largely the result
of the production of volatile substances such as geosmin. Streptomycetes play a major
role in mineralisation and can aerobically degrade resistant substances such as pectin,
lignin, chitin, latex and others. They are best known, of course, for their synthesis of a vast
array of antibiotics. S.somaliensis is the only known streptomycin to be pathogenic to
humans causing actinomycetoma, which is an infection of the subcutaneous tissues.
2.2 Eukaryotes
2.2.1 CeII structure and function
The eukaryotic cell is structurally more complex than the prokaryotic cell. They show to
varying degrees localisation of cellular functions in distinct membrane-enclosed
intracellular structures called cell organelles. n general, the sizes of eukaryotic cells are
much greater than prokaryotic cells, although some eukaryotic cells are as small as large
prokaryotic cells. The main general difference would be summarised as follows:

CeII waII: some eukaryotic cells have cell walls. Their structure consists of two main types
of components: a network of microfibrils that gives rigidity to the cell wall, and a substance
within which the microfibrils are embedded. The composition of these materials varies
according to the type of organism.
EndopIasmic reticuIum: a complex membrane system extending throughout the
cytoplasm, dividing it into compartments and channels. t serves as a barrier between the
various organelles and keeps them in constant relative position. The endoplasmic
reticulum specialises in the transport and synthesis of lipids and membrane proteins.

NucIeus: this is a prominent, usually circular body surrounded by a double membrane, the
nuclear envelope. The nuclear substance consists of DNA in the form of chromosomes,
RNA and proteins. Within the nucleus there are one or more dense bodies called nuclei,
which are packed with RNA, and possibly are sites of ribosomal RNA synthesis.
GoIgi Apparatus: a membraneous organelle made up of a group of flattened disclike
sacs, arranged in stacks like pancakes and surrounded by tubules and small vesicles. t
transports proteins and polysaccharides out of the cell and between other organelles.
Lysosomes: organelles which store enzymes for intracellular digestion.
Peroxisomes: organelles in which peroxides are generated and degraded
Mitochondria: organelles enclosed within a double membrane function as the principal
sites of energy production in cellular processes. Mitochondria may have many shapes, but
most often they are rod-shaped structures. The mitochondrial membrane is constructed in
a manner similar to other membranes, a bilayer formed of phospholipid with proteins
embedded in the lipid layer. Mitochondrial membranes lack, however, sterols and thus is
much less rigid that the plasma membrane. Mitochondria usually possess a complex
system of inner membranes, which are called cristae and are unique to mitochondria.
Within the inner compartment formed by the cristae is the matrix, which is gellike and
contains large amounts of proteins.
ChIoropIasts: organelles in plant cells, which contain the pigment chlorophyll and are
responsible for the conversion of light energy into chemical energy. Each chloroplast has
an outer membrane, within which are a large number of internal membranes called
photosynthetic lamellae or thylakoids with which the chlorophyll is associated. n the
green algae, the thylakoids are usually associated in discrete structural units called grana,
and in this respect the green algae are similar to higher plants, where the thylakoids are
also arranged in grana. Among the algae, grana are found only in the Chlorophyceae.
VacuoIe: membrane-bound space, containing dilute solutions of various substrates.
FIageIIa and CiIia: appendages of the cell, more complex than those of prokaryotes,
responsible for the movement of cells.
PIasma membrane: in principle similar to the prokaryotes, but contain sterols, which make
the membrane much more rigid.
The inclusion of DNA in the nucleus of eukaryotic cells separates two crucial steps in gene
expression: transcription and translation. Transcription is the copying of DNA sequences
into RNA sequences, which occurs in eukaryotes in the nucleus. Translation occurs when
RNA direct the synthesis of specific proteins and this occurs in eukaryotes in the
cytoplasm. n comparison, prokaryotes have no such compartmentalisation and thus
translation of RNA sequences into protein begins as soon as they are transcribed.

2.2.2 AIgae
The term algae refers to a large, morphologically and physiologically diverse assemblage
of organisms containing chlorophyll and carrying out an oxygen-evolving type of
photosynthesis. Although most algae are of microscopic size and hence are clearly
microorganisms, a number of forms are macroscopic, some of the seaweeds growing in
length to over 3 meters. Algae show considerable diversity in the chemistry of their cell
wall. n most cases, the cell wall consists basically of cellulose and is usually modified by
the incorporation of other polysaccharides. The composition makes the cell wall relatively
easily digestable, one of the reasons for being used as a food in a number of countries.
Algae are ubiquitous in habitat, as long as sunlight is available together with moisture and
simple nutrients. Algae may be unicellular or multicellular with some being microscopic,
others are macroscopic.
Algae reproduce by asexual or sexual means. They use many modes of each type of
reproduction. Some algae have complex life cycles, which include both asexual and sexual
modes.
The capacity of algae to remove carbon dioxide and produce oxygen and to carry out
photosynthetic activity, together with the ease of cultivation and unicellular forms, make
them extremely attractive for use in space travel and environmental biotechnology. Their
high protein and low nucleic acid content is a very attractive source of food.
Algae most commonly occur in water in which they may be suspended (planktonic) or
attached and living at the bottom (benthonic). PIankton consists of free floating, mostly
microscopic organisms and is made up of algae and plants. Algae also associate with
fungi to form lichens. They are divided into 7 divisions, Chlorophyta, Charophyta,
Euglenophyta, Chrysophyta, Phaeophyta, Rhodophyta, and Pyrrophyta.

2.2.3 Yeast
Yeasts belong systematically to the molds or filamentous fungi, that form a large group of
eukaryotic organisms lacking chlorophyll. The more than 500 known species of yeasts are
in the class of the Actinomycetes and are classified taxonomically according to their mode
of reproduction into 4 main families: Encomycetaceae, Saccharomycetaceae,
Cryptococcaceae and Sporobolomycetaceae.
Most yeasts are unicellular like the bacteria, but they are larger, usually from 6-12
micrometers. The cell wall contains glucans and mannans. The respiratory enzymes are
situated in the mitochondria and the fermentation enzymes reside in the cytoplasm. Yeasts
reproduce by asexual and sexual means by budding or spore formation.
Because of their haploid and diploid character, yeasts have been used extensively in the
past for genetic selection, eg wine yeasts, brewer's yeast, baker's yeast etc.
Yeasts were exploited for thousands of years in the making of alcoholic beverages from
carbohydrates under anaerobic conditions. When grown aerobically, a much broader
range of compounds can be exploited as substrates ranging from n-alkanes in the
hydrocarbon group or organic compounds to ethanol in the small molecule compound
region, depending on the yeast species. This characteristic is also being used for microbial
protein production.

2.2.4 Fungi or mouIds
The fungi are strict aerobes. n addition to true nuclei like the yeast, the fungi are usually
characterised as structures where the vegetative body of somatic tissue is filamentous and
branched.The vegetative structure of a mold is often called a thallus. The most typical
fungal thallus is composed of filaments, which are usually branched. Each individual
filament is called a hyphae, and a mass of hyphae is called a mycelium. Filamentous
growth occurs by continuous expansion of hyphal tips.
Fungi are broadly classified into four groups: Phycomycetes, Ascomycetes,
Basidiomycetes and Deuteromycetes. The most important features for this classification is
the type of reproductive spores formed.
Phycomycetes, eg Mucor, Rhizopus etc, are generally considered to represent the most
primitive group, since their hyphae have no septe or cross walls. Their asexual spores are
enclosed in a sac, the sporangium, which bursts upon maturation. The spores released
initiate new growth. n sexual reproduction, two hyphae (gametangia) join and form a
zygo-spore, which is diploid.
Ascomycetes, eg Penicillium, Aspergillus etc, have septa in their hyphae. Asexual spores
are always outside the hyphae and are called conidiospores. These spores sit on hyphal
extensions, called conidiophores. Sexual recombinatoin results in the formation of either
four or eight spores in a sac, called ascus.
Basidiomycetes are the well-known mushrooms. They have septa in their hyphae.
Sexual fusion results in the formation of macroscopic structures called fruiting bodies. The
spores formed are called basidiospores, because they are formed at the tip of a
differential portion of the hyphae called a basidium. Asexual reproduction is rare.
Deuteromycetes [often referred to as Fungi imperfecti] are fungi for which no sexual form
has yet been found.
Classification is based on several features of which the most important is the type of
reproductive spores formed, both asexual and sexual. Asexual spores are usually resistant
to drying or radiation, but are not very heat resistant and exhibit no dormance. They are
able to germinate when moisture becomes available, often even in the absence of
nutrients. Sexual spores are usually more resistant to heat, although no fungus spore
shows the extreme heat resistance characteristic of bacterial endospores. Sexual spores
also exhibit often dormancy. They require some sort of activation for germination, such as
mild heat or certain chemicals.
Fungi have tough cell walls made up of variously linked polymers of glucose (known as
glucans), of glucosamine (chitosan) and N-acetylglucosamine (chitin).
Fungi are well digestable and have a high protein and low nucleic acid content and thus
are often used in food fermentation and microbial biomass production.
Phycomycetes are generally considered to be the most primitive of all fungi. The lack of
cross-walls or septa leads to their classification as non-septate fungi. The hyphae contain
many nuclei and the organisms are coenocytic, which means without cells. Asexual
reproduction occurs by the differentiation of certain parts of the hyphae, which support
specialized structures called sporangia containing sporangiospores. t is characteristic
that their asexual spores are enclosed in a sac, the sporangium. Sexual reproduction
involves the joining of two specialised hyphae called gametangia with the formation of a
new structure, the zygospore, between them.
The ascomycetes contain both fungi as well as yeasts. The molds appear to have septa
and therefore are traditionally referred to as septate fungi. Asexual reproduction consists
of the formation of distinct conidiospores on specialised hypae extensions called
conidiophores. n contrast to the Phycomycetes, the asexual spores of the Ascomycetes
are always external and never enclosed. Sexual recombinaiton is usually heterothallic
and results in the formation of either four or eight spores in a sac called an ascus, hence
the name ascospores.
Basidiomycetes are molds. The hyphal structure is similar to that of the ascomycetes.
Asexual reproduction is usually confined to vegetative haploid mycelial growth and is rare.
Most Basidiomnycetes are heterothallic and their sexual fusion results in the formation of
macroscopic structures, the fruiting bodies. The most common examples are the
mushrooms. The spores formes are called basidiospores, because they are formed at
the tip of a differentiated portion of the hyphae called a basidium.
Deuteromycetes are septate fungi for which no sexual form has been observed. Since the
form of sexual spores is crucial for classification, it is a group set up as a provisional class
for new isolates with the expectation that each member would be transferred to the
Ascomycetes or Basidiomycetes once the sexual form was observed.
Fungi are important to humans in both beneficial and harmful ways. With bacteria, fungi
act as decomposers, a role of enormous significance. They degrade complex organic
materials in the environment to simple organic compounds and inorganic molecules. Some
of them, in particular the mushrooms are of particular interest for their capability to
separate lignin from cellulose using the unique enzyme ligninase. Thus they are very
important for composting and others are excellent nutritional food for humans.
Fungi are also the major cause of plant diseases. Over 5000 species attack economically
valuable crops and garden plants, and cause diseases in animals and humans.
Fungi are also used in food fermentation industries such as dairy (cheese) and traditional
food such as tofu, tempeh, and in commercial industries for organic acid (citric acid) and
certain drug (cortisone) production as well as the manufacture of antibiotics (penicillin,
griseofulvin) and the immunosuppressive drug cyclosporine.
3. OsmoreguIation
Most free-living organisms live in an environment with a water concentration considerably
greater than that inside the cell. Since the cytoplasmic membrane is freely permeable to
water, but not to many solutes, there is a tendency for water to enter the cell. Unless this
tendency is counterbalanced in some manner, the cell swells and eventually undergoes
osmotic lysis. n many algae, fungi and most bacteria the danger of osmotic lysis is
prevented mechanically by enclosure of the cell in a rigid cell wall of sufficient tensile
strength to counterbalance water pressure and hence prevent lysis. All but one small
group of eubacteria possess a characteristic polymer murein, a form of a peptidoglycan.
The walls of archaebacteria and algae and fungi are more variable. Murein is never found
outside the eubacteria.
Most bacteria that lack a rigid cell wall are osmotically sensitive and hence confined to
environments of high osmolality.
n all cellular organisms the genetic information is stored as a linear series of bases in
desoxyribonucleic acid or DNA. The DNA of cells is a souble-stranded helix in which the
two strands wind around each other, making one complete turn about every 10 base pairs.
The two strands are held together by hydrogen bonding between the bases adenine and
thymine, and between guanine and cytosine. Each strain thus contains the information
necessary to specify its complementary strand, a feature of central importance in both the
replication and expression of DNA. This basic genomic structure is common to all cells.
There are, however, differences among the major groups with respect to the organisation
of their chromosomes.
The genome of the eukaryotic cells is always distributed over several chromosomes.
Some of these chromosomes are circular and similar to those found in eubacteria, and
they are housed in the mitochondria and chloroplasts. However, the bulk of the DNA is
contained within the nucleus, and is always distributed among several chromosomes, each
of which contains a single linear molecule of double-stranded DNA.
Within the non-dividing nucleus, the eukaryotic chromosomes are dispersed as long,
threadlike strands with a distinct substructure, a string of nucleosomes. Each of these
nucleosomes is composed of nine molecules of protein, called histones, and about 165
base pairs of DNA. Histones are DNA-binding proteins of relatively low molecular weight,
with a high content of base amino acids. Most eukaryotic cells have five different types of
histone, of which four comprise the nucleosome core, whereas the fifth histone termed H
is present in one copy per nucleosome and apparently binds to the DNA where it enters
and exits the chromosome.
Unlike the eukaryotic genome, the eubacteria contain a single chromosome that is circular
and covalently closed except during replication. t is attached to the cell membrane during
replication and segregation. n addition to the chromosome, a variable number of
plasmids may be present. Like the chromosome, the plasmids are covalently closed
circular molecules of DNA, vbut hey are not necessary for growth under all conditions and
they are small compared to the chromosome.
The eubacteria contain a single type of histonelike protein, called HU [in Escherichia coli].
Although these proteins share with eukaryuotic histones the properties of low molecular
weight and a high content of base amino acids, their ability to bind DNA at physiological
ionic strength is still disputed and uncertain.
The condensation of DNA by wrapping around a proteinaceous core appears also to
characterize the archaebacteriaI chromosome.
5. Viruses
One class of microorganisms, the viruses, are acellular. They differ from cellular
organisms in structure, chemical composition and mode of growth.
The viruses are obligate parasites, capable of development only within the cells of
susceptible host organisms. Viral hosts include almost all cellular organisms, prokaryotes
as well as eukaryotes. Viruses are transmitted from cell to cell in form of small infectious
particles known as virions. Each virion consists of a core of nucleic acid, enclosed within
a protein coat or capsid, which normally is composed of a fixed number of identical protein
subunits, the arrangement of which confers on the virion its external form. Many of those
that infect animals are enclosed in lipoprotein membranes, usually derived from the host
cell nuclear or cytoplasmic membrane. Certain of those that infect prokaryotes have
special proteinaceous tail structures attached to the capsid, which function in the
attachment of the virion to the host cell, and the introduction of viral nucleic acid into the
host.
The core of the virion contains only one kind of nucleic acid. Depending on the virus, it
may be double-stranded or single-stranded DNA or double-stranded or single-stranded
RNA, but in all cases it provides the genetic information required for the synthesis of viral
components and their assembly into new new virions by the infected host cell.
Although all viruses are dependent on host cells for their development, the extent and
nature of this dependence varies. The simplest viruses contain very little genetic
information sufficient to code at most for three proteins. n such cases, the genetic
information and enzymatic machinery of the host cell play the predominant role in viral
synthesis. The largest viruses contain genetic information sufficient to code for as many as
500 different proteins, including many enzymes specific to viral synthesis. n all cases,
however, the provision of energy and low molecular weight precursors of proteins and
nucleic acids, together with much of the machinery for protein synthesis is assured by the
host cell. ntracellular maturation of the virions is followed by their release from the cell,
which is generally killed.
6. Literature
BaIows, A., Trper, H.G., Dworkin,M., Harder,W. and SchIeifer, K.H. 1992 - The
prokaryotes, 2nd edition, Springer Verlag, Heidelberg

BertoIdo,C. and Antranikian,G. 2003 - Biotechnology of Archae. n Biotechnology
[H.W.Doelle, E.J.DaSilva,eds.], Enzyclopedia for Life Support Systems, EOLSS
Publishers, Cambridge, UK

Chang, S.T. and MiIes,P.G. 1984 - A new look at cultivated mushrooms. BioScience 34,
358-362

DoeIIe,H.W. 1994 Microbial Process Development. Scientific Publishers, Singapore

HoIt,G.J. (edt.) 1989 - Bergey's manual of systematic bacteriology . Williams & Wilkins,
Baltimore

KandIer,O. and ZiIIig,W. (eds.) 1986 - Archaeobacteria. Gustav Fischer Verlag, New York

Knig, H. 1993 - Methanogens. n Biological Fundamentals.(H.Sahm, ed). Biotechnology,
2nd edition, Vol. 7, VCH, Weinheim, Germany

Lee,R.E. 1989 - Phycology, 2nd edition, Cambridge University Press, New York

Moore-Landecker, E. 1991 - Fundamentals of fungi. 3rd edition, Prentice Hall, Englewood
Cliffs, N.J.

Prescott, L.M., HarIey,J.P., and KIein,D.A. 1993 Microbiology. 2nd edition, Wm.C.Brown
Publishers, Oxford

SchIegeI, H.G. and Bowien,B. 1989 - Autotrophic bacteria. Science Tech. Publishers,
Madison, Wisconsin

Rogers,L.J. and GaIIon,J.R. (eds.) 1988 - Biochemistry of the algae and cyanobacteria.
Oxford University Press, New York

Stanier,R.Y., Ingraham,J.L., WheeIis,M.L. and Painter,P.R. 1987 - General
Microbiology, 5th edition, MacMillan Education Ltd, London
Chapter 7
MicrobiaI CeII CuItivation Systems
[The content of this chapter was an integrated part of every Unesco sponsored training course outlined in
chapter1@
Content:
1. Introduction
2. Batch CuItivation System
4. Fed-Batch CuItivation System
7. SoIid State and SoIid-Substrate CuItivation System
7.1 PrincipIes
7.2 GeneraI Features
7.6 AppIications of SSC
8.1 AIginate
8.2 Carrageenan
8.3 Ion Exchange Resins
8.4 PoIyurethane Foam
8.7 Passive ImmobiIisation
8.9 Biosensors
9. References

1. Introduction
When microbial cells are inoculated into a nutrient broth and incubated at a suitable
temperature, a sequence of changes occur. These changes are reflected in an increase of
biomass, which in the case of bacteria and yeast normally result in an increase of cell
numbers, whereas algae and fungi form extensive mycelia. The latter may also occur with
bacteria in media of nitrogen deficiency. n order to be able to characterise and describe
the behaviour of various organisms under a variety of conditions and to find optimal
cultivation conditions, it is necessary to use some form of mathematical description.
2. Batch cuItivation system
Let us assume that a bacterial culture is growing at and under ideal conditions. Under
these conditions we obtain balanced growth and the culture mimics a first order
autocatalytic chemical reaction. This means that the amount of living matter or number of
cells increases not in proportion to time, but in accordance to a geometric progression with
time. t multiplies itself by a constant factor in each successive unit of time

f the number of cells at inoculum is expressed as N
0
, the number of cells, N, after n
divisions will be
N = N
0
x 2
n
whereby n expresses the number of multiplications (generations), N
0
the number of cells
immediately after inoculation and N the final number at the time of harvest and/or analysis.
f we use a spectrophotometer for the analysis of biomass concentrations, N is expressed
as X, thus
X = X
0
x 2
n
[1]
n rearranging this equation, it is possible to determine the number of generations or cell
divisions
Iog X = Iog X
0
+ nIog2 [2]

Iog X - Iog X
0

n = ---------------------- [3]
Iog 2

since log 2 = 0.301,
n = 3.32(IogX - Iog X
0
[4]
f under these ideal conditions the time factor is included, then we arrive at the multipli-
cation rate, r, of the culture or the rate of formation of generations per unit time (h
-1
)
n
r = -------------- [5]
t
1
- t
2

or
3.32[IogX - IogX
0

r = -----------------------------------
t
1
- t
2

Another frequent expression is the generation time, g, or doubling time, t
d
, that
expresses the time it will take for a culture to double its population. This doubling time is
mostly expressed in minutes:
t
g = t
d
= ---- [6]
n

1
= ----- [7]
r

All the above equations express the growth of the microbial population in a certain time
interval, but not the rate of growth itself.
f all the requirements for growth are satisfied, then during an infinitely small time interval
(dt) one expects the increase in biomass (dX) to be proportional to the amount of biomass
present and to the time interval:
dX
----- = kX
dt

The differential coefficient (dX/dt) expresses the population growth rate, whereas the
constant of proportionality, k, relates the rate of increase of any given cellular component
to the amount of that cellular component, or in the case of biomass or cell numbers to the
specific parameter
dX
----- = X [8]
dt

dX 1
= ----- -- [9]
dt X

and is termed the specific growth rate with the dimension of reciprocal time (h
-1
).
t should always be recognised that the population growth is expressed in two different and
distinct terms:
growth rate = rate of biomass increase with time

dX
growth rate = -------
dt

specific growth rate = rate of biomass increase with time per unit of biomass

1 dX
= ---- ----
X dt

When is constant, the integration of dX/dt = X gives:
In X = In X
0
+ t [10]
where X
0
is the biomass at t = 0. f lnX is plotted against time, the resulting straight line
expresses

Figure 1: Logarithmic growth determination
The slope can be determined by the relationship
BB
1
CC
1

tan = -------- = ------ [11]
AB
1
AC
1

There also exists a relationship between the specific growth rate and the earlier developed
doubling or generation time. n putting X = 2X
0
and t = g
In2 0.693
g = ------ = ------- [12]

Growth, which accords with this law is called constant, exponential or logarithmic
growth. The basic measure of the rate of growth is the specific growth rate. The law of
exponential growth must be followed when the environmental conditions are constant and
the constitution of the biomass remains constant.
f the growth does not accord with this law, it indicates that either one or both of the two
qualifying conditions are not being met. Probably the most common first cause of failure to
maintain constant exponential growth is a change in the environment. n batch cultures,
this is unavoidable sooner or later and limits this particular cultivation technique.
Growth, of course, depends on the energy and carbon obtained from the nutrient. When
growth is limited by a particular nutrient, there is a fixed linear relationship between the
concentration of that limiting nutrient initially present in the medium and net growth. The
mass of cells produced per unit of limiting nutrient is accordingly a constant, the growth
yieId (Y). This value of Y can be calculated from single measurements of total growth by
the equation
X - X
0
dX
Y = ---------- = ---- [13]
S
0
- S dS

where X is the dry weight/ml and S represents the concentration of the limiting nutrient.
The growth yield is important as a means of expressing the quantitative requirements of an
organism. Monod first showed in 1942 that in bacterial cultures, when the conditions are
maintained constant, the growth yield is a constant and reproducible quantity

X - X
0
= Y(S
0
- S) [14]

f the substrate is growth limiting, S 0 as soon as the culture has reached its
maximum biomass, Xm, thus

X
m
- X
0
= YS
0
[15]

Hence, for a growth limiting substrate, a plot (Figure 2) of Xm against S
0
should be a
straight line with the slope Y

Figure 2: Correlation between maximal growth and substrate concentration

The rate of consumption of the substrate in a particular moment is given as
dS
----- = qX [16]
dt

where X is the biomass and the coefficient q is known as the metabolic quotient or the
specific metabolic rate. The metabolic quotient is analogous to an enzyme activity. f the
biomass constitution is constant and the environment is constant, then q must also be
constant.
Since both can be substituted and give

dX
Y = -----
dS
and
dX
X = -----
dt

X
dS = ----- dt [17]
Y
or

dS X
----- = ------ [18]
dt Y

q = ----- [19]
Y

This equation is used to estimate the demands for substrates at different growth rates.
The occurrence of constant exponential growth in batch cultures shows that the growth
rate may be virtually unaffected by substrate concentration over a wide range, that is, the
growth process shows zero order kinetics as is known to occur during enzyme reactions.
Since the velocity of chemical reactions is determined by the concentration of the
reactants, the apparent paradox can be explained by the action of permeases or other
catalysed transport systems, which are capable of maintaining saturating intracellular
concentrations of nutrients over a wide range of external concentrations. Nevertheless at
extremely low concentrations of external nutrients, the permease systems are no longer
able to maintain saturating intracellular concentrations and the growth rate falls. At this
point, substrate consumption for growth follows that of enzyme kinetics and a typical
hyperbola is obtained if one plots growth rate against substrate concentration, which fits
the equation
S
=
max
------------ [20]
(K
s
+ S)

where Ks, called the saturation constant (Figure 3), is equivalent to the Michaelis-Menten
constant, being numerically equal to the substrate concentration supporting a growth rate
equal to 0.5
max
.
To give some examples, the value of K
s
for glucose utilisation by E.coli is 0.18 g/ml. n the
case of amino acids the values are even lower. These very low values are attributable to
the high affinities characteristic of many bacterial permeases.

Figure 3: Calculation of the Saturation Constant K
s

For a more exact determination of the values K
s
and m, the method of Lineweaver and
Burke (Figure 4) can also be used and hence a plot of 1/ against 1/S should give a
straight line with an intercept on the ordinate 1/
max

Figure 4: Use of the Lineweaver-Burk plot for maximal growth rate calculations

n order to fully characterise the growth in a batch culture system, one has to determine
therefore the following parameters:

1. specific growth rate,
2. generation or doubIing time, g or t
d

3. growth yieId, Y
4. the metaboIic quotient, q.

These parameters are necessary before any changes in environment or cultivation are
being made.
n batch culture systems, biomass and product formation are limited to a certain period of
time it takes the microbes to exhaust the substrate or be restricted owing to changes in
environmental conditions.
f one wants to maintain a microbial population in a state of exponential growth over a long
period of time, a change in cultivation technique is necessary. The appropriate system is
termed a continuous culture system.
Such a system (Figure 5) requires the connection of a medium reservoir to the fermenter
vessel and an overflow device in the fermenter vessel connected to a collecting vessel.

Figure 5: General Diagram of a Continuous Culture Flow System

Once growth has been initiated, fresh medium is continuously supplied from the reservoir
and the total volume of the fermenter vessel is maintained constant by allowing the excess
volume to be removed continuously through the overflow device. The ratio of inflowing
medium per hour to the growth vessel volume is called dilution rate, which is expressed
as
F
D = --- [21]
V

whereby D is the dilution rate, F equals the flow rate set by the operator and V represents
the total volume in the fermenter vessel.
f one starts a continuous culture system from a batch culture system, the following three
stages could be obtained (Figure 6):

Figure 6: Start of a continuous culture system from a batch culture

1. the rate of washout of biomass will exceed the rate of growth, so that the biomass
concentration will decrease and the growth-limiting substrate concentration will tend
towards S
0
;
2. the rate of washout of biomass will exactly balance the growth rate, that is the organism
will grow with its specific growth rate at the maximum. n this case there will be a steady
state in which the biomass and growth-limiting substrate concentration remain constant;
3. the rate of washout of biomass is less than the maximum specific growth rate. n this
case the biomass concentration will continue to increase. Eventually, the decrease in the
growth-limiting substrate concentration must decrease the specific growth rate until the
biomass growth rate equals the washout rate. Then there can be no further change in the
concentrations of biomass and growth-limiting substrate. A steady state is reached. This
exemplifies best the self-regulatory capacity of the continuous cultivation system.

The essential feature of a continuous culture is that microbial growth takes place under
steady state conditions. Derivations of equations describing the steady state growth
occurring in continuous culture systems are for simple equations based on a consideration
of

(a) the kinetics of a continuously flowing system;
(b) the kinetics of bacterial growth as described for the batch cultivation system
f the dilution rate D is kept constant, the so-called growth-limiting substrate must give an
equal value to the specific growth rate, therefore
D = [22]

Such a steady state can be maintained for an infinitely long period.
The object of the quantitative theory is to predict the values of the growth rate and the
concentrations of biomass and substrate under different conditions.
f we take equation [8] from the batch culture cultivation system and adapt it to the
continuous flow system, we have to compensate this formula by the outflow of biomass
with the medium, DX, and obtain
Increase = growth - outfIow
dX
----- = X - DX
dt

= X( - D) [23]

t follows from this relationship, that in the steady state, when dX/dt = 0,
= D

Only under these conditions will the biomass remain constant.
As bacterial growth is dependent on substrate concentration, it is necessary to consider
also the effects of the dilution rate on the concentration of growth-limiting substrate (S) in
the culture. The net change in substrate concentration is

Increase = input - outfIow - consumption
dS X
------ = DS
0
- DS - --------
dt Y

= D(S
0
- S) - X/Y [24]

n the steady state, dX/dt = dS/dt = 0 and the steady state value of X and S are given by
( - D)X = 0 [25]
D(S
0
- S) - X/Y = 0 [26]
To obtain X and S, one simply substitutes for the specific growth rate

m
S
= ---------
(S + K
s
)
and obtains
D(S
0
- S) = X/Y
m
S/(S + K
s
) [27]
Now we substitute = D, and we obtain for the steady state:
X = Y(S
0
- S) [28]
S = K
s
D/(
m
- D) [29]

n substituting S from equation [29] into equation [28], the formula for the self-regulating
capacity of the continuous culture system is obtained as:
X = Y[S
0
- K
s
(D/
m
- D)] [30]
As the values K
s
,
max
, and Y are determined from the batch cultivation systems and thus
become constants in the continuous cultivation system, it is possible to plot the self-
regulating capacity of the continuous cultivation process (Figure 7).

Figure 7: Self-regulating capacity of a continuous culture system

By varying S
o
or D experimentally, a great number of steady states can be obtained,
restricted only by the concentration of S remaining at a level where it is the limiting factor
in the growth of the bacterial culture.
Continuous cultivation systems can be operated as chemostats or as turbidostats.

n a chemostat the flow rate is set at a particular value and the rate of growth of the
culture adjusts to the flow rate.
n a turbidostat the system includes an optical sensing device which measures the
density or absorbency of the culture in the growth vessel. The electrical signal from this
device regulates the flow rate. Thus the absorbency of the culture controls the flow rate
and the rate of growth of the culture adjusts to this flow rate.
As a practical matter, chemostats and turbidostats are usually operated at different dilution
rates. n the chemostat, maximum stability is attained within a range of dilution rates over
which cell concentration changes only slightly with changes in dilution rate, ie, at low
dilution rates. n contrast, in the turbidostat maximum sensitivity and stability are achieved
at high dilution rates within a range over which culture biomass changes rapidly with
dilution rate. A turbidostat can be operated at
max

by setting an absorbency value that
maintains a concentration of cells insufficient to cause any nutrient to fall to a growth-
limiting concentration.
Continuous culture systems offer two valuable features for the study of microorganisms.
They provide a constant source of cells in an exponential phase of growth, and they allow
cultures to be grown continuously at extremely low concentrations of substrate. Growth at
low substrate concentrations is valuable in studies on regulation of synthesis or catabolism
of the limiting substrate, in selection of various classes of mutants, and in ecological
studies. The disadvantage of this system is, of course, the possibility of mutant selection
during metabolic studies.
Let us now briefly return to the relationship of
q = /Y
n dealing with the chemostat it was mentioned that Y is constant and independent of the
growth rate. This means then that and q must have the same relationship at all dilution
rates
D = = Yq

Under very low dilution rates with D 0, and q would therefore be expected to approach
zero also with Y remaining constant.
f one plots D against q, a straight line should eventuate with its extrapolation through the
point of origin. This, however, does not occur. Although the relationship between D and q
is indeed a linear function, an extrapolation to D = 0 , the lines do not pass through the
point of origin (Figure 8), but intersect the ordinate at finite values of q:

Figure 8: Relationship between the metabolic rate q and the dilution rate D in a
continuous culture system

A very similar situation exists with Y, since Y = /q, or at steady state, where = D, Y =
D/q.
These relationships indicate that a certain proportion of the substrate, and under aerobic
conditions also of oxygen, was required for some growth-dependent function. f the limiting
nutrient concentration is the energy source for the culture, growth ceases at very low
dilution rates because a certain amount of energy, termed maintenance energy, is always
used for the purposes other than growth, including motility, DNA repair, and the transport
of nutrients into the cell against a concentration gradient etc. When the rate of entry of the
source of energy is insufficient to supply more energy than that required for maintenance,
growth cannot occur. Maintenance energy can be mathematically expressed as
dX
----- = X - aX
dt
where a is the specific maintenance rate constant. Normally these rates are very low, but
can become significant under unfavourable conditions. This, of course, introduces a
certain complication into the concept of Y. Rather than redefine Y for the special case of a
substrate that serves as an energy source, a new parameter can be used as Yg, that fits
the original definition of Y and leaves the term Y reserved to describe substrate used for
growth and maintenance. This leads to a new parameter, maintenance coefficient (m) to
describe maintenance
dS dS dS
----- = ---- + ----
dt dt
g
dt
M

and

1 m 1
---- = ---- + ----
Y Y
g

The maintenance coefficient, m, and the specific maintenance rate, a, are related by the
equation
m = a/Y
g

This mathematical description of bacterial growth is the only way to evaluate the
organism's behaviour on certain substrates. t is also a means for comparison using
different substrates or culture conditions as well as different microbial populations. The use
of the basic description further allows the prediction of growth, substrate utilisation, product
formation and a bioenergetic evaluation for optimising microbial processes.
4. Fed-Batch CuItivation System
n between these two different kinds of cultivation systems, batch and continuous, there
exists a third extremely versatile technique referred to as fed-batch cultivation. This term
refers to a cultivation system, whereby a batch culture is either fed continuously or
sporadically with nutrient medium without keeping the growth vessel volume constant. The
latter system is also called pulse-feeding. f a portion of the culture is withdrawn at set
intervals, the system may become a repeated fed-batch system. n contrast to the
continuous culture system, the fed-batch culture does not operate with a constant volume
and thus does not reach a steady state, as the actual volume V is not constant and may
vary according to the volume of F added:
dV
---- = F
dt

dX dX dV
------ = V---- - X------ /V
2

dt dt dt

dS X
----- = FS
0
- --------
dt Y

Therefore, fed-batch cultures could be called a growth system in a quasi-steady state
dX
----- = FYS
0

dt

X = X
0
+ FY(S
0
- S)

X
max
= X
0
+ FY(S
0
- S)

Note, that the main difference between the fed-batch and continuous culture is the
changing value for the actual volume V.
There are, of course, a large number of variations within the continuous and the fed-batch
culture systems. Whereas the former may have multi-stage systems or a continuous train
system of a number of reactor vessels before collecting the final effluent, fed-batch culture
systems can be continuously fed with intermittent withdrawals (with varying volumes being
withdrawn), pulse fed with constant or intermittent withdrawals and so on.
The application of this culture system is on the increase as

1. restriction of the rate substrate utilisation by means of substrate feed rate is a mean of
overcoming catabolite repression of substrate;
2. regular or irregular withdrawal is a mean of overcoming endproduct inhibition;
3. pulse feeding allows high substrate concentration feed for regulation of possible by-
product formations.

Fed-batch culture is technically simpler than chemostat culture, because the need to keep
the culture volume constant, which often is the most exacting part of the chemostat
culture, is eliminated. The most important feature of a fed-batch culture is that it is an
unique means of realising transient conditions between fixed growth rates. There is ever
increasing evidence becoming available, that the maximum rate of some processes can be
achieved only transiently.
A recycling of cells from the chemostat effluent provides a means of continuously
inoculating the vessel. Such recycling often adds stability to the system and minimises the
effect of process perturbations. t allows a chemostat to be operated at higher steady state
cell concentrations than in a chemostat without recycling (Figure 9).

Figure 9: Generalised Diagram for a Cell Recycling System

The nomenclature is the same as for the chemostat except for two additional parameters,
a, the recycle ratio, and C, the concentration factor:
Cell maintenance and death are assumed to be small.
Solving the equation for the steady state where dX/dt = 0, and substituting D = F/V,
= (1 + a - aC)D
With recycle therefore, D does not equal anymore , since (1 + a - aC) is aIways Iess
than 1, the concentration factor 1 is greater than 1, and is Iess than D.
As a consequence of the cell recycle, it is possible to increase the overall productivity of
the system because a dilution rate greater than the maximum specific growth rate may be
employed.
Recycling systems can be very expensive in certain industries, so can be the requirement
for each batch inoculation.
The inoculum cascading technique has been trialed and found suitable for certain
processes, whereby one batch is inoculated from the previous batch fermentation. Timing
and culture conditions are, however, of greatest importance. Often, inoculum cascading
has to be performed after only a short fermentation time to avoid carry-over of lysed or
dead or contaminating cells.
7. SoIid-state and SoIid-substrate CuItivation System
7.1 PrincipIes
. Solid substrate fermentations [SSF] are difficult to define precisely. They are generally
characterised by the growth of microorganisms on water insoluble substrates in the
presence of varying degrees of free water. Some researchers proposed the term solid
state fermentation for all those processes which utilise water insoluble materials for
microbial growth in the absence of free water. With increasing amounts of free water,
solid-substrate fermentations progress from solid-state through slurry to fermentations of
suspensions of solid particles.
The division between solid state and other solid substrate fermentations is not easy to
define precisely as free water becomes apparent in different substrates at different water
contents, eg wheat straw at 75% moisture content, in maple bark at 40% moisture content.
There is no simple measured parameter which distinguishes between them, although the
water activity (aw) is a potential candidate.
Problems therefore still arise in following the literature, as various other terms have been
used to describe solid-state fermentations, eg solid-substrate fermentation, solid-state
cultivation, solid-state processes, moist solid fermentation, surface culture, and koji
fermentation.
Since the word 'fermentation' itself has become very vague and unspecific terminology in
industry, and since solid-state as well as solid-substrate fermentations are really different
cultivation techniques (eg. Agar plates) and not new processes, the terms soIid-state
cuIture or soIid-substrate cuIture would be much more appropriate. Thus to avoid
confusion, the words culture and cultivation have been substituted for the word
'fermentation' to stress the analogy of solid state culture (SSC) to submerged liquid culture
(SLC) as a cultivation technique (Mitchell 1993).
The steps involved in an SSC process consist basically of (Lonsane et al. 1985):

1. the preparation of a solid substrate, often with pretreatment to decrease the particle size
or increase the availability of nutrients in the substrate to the organism. Additional nutrients
may be added and the pH adjusted at this stage;
2. a cooking step which sterilises or at least pasteurises the substrate, and causes the
absorption of water into the substrate particles;
3. the raising of a suitable inoculum, either by traditional techniques or pure culture
techniques;
4. the inoculation of the moist solids;
5. the incubation in appropriate culture vessels;
6. the maintenance of optimal conditions as far as possible;
7. the harvesting of solids;
8. the drying of the solids or extraction of the product from the solids;
9. further downstream processing, if necessary.
7.2 GeneraI features
. The efficiency, productivity and economy of SSC are affected by various factors. The
single most important feature is the moisture content of the medium, which makes SSC
fundamentally different from submerged liquid culture Water is essential for microbial
growth. t is present in the substrate in various degrees of tightness of adsorption and
there may even be some free water in the particle interior and on the solid-substrate
surface. The restricted amount of water available has several consequences:

a) the water activity of solid substrates can be significantly below 0.99, especially in solid-
state cultures where free water is virtually absent. This tends to favour filamentous fungi
many of which grow well between water activities of 0.93 and 0.98. For most bacteria and
yeast, growth is optimal above a water activity of 0.99. Therefore many SSC applications
involve fungi;
b) heat transfer tends to be restricted, which can lead to severe overheating problems at
large scale. Evaporative cooling is probably the most effective cooling method for SSC,
although this will even further reduce water availability;
c) substrates are more concentrated in SSC than in SLC. n SLC, water typically
comprises 90-99% of the total mass, whereas in SSC it may comprise only 10-85%.
Concentration of soluble substrates and products can reach high levels, potentially
exacerbating inhibitory and repressive effects. However, recently it was demonstrated that
SSC has the ability to overcome catabolic and endproduct repression in the production of
a bacterial alpha-amylase.
Solid substrates used in SSC are generally unrefined materials of agricultural origin. Often
substrates are simply moistened with water, although some pretreatment may be required.
Since solid substrates are relatively unprocessed, they are structurally and nutritionally
complex. The heterogeneity makes the study of SSC difficult because it makes complete
characterisation impossible and may also interfere with reproducibility. The presence of
high and low molecular weight macromolecules requires the synthesis and excretion of
extracellular hydrolytic enzymes by the microorganisms. The solid nature of the substrate
has several consequences:
1. The substrate mass is not evenly distributed in space, but contains interparticle spaces.
n solid-state culture these are continuous with the external gas phase, while in slurry
culture they are filled with liquid. This uneven distribution means that some probes (eg. for
pH) used for SLC cannot be used because proper contact with the substrate cannot be
ensured.
2. the handling of solids requires specialised equipment. Solid and slurry flow dynamics
are complex and poorly understood. Mixing of solids during cultivation requires specialised
reactor and agitator designs to prevent the substrate from being compacted;
3. mixing cannot be achieved at scales below the particle size without the destruction of
the particle. Within solid substrates therefore, mass transfer is restricted to diffusion;
4. microbial growth is largely restricted to the surfaces of solid substrates by the availability
of oxygen and since the solid matrix restricts oxygen transfer, the effects of the
microorganisms on the substrate are concentrated at the surface, which leads to a
concentration gradient of substrates, metabolic products, and pH within the solid particles.

. Many bacteria, yeasts, and fungi are capable of growth on solid substrates and find
therefore application in SSC processes. Amongst these microorganisms, filamentous fungi
are the best adapted for these processes and dominate in the research presently carried
out around the world.
Many important microbial processes in submerged liquid culture are performed with pure
cuItures. The substrate is sterilised and then inoculated with a single microbial species.
Strict aseptic conditions are required and steps must be taken to prevent contamination.
However, in the case of SSC, only a minor proportion of the processes are operated with
pure cultures under strict aseptic conditions. The range of cuIture types commonIy
practised in SSC are (Mitchell 1992):
a) Single organism culture processes: Many SSC processes (especially non-traditional
processes) are inoculated with a single microbial species. However, strict aseptic
procedures are often not used, thus the control of contaminants relies either upon
selection for the desired organism by the culture conditions which prevail in SSC or simply
by the initial numerical superiority of the inoculated microorganism. This selective pressure
in SSC makes it useful for application by unskilled operators.
b)Defined mixed culture: A defined mixed culture involves the inoculation of the
substrate with more than one pure culture, so that both organisms grow simultaneously.
Mixed cultures of Trichoderma reesei or Chaetomium cellolyticum with Candida lipolytica
resulted in increased protein production from wheat straw. The reason of the increase was
suggested to be the removal of glucose, which may act as a catabolic repressor of the
fungal cellulases. Although there are no reports of defined mixed cultures for SSC of
starchy substrates, it was found that the amylase of Aspergillus was inhibited, not
repressed, by glucose. This inhibition could be relieved by a co-culture with
Saccharomyces cerevisiae. Thus, defined mixed culture in SSC has not been carried out
extensively and has the aim of preventing the inhibition or repression of hydrolytic
enzymes.

c) Sequential culture is basically a type of mixed culture whereby the second organism is
inoculated after growth of the first organism has ceased. f wheat straw was first inoculated
with a culture of Chaetomium cellulolyticum or Trichoderma reesei followed by Candida
utilis 72 hours later, the protein content was significantly enhanced compared to
simultaneous inoculation. By 6this time the fungus had ceased growth but it had not
utilised all the reducing sugars which were liberated from the straw. These excess sugars
provided the substrate for C.utilis. When the latter was added at the beginning of the
culture, the enhancement of protein production was less effective, presumably because
the yeast competed with the fungus for the limited amount of sugars available, retarding
fungal growth and cellulase production.
A novel method of sequential culture tried to maximise first the production of cellulolytic
enzymes on rice straw and wheat bran. After the initial fermentation the enzymes were
extracted with water and the solid residue was dried and then re-supplemented with a
mineral solution and re-inoculated. The same fungal strain could be grown in five
successive cultivations on the same substrate to maximise substrate utilisation and
enzyme production. Even higher enzyme yields were obtained when five different fungal
strains were cultured successively on the substrate.

d) Undefined mixed culture. All these previously described culture methods involve the
use of well-defined and pure inocula. The term undefined mixed culture will be used to
describe processes in which the substrate is inoculated with an undefined mixture of
microorganisms. Examples of such inocula are those which are prepared by traditional
methods which favour a specific organism or group, the natural microflora of the substrate
itself, or inocula consisting of natural sources of mixed populations of microorganisms.
These processes often rely on the fermentation conditions favouring the growth of a single
organism or a group of organisms. As the fermentation proceeds the conditions may
change and succession of several microbial populations may occur.
The importance of these undefined mixed cultures can be seen in producing subtle
combinations of flavour which make fermented food more desirable than when produced
by pure culture with the dominant microbe. Since pure inocula are not used, undefined
mixed cultures may be difficult to characterise. n characterisation of the microflora, minor
members may be swamped out being the predominant organism in isolation procedures.
Alternatively, some members of the microflora may rely on others for metabolite excretion
or polymer hydrolysis and therefore might not grow or might grow very poorly in pure
culture during isolation procedures. Studies of processes involving natural microflora
therefore are hampered by the inability to measure the growth of individual strains, or by
complex interactions between the microbes present. Oriental fermented foods have been
traditionally by this method for thousands of years. solation of the predominant organism
and its pure culture was not applied to the production of these foods until the 20th century.

The main advantage of culture methods which involve more than one microorganism is the
increased substrate utilisation due to the pooling together of the degradative abilities of a
number of microorganisms. Therefore, when using mixed cultures, organisms should be
chosen which do not compete for the same substrate but rather utilise different substrates.
Also one microorganism might remove a substrate causing inhibition or catabolite
repression or another microorganism. Finally, mixed cultures tend to be more resistant to
contamination, which is mainly due to the increased competition that a contaminant
experiences.
The importance of the inoculum has been recognised by many researchers. Since most
applications of SSC involve fungi, spore inocula are commonly used. A spore inoculum
does not always give the best results, although it is usually the most convenient to use.
Spore inocula allow a greater flexibility in coordination of inoculum preparation with the
cultivation process. Spores also retain viability for longer periods than fungal mycelium and
therefore the spores can be stored and used when required. A disadvantage of spores is
that they are metabolically dormant. Therefore, all the necessary metabolic activities must
be induced and the appropriate enzyme systems synthesised before the fungus can begin
to utilise the substrate and commence growth.
nocula may be produced either in SSC or SLC. Sporulation of fungi is generally better on
solid media than in liquid media. n any case, liquid inocula can easily be prepared from a
sporulating solid culture by suspending the spores in a liquid medium. This spore inoculum
may then be pregerminated if desired.
The even distribution and the density of the inoculum are also important. Liquid inocula are
more easily distributed evenly than solid inocula. noculum density is also an important
consideration since overcrowding of spores can inhibit germination and development.
As with submerged liquid culture, reactor design is a vitally important factor which
determines the efficiency of SSC processes (Mitchell et al. 1992). Unfortunately, the
design of solid-state bioreactors has to date been almost entirely empirical.
Tray bioreactors are the simplest SSC bioreactors with the following basic
characteristics:

a) a relatively thin layer of substrate is spread over a relatively large horizontal area;
b) there is no forced aeration, although the base of the tray may be perforated and air may
be gently circulated around the tray;
c) mixing, if done at all, is done intermittently by simple automatic devices or manually;
d) temperature may vary with the ambient or the tray may be placed in a temperature
controlled cabinet or room.
Tray bioreactors have been used successfully at laboratory, pilot, semi-commercial and
commercial scale. A major disadvantage of tray bioreactors is that at large scale
operations are not easily automated and therefore tend to be labour intensive.
Packed bed bioreactors are characterised by having a static substrate supported on a
perforated base plate through which forced aeration is applied. Many variations of this
basic design are possible, but the typical design is a tall thin cylindrical column with the
forced aeration applied at the bottom.
The advantage of these packed bed bioreactors is that they remain relatively simple while
allowing better process control than is possible with trays, in particular temperature and
humidity. Disadvantages associated with these bioreactors include difficulties with the
emptying of the final product, non-uniform growth, poor heat removal and problems with
scale-up.

Rotating drum bioreactors have the following characteristics:
a) a horizontal or inclined cylinder;
b) rotation of the cylinder around its central axis to cause a tumbling motion of the
substrate, which may be aided by baffles;
c) aeration, if supplied, is with low pressure air fed into the reactor headspace.
The mixing provided by the tumbling action in rotating drum bioreactors is relatively gentle,
and of all the methods of automated mixing should cause the least damage to
microorganisms or to the substrate structure.
Problems may arise due to either the agglomeration of substrate particles or particle
attrition. n addition, temperature control is quite difficult, since it is difficult to water-jacket
the moving body of the bioreactor.

Stirred bioreactors are of two main types depending on whether the axis of the bioreactor
is horizontal or vertical. Horizontal stirred bioreactors are quite similar to rotating drum
bioreactors except that the mixing is provided by an internal scraper or paddles, rather
than by the rotation of the body of the bioreactor. Vertical stirred bioreactors (eg NRA-
Dijon reactor) are often subjected to forced aeration. They differ from packed bed
bioreactors by the fact that they are agitated, which may either be continuous or
intermittent.

Air-solid fluidised bed [ASFB] reactors have the following basic characteristics:

a) relatively tall column;
b) a perforated base through which air is blown at sufficient velocity to make the substrate
particles buoyant;
c) a specialised stirring device may be included to aid in agitation of the solids.
Special features and advantages of the ASFB technology include:
1. very good aeration is provided, enabling good growth of aerobic microorganisms.
2. overheating is not a problem since metabolic heat is removed by the airstream;
3. gaseous and volatile metabolic products are quickly eliminated, which reduces inhibitory
effects;
4. in the production of products, such as single cell protein, drying of the product can take
place in the column itself;
5. highly effective mixing is achieved, avoiding problems of gradients of temperature and
moisture content within reactors, and enabling superior control of process parameters;
6. higher productivities can be obtained than in traditional SSC processes, resulting in
savings of plant space and operating costs.

Further developments in reactor designs are on their way.
7.6 AppIication of SSC
The applications of SSC can be arbitrarily divided into socio-economic and profit-
economic applications. A profit-economic application will seek maximising profit, and will
often produce products which are not so much essential but rather desirable 'luxury
comforts' with a primary aim for commercial profit.
Socio-economic applications of SSC off the potential of significantly raising living
standards with only a low technology input requirement. n fact, many of the current
applications of SSC come from developing countries. These processes commonly involve
the disposal of solid agricultural or domestic wastes, or the improvement of the nutritional
value or flavour or keeping qualities of agricultural products or byproducts to make them
suitable for use as human food or animal feed. Classical examples of socio-economic
applications of SSC include composting of waste or municipal refuse, ensiling of grasses
and upgrading of lignocellulosic products or staple food.
n socio-economic applications the inoculum preparation and the cultivation itself are not
carried out aseptically. The inoculum may be prepared according to traditional recipe, or
may simply be the microflora of the substrate itself. t is essential that the inoculum can be
prepared simply and maintained easily. Sterilisation is not performed, although
pasteurisation may be achieved if the substrate is cooked prior to inoculation. These
processes must use locally available substrates, which, if they cannot be stored, may be
seasonal in supply. A minimum control is exerted over culture conditions.
Pure cultures are commonly used in profit-economic applications of SSC, therefore both
fermenter design and process operation must prevent contamination. Control is usually
exerted over the process conditions, especially temperature, amount of aeration and
moisture. A range of products may be produced and may require extraction from the
substrate.
Most of the processes using the solid-substrate cultivation technique are commercialised
in SEAsia, African and Central American countries. Nevertheless, a resurgent of interest
has occurred in Western and European countries over the past 10-15 years in response to
the ever rising demand for economy in processes.

[This part has been adapted from the publication by Doelle et al. 1993]
All of these mentioned types of cultivation techniques above have been developed to
overcome certain problems in biomass or product formation, not only using
microorganisms, but also in the case of plant and animal cell cultivation. Many variations
exist of each of these systems and there is no doubt that many more variations will be
designed in future. t is one of the most difficult problems in microbial process development
to find or design a cultivation technique which results in the eventual goal, as an ideal
cultivation technique should also minimise waste.
A very dangerous and also costly waste or pollutant in the outflowing medium is the
catalyst itself. Firstly, every microorganism has to be regarded as a potential health hazard
and thus should be destroyed before the effluent is released. This is particularly important
for the newly genetically engineered microorganisms. Secondly, the value ofm a biological
catalyst is at least equal to the value of the nutrients consumed in growing the cells, which
means that its waste can be very costly.
There exist at the moment two ways to limit the amount of catalyst that is lost. One way is
to recycle the catalyst from the overflow back into the fermenter or bioreactor. Such
recycling systems have been tried with various degrees of success. n general they can be
difficult and costly systems, particularly with bacteria, since the cells are often damaged
and the recycling system is open to contamination with foreign microorganisms. The
second alternative is, of course, to keep the catalyst in the bioreactor. The most common
technique is the use of packed bed systems, which use a solid support in which the cells
are encouraged to grow.
mmobilisation techniques have revolutionised the use of microorganisms for waste
treatment and in the industrial production of chemicals, drugs, food and other products.
mmobilised systems are of particular use in continuous systems, where their application
results in better process control, reduced operational cost, minimum down time, reduced
lag periods and better product uniformity (Brodelius and Vandamme 1984). t is also
possible to achieve increased cell densities, which allow continuous fermenters to run at
higher dilution rates with higher reaction rates, product yields and use of more
concentrated substrate stream (Kolot 1988). These advantages are economically
important, because the biocatalyst can be reused, the scale of the reactor, and thus the
overall operational size, can be reduced with less waste in the effluent stream.
The five main methods that have been used for cell immobilisation are entrapment,
adsorption, aggregation/flocculation, covalent coupling, and microencapsulation. Each
method has its advantages and disadvantages, and not all of these methods may be
appropriate for the immobilisation of a particular strain. n the case of Zymomonas mobilis,
the support must be inert to action by ethanol, low pH, disruption by CO
2
, and of low cost.
Z.mobilis has been immobilised by (1) occlusion in alginate and carrageenan gels; (2)
adsorption to various materials such as ion exchange resins and polyurethane foam; (3)
flocculation; and (4) covalent coupling.
8.1 AIginate
Alginate is probably the most widely used method of immobilization. t is a polysaccharide
isolated from kelp, which is a copolymer of beta-D-manuronate and alpha-L-gularonate
joined by 1,4-linkages (Brodelius and Vandamme 1984). Beads of immobilised cells are
obtained by ionotropic gelation, whereby a solution of alginate and cells is dropped into a
medium containing CaCl
2
. The immobilisation of Z.mobilis with alginate resulted in a
complete substrate utilisation with some of the highest ethanol productivities.
A number of research groups have chosen to model various aspects on immobilised Z.
mobilis fermentation using cells immobilised in alginate as the model system. A
comparison of the model with actual experimental results exhibited average deviations of
30-40% with the predicted glucose profiles within the fermenter (Melick et al. 1987).
The diffusivity of substrate and products in the catalyst particles is largely influenced by the
cell density, which has to be taken into account to predict the overall reaction rate of
alginate-immobilise cells (Sakaki et al. 1988).
8.2 Carrageenan
Carrageenan is a polysaccharide extracted from seaweed (Chibata et al. 1986), consisting
of beta-D-galactose-4-sulfate and 3,6-anhydro-D-galactose units. t forms rigid gels by
cooling a heated suspension or contacting it with a solution containing a gel-inducing
agent such as K
+
, NH
4
+
, Ca
2+
, Cu
2+
, Mg
2+
, Fe
3+
amines, or water-miscible organic solvents.
n the case of Z. mobilis, lower productivity has generally been obtained when compared
with alginate.
8.3 Ion exchange resins
on exchange resins have been used by many researchers for immobilisation, but had in
the case of Z. mobilis difficulties with gas disruption, channelling, and plugging problems
associated with excessive filamentous growth (Krug and Daugulis 1983). n general, the
immobilisation of cells on ion exchange resins is a method that has had limited use with Z.
mobilis. The choice of an appropriate resin may be an expensive and time-consuming
task, as each resin has a different capacity to adsorb cells and some may not even
retainthe cells under fermentation conditions. However, ion exchange immobilisation does
not suffer from mass transfer problems.
8.4 PoIyurethane foam
Polyurethan foam is a versatile, inert, low-cost immobilisation support that has been used
successfully to immobilise Z.mobilis (Amin & Verachtert 1982). t relies on the ability of the
cell to adsorb to the foam surface and to grow within the porous structure. Amin and co-
workers (1987) immobilised Z.mobilis in 1 cm
3
polyurethane foam cubes, which were fixed
onto steel rods and placed in a glass column reactor. The productivity of this bioreactor
was only one-third of that obtained with Z.mobilis immobilised in foam discs within a
vertical rotating reactor (Amin & Doelle 1990).
Cell aggregation and/or flocculation is a relatively simply method in particular with
flocculating cells, as a heavy floc can easily be separated from the liquid phase and cell
washout is less likely. As no solid support is required, the cell density that can be achieved
is higher. Flocculated cells are particularly compatible for use in fluidized bed bioreactors
(Scott 1984).
Cells that are not naturally flocculating can be artificially induced to pellet, cross-link, or
flocculate. Cross-linking as an immobilisation technique has not been used to a great
extent as in most cases, it results in cell death. Cell flocculation can also be induced by the
addition of polyelectrolytes such as titanium hydrous oxide or synthetic flocculants with
chitosan.
Covalent coupling is the direct linkage of cells to an active support (Cheetham 1980). n
most cases, this procedure results in cell death as the coupling reagents can be highly
toxic. On the other hand, these systems have the advantage of not suffering from any
diffusional limitations and are less prone to release cell progeny into the medium.
8.7 Passive immobiIisation
Passive immobilisation is an immobilisation technique whereby cells are immobilised by
passive adsorption onto or into a huge variety of supports such as alumina pellets,
activated carbon, borosilicate glass fiber pads, polyester fibers, vermiculite, sponge and
mesh, polystyrene and many other materials.
The advantage of using any of these supports are that they involve relatively simple
techniques and they are inexpensive, reusable, and available in abundance. However,
these passive immobilisation techniques have apparently received less attention because
of the doubtful stability of adsorbed biomass to withstand pH and temperature changes,
variations in media composition, high flow rates, or periodic starvation from the substrate.
A number of research groups found that passive immobilisation could be enhanced if
filamentous growth was abundant.
mmobilised bioreactor design is very important and can be a major factor contributing to
the overall efficiency of the immobilised cell systems.
The fixed bed bioreactor is the most frequently used type of immobilised cell bioreactor
(Kolot 1988). A columnar reactor has the advantage over a mixed reactor because of its
plug flow or multistage characteristics. t also has serious limitations, as it can be unstable
during long-term operations because of continuous biomass accumulation, mass transfer
limitations, and CO2 holdup, which becomes responsible for channelling and the creation
of dead spaces and even matrix disruption.
n an attempt to reduce product inhibitory effects, a simultaneous separation process was
incorporated where volatile ethanol is stripped out of the broth by a gas that is passed
through the top segment of the fixed-bed reactor. This increased the volumetric
productivity in the case of Z. mobilis dramatically from 23.7 g/l/h to 41.4 g/l/h.

The Stirred Tank Reactor [STR] has been used by only a few researchers, as they
promote good mixing but they are not very suitable for heavy immobilised cell
preparations. Fast stirring results in high sheer stress, thus increasing cell leakage from
alginate or carrageenan beads, cell detachment from ion exchange resins and floc
disruption.

The concept of the Rotating Disc Reactor [RDR] is relatively new (Parakh et al. 1989)
and consists of immobilised cell units such as polyurethane foam sheets or fiber discs
being attached to a rotating shaft. The RDR is slowly stirred, thus allowing good mixing
and removal of dead cells, debris and evolved carbon dioxide.
n Brisbane (Amin and Doelle 1989;1990) we developed and maintained successfully a
constant immobilised cell culture that achieved ethanol concentrations of 120 g/l and a
productivity of 13 g/l/h in repeated batch operation. n continuous operation, we attained
80 g/l ethanol and a productivity of 63 g/l/h.
With a Fluidized Bed Reactor system, the substrate or gas is passed at high velocity up
through a reactor containing folcs or beads of cells. Such bioreactors promote good mass
transfer, the dead cells are removed from the system, and large volumes of carbon dioxide
can be released without channelling. n bioreactors, fkuidization avoids problems such as
contamination, shear damage, and limits to scale-up, associated with impeller shafts and
blades in stirred bioreactors.
n a comparative study between an ordinary stirred tank bioreactor and a fluidised
bioreactor with Z. mobilis cells immobilised in alginate (Margaritis and Wallace 1982), it
was found that with the fluidised system, the beads were subject to much lower sheer
rates, resulting in about 100times less cell leakage and a 64% higher maximum rate of
ethanol production.
8.9 Biosensors
A biosensor is an analytical device that incorporates biologically acive sensing material
with a transducer, which amplifies the biological recognition response to a specific
chemical species into an observable electrical signal (Turner et al. 1989).
A microbial sensor consisting of immobilised living whole cells of Brevibacterium
lactofermentum, for example, and an oxygen electrode was constructed for the continuous
determination of total assimilable sugars (sucrose, glucose and fructose) and a microbial
sensor consisting of immobilised whole cells of Pseudomonas fluorescens and an oxygen
electrode was developed for the determination of glucose (Karube et al. 1979). Biosensors
have exploded into the field of biotechnology and medicine using either whole cells or
enzymes for the determination of chemical compounds.
9. References
Amin,G. and Verachtert,H. 1982 Comparative study of ethanol production by immobilised
cell systems using Zymomonas mobilis or Saccharomyces bayanus. Europ. J. Appl.
Microbiol. Biotechnol. 14, 59

Amin,G. and DoeIIe,H.W. 1989 Vertical rotating immobilised cell reactor of the bacterium
Zymomonas mobilis for stable long-term continuous ethanol production. Biotechnol.
Techniques 3,95
Amin,G. and DoeIIe, H.W. 1990 Production of high ethanol concentrations from glucose
using a vertical rotating immobilised cell reactor of the bacterium Zymomonas mobilis.
Acta Biotechnol. 10,35

Amin,G. and DoeIIe,H.W. 1990 Production of high ethanol concentrations from glucose
using Zymomonas mobilis entrapped in a vertical rotating immobilised cell reactor.
Enzyme Microbiol Technol. 12, 443

BrodeIius,P. and Vandamme,E.J. 1984 mmobilised cell systems. n 'Biotechnology' , Vol
7b (H.J.Rehm, G.Reeds,eds.), Verlag Chemie GmbH, Weinheim, pp405

Chibata,I., Tosa,T., and Sato,T. 1986 Methods of Cell mmobilisation. n 'Manual of
Industrial Microbiology and Biotechnology ' [A.L.Demain & N.A.Solomon, eds], p. 217,
American Society for Microbioology, Washington

Cheetham, P.S.J. 1980 Developments in the immobilisation of microbial cells and their
applications. n ' Topics in Enzyme and Fermentation Biotechnology ' Vol 4 [Wiseman,A.,
ed.]. John Wiley, New York

DoeIIe,H.W. 1975 Bacterial Metabolism. Academic Press, New York

DoeIIe,H.W. 1991 Microbial Process Development. n 'The Importance of Microbiological
Biotechnology for Community and Economic Development' , Unesco-MRCEN Regional
Training Course, Motupore sland, Univesity of Papua New Guinea, September 1991

DoeIIe,H.W. 1994 Microbial Process Development. World Scientific Publishers, Singapore

DoeIIe,H.W. 1997 Solid State Cultivation: Basic concept and strategies. n
'Treatment and Utilisation of Agro-Industrial Waste for a Cleaner Environment and
Sustainability', CRO/UNESCO/MRCEN/BOTEC nternational Regional Training Course,
Prince of Songkla University, Hat Yai, Thailand, August 1997

DoeIIe,H.W., Kirk,L., Crittenden,R., Toh,H. and DoeIIe, M.B. 1993 Zymomonas mobilis
Science and Industrial Application. Critical Revs. Biotechnol. 13, 57-98

Hamer,G. 1997 Theoretical analysis of microbial growth & . n 'Treatment and Utilisation
of Agro-Industrial Waste for a Cleaner Environment and Sustainability'
CRO/UNESCO/MRCEN/BOTEC nternational Regional Training Course, Prince of
Songkla University, Hat Yai, Thailand, August 1997

Karube,I., Misuda,S and Suzuki,S. 1979 Glucose sensor using immobilised whole cells
of Pseudomonas fluorescens. Europ. J. Appl. Microbiol. Biotechnol. 7,343

Karube,I. And Suzuki,M. 1990 Construction of microbial sensors. n 'Biosensors: A
Practical Approach' [A.E.G.Cass, ed.]. p. 157. RL Press, New York

KoIot,F.B. 1988 mmobilised Microbial Systems: Principles, Techniques and ndustrial
Applications. Robert E.Krieger Publishing Comp., Malabar

Krug,T.A. and DauguIis,A.J. 1983 Ethanol production using Zymomonas mobilis
immobilised on an ion exchange resin. Biotechnol. Lettrs. 5, 159
Lonsane,B.K., GhiIdyaI,N.P., Budiatman,S. & Ramakrishna,S.V. 1985 Engineering
aspects of solid state fermentation. Enzyme Microbiol. Technol. 7, 258-265

Margaritis,A. and WaIIace,J.B. 1982 The use of immobilised cells of Zymomonas mobilis
in a novel fluidised bioreactor to produce ethanol. n 'Fourth Symp. Biotechnol. Energy
Production and Conservation ' [Scott,D.C., ed.], John Wiley, New York

MeIick,M.R., Karim,M.N., Linden,J.C., DaIe,B.E. and MihaItz,P. 1987 Mathematical
modelling of ethanol production by immobilised Zymomonas mobilis in a packed bed
fermenter. Biotechnol. Bioeng. 29, 370

MitcheII,D.A. 1992 Microbial Basis of Processes. n ' Solid Substrate
Cultivation'(H.W.Doelle, D.A.Mitchell, C.A.Rolz, eds.), p. 17-28, Elsevier Applied Science,
London

MitcheII,D.A. and Lonsane,B.K. 1992 Definition, Characteristics and Potential. n 'Solid
Substrate Cultivation' (H.W.Doelle, D.A.Mitchell & C.A.Rolz, eds), p.1-16, Elsevier Applied
Science, London

MitcheII,D.A., Lonsane,B.K., Durand,A., Renaud,R., AImanza,S., Maratray,J.,
Desgranges,C., Crooke,P.S., Hong,K., Tanner,R.D. & MaIaney,G.W. 1992 General
Principles of Reactor Design and Operatoin for SSC. n 'Solid Substrate Cultivation',
(H.W.Doelle, D.A.Mitchell & C.A.Rolz, eds.), p.115-140, Elsevier Applied Science, London

Parekh,S.R., Parekh,R.S. and M.Wayman 1989 Ethanolic fermentation of wood-derived
cellulose hydrolysates by Zymomonas mobilis in a continuous dynamic immobilised
biocatalyst bioreactor. Proc. Biochem. 24, 88

Pirt,S.J. 1975 Principles of Microbe and Cell Cultivation. Blackwell Scientific
Publications, London

Sakaki,K., Nowaqwa,T. and Furusaki,S. 1988 Effect of intraparticle diffusion in ethanol
fermentation by immobilised Zymomonas mobilis. Biotechnol. Bioeng. 31 , 603

Scott,C.D. 1984 Ethanol production in a fluidised bed bioreactor utilising flocculating
Zymomonas mobilis with a biomass recycle. Biotechnol. Bioeng. Symp., 13, 287

Turner,A.P.F., Karube,I. And G.S.WiIson 1989 Biosensors Fundamentals and
Applications. Oxford University Press

Deputy-Director, MIRCEN-Biotechnology Brisbane and the Pacific Regional Network;
Chapter 8
Thermodynamics, SoIute Transport and Enzyme
CataIysis

Content:
1. Concept of thermodynamics of bioIogicaI systems
1.2.1 Proton-transIocating eIectron transport chain
2. Membrane and SoIute Transport Mechanisms
2.1 Passive Diffusion
2.2 FaciIitated Diffusion
2.3 Active Transport
2.4 Group TransIocation
3. Concepts of MetaboIism
3.1 Photosynthesis
3.3 Anaerobic Respiration
3.4 Fermentation
5. BibIiography

1. Concept of thermodynamics of bioIogicaI systems
One of the most fundamental properties of living cell systems is their ability to utilise and
transform energy. This energy occurs in a number of forms:

Mechanical Energy is developed during cellular movement, beating of flagella, re-
organisation of intracellular structures such as mitochondria, and alteration of cell shape;
Electrical Energy is produced when electrons move from one place to another, usually
expressed as a flow of current between two points due to a difference in voltage.

Electromagnetic Energy occurs in the form of radiation, and in biology the most
significant is that from visible or near-visible light, such as radiation from the sun for
photosynthetic organisms. Some organisms release energy and glow, which is referred to
as bioluminescens. They produce light energy.

Chemical Energy is the energy that can be released from chemical reactions;

Thermal Energy or heat is produced as part of the normal energy transformation
processes and occurs as waste energy released into the surroundings;

Atomic Energy is contained within the structure of atoms themselves and is released in
the form of atomic radiation, which can not be utilised by living organisms.

Since growth can be defined as the orderly increase of all chemical components, it is the
chemical form of energy which is of greatest importance for the understanding of microbial
growth and metabolism.

Microbial metabolism consists of thousands of individual chemical and enzyme-
catalysed chemical reactions. These chemical reactions in living organisms occur in
characteristically organised sequences, called metabolic pathways. There are two main
types of metabolic pathways:

1. pathways which lead from large [low oxidative state] to smaller molecules [high
oxidative state], which are called catabolic pathways or cataboIism

2. pathways which lead from small [high oxidative state] to large molecules [low oxidative
state] essential for the formation of cellular material, which are referred to as anabolic or
biosynthetic pathways or anaboIism.

The main concept of catabolism is therefore to provide the cell with small molecules or
precursors suitable for biosynthesis of all major chemical constituents of the living cell and
with reductant and energy as well to carry out these endergonic and reducing reactions
leading towards compounds of low oxidative state. Whereas all catabolic pathways are
therefore oxidative and thus energy-producing, the biosynthetic pathways are reductive
and energy-consuming. Metabolism consists therefore entirely of energy transformation
and transfer mechanisms, which are based on thermodynamics.
The amount of energy involved in a chemical reaction is expressed in terms of gain or loss
of energy during the reaction. The First Law of Thermodynamics is the law of
conservation of energy and states that the total amount of energy in nature is
constant. This means that, if heat [q] is added to a system of a given energy content, it
must appear as a change in the internal energy [E] of the system or in the total work
performed by the system on the surrounding [w]
q = E + w'
E = q - w'

Fig. 1: Generalised scheme for metabolic energy formation and usage

Such an addition of heat results in many instances in a change of volume [V] at a constant
pressure [P]
q = E + PV + w'
whereby the expression E + PV, representing the change of the heat content or
enthaIpy, can be replaced by H
H = E + PV
= q - w'
At any temperature, PV = nRT, where n represents the number of moles and R is the gas
constant [= 1.987 cal.moldegree
-1
] with T signifying the absolute temperature

H = E + nRT

This expression reveals that each chemical reaction proceeds to completion with a definite
heat of reaction that is quantitatively related to the number of molecules reacting. The
energy unit for its measurement is the calorie, which is defined as the quantity of heat
energy necessary to raise the temperature of 1 g water by 10
0
C. One calorie is equal to
4.184 joules.

ExampIe:
C
6
H
12
O
6
6 CO
2
+ 6 H
2
O
H = -673 kcaI = -2,815.8 kJ/moI

The negative sign indicates an exergonic reaction.
Whereas the first law of thermodynamics implies only that there is a quantitative
correspondence between different kinds of energy and that the energy content in nature is
constant, it is the Second Law of Thermodynamics which states that all physical and
chemical processes proceed in such a direction that the randomness or entropy of
the universe increases to the maximum possible, at which point there is an
eqiulibrium. Since the entropy has the symbol S,

q
S = ----
T
q = TS

f one combines the equations of the first and second law of thermodynamics,
H = TS - w'
n considering these thermodynamic relationships one should always be aware that the
main interest in biological reactions is not in reactions at equilibrium, but in those
proceeding in the direction that approaches equilibrium. The tendency to seek the
position of maximum entropy is the driving force of all processes, and heat is either given
up or absorbed by the surrounding system to allow the system plus surrounding to reach
the state of maximum entropy.
These changes in heat and entropy are related by the free energy, G
G = H - TS
and since H = TS - w',
then G = - w'
which expresses the energy released that is available to do useful work. The determination
of G depends on accurate measurements of either the equilibrium constant, K, of a
reversible reaction or its electromotive force. The equilibrium constant is very difficult to
determine, since often no adequate analytical methods are available to analyse the
reactants and product concentrations.
Since metabolism consists of sequences of oxidations and reductions, the measurement
of the electromotive force is the choice for establishing G.
This electromotive force is the algebraic difference of the potentials of two half-cells. n
order to understand this definition, it is necessary to realise that energy-yielding reactions
within the cell are of the nature of oxidations. An oxidation is generally defined as the loss
of electrons and reduction as the gain of electrons
H
2
+ 2e
-
2 H
+

Electrons released by an oxidation MUST be accepted by an oxidising agent, which itself
will be reduced. Such a transfer of electrons, according to modern theory, establishes an
electric current, since the electron donor possesses a characteristic electron affinity. t
should therefore be possible to obtain direct proof of the transfer of electricity in oxidation-
reduction reactions under suitable experimental conditions. This transfer could be a
quantitative measure of the tendency of substances to donate or accept electrons and thus
a means for calculating free energy changes for oxidation-reduction reactions. This
quantitative measure is termed an oxidation-reduction potential.
The measured potential difference [E
h
] of an oxidation-reduction system is expressed by
the NERNST equation
RT [A
ox
]
E
h
= E
0
+ ----- In -------
nF [A
red
]

where E
0
is the standard electrode potential, R is the gas constant (= 8.314 J degree
-1
mol
-
1
), n represents the number of electron involved in the reaction, F is the Faraday constant
(= 96,494 coulomb) necessary to convert one equivalent of ions, and [A
ox
] and [A
red
] are
the activities of the oxidised and reduced form of the oxidation-reduction system.
The standard electrode potential [E
0
] is the potential of an electrode in equilibrium with a
unity activity of its ions. This value is characteristic for each oxidation-reduction system
and gives a measure of the relative ability of that system to accept or donate electrons in
oxidation-reduction reactions.
The free energy change associated with an oxidative reaction may now be calculated from
the standard electrode potentials of the two reacting systems. For this, the NERNST
equation for the two systems has to be incorporated into the standard free energy equation

RT
Since G = RTInK and E
0
= ---- InK
nF

nFE
0
= RTInK,

therefore. -G = nFE
0

Let us demonstrate these calculations on an exampIe:

MaIate + cyt c oxaIacetate + cyt c

malate/oxalacetate has a E
0
= - 0.17V
cyt. c
ox
/cyt. c
red
has a E
0
= + 0.22V
therefore, G = - nFE
0

= - 2 x 96,500 x [0.22 - (-0.17 V)]
= - 75.34 kJ/moI

Since electrons move only to a more positive redox system, the greater the difference
between the systems, the greater is the oxidising ability of the system. Energy is released
in direct proportion to the difference in E
0
values.
Principles of electron transfer and transport The simplest way to think about oxidation-
reduction reactions in biological systems is in terms of eIectron donors and eIectron
acceptors. The energy source donates electrons and becomes oxidised, whereas the
oxidising agent accepts electrons and becomes reduced. Since microorganisms can
obtain their energy required from a considerable number of diverse and varied reactions,
electron donors and acceptors play a most important role in catabolic processes.
A convenient way of viewing such electron transfers in oxidation-reduction reactions is to
imagine a vertical redoxpotential tower (Fig. 2)

Figure 2: Redoxpotential tower and its relation to different modes of metabolism

This tower represents the range of redoxpotentials from the most negative to the most
positive. Molecular hydrogen and organic compounds with carbonyl groups have a very
negative [approx. -0.4V] and molecular oxygen as the best oxidising agent has the most
positive potential [+ 0.81V]. f both would act as electron donor and acceptor, the bridging
gap of 1.2V would give a G
0
of 238.5 kJ/mol, causing certain death to any cell in the
biological system.
n order to avoid cell death and energy waste through the release of excessive heat, the
cells possess a number of redox systems or electron carriers, which shuffle the electrons
from the low to the high redox potential system. This arrangement is termed the electron
transport chain. The importance of this electron transport chain with its redox cascades
lies in the possibility of transforming the free energy obtained in every step into chemical
energy and of storing the small energy parcels released. Of all energy carriers, the most
important is ATP (adenosine triphosphate), which means that the free energy from a
chemical reaction can be trapped or conserved through the formation of an energy-rich
intermediate and is located in the phosphate bonds of ATP. The approximate value for the
free energy change of ATP hydrolysis at pH 7.0 and 25
0
C under standard conditions was
calculated as G
0
= -33.47 kJ/mol or -8 kcal/mol.
The energy released from the electron shuttles outlined in the four different metabolic
modes are stored in the form of ATP. The mechanism that leads from the release of
energy to the readily formed storage product ATP or the necessary energy-coupling of the
various energy-dependent functions follows the chemiosmotic coupIing hypothesis.

Figure 3: General diagram of protonmotive force in the bacterial cell membrane
The characteristic feature of the chemiosmotic coupling hypothesis is the consideration
given to the asymmetrical orientation of membrane-bound enzymes catalysing vectorial
reactions that bring about the translocation of molecules, ions and chemical groups across
the cellular membrane. This feature postulates that the inner membrane, in which the
photosynthetic centers, the respiratory chains and coupling devices are located, is
essentially impermeable to most ions, including OH
-
and H
+
. n consequence the
membrane or the barrier protein has a low electrical conductivity. The vectorial reactions
would therefore lead to the separation of electrical charges within and across the
membrane and their recombinations underlie the performance of osmotic, chemical and
mechanical work. These postulates indicate that the chemiosmotic coupling hypothesis
requires in its simplest form the coexistence of a proton-translocating ATPase in a
membrane that is essentially impermeable to ions.

1.2.1 Proton-transIocating eIectron transport chain

The proton-translocating electron transport chain is an alternating sequence of hydrogen
carriers and electron carriers, which are arranged across the membrane in loops. The
oxidoreductions, causing hydrogen and electron transport through the electron transport
chain in the forward direction, result also in the translocation of protons from the inner to
the outer phase. The net result of such oxido-reduction sequence is the appearance of 2
H
+
on the outside and the disappearance of 2 H
+
from the inside of the membrane.
The translocation of protons in one direction is equivalent to the appearance of OH
-

movement the other way. This creates in addition to the pH difference, an eIectricaI
potentiaI difference, which combines as the protonmotive force, which tends to drive
the translocated protons back into the cell.

Figure 4: Formation of an energised cell membrane: Protonmotive Force

The proton-translocating membrane-bound ATPase complex catalyses the vectorial
reaction
ATP + H
2
O + 2 H
+
ADP + Pi + 2 H
+

whereby the thermodynamic equilibrium is strongly in favour of ATP hydrolysis. The
proposed stoichiometry is 2 H
+
translocated per molecule of ATP. n order to obtain ATP
synthesis, a special mechanism is required to drive the reaction in the opposite direction.
According to the chemiosmotic coupling theory, it is the gradient of pH and of the electrical
potential generated by the electron transport chain, which reverses the direction of the
ATPase catalysis from ATP hydrolysis to ATP synthesis. f ATP hydrolysis is coupled to
proton-translocation from the inner to the outer phase, ATP synthesis is coupled to inward
proton-translocation driven by the protonmotive force generated by the oxidoreduction
reactions.

Figure 5: Net H+ into the cell: Dissipation of energised cell membrane: ATP synthesis
[Aerobic System]

The important feature of the ATPase complex is that the enzyme is fully reversible and not
only acts as an ATP synthase during oxidative phosphoryIation, but can also catalyse
the formation of a protonmotive force at the expense of ATP hydroIysis in the absence
of oxidative phosphoryIation [substrate phosphorylation] or an electron transport
chain.

Figure 6: Formation of energised cell membrane by ATP hydroIysis: Protonmotive Force
[Anaerobic system]

n the latter case, oxidative phosphorylation is replaced by substrate phosphoryIation,
an energy conservation system operative under both aerobic and anaerobic conditions
and dominantly present under fermentation conditions.
n substrate phosphorylation, the oxidation of a substrate is followed by an energy transfer,
whereby ATP is synthesised and again hydrolysed without the involvement of intermediate
electron carriers, but specific enzymes and catalysts instead

Figure 7 : Substrate phosphorylation in anaerobic systems

The total energy coupling through the ATP system can now be proposed.
Biological fuel molecules such as carbohydrates, lipids and proteins contain high levels of
chemical energy because of their degree of structural order. During catabolism they are
degraded, i.e. oxidised, into smaller molecules such as carbon dioxide and water,
alcohols, acids etc. As a result of this transformation, which means an increase in
randomness or entropy of their constituent atoms, the fuel molecules undergo a loss of
energy, ie. that form of energy capable of doing work or being conserved in the form of
ATP at constant temperature and pressure.
2. Membrane and SoIute Transport Mechanisms
Substrates undergoing catabolic reactions must enter the cell before catalysis can occur,
since the large majority of catalytic enzyme proteins are found inside the cell. Four major
transport processes are known that could be responsible for the transport of molecules
across the cell membrane.
2.1 Passive Diffusion.
Net flow of a solute by passive diffusion occurs only in response to a difference in its
concentration across the cell membrane (concentration gradient) and as a result of such a
flow, the difference diminishes. The rate of flow is a direct function of the magnitude of the
gradient and does not approach a limiting value even when the concentration difference is
great. Passive diffusion occurs when there are regions of the membrane through which a
particular solute can pass freely, much as small molecules can pass through the artificial
membrane used for dialysis. Water and certain gases, such as oxygen and nitrogen are
the principle nutrients that cross the cell membrane by passive diffusion. The speed of
diffusion depends very much on the Brown's molecular movement. Normally only water
can enter the cell via this system.

2.2 FaciIitated Diffusion.
A very much related transport mechanism to passive diffusion is the facilitated diffusion.
As the name indicates, the diffusion in or out of the cell of certain compouns to which the
cell is otherwise impermeable is mediated or facilitated by specific membrane proteins, the
presence of which are induced by their substrate.
These proteins, collectively known as permeases or carrier proteins, bind to their
substrate on the membrane's outer surface and, by mechanisms still largely unknown,
mediate their passage through the membrane to the inner surface where the carrier-
substrate complex dissociates, releasing the substrate into the cytoplasm. t is thought that
this transport is further facilitated by the existence of a protonmotive force through proton
carriers (see active transport). The proteins involved are therefore enzymes that catalyse
the general reaction:
substrate
(outside)
substrate
(inside)

Facilitated diffusion is similar to passive diffusion in the sense that the substrate moves
down a concentration gradient; neither of these processes require the expenditure of
metabolic energy. t differs from passive diffusion by its enzymatic nature: the process is
rapid; the carrier proteins are often inducible; the rate of reaction approaches a limiting
value with increasing concentrations of substrate, ie. it obeys normal enzyme kinetics
[Michaelis-Menten] .
2.3 Active transport.
n prokaryotes, the energy-coupIed transport systems are dominant. The mechanism of
transport known collectively as active transport permits a solute to enter the cell against a
thermodynamically unfavourable gradient of concentration. These mechanisms create
concentrations of solutes within the cell that can be several hundred to thousand times
greater than those outside. The prevalence of active transport systems in bacteria can be
correlated with the facts that bacteria frequently occur in dilute chemical environments but
nevertheless exhibit rapid rates of metabolism.
Active transport systems appear to function as facilitated diffusion systems coupled to a
source of metabolic energy, thereby permitting accomplishment of the chemical work
necessary for creating and maintaining a concentration gradient across the cell
membrane.
Several sources of metabolic energy drive the cell's various active transport systems:

1. the electrostatic or pH gradient components of the protonmotive force;

2. secondary gradients (eg. of ions such as Na
+
) derived from the protonmotive force by
other active transport systems, and ATP.

The establishment of a protonmotive force by proton extrusion associated with the
passage of electrons through a membrane-bound transport chain or by hydrolysis of ATP
by the membrane-bound ATPase is sometimes termed primary active transport. The
protonmotive force as mentioned earlier is a source of energy that can drive the ATP
synthesis at the membrane-bound ATPase site; it can also drive molecules across the cell
membrane. Such movement of a molecule across the cell membrane at the expense of a
previously established gradient of another molecular species is termed a secondary
active transport.
There are three types of secondary active transports: symport, antiport, and uniport:
Symport is the simultaneous transport of two molecules by the same carrier; one

Figure 8 : Secondary active transport: Symport

molecule flows down its previously established gradient and the other flows with it.

Antiport is the simultaneous transport by the same carrier of two molecules in the

Figure 9 : Secondary active transport: Antiport

opposite direction across the membrane, one which flows down its concentration gradient,
thus exchanging one gradient for another.

Uniport is the flow of ions driven directly by an electrostatic gradient.

Figure 10: Secondary active transport: Uniport

Many active transport systems of gramnegative bacteria are associated with a so-called
binding protein, which are located in the periplasmic space. This binding protein transport
system utilises proteins as essential components of the transport system. These proteins
have no catalytic activity, but they form tight complexes with specific nutrients, such as
amino acids, sugars etc. These proteins work in conjunction with permeases.
All these transport mechanisms considered so far catalyse the movement of chemically
unmodified nutrients across the cell membrane, eg. glucose is transported across the
cellular membrane and released inside the cell as glucose. Bacteria, however, possess
other transport systems which , during the process of of transport convert the nutrient to a
chemically modified form. The passage of the substrate across the membrane occurs
concomitantly with and as a consequence of the chemical transformation of the substrate.
This is one way of assuring unidirectional flow of the solute and preventing exit via the
same specific carrier. As mentioned under active transport, the chemically modified
nutrient inside the cell can greatly exceed the concentration of free nutrient in the medium.
2.4 Group transIocation.
Group translocation mechanisms differ fundamentally from true active transport because
they do not establish a concentration gradient of a molecule species across the cell
membrane. One compound is found in the external environment, a chemically modified
form of it is found inside the cell. Group translocation mechanisms are particularly
conserving metabolic energy. The chemical change of the substrate that occurs on its
entry into the cell requires the expenditure of energy in form of a high-energy phosphate
bond, but this change is also required for the substrate's further metabolism. t is part of a
substrate phosphorylation. Transport is therefore accomplished by a reaction that would
also occur intracellularly even if the substrate were brought into the cell by another energy-
requiring mechanism.
One example of a group-translocating system widely distributed in bacteria is the
phosphotransferase system. t mediates the transport of many sugars and sugar
derivatives, which are phosphorylated during the process, entering the cell as sugar
phosphates.
The phosphoenolpyruvate-phosphotransferase (PTS) system appears to be confined to
anaerobic and facultative anaerobic bacteria. t replaces the phosphoryl donor ATP in an
ATP-dependent sugar kinase with phosphoenolpyruvate. The system is generally
considered to consist of four proteins, arranged in the following sequence:

PEP + enzyme I P-enzyme I + pyruvate
P-enzyme I + HPR P-HPR + enzyme I
P-HPR + enzyme III HPR + P-enzyme III
P-enzyme III + sugar sugar-P + enzyme III
enzyme II

Each PTS is reasonably complex involving the sequential action of all four distinct
phosphate-carrying proteins. The last member of the chain, enzyme , is located within the
membrane and serves as the carrier protein for the sugar substrate. The penultimate
member of the chain is a membrane-associated protein, enzyme , that catalyses the
transfer of a phosphate group to the entering sugar. Only two of the four proteins are
specific, E- and E-. Their synthesis is usually induced by the presence of that sugar in
the cell's external environment.
Over the past decades, other examples of group transIocations have been proposed.
Among these is the coenzyme A transfer system mediated by acetyI-CoA
synthetase for the uptake of fatty acids.
t should finally be mentioned here that the PEP-phosphotransferase system has been
suggested to play a very significant role apart from its sugar transport. It not onIy
cataIyses the uptake of its own sugar substrates, but it aIso reguIates the uptake of
other carbohydrates. This second function encompasses the regulation of flagella
synthesis, transmembrane transport, cyclic-3'5'-monophosphate synthesis and catabolic
enzyme synthesis. Therefore the additoin of a sugar substrate of the PEP-
phosphotransferase system to a bacterial suspension simultaneously inhibits the activities
of adenylate cyclase (cyclic-AMP synthesising enzyme) as well as a member of bacterial
permeases.
A number of macromolecules [eg. sucrose, starch, cellulose etc] that cannot pass the cell
membrane are nevertheless utilisable as substrate for growth. These substrates are
enzymatically hydrolysed in the external medium by enzymes secreted by the cell. The
hydrolytic products of these substrates then enter the cell by specific transport systems.
With all the nutrients required inside the cell, one can now proceed to the events
occurring inside the cell with all its metabolic activities.

3. Concepts of MetaboIism.
3.1 Photosynthesis
The first mode of energy producing systems is photosynthesis. There is almost no doubt,
that photosynthesis is the basic process of life. t is the process by which plants and
certain bacteria, termed phototroph, convert radiant energy in the form of light into
metabolic energy [ATP] and reducing power [NAD(P)H+H
+
]. n contrast to plants and
algae, photosynthetic bacteria do not require oxygen, do not liberate oxygen and therefore
are of anaerobic nature. The difference between the oxygenic and anoxygenic
photosynthesis lies in the oxidation of water to oxygen and hydrogen, a reaction which
does not proceed spontaneously and requires energy. This energy comes from a second
photosystem. Oxygenic photosynthesis has therefore two photosystems, whereby
only the first reaction center mediates phosphorylation (energy production) and the second
drives the photochemical oxidation of water (reductant production). Bacteria are lacking
this second photosystem and must obtain their reductant in a different way using oxidation
reactions.

Light-harvesting pigments can include chlorophylls, carotenoids, and phycobiliproteins,
which function to absorb light energy and transmit this energy to the photosynthetic
reaction centre. The particular set of light harvesting pigments, that comprise an antenna
system, are group specific and their cumulative light absorptive properties determined the
range of wavelength of light over which photosynthesis occurs. As a consequence of these
variations in the composition of the antenna system, phototrophs collectively are capable
of utilising as an energy source all radiant energy that falls in the wavelength from visible
to near infrared light.

Radiant energy is always transferred in discrete packets known as photons, the enegry
content of which is inversely related to wavelength. Wavelengths longer than 1200 nm,
infrared region and beyond, have such a small energy content that the absorbed energy is
immediately converted to heat as it cannot mediate chemical changes. Wavelengths less
than 200 nm, the range of X rays and cosmic rays, are termed ionising radiation because
their energy content is so high that molecules in their path are immediately ionised.
Between these two extremes lies the region of electromagnetic radiation that is capable of
mediating biological reactions. t is this portion that can serve for the performance of
photosynthesis and that constitutes the major portion of the energy content of solar
radiation at the earth's surface.
The chlorophylls, of which at least seven kinds occur in various groups of phototrophs,
absorb light intensely in two regions of the spectrum, the violet around 400 nm, and the
red or near infrared around 600-1100 nm.
Carotenoids have a single broad region of absorption between 450 and 550 nm.
Phycobiliproteins absorb from 550 to 650 nm, between the major absorption regions of a
chlorophyll.

The photochemical reaction center contains the site where a molecule of chlorophyll
becomes photoactivated and oxidised by donating an electron to a carrier molecule.
Chlorophyll molecules in the reaction center differ from those in the antenna in two
important aspects

1. they are associated with certain proteins that interact with them in a manner that
decreases the energy required to raise them to the activated state;
2. they are in close proximity with carrier molecules that can accept an electron from them
when they are activated.

The more numerous chlorophylls in the antenna therefore absorb energy and transfer their
absorbed energy by a process called inductive resonance to an adjacent pigment and
eventually to the reaction center. The energy required to activate a molecule of chlorophyll,
designated P, in a reaction center can be reckoned by the maximum wavelength of a
photon that can bring it about. Thus a reaction center bacteriochlorophyll that is activated
maximally by photons of the wavelength 870 nm is designated P
870
.
All organisms capable of carrying out photosynthesis therefore possess a so-called
photosynthetic apparatus consisting of three essential components:

1. the Iight-harvesting pigments located in that part called antenna, which are
predominantly carotenoids although chlorophylls can be included. Their cumulative light-
absorptive properties help the reaction center to function;

2. the photosynthetic reaction center, which contains chlorophyll molecules in a special
state;

3. the eIectron transport chain, which accepts the electrons from the reaction center and
passes these through a number of intermediate oxidoreduction carriers for ATP formation.

The simplest pattern of electron flow is, when the reaction center chlorophyll in its
photoactivated and oxidised state serves respectively as both an electron donor and
acceptor for an electron transport chain. Such a system is called cycIic
photophosphoryIation, whereby electrons are passed through a closed loop of electron
carrier molecules back to the chlorophyll molecule, now an oxidant, that lost the electrons
in the primary photochemical reaction (Figure 11).

Figure 11: Cyclic Photophosphorylation

Since no electrons can be removed from this cycle for reducing power [NAD(P)H
2
]
formation, a second source of electrons and protons is required, which in oxygenic
photosystems is water, but in anoxygenic photosystems is an inorganic compound. The
latter redoxpotentials are, however, in the region of +0.2V. The photochemical center with
its energy forming capability reduces the redoxpotential and makes it possible for the
electrons to combine with the low potential ferredoxin-NAD(P) reductase in a non-cycIic
photophosphoryIation to form reduced pyridine nucleotides. These reduced pyridine
nucleotides can therefore be obtained either through hydrogen supply or the
photoproduction of hydrogen from inorganic compounds (Figure 12).

Figure 12: Non-cyclic photophosphorylation

The individual components of the electron transport system differ according to the three
major families of the photosynthetic bacteria
HaIobacterium Photosynthesis
The halophilic bacteria are a major group of the archaebacteria with six genera in one
family, Halobacteriaceae. The halophilic bacterium Halobacterium aslinarium normally
depends on respiration for the production of enegry. However, under conditions of low
oxygen and high light intensity, they are capable of synthesizing a deep purple pigment
called bacteriorhodopsin, which closely resembles the pigment rhodopsin.
Bacteriorhodopsin's chromophore is the carotenoid derivative retinal that is covalently
attached to the pigment protein by a Schiff base with the amino group of lysine. The
protein extends through the plasma membrane, and its retinal rests in the center of the
membrane. They aggregate in the membrane and form crystalline patches, which give the
appearance of purple membrane patches.
Bacteriorhodopsin functions as a light-driven proton pump. When retinal absorbs light, the
Schiff base loses a proton, which is moved across the plasma membrane to the
periplasmic space during these alterations. The light-driven proton pumping generates a
pH gradient that can be used to power the synthesis of ATP by a chemiosmotic
mechanism.
The photosynthetic capacity is particularly useful to Halobacterium because oxygen is not
very soluble in concentrated salt solutions and may decrease to an extremely low level in
its habitat. When the surroundings become temporarily anaerobic, the bacterium uses light
energy to synthesize sufficient ATP to remain alive until the oxygen levels rise again.
Halobacterium is an aerobic organism, as it requires oxygen for continued retinal
synthesis, but it can simply survive under oxygen limitations using this type of
photosynthesis.
Aerobic respiration (see also chapter 9) involves all chemical energy-yielding reactions
that require molecular oxygen as the final electron acceptor [0.5 O
2
/H
2
O = +0.81 V). n the
vast majority of cases, the substrate used is of organic nature, that is organic compounds
containing carbonyl or carboxyl groups with a redox potential as low as -0.4 V. The E
0
of
such a system would therefore be 1.21 V and equivalent to a G
0
of - 55 kcal or 229.9
kJoules. This is the highest energy producing system of a bioIogicaI ceII. Such
systems can onIy be utiIised by chemoorganotrophic microorganisms.
The efficiency of the chemical work can be calculated as follows:

1. theoreticaI

C
6
H
12
O
6
+ 6 O
2
6 CO
2
+ 6 H
2
O
G
0
= - 2,876.5 kJ/moI = - 686.0 kcaI/moI

2. experimentaI

C
6
H
12
O
6
+ 36 ADP + 36 Pi 6 CO
2
+ 36 ATP + 42 H
2
O
G
0
= - 1,726.3 kJ/moI = - 413 kcaI/moI

Efficiency:
36 x 7.3
------------ x 100 = 38 %
686

f one assumes therefore that the formation of 1 mole of ATP requires 7.3 kcal, the
efficiency of aerobic respiration is around 385, with restly 62% energy being released into
the surrounding.
n some instances, the substrates used for energy production are inorganic compounds,
such as ammonia, hydrogen sulfide and others, whose redoxpotential is between -0.2 and
+0.2 V. The potential difference to the molecular oxygen couple as final electron acceptor
is therefore much smaller and less energy is available for growth. Such systems can onIy
be utiIised by chemolithotrophic microorganisms.
3.3 Anaerobic Respiration.
This mode of energy production involves all chemical energy-yielding reactions that
require inorganic compounds other than molecular oxygen as final electron acceptors,
whereas the electron donors can be any organic compound. The onIy and cardinaI
difference between aerobic and anaerobic respiration is therefore the change in
finaI eIectron acceptor, which in turn also shortens the potential difference between
electron donor and electron acceptor couple by approximately 0.6 V. The potential
difference, however, is still large enough for electrons to be mediated by a number of
electron carriers. Anaerobic respiration occurs mainly in the soil [denitrification;
desulfurication] and is can be carried out by facuItative aerobic or anaerobic
microorganisms.
3.4 Fermentation
Fermentation involves all chemical energy-yielding reactions that require organic
compounds as final electron acceptor couple. This system therefore has an organic
compound for both eIectron donor and acceptor (see aIso chapter 10), although two
different organic compounds have to be involved. The hydrogens and electrons are
shuffled via the NAD(P)
+
/NAD(P)H+H
+
system with no electron carrier between electron
donor and acceptor. It is this system, whereby substrate phosphoryIation prevaiIs
and the Ieast energy production.
The efficiency of the chemical work is therefore
TheoreticaI
C
6
H
12
O
6
2 C
2
H
5
OH + 2 CO
2

G
0
= -236.2 kJ/moI = - 56.5 kcaI/moI
ExperimentaI
C
6
H
12
O
6
+ 2 ADP + 2 Pi 2 C
2
H
5
OH + 2 CO
2
+ 2 ATP
2 x 7.3
--------- x 100 = 26%
56.5

These are the four principal modes of energy production in biological cell systems.
A high negative value of G indicates that a chemical reaction is likely to proceed
spontaneously and that the product will greatly exceed the reactants at equilibrium (see
Section 2.3). However, it does not guarantee that the reaction will proceed with
measurable speed. There exists a kind of energy barrier that must be overcome before the
reaction can proceed. The number of molecules which can cross this barrier is a function
of both the temperature and the respective activation energy barrier. The rate constant k
for the reaction can be expressed as:
k = Ae
- Ea/(RT)

where R is the gas constant, T is the absolute temperature, Ea represents the activation
energy and A is a constant related to the efficiency of collision. Whereas chemists can
easily change the temperature and add a catalyst to reduce Ea, the cell in general
depends solely on the presence of a suitable enzyme in order to achieve sufficiently high
reaction rates.
Enzymes are true catalysts because they do not influence the point of equilibrium of the
reaction they catalyze, nor are they used up during catalysis. Every enzymatically
catalyzed reaction keeps on reacting until the equilibrium state is obtained. This is one of
the basic Iaws of enzymoIogy. The only possibility for a reaction to continue beyond
equilibrium occurs in coupled reactions, where the product of the first reaction is
immediately catalyzed to a further product with the help of a different enzyme, as is the
case in cell metabolism . Therefore an organism will never be close to a chemical
equilibrium stage, as a system in equilibrium cannot perform any work.
All enzymes are proteins and all methods to separate and purify enzymes are the same as
for protein. Apart from the protein, many enzymes contain also a 'prosthetic group'. The
letter may be removed reversibly, and in such cases the protein part is called apoenzyme
and the prosthetic group coenzyme.
Substrate concentration is one of the most important factors that determine the velocity of
enzymatic reactions. The enzyme first forms a complex with its substrate, which
subsequently breaks down resulting in the free enzyme and the product of the reaction:

Enzyme + substrate enzyme-substrate product + enzyme
k
1
k
2

E + S ES P + E
-k
1

f one starts with a given amount of enzyme and raises the substrate concentration
gradually, more and more enzyme will be converted into the complex ES. The equilibrium
constant can therefore be calculated from

[E] + [S]
K
eq
= ------------------
[ES]

The enzyme is then saturated and the reaction rate is maximal. When the velocity is
plotted against the substrate concentration, a section of a rectangular hyperbola is
obtained (Figure 13).

Figure 13: Hyperbolic substrate concentration curve of an enzymatic reaction. Direct
Michaelis-Menten plot

Under conditions where the enzyme is in a low, fixed concentration in comparison to the
substrate, a steady state is assumed, eg the formation of ES is balanced by the rate of
product formation. We obtain the MichaeIis-Menten equation for the initial rate, which is
the basic equation of enzyme kinetics:
V
max
[S] k
cat
[E
0
][S]
V = --------------- = -----------------
K
m
+ [S] K
m
+ [S]

which means that the initial rate (v) is proportional to the total enzyme concentration [E
0
],
whereas v follows saturation kinetics with respect to the substrate concentration. V and K
m

can easily be determined since

K
m
= [S] at haIf maximaI veIocity
n other words, that substrate concentration at which half maximal reaction velocity is
reached equals the dissociation constant of ES. This constant K
m
is called the MichaeIis-
Menten constant.
Since saturation cannot be fully attained, neither V
max
nor K
m
can be directly deduced from
Figure 13. This is the reason for transforming the Michaelis-Menten equation into a linear
form. The most widely used are the Lineweaver Burk plot (Figure 14) and the Eadie-
Hofstee plot (Figure 15) or the computer assisted methods for fitting procedures.

Figure 14: Reciprocal plot according to Lineweaver-Burk

Figure 15: Reciprocal plot according to Eadie-Hofstee

Enzymes frequently catalyze the reaction of two or more different molecules, actually
multi-substrate reactions are very common enzyme mechanisms. These reactions fall into
two major classes:
a) sequential mechanisms, whereby all reactions combine with the enzyme before the
reaction takes place and any products are released;
b) so-called 'ping-pong' mechanisms, in which one or more products are released
before all substrates are bound. These types of reactions can also be graphically
analyzed using appropriate reciprocal plots.
Another very important feature of a particular enzyme is the interaction with specific
inhibitors. nhibition may either be reversible or irreversible, the latter being brought about
by covalent binding. The analysis of reversible inhibition is very common and can be either
of a competitive or non-competitive nature. The former is due to a competition of the
inhibitor with the substrate at the active site of the enzyme owing to structural similarity. n
this case V
max
stays unchanged but K
m
is altered. n non-competitive inhibition, V
max

changes, but Km stays the same.
The reaction rate of an enzyme catalyzed reaction is also used to define 'enzyme units'.
One unit (U) of any enzyme is defined as that amount that will catalyze the transformation
of 1 mole of substrate per minute, or frequently the term 'cat' is being used, which
transforms 1 nmole per second. Specific activity is expressed as units of enzyme per
milligram of protein. The concentration of an enzyme in solution should always be
expressed as U ml
-1
.
n view of the vast number of enzymes known, a system of classification is essential (see
also chapter 12). t is important therefore to know the principles on which the UB
[nternational Union of Biochemists] has based its classification scheme. Each enzyme is
classified by four (4) numbers. The first number indicates to which of the six classes the
particular enzyme belongs to (eg, 4, ligase), the second number indicates the subclass (eg
4.1, carbon-carbon lyase), the third number divides subsubclasses (eg 4.1.2, enzyme is an
aldehyde lyase) and the final number is the serial number of the enzyme (eg 4.1.2.13,
fructose bisphosphate aldolase). For most enzymes there exist two names, the systematic
one (see above) and a trivial or common name (eg zymohexase, aldolase). The
characteristic of each enzyme is the prefix -ase at the end of the name.
5. References
BaiIey,J.E. & D.F.OIIis 1986 - Biochemical Engineering Fundamentals, McGraw-Hill Book
Comp., New York
Broda,E. 1975 - The Evolution of the Bioenergetic Processes. Pergamon Press, Paris
CarafoIi,E. & A.Scarpa (eds.) 1982 Transport ATPases. New York Academy of Sciences ,
New York
DoeIIe,H.W. 1975 - Bacterial Metabolism, Academic Press, New York
DoeIIe,H.W. 1990 - Australian Academia of Sciences/Academia Sinica/Unesco/MRCEN
Training Course on Microbial Process Development. nstitute of Microbiology, Academia
Sinica, Beijing, PR China, Oct. 25 - Nov. 17
DoeIIe, H.W. 1991 - Uneso/MRCEN Regional Training Course on The Importance of
Microbiological Biotechnology for Community and Economic Development. Motupore
sland, University of Papua New Guinea, Sept. 2-7
DoeIIe,H.W. 1992 - African Regional Network for Microbiology & FADB nternational
Workshop on Fermentation Technology, Awka & Enugu, Nigeria, Sept. 7-19
DoeIIe,H.W. 1992 - Unesco/MRCEN-Regional Training Course on Fermentation
Technology for the Conservation of the Environment, Shanghai nstitute of ndustrial
Microbiology, Shanghai, PR China, Nov. 2-13
DoeIIe,H.W. 1994 - Unesco/MRCEN/CRO Regional Training Course on Microbial
Process Development and the Ecological Environment in relation to the Development of
Fermentation Industries. Food ndustries Research nstitute, Hanoi, Vietnam, Oct. 24- Nov.
2
DoeIIe, H.W. 1994 - Microbial Process Development. World Scientific Publisher,
Singapore
DoeIIe,H.W. 1998 - Unesco/CRO/MRCEN/OBB Regional Training Course on Current
Trends in Microbial Technology for a Sustainable Environment: Exploring Microbial
Diversity for Novel Processes. University of Kuala Lumpur, Malaysia, October 12-24
KIotz,M.I. 1967 - Energy changes in biochemical reactions. Academic Press New York
Kummerow,F.A., G.Benga & R.P.HoImes 1983 - Biomembranes and cell function. New
York Academy of Sciences
Morowitz,H.J. 1970 - Entropy for Biologists: An introduction to thermodynamics.
Academic Press nc., New York
NicoIIs,D.G. 1982 - Bioenergetics - An introduction to the chemiosmotic theory. Academic
Press nc., London
Rosen,B.P.(ed.) 1978 - Bacterial Transport. Marcel Dekker, nc., Basel

Horst W. DoeIIe
Past Chairman, nternational Organisation for Biotechnology and Bioengineering

Chapter 9
Basic Strategies of Energy MetaboIism under Aerobic
Conditions
1. Introduction
2.1 PoIymer hydroIysis
2.1.1 Starch hydroIysis to gIucose
2.1.2 CeIIuIose hydroIysis to gIucose
2.1.3 Protein hydroIysis to amino acids
2.1.4 Fat hydroIysis to fatty acids
2.2 Monomer UtiIisation
2.2.1 Carbohydrate utiIisation
2.2.2 Amino acid utiIisation
2.2.3 Fatty acid utiIisation
3. Non-renewabIe Substrates
3.1 Hydrocarbon utiIisation
3.2 SingIe carbon compound utiIisation
4. BibIiography
1. Introduction
Many microorganisms generate energy during the metabolism of organic compounds.
These reactions involve oxidation-reduction processes accompanied by the release of
energy, some of which is conserved in high-energy phosphate bonds [ATP]. The particular
compound may be oxidised either completely to carbon dioxide (CO
2
) or only partially, but
in either case the compound is degraded into smaller and simpler products. The process
involved in the breakdown of compounds are collectively called catabolism, and the
enzymatic reactions involved in breakdown are called cataboIic reactions.
n order to grow, cells have also to build up a vast array of chemical substances of which
they are composed. These substances often quite complex are synthesised from simpler
molecules by processes called anabolism. The enzymatic reaction involved in anabolism
are often referred to as biosynthetic reactions. These biosynthetic reactions are often
energy-requiring, and ATP formed during catabolic reactions is used up during
biosynthetic reactions. Collectively, cataboIic and biosynthetic reactions are called
metaboIic reactions. Metabolism refers therefore to the whoIe array of degradative
and biosynthetic reactions taking pIace within the ceII.
Although energy is required in certain key biosynthetic reactions, the focus of biosynthesis
is not on energy, but rather on carbon and on the intermediates occurring in the build-up of
cell constituents from simple starting materials. These intermediates are often formed as a
result of catabolism, but also serve as starting materials in biosynthetic reactions. Some of
the starting compounds for biosynthesis are formed by simple depolymerisation of large
molecules. For example, protein may be hydrolysed to amino acids, which can be directly
used in new protein biosynthesis provided the cell possesses the particular transport
systems for these amino acids.
The relationships between catabolism and anabolism occur not only at the common
intermediates. Certain pathways play dual roles, as they function in both anabolism and
catabolism. For instance, the tricarboxylic acid cycle, as will be demonstrated later, is
involved not only in the oxidation of pyruvate and acetyl-CoA to carbon dioxide for energy
production, but also in the generation of a number of intermediates such as succinyl-CoA,
oxalacetate and ketoglutarate, which serve as starting points for the synthesis of amino
acids, porphyrins and other compounds necessary for growth. A pathway that serves the
duaI function of cataboIism and anaboIism is caIIed an amphibolic pathway. Many
pathways are amphibolic.Thus ketoglutarate may be oxidised to succinate, leading to
energy generation, or it may be converted to the amino acid glutamate for use in protein
biosynthesis. Obviously the cell has to make a choice. A given molecule at a given
moment may go to catabolic or anabolic sequences; it cannot do both simultaneously.
However, if one has a cyclic mechanism like the TCA cycle, any compound taken up for
biosynthetic reactions would endanger further functioning of the cycle as links in the cycle
would be deleted. This deficiency is overcome by replacement of oxalacetate by ancillary
synthetic reactions that come out of the main cycle. Reactions of this type have been
grouped under the term anapIerotic, meaning 'replenishing' reactions.
One key regarding the contrasts between anabolic and catabolic reactions is that even
where the same reactants are involved, the enzymes acting in anabolic reactions are often
different from those involved in catabolic reactions.
n eukaryotes, a further difference between anabolism and catabolism is in the cellular
localisation of the processes. Many catabolic reactions are localised in the mitochondria
and microbodies, whereas anabolic reactions are primarily cytoplasmic. At least one
advantage of having separate sites for catabolic and anabolic reactions is that both can
occur at the same time without confusion.
n prokaryotes, where compartmentation is less structured, control of reactions occurs
mainly at the level of single enzymes.

The schematic representation of aerobic catabolism [Fig. 1] exhibits the strict uniformity
which exists in the utilisation of carbon compounds - renewable, non-renewable or waste
substrates - as complex as they may occur in nature. All complex polymers, such as
cellulose, sucrose, polysaccharides etc are broken down to their respective monomers first
before they enter the cell and join into a common pathway at various stages. t should also
be realised that every large group of similar organic nature, enters at a specific stage the
common pathway. This unifying biochemical concept demonstrates the economics of
energy production and cellular biosynthesis of microorganisms. The center core of
catabolism leads from glucose, a 6-carbohydrate, to pyruvate, a C-3 compound, and from
here through the cyclic mechanism, the tricarboxylic acid cycle. The latter releases carbon
dioxide [CO
2
] and water. This core of aerobic catabolism, in particular the TCA cycle
makes certain that the substrate carbon chain is oxidised to the smallest carbon
compound possible with the electrons from the individual oxidations moving along the
electron carbon chain to produce energy in form of ATP and water by the reduction of
oxygen. As long as oxygen is available, this core pathway system produces not only the
maximum energy possible in a single oxidation step, but also consists of the maximal
number of possible steps.

Figure 1: Schematic diagram of aerobic catabolism

Furthermore, Figure 1 also indicates that all other complex compounds, such as fats,
nucleic acids and hydrocarbons enter the aerobic pathway at various stages and join the
TCA cycle for maximal energy production.
Under maximaI oxygen avaiIabiIity, microorganisms wiII aIways have CO
2
and water
as their finaI products. ResiduaI organic compounds wiII onIy appear therefore,
when oxygen avaiIabiIity is Iimited [anaerobic respiration and/or fermentation] or in
cases of incompIete oxidations.
2.1 PoIymer hydroIysis.
Organic substances in nature are in abundance in the form of polymers. Under this term
we understand molecules consisting of basic monomeric units linked together. For
example, if the monomeric structure consists of carbohydrates, one uses the term
polysaccharides. These polymers cannot penetrate the cell membrane and must be
broken down first into small transportable molecules, which in many cases are the
monomers. This enzymatic process of cutting the chains into their basic components is
generally referred to as hydrolysis and the corresponding enzymes are therefore called
hydrolytic enzymes or hydrolases. The action of these hydrolytic enzymes is of extreme
importance and often does not underlie specific control systems, but exhibit reaction rates
completely independent from the monomeric cell uptake or transport rates. They play a
significant role in the macroscopic degradations such as food spoilage and waste
treatment apart from the desirable ageing of meat, curing cheese, preventing beer haze
and many other processes. The isolation and purification of these enzymes is a large
commercial enterprise.

There are two polymers which become of increasing interest because of their abundance
availability in nature: starch and ceIIuIose. Both polymers are polysaccharides. Which
means there monomers are sugars, or in this specific case, glucose. t is of interest to
realise that although these two polymers are formed from the same monomer, they
possess vastly different properties. Starch is readily digestible by many microorganisms
and is a prime energy source, whereas cellulose, the key constituent of plant cell walls, is
more difficult to digest whether in its pure form or linked with the heteropolymer lignin.

2.1.1 Starch hydroIysis to gIucose
The chemical structure of starch is essentially the same throughout nature. The naturally
occurring starch is a mixture of two polysaccharides, both of which are polymers of
glucose. The major component is amyIopectin with a branched structure and the minor
component is amyIose, which is a linear macromolecule. AmyIose is a flexible, linear
chain molecule of 500 or more glucose units and gives a blue colour with iodine. The
glucose units are joined by alpha-1,4-glycosidic linkages. The linear portion of amylopectin
is identical to amylose, but every 20-30 glucose units a branching occurs by an alpha-1,6-
glycosidic linkage. Amylopectin gives a red to purple colour with iodine.
Starch hydrolases degrade the polysaccharide to soluble, low molecular weight products
such as glucose. These enzymes were originally called diastases, but now they became
amyIases:

1. aIpha - amyIase or alpha-D-(1-->4)-glucan-4-glucanohydrolase [EC 3.2.1.1], which
splits the bonds in the interior of the substrate and could be referred to as endoamylase.
The results of the enzyme action is the formation of D-glucose, maltose, and small
amounts of dextrins;

2. beta - amyIase or alpha-D-(1-->4)-glucan maltohydrolase [EC 3.2.1.2], which
hydrolyses units from the non-reducing end of the substrate and could be referred to as
exoamylase. This enzyme has not been found in microorganisms, but can be obtained
from plants.

Both alpha- and beta-amylases are not able to hydrolyse the alpha -D-(1-->6) glycosidic
bond of amylopectin.

3. amyIogIucosidase, gIucoamyIase or alpha -1,4-glucan glucohydrolase [EC 3.2.1.3)
removes the glucose units from the non-reducing end of the polymer. t hydrolyses
amylopectin and amylose completely to glucose and is capable to hydrolyse the alpha -D-
(1-->4) as well as the alpha-D-(1--->6) glycosidic bond.

Amongst the three different amylases known, only one is capable to break both types of
glycosidic linkages and this is glucoamylase [EC 3.2.1.3]. Only this particular enzyme is
able to convert all the starch into glucose without any other product being formed.

2.1.2 CeIIuIose hydroIysis to gIucose
Cellulose is the largest biological component of the renewable resources available on
earth. However, most of the cellulose occurs in a lignocellulose complex containing large
fractions of crystalline cellulose. The resistance of these natural forms of cellulose to
chemical and microbial attack has sofar limited its use. The hydrolysis of cellulose by
cellulolytic enzymes is therefore under intensive studies in order to develop a process for
the production of sugars. Although cellulose consists of glucose units as does starch, the
bonding arrangement of these glucose molecules is quite different. n cellulose, the
glucose units are linked by beta -(1-->4) linkages. The hydrolysis products are therefore
glucose and cellobiose. At least four enzymes are known to be involved in the cellulose to
glucose conversion:

1. ceIIobiohydroIase, which splits cellobiose from the non-reducing end of the cellulose
chain;
2. exogIucanase, which splits glucose from the non-reducing end of the cellulose chain;
3. ceIIobiase, which hydrolyses cellobiose to glucose;
4. endogIucanase, which hydrolyses long polymers into oligosaccharides.

Our present knowledge on cellulose degradation is still relatively scanty, but it is known
that cellobiose is the first product of cellulose hydrolysis and is a potent inhibitor of
cellulose hydrolysis. t is therefore important that the enzyme cellobiase is

present to remove cellobiose from the medium. The complexity of the system certainly
awaits further intensive research, in particular the removal of the heteropolymer lignin from
the lignocellulose, as cellulose in nature is surrounded by a lignocellulosic ring enclosure.
Sofar, only certain fungi species [eg. mushrooms] are known to be able to split this ring
(see mushroom production).
2.1.3 Protein hydroIysis to amino acids
The ability to break down proteins is very similar to the above described polymer
degradation. n the case of proteins, the enzymes involved are proteolytic enzymes,
proteases and proteinases, which are classified according to their attacks on the
individual proteins and polypeptide chains. They hydrolyse peptides and are able to break
down the chain, liberating di- or tripeptides, which the di- and tripeptidases utilise further
forming free amino acids. These monomers are now able to diffuse or be transported into
the cell.
2.1.4 Fat hydroIysis to fatty acids
Fats are another major carbon source for microorganisms. The majority of fats are simply
triglycerides, that is glycerol substituted fatty acids:
CH
2
- OOC
n
H
n

|
CH - OOC
n
H
n

|
CH
2
- OOC
n
H
n

Similar to the cellulose, starch or protein polymers, fats have to be converted into
monomeric substrates, which are fatty acids and glycerol. This hydrolysis is carried out by
Iipases outside the cell. These lipases separate the fatty acids from the glycerol moiety
resulting in glycerol plus 3 fatty acids. Under the term lipase one understands a
conglomerate of different hydrolytic enzymes or esterases, which specifically act upon the
different glycerol-substituted fatty acids.
The lipase action can occur with a simultaneous fatty acid activation to its CoA-derivative,
as it occurs in the prokaryotes or can simply lead to a fatty acid followed by an acylation
involving carnitine, which serves as a carrier of acyl-groups into and out of mitochondria,
as it occurs in the eukaryotes including mammalian cells.
n bacteria, the first attack on the triglyceride is catalysed by a diglyceride acyltransferase,
whereby the fatty acid in the 3-position of the glycerol is released and immediately
converted into its CoA derivative (Figure 2). The so formed alcohol group is then
phosphorylated before the other two fatty acids are released. This phosphorylation step is
catalysed by the phosphatidate phosphorylase (EC 3.1.3.4), which incorporates 1
molecule inorganic phosphate into the diglyceride, thus forming phosphatidic acid.
The two residual fatty acids on the phosphatidic acid molecule are now removed and
transformed to their respective CoA-derivatives by glycerophosphate acyltransferase (EC
2.3.1.15). The endproducts of these reactions are therefore the monomers
glycerophosphate plus 3 fatty acid CoA esters. These compounds can now be transported
into the cell.

FATS
[trigIycerides]

CoA

fatty acid-CoA

1,2 - digIyceride

Phosphatidic acid

gIyceroI-P fatty acid-CoA

Figure 2 : Fat hydrolysis to glycerol and fatty acids

2.2 Monomer utiIisation

2.2.1 Carbohydrate utiIisation
With starch and cellulose hydrolysed to their monomeric glucose molecules, one can now
return to the scheme outlined in Figure 1.
A closer look at the initial section of this core pathway [Fig. 3] system indicates, that the
cell has a choice of three different ways of glucose to pyruvate conversion:

1. The Embden-Meyerhof-Parnas [EMP] pathway, often referred to as glycolytic
pathway;

2. The Hexose Monophosphate [HMP] pathway, often referred to as pentose or ribose
phosphate pathway;

3. The Entner-Doudoroff [ED] pathway, which so far has only been found in bacteria.

All three pathways have a great number of intermediates in common. There are, however,
some enzymes that are characteristic for the individual pathways, as they occur only in
that particular pathway and not in any of the others. These so-called key enzymes are
phosphofructokinase [EMP pathway], 6-phosphogluconate dehydrogenase [HMP
pathway] and 2-keto-3-deoxy-6-phosphogluconate aldolase [ED pathway]. Which play
an important role in the differentiation between the pathways. Each of the three pathways
could serve certain aspects of metabolism.

Figure 3: Pathways of glucose metabolism

The EMP pathway provides the greatest amount of energy as ATP (substrate-level
phosphorylation), but does not produce the important precursors or intermediates for
purine and pyrimidine biosynthesis, ribose 5-phosphate and erythrose 4-phosphate. t can
therefore be assumed that microorganisms using the EMP pathway must have specific
growth factor requirements for purines, pyrimidines, pentoses in order to built their nucleic
acids (DNA, RNA) and aromatic amino acids. n general, microorganisms that grow only
on complex media, eg. media containing meat extract and yeast extract, use this particular
pathway.
n contrast, the HMP pathway produces all the precursors necessary for purine, pyrimidine
and aromatic amino acid biosynthesis [Fig. 4], but produces only half the amount of ATP.
This pathway does not produce pyruvate directly, thus it possesses part of the EMP
pathway enzyme system. t is, therefore not surprising that both pathways may be present
to a varying degree in a great number of microorganisms. The ratio of usage or carbon
flow via the EMP and HMP pathways can vary greatly depending on environmental
conditions.
The ED pathway is linked to part of the HMP, since the HMP is present but functions in
reverse. The direct formation of pyruvate, production of 1 mol ATP as well as the
precursors for nucleic acid and amino acid biosynthesis, makes this pathway independent
of the other two and the one preferred by most strictly aerobic microorganisms. t does not
occur in eukaryotes.

The HMP pathway exists in two forms, the complete and the incomplete cycle. The
compIete HMP pathway represents what is called the 'pentose shunt' and has the
following sum of reactions:

GIucose + 12 NADP
+
+ 7 H
2
O + ATP 6 CO
2
+ 12 (NADPH+H
+
+ H
3
PO
4
+ ADP

Oxidative microorganisms, however, can also use the incompIete or partIy compIete
HMP pathway in order to produce pyruvate from glyceraldehyde 3-phosphate, utilising the
same enzymes as they occur in the EMP pathway. The sum of reactions of this incomplete
cycle would be

3 gIucose + 6 NADP
+
+ ATP 2 fructose 6-P + gIyceraIdehyde 3-P + 3 CO
2

+ ADP + 6 (NADPH+H
+
) + H
3
PO
4

n comparing the usefulness of both the complete and partly complete HMP pathway, it
could be envisaged, that oxidative microorganisms most certainly would utilise the partly
complete pathway in order to obtain energy via pyruvate and the TCA cycle. Those
microorganisms which metabolise glucose mainly via the EMP pathway under anaerobic
conditions, would utilise the complete HMP pathway only for the purpose of producing the
precursors for purine, pyrimidine and aromatic amino acid biosynthesis (Figure 4).
The reasons for the individual choices are not clear. t was originally thought that oxygen
may play an important role in the selection of pathway usage. The majority of anaerobic
bacteria were found to contain the EMP pathway, eg clostridia, enteric bacteria,
spirochaetes and sarcinae, those which are facultative anaerobes found to contain a
combination of EMP and HMP pathways, eg Escherichia coli, and strict aerobes found to
contain almost exclusively the ED pathway, eg pseudomonads and Rhizobium. However,
the discovery that Zymomonas can use only the ED pathway anaerobically as we'll as
aerobically, Clostridium aceticum a modified ED pathway with gluconate as carbon source,
and that homofermentative lactobacilli can use the EMP pathway even under aerobic
conditions as Ferrobacillus ferro-oxidans does, cast doubt upon these early assumptions.
f the microorganisms are able to utilise glucose via three different pathways and the
carbon flow is proportionally equal, certain control functions must exist for the arrangement
of carbon flow.
The major intermediate for the distribution of the glucose carbon between the EMP and
HMP pathway is undoubtedly gIucose 6-phosphate (Doelle et al. 1982). t is known that
20-30% of glucose is utilised by the facultative anaerobes through the HMP pathway under
anaerobic conditions. The first enzyme of the HMP pathway, gIucose 6-phosphate
dehydrogenase, is inhibited in an allosteric manner by NADH+H
+
, which can not be
reversed by NAD or AMP. With this observation, NADH+H
+
becomes almost an universal
inhibitor of most of the NAD(P)-dependent enzymes in the enteric group. This enzyme is
not affected by ATP, which is in contrast to the same enzyme in pseudomonads, which do
not use the EMP pathway under any conditions.

gIucose 6P
GIucose 6-P + NADP
+
gIucono-Iactone 6-P + NADPH+H
+

dehydrogenase

Figure 4: Biosynthetic precursor formation for biosynthesis [Schlegel et al.1985]

The second important enzyme in the HMP pathway is the 6-phosphogIuconate
dehydrogenase, which can be inhibited by fructose 1,6-bisphoshate [FDP]:

6-P gIuconate
6-phosphogIuconate + NADP
+
ribuIose 5-P + NADPH+H
+

dehydrogenase

The third important enzyme is phosphofructokinase occurring in the EMP pathway,
which is under strict ATP control:

phosphofructokinase
fructose 6-phosphate + ATP fructose 1,6-bisphosphate + ADP

Phosphofructokinase is well known for its sensitivity to ATP under anaerobic conditions. t
is an allosteric enzyme and it appears that only organisms using the EMP and HMP
pathways have an allosteric phosphofructokinase. We know, of course, that
phosphofructokinase also exists in a non-allosteric form.
Thus, after NADH+H
+
and fructose 1,6-bisphosphate, ATP is the third compound that
could influence the distribution of the glucose carbon.

A possible fourth important enzyme is related to the catalytic action of gIyceraIdehyde 3-
phosphate dehydrogenase, which only functions in the presence of NAD
+
, but can be
affected also by ATP:

GIyceraIdehyde 3-phosphate + NAD
+
2,3-bisphosphogIycerate

This enzyme is affected therefore not only by NADH+H
+
or the availability of NAD
+
, but
also by ATP.

f one summarises all these control effects, one could draw a picture of carbon flow
regulation. Under strict aerobic conditions and low glucose concentrations [eg 0.1%],
aldolase activity would be very low, which would indicate a channelling of glucose carbon
through the HMP pathway. Such a flow would soon lead to an accumulation of fructose-
bisphosphate, which would soon inhibit glucose 6-phosphogluconate dehydrogenase
activity, making this enzyme the pacemaker of the HMP pathway.. The resulting increased
glucose concentration would cause an increase in aldolase activity and thus open the EMP
pathway for NADH+H
+
production, which in turn inhibits glucose 6-phosphate
dehydrogenase. As the fructose bisphosphate concentration is lowered, the inhibition of
phosphogluconate dehydrogenase disappears and glucose 6-phosphate dehydrogenase
will be the pacemaker of the HMP pathway.
t therefore appears that the glucose concentration could also be responsible for the
selection of either pathway. t is now assumed that the above type of regulation may be
responsible for the carbon flow via one, two or all three pathways and also could
determine the proportion of each participating pathway.
At the pyruvate level, the pathway now divides, depending upon the mode of energy
metabolism.

Under aerobic conditions, pyruvate is oxidised via the TCA [tricarboxyIic acid] cycIe
into water and carbon dioxide (Fig. 5 ). This cycle is the most important energy-
producing reaction sequence, as it produces 32 of the 38 moles ATP possible from
1 mol glucose.

Figure 5: The Tricarboxylic Acid Cycle [TCA cycle]

The TCA cycle, however, is not only a catabolic or energy producing, but rather an
amphibolic pathway, as it also supplies a number of precursors for amino acid
biosynthesis. t also serves as an entrance for hydrocarbon, fat and lower molecular
weight compounds such as acetate or dicarboxylic acid utilisation pathways. Since these
lower molecular weight compounds consist of two-, three- or four-carbons, the precursors
for DNA and RNA biosynthesis are not available to them.

Those microorganisms growing on Iower moIecuIar weight compounds,
hydrocarbons and fats must therefore possess a mechanism, whereby they use not
onIy the TCA cycIe as energy producer and to make intermediates avaiIabIe for
protein biosynthesis, but aIso to conserve and add carbons in order to form the
necessary pentose phosphates. This mechanism consists of two anaplerotic
sequence reactions forming the so-caIIed glyoxylate cycIe and a new route referred
to as gluconeogenesis, whereby the Iatter is simpIy a reversaI of the EMP pathway
(Fig. 6).

Figure 6: Gluconeogenesis

The use of the TCA cycle as energy producing and the glyoxylate cycle as energy
consuming pathway requires strict energy regulation. One enzyme of the TCA cycle,
isocitrate dehydrogenase, is the control point. f there exists an overproduction of ATP,
the enzyme is inhibited leading to the accumulation of oxalacetate and pyruvate, which in
turn induce the isocitrate lyase and malate synthase to start the glyoxylate cycle activity.
Once the biosynthetic reactions have used most of the ATP available, the reactions of the
TCA cycle again become dominant. The glyoxylate cycle is simply a short-cut TCA cycle to
avoid the carbon dioxide producing steps.

2.2.2 Amino Acid utiIisation
n most cases, the amino acids produced from proteins by enzymes called proteases or
added to the environment are first converted into the corresponding keto acid, a reaction
which can occur in three different ways:

1. oxidation by cytochrome-Iinked oxidases, which are flavoprotein enzymes and are
stereospecific for amino acids with the D-configuration. Here the amino acid forms first an
imino acid as intermediate:

R-CHNH
2
-COOH + 0.5 O
2
R-CNH-COOH R-CO-COOH + NH
3

These amino acid oxidases are frequently suppressed as soon as a second substrate
such as glucose is added to the medium.
2. transamination, whereby the amino group is transferred to a keto acid, which in turn
becomes a new amino acid. These transaminations occur most frequently with amino
acids having the L-configuration:

3. oxidation by NAD(P)-Iinked dehydrogenases to make keto acids available for
transaminations:

CH
3
-CHNH
2
-COOH + NAD
+
+ H
2
O CH
3
-CO-COOH + NADH+H
+
+ NH
3

Other amino acids utilise different pathways depending upon their stereospecific
configuration. Under aerobic conditions, all pathways lead to the TCA cycle (Figs. 7 and 8)
and glyoxylate cycle, which are connected with gluconeogenesis for obtaining all
precursors for biosynthesis.

Figure 7: Amino acid metabolism

Figure 8: Threonine metabolism

2.2.3 Fatty Acid utiIisation
The fatty acid CoA esters produced through lipase action from the fats enter now a cyclic
oxidation pathway. t is important to realise that all fatty acids in the medium and outside
the TCA and glyoxylate cycles have first to be activated before they can enter any
degradative pathway.
n regard to the further degradation, some basic principles exist for fatty acid oxidation, as
they may undergo an alpha-, beta- or omega-oxidation.
Alpha - oxidation of long chain fatty acids occur at the second position of the chain. The
appropriate enzyme system catalyses the 2-hydroxylation of the particular acid. The result
of such oxidative reaction is an odd-numbered fatty acid, since a decarboxylation of the -
hydroxy fatty acid usually occurs resulting in a CO
2
release:

R-CH
2
-CH
2
-CH
2
-COOH R-CH
2
-CH
2
-CHOH-CO)H R-CH
2
-CH
2
-CO-COOH

R-CH
2
-CH
2
-COOH + CO
2

Beta - oxidation of long chain fatty acids is the best known and possibly the most
frequently occurring one. The -oxidation mechanism results in the continual removal of
acetyl-CoA (C
2
-unit), using a hydroxylation of the third position of the chain.
Omega - oxidation of long chain fatty acids involves the conversion of the acids to the -
hydroxy group, that are subsequently converted to ,-dicarboxylic acids. These oxidations
occur predominantly as mixed function oxidase systems during hydrocarbon metabolism.
Once formed, the dicarboxylic acids may be shortened from either end of the molecule
using the -oxidation sequence.
The fatty acid cycle pathway is essentially a beta -oxidation pathway. The initial step is the
activation of the fatty acid by its transformation into the corresponding CoA thioester. This
is an ATP requiring step catalysed by the appropriate acyl-CoA synthetase or in the case
of triglycerides, the appropriate acyltransferase.
The CoA ester is now converted to its unsaturated form in an oxidation step catalysed by
an acyl-CoA dehydrogenase
R-CH
2
-CH
2
-CO-ScoA + A R-CH=CHCO-SCoA + AH
2

whereby A represents a hydrogen acceptor such as NAD
+
or FAD
+
. The formation of this
double bond is the first step towards the release of the C
2
-unit acetyl-CoA. The
unsaturated fatty acid is now hydrated:

R-CH=CH-CO-SCoA + H
2
O R-CHOH-CH
2
-CO-SCoA

a reaction catalysed by an enoyl-CoA hydratase and further oxidised by a 3-hydroxyacyl-
CoA dehydrogenase to form a new carbonyl group

Both of these latter reactions are stereospecific and the conversion only occurs at that
particular position of the various fatty acids, independent of the length of the chain.
With the formation of the new carbonyl group, the terminal -oxidation reaction can occur,
whereby acetyl-CoA is split off and the residual fatty acid is again activated. Acetyl-CoA
acetyltransferase catalyse such reactions:

The so activated, but 2 carbon shorter fatty acid reenters the cyclic pathway, a
performance repeated until the saturated fatty acid is completely converted to C
2
-units. n
the case of an odd-numbered saturated fatty acid, the last compound would be a C
3
-unit or
propionyl-CoA.

The acetyI-CoA compound can now enter the TCA cycIe for energy and biosynthetic
precursor formation, using the TCA cycIe for energy production, the gIyoxyIate and
gIuconeogenesis for precursor biosynthesis in order to satisfy growth demands and
guarantee optimaI growth conditions.

3. Non-RenewabIe Substrates
3.1 Hydrocarbon utiIisation
Hydrocarbons can serve as excellent carbon sources in the aliphatic as well as aromatic
form. The n-alkanes of the aliphatic hydrocarbon group are most commonly converted via
the primary alcohol to the corresponding fatty acid and join the -oxidation cycle as
described under fatty acid metabolism:

The initial monoterminal methyl-group oxidation requires the active incorporation of 0.5
molecule of oxygen. These reactions of oxygen incorporation are catalysed by special
enzymes referred to as oxygenases. n n-alkane oxidation, a mono-oxygenase or
hydroxylase is involved. n comparison, n-alkane degradation is only a slightly extended
fatty acid metabolism, although a significantly higher amount of oxygen is required owing
to the conversion of the methyl- to a carboxyl-group.

Hydrocarbons of the aromatic type follow an entirely different, yet unique and uniform
biochemical concept. The great majority of aromatic hydrocarbons, irrespective of the
number of benzene rings, converge in their oxidative metabolism to three major
intermediates, catechoI, protocatechuate or gentisate. The ring of these last
intermediates is cleaved either between or outside adjacent hydroxyl groups. The former is
referred to as ortho- and the latter to as meta-cleavage. n both cases, the active
incorporation of a full oxygen molecule is required and catalysed by so-called
dioxygenases or simply oxygenases (Fig. 9). t is again this active incorporation of oxygen
into the benzene ring which creates the high demand for oxygen to obtain optimal growth
conditions.

Figure 9: Hydrocarbon metabolism using ortho- and meta-fission

Both cleavages lead to straight chain compounds, which finally enter the TCA cycle at the
level of acetyl-Coa, succinate or malate and fumarate. f one compares the oxygen
requirement for optimal growth, the demand increases from
carbohydrate < fats < aIkanes < aromatic hydrocarbons

t should be obvious therefore, why the oxygen demand for the production of the same
biomass increases depending upon the substrate used.

3.2 SingIe Carbon Compound utiIisation
The smallest carbon substrate is undoubtedly the single carbon compound,
Microorganisms, which have the ability to derive both carbon and energy from the
metabolism of one-carbon compounds containing a methyl-group or of compounds
containing two or more methy-groups that are not directly linked to one another (eg CH
3
-
O-CH
3
) are called methophiIic. Methane and methanol are the most commonly used
substrates for single cell protein production plants. The prokaryotic methophiles fall into
two primary physiological sub-groups, the methanotrophs and the methylotrophs.

Methanotrophs are able to grow at the expense of methane and are more or less
frequently employed in the coal mining industry to clean up gas pockets. Many are also
able to utilise methanol, formaldehyde, or dimethylether, but only a few are capable of
utilising a wider range of organic compounds.

MethyIotrophs are generally more versatile nutritionally, being frequently capable of
growing with a variety of organic compounds, but cannot use methane as carbon and
energy source.
The oxidation of methane leads via methanol to carbon dioxide. Although this complete
oxidation is of great importance for energy production, it does not provide the organism
with any precursor for biosynthesis.

Figure 10: Single carbon compound metabolism

These precursors are obtained via a very unique carbon assimilatory pathway with
formaldehyde (HCHO), which is an intermediate in the methane to carbon dioxide
oxidation, being incorporated into either ribulose 5-phosphate or the serine pathway. Both
pathways build up the carbon chain for the biosynthesis of RNA, DNA and all the main
amino acids for protein biosynthesis.

The first step in methane utilisation is the oxidation of methane. This reaction requires the
active incorporation of an oxygen atom into the methane molecule. This incorporation
produces the first intermediate methanol. t is the additional oxygen requirement which,
similar to hydrocarbon utilisation, increases the normal oxygen demand in single cell
protein production systems. The oxidation steps from methanol to carbon dioxide are
catalysed by respective dehydrogenating enzymes, whereby the electrons are transferred
to he aerobic electron transport chain for ATP energy production. All cell constituents
necessary must therefore be obtained through either of the three assimilatory pathways.
A variety of bacteria are capable of oxidising carbon monoxide to carbon dioxide
(carboxydobacteria). Although this is a strongly exergonic reaction, not all of these
bacteria are able to grow at the expense of carbon monoxide. The biochemistry of carbon
monoxide oxidation is still poorly understood. At least some carboxydobacteria
appear to utilise a soluble carbon monoxide oxidoreductase which catalyses the reaction

The electron acceptor in this reaction is an unidentified component of the electron
transport chain with a redox potential of about zero mV, perhaps of the ubiquinone or
cytochrome b-type.

This concIudes our aerobic metaboIism part which shouId demonstrate that
microorganisms are capabIe of oxidising aImost every naturaI compound in
existence.

4. References
BaiIey,S.E. and OIIis,D.F. - 1986 - Biochemical Engineering Fundamentals. McGraw Hill
Book Comp., New York
Brock,T.D., Smith,D.W. and Madigan,M.T. - 1984 - Biology of Microorganisms, 4th ed.,
Prentice Hall
Brode,E. - 1975 - The evolution of the Bioenergetic Processes. Pergamon Press
Dijkhuizen,L. - 1993 - Methylotrophs. n 'Biotechnology, Vol. 1 (H.Sahm, ed.), pp 256-284.
VCH Weinheim
DoeIIe,H.W. 1975 - Bacterial Metabolism. 2nd ed., Academic Press, New York
DoeIIe,H.W. 1994 - Microbial Process Development. World Scientific Publishers,
Singapore
Hobson,P.N. and WheatIey,A.D. - 1993 - Anaerobic Digestion: Modern Theory and
Practice. Elsevier Applied Science, London
Konig,H. - 1993 - Methanogens. n 'Biotechnology', Vol. 1 (H.Sahm, ed.), pp 251-264.
VCH Weinheim
Kraemer,R. and Sprenger,G. - 1993 - Metabolism. n 'Biotechnology', Vol. 1 (H.Sahm,
ed.),pp 47-110. VCH Weinheim
Sahm,H. (ed.) - 1993 - Biological Fundamentals. n 'Biotechnology' Vol. 1, 640 pp. VCH
Weinheim
SchIegeI,H. - 1981 - Allgemeine Mikrobiologies. Georg Thieme Verlag, Stuttgart
Stanier,R.Y., Ingreham,J.L., WheeIis,M.L., Painter,P.R. - 1987 - General Microbiology.
5th ed., MacMillan, London

Horst W.Doelle, Sc, DSc hc]
Deputy-Director, 0,5&(1-Biotechnology, Brisbane and PaciIic Regional Network;
Past Chairman, International Organisation oI Biotechnology and Bioengineering
Chapter 10
Basic Strategies of Energy MetaboIism under Anaerobic
Conditions [Fermentation]
Content:
1. Introduction
2.1 EthanoI formation
2.2 Acetone, butanoI formation
2.3 Organic acid formation
2.3.1 Propionic and succinic acid formation
2.3.2 MaIo-Iactic acid fermentation
2.3.3 Formation of diacetyI, acetoin and butanedioI
3. Protein and Amino Acid Fermentation
3.1 SingIe amino acid fermentation
3.2 Pairs of amino acid fermentation
3.3 Fermentation of a singIe amino acid in combination with a keto acid
4. Fatty Acid Fermentation
5. BibIiography
1. Introduction
Most of the organic carbon and nitrogen substrates mentioned in the last chapter can also
be utilised in the absence of oxygen. This type of metabolism is referred to as
fermentation. The microorganisms that carry out fermentations are either facultative or
obligate anaerobes. The former grow as aerobic heterotrophs in the presence of oxygen
and carry out fermentation in the absence of oxygen. n contrast, obligate anaerobes are
not able to synthesise the components of an oxygen-linked electron transport system and
are not able to grow under aerobic conditions. The most characteristic difference between
respiratory and fermentative metabolism is therefore ATP energy production. Without an
electron transport system, NADH
2
oxidation is difficult since the electrons can not be
accepted by a more positive redoxpotential carrier. The withdrawal of oxygen as the final
electron acceptor leads therefore to an accumulation of NADH
2
, which in turn results in a
cessation of the energy-producing TCA cycIe. Facultative and obligate anaerobes must
seek alternative electron acceptors, which under anaerobic conditions are organic
substances of similar redoxpotential. n order to reoxidise NADH
2
, anaerobes carry out a
variety of oxidation-reduction reactions, which yield less ATP energy and organic
endproducts. n dealing with fermentation processes one has therefore to realise that the
number of cells obtained per mol of substrate is much smaller than under aerobic
conditions and that, in addition to the cell material, large amounts of organic endproducts
are formed.
The strict correlation between energy and biomass formation and the strict oxidation-
reduction balance can be used as process control parameters:

The anaerobic mode of growth poses a special problem in a great number of heterotrophic
microorganisms, because their overall ATP requirement for biosynthesis can only be
satisfied by the degradation of a relatively large quantity of an organic compound that
serves as the energy source. This necessitates control mechanisms for the electron flow,
the result of which is the ability of many such microorganisms to dispose of 'excess'
electrons in the form of hydrogen through the activity of a special enzyme called
hydrogenase.
This phenomenon is widespread and could be exploited in biotechnology. t was also
mentioned earlier that anaerobic growth can be facilitated by the addition of growth factors.
Fermentations are usually classified according to the main fermentation
endproducts

Figure 1: Endproducts from glucose fermentation
The generation of these individual endproducts maintains the cellular redox balance in the
absence of oxygen or other terminal electron acceptors.
The details of these endproduct formations will be dealt with under Product Formation.
2.1 EthanoI formation
Traditional ethanol fermentation is carried out by yeasts, particularly Saccharomyces
cerevisiae. The yeasts degrade glucose via the EMP pathway to pyruvate, which is then
decarboxylated (pyruvate decarboxylase) to acetaldehyde. The action of an alcohol
dehydrogenase produces ethanol:
CH
3
-CO-COOH CO
2
+ CH
3
CHO
CH
3
CHO + NADH+H
+
CH
3
CH
2
OH + NAD
+

The situation is quite different in the case of the bacterium Zymomonas mobilis, which is
one of the very few bacteria possessing the enzyme pyruvate decarboxylase and therefore
also converts glucose directly into ethanol, but uses the ED pathway. Whereas yeasts
require some oxygen for growth, Zymomonas mobilis is an anaerobic bacterium and grows
very well under anaerobic conditions. Ethanol production is therefore much faster and
more economical.
The majority of bacteria lack the enzyme pyruvate decarboxylase and replace this enzyme
by enzymes which do not produce acetaldehyde as first reaction product. The enteric
group of bacteria such as Escherichia coli carry out a phophoroclastic split producing
acetyl-CoA and formate, which lead to the production of formate, acetate and ethanol. n
addition, a large number of enteric bacteria also possess the enzymes formic
hydrogenlyase, converting formate into hydrogen and carbon dioxide, lactate
dehydrogenase, converting pyruvate to lactic acid and the reductive carboxylic acid cycle
to produce succinate. This is the reason why this bacterial group is being referred to as
'mixed acid fermenters' (Figure 2).

Figure 2: Mixed Acid Fermentation

2.2 Acetone and ButanoI formation
The production of acetone,butanol, butyric acid and isopropanol by fermentation is an old
industrial process. The bacteria that carry out a fermentation of this kind belong to the
genera Clostridium and Butyribacterium. The overall pathway of these clostridia is initiated
by a conversion of sucrose to pyruvate (Figure 3)

Figure 3: Acetone-Butanol Fermentation

through the EMP pathway. The breakdown of pyruvate is characteristic for clostridia and is
often referred to as the 'clostridial type'. Pyruvate is decarboxylated using a
phosphoroclastic reaction distinctly different yet similar to the one described for enteric
bacteria. The similarity lies in the products formed [acetyl-CoA, hydrogen and carbon
dioxide], but the enzyme employed is a pyruvate-ferredoxin oxidoreductase (Figure 4):

Figure 4: Action of pyruvate-ferredoxin oxidoreductase

Acetyl-CoA is the branching point for all the endproducts as can be seen in Figure 3.
2.3 Organic acid formation
Anaerobic chemoheterotrophs are able to produce a large array of organic acids, many
being of extreme commercial value. Citric acid, lactic acid, acetic acid, gluconic acid and
itaconic formations are described in details under Product formatoin and thus will not be
considered here.
2.3.1 Propionic and succinic acid formation
Propionic acid and succinic acid formation are very important in cheese manufacturing.
Both are products either of carbohydrate or lactate fermentation by Megasphaera elsdenii,
Clostridium propionicum, Propionibacterium pentosaceum and P.shermanii. Whereas
propionibacteria prefer glucose as carbon and energy source, Megasphaera and others
have lost this ability and use lactate as their carbon and energy source:

1.5 gIucose 2 propionate + acetate + CO
2

3.0 Iactate 2 propionate + acetate + CO
2

Whereas those microorganisms using glucose and lactate as carbon source possess the
propionate-succinate pathway (Figure 5), the lactate users possess the acrylate pathway
of propionic acid formation (Figure 6). Glucose is utilized to pyruvate via the EMP pathway.
Once pyruvate is formed, the pathway splits into two branches, one terminating in the
formation of acetate and carbon dioxide, whereas the second branch produces propionic
acid in a cyclic mechanism. The key enzyme of this cyclic pathway is DS-methylmalonyl-
CoA:pyruvate transcarboxylase, which transfers a carboxyl group from the DS-
methylmalonyl-CoA to pyruvate forming propionyl-CoA and oxalacetate:

COO-CH
3
-CHCO-SCoA + CH
3
-CO-COOH
CH
3
-CH
2
-CO-CoA + COOH-CH
2
-CO-COOH

Figure 5 : Propionate-succinate pathway in propionibacteria.
1 - lactate dehydrogenase; 2 - pyruvate-ferredoxin oxidoreductase;
3 - phosphoacetyl transferase; 4 - acetate kinase;
5 - Ds-methymalonyl -CoA-pyruvate transcarboxylase;
6 - malate dehydrogenase; 7 - fumarase; 8 - fumarate reductase;
9 - succinyl-CoA transferase; 10 - LR-methylmalonyl-CoA mutase;
11 - methylmalonyl-CoA racemase; 12 - pyruvate-phosphate dikinase;
13 - PEP-carboxytransphosphorylase.

Figure 6 : Acrylate pathway for propionate formation
1 - lactate racemase; 2- CoA transferase; 3 -acyl-CoA dehydrogenase;
4- D-lactate dehydrogenase; 5- pyruvate-ferredoxin oxidoreductase;
6 - phosphotransacetylase; 7 - acetate kinase

The rest of the pathway is a reversed TCA cycle up to succinate and leads from here back
to DS-methylmalonyl-CoA, whereby a CoA transferase transfers the CoA from propionyl-
CoA to succinate forming succinyl-CoA and propionate.
n the case of lactate as carbon source, propionibacteria introduce two further enzymes.
The first enzyme is a lactate dehydrogenase converting lactate to pyruvate and the second
is a pyruvate-phophate dikinase, producing phosphoenolpyruvate from pyruvate. All other
reactions would be identical to the ones described above.
Clostridium propionicum and Megasphaera possess an entirely different mechanism of
propionate formation from lactate (Figure 6).

2.3.2 MaIo-Iactic fermentation
The utilization of malic acid to lactic acid is carried out by a large number of lactic acid
bacteria and is of extreme importance in the wine industry [see also Product formation].
The actual metabolic process is thought to be either a conversion via pyruvate, which
would necessitate the presence of a specific L(+)-lactate dehydrogenase:
COOH-CH
2
-CHOH-COOH + NAD
+
CO
2
+ COOH-CO-CH
3
+ NADH+H
+

COOH-CO-CH
3
+ NADH+H
+
COOH-CHOH-CH
3
+ NAD
+

or directly converts malic into lactic acid as is the case with the most prominently used
Leuconostoc oenos.

2.3.3 Formation of diacetyI, acetoin and butanedioI
n addition to the usual endproduct lactate, some lactic acid bacteria, eg Streptococcus
cremoris, are able to utilize citrate as carbon source with the endproducts acetoin and
diacetyl (Figure 7). The adaptability to milk, which contains close to 1gl-1 citrate, is
commercially utilized in the butter manufacturing industry. Diacetyl represents the
characteristic flavour of butter.

Figure 7: Diacetyl and acetoin formation from citrate.
1 - citrate lyase; 2 - oxalacetate decarboxylase;
3 - lactate dehydrogenase; 4 - pyruvate dehydrogenase;
5 - diacetyl synthase; 6 - acetoin dehydrogenase

The enteric group of bacteria does not possess an acetoin dehydrogenase and has to
derive acetoin and subsequently butanediol via a different pathway (Figure 8). These
bacteria utilize glucose to pyruvate via the EMP pathway, which is converted to acetyl-
lipoate before a condensation step with a second pyruvate molecule forms acetolactate.
Acetolactate decarboxylase removes one molecule of CO
2
and forms acetoin. t is
important to note that these microorganisms are not able to form diacetyl as they lack the
corresponding enzyme, which is substituted by a butanediol dehydrogenase to form
butanediol. The formation of butanediol and acetoin seems to be strictly pH dependent in
the enteric group of microorganisms. f the pH rises above 6.3, acetate and formate
accumulate instead.

Figure 8: Butanediol formation from glucose

3. Protein and amino acid fermentation
As was the case under aerobic conditions, sugars and organic acids are not the only
substrates for microorganisms. Proteins and amino acids are excellent carbon, nitrogen
and energy sources, particularly for the proteolytic clostridia. Proteases are replaced by
proteolytic enzymes called peptidases and break the proteins anaerobically into their
monomeric structure, the amino acids. The catabolism of amino acids is carried out by a
great number of anaerobic microorganisms. t takes place either with single amino acids,
pairs of amino acids or with one amino acid in conjunction with a keto acid. Everyone of
these pathways leads to a corresponding fatty acid, often referred to as 'volatile fatty acid',
which form an excellent substrate for the acetogenic bacteria during methane formation .
3.1 SingIe Amino Acid fermentation
A number of single amino acids can serve as energy and carbon source for anaerobes.
Most of these processes involved liberate ammonia, which are refereed to as
deaminations. These deaminations proceed in several ways, which differ according to the
enzymatic constitution of the organism and the conditions of the medium:

1. reductive deamination results in the corresponding saturated fatty acid and ammonia
catalysed by dehydrogenases with hydrogen as electron donor
RCH
2
-CH-COOH + 2 H
+
RCH
2
-CH
2
-COOH + NH
3

NH
2

2. de-saturation deamination results in the corresponding unsaturated fatty acid:

RCH
2
-CH-COOH RCH
2
=CH-COOH + NH
3

NH
2
3.2 Pairs of amino acids fermentation
Many clostridia, growing on protein hydrolysates or amino acid mixtures, appear to obtain
most of their energy by a coupled oxidation-reduction reaction between two suitable amino
acids. This coupled decomposition is commonly referred to as the StickIand Reaction,
since Stickland discovered this mechanism in 1934 (Figure 9).

Figure 9: Stickland Reaction of pairs of amino acids
The characteristic feature of this Stickland Reaction is that single amino acids are not
utilised appreciably, but appropriate pairs are decomposed very rapidly. One member of
the pair is oxidised while the other is reduced.
3.3 Fermentation of a singIe amino acid in combination with a keto acid.
The second amino acid in the Stickland reaction can be replaced by a keto acid.
Clostridium propionicum uses the Stickland reaction to metabolise -alanine to propionic
acid, with pyruvate playing a key role in the catalytic function of the two cycles. Amino acid
utilisation under anaerobic conditions therefore results in corresponding volatile fatty acid
production.
4. Fatty Acid Fermentation and methane formation
Whereas most of the carbohydrates are utilised into acetate and carbon dioxide, fat and
protein metabolism in general form higher volatile and non-volatile fatty acids such as
propionic, butyric, valeric and other acids. Although most of the volatile fatty acids escape
into the air, in certain anaerobic environments, these volatile fatty acids, which normally
exhibit a very strong odour, can be broken down to acetate, carbon dioxide and hydrogen.
Such a special environment is the anaerobic digester or the methanogenic environment. t
is therefore not unusual for the methanogens, which can only utilise acetate and carbon
dioxide (see section 4.4), to co-exist in close association with another metabolically
specific bacterial group, often referred to as the acetogenic bacteria. This close
association is called syntrophism and is based upon closely integrated biochemical
features of the two bacterial genera in this anaerobic consortium. One member is called
'methanogens' (see section 4.4), because they generate methane, whereas the other
members are called 'acetogens' as they are producing hydrogen plus acetate. The most
important feature in this syntrophism is the interspecies hydrogen transfer, without
which no methane can be formed.
t is therefore the role of the acetogens to degrade higher fatty acids to hydrogen and
acetic acid:
Syntrophobacter wolinii: Propionate acetate + CO
2
+ H
2

Syntrophobacter wolfei: Butyrate acetate + CO
2
+ H
2

Syntrophonomas wolfei: Higher fatty acids acetate + CO
2
+ H
2

Consequently, acetogens are capable of converting the products of other microbial
degradation reactions to substrates easily metabolisable by the methanogens and thus
remove the bad odour of the volatile fatty acids.

Figure 10: Methane formation from fatty acids by methanogenic bacteria
Methane is the most reduced organic compound and its production is becoming a very
important industrial enterprise (see Product Formation). Biological methane formation is a
geologically important process that occurs in most anaerobic environments where organic
matter undergoes decompositoin: swamps, lake sediments, the rumen of sheep and cattle,
and anaerobic sewage digesters. Methane is essentially a conversion of hydrogen and
carbon dioxide by methanogenic bacteria, which are probably the most strictly anaerobic
bacteria known and are therefore extremely oxygen sensitive. The methanogenic bacteria
are very specialized microorganisms with methane or methane plus carbon dioxide,
sometimes with up to 1 percent H
2
S as their only endproduct. They are morphologically a
very diverse group of microorganisms and form part of the archaebacteria.
Methanogens contain several cofactors not found in any other bacteria.. Three of them,
methanopterin (MP), methanofuran (MF), and CoM are carriers of the C-1 unit during its
reduction from carbon dioxide to methane. Factor-420 probably functions as a hydrogen
carrier in these reductions, and factor-430, an unusual nickel-tetrapyrrol, is the prosthetic
group of methyl-CoM reductase, the last enzyme in the reduction pathway (Figure 10).
5. BibIiography
Please refer to the references in Chapter 9.

Deputy-Director, MIRCEN-Biotechnology Brisbane and the Pacific Regional Network;
Chapter 11
Basic Strategies for Biosynthesis [anaboIism] of
CeIIuIar Components and MetaboIic ReguIation
Content:
1. Introduction
2. Autotrophic Carbon AssimiIation
3. Heterotrophic Carbon AssimiIation
3.1 Formation of proteins
3.2 Formation of RNA and DNA
3.3 Formation of Iipids
3.4 CeII waII formation
4.1 Transcription
4.2 TransIation
4.3 Activation
4.4 Inititiation
4.5 EIongation
4.6 Termination-reIease
4.7PoIypeptide foIding and formation of functionaI protein
5.1 Enzyme activity reguIation
5.2 Enzyme synthesis reguIation
5.3 CataboIite repression
6. BibIiography
1. Introduction
The microbial cell consists of five major types of macromolecules: proteins,
poIysaccharides, Iipids, RNA and DNA. These macromolecules consist of the respective
monomers, amino acids, sugar phosphates, fatty acids, ribonucleotides and
desoxyribonucleotides. All of these monomers the cell has to provide or to synthesise.
The enzymatic reactions involved in biosynthesis or anabolism are often called
biosynthetic reactions. Biosynthetic reactions are often energy-requiring, and ATP formed
in catabolic reactions is sued for this purpose. Although energy is required in certain key
biosynthetic reactions, the focus of biosynthesis is not on energy, but on carbon and the
pathways involved in the build-up of the macromolecules mentioned from simple starting
materials. All of these simple starting materials are organic compounds and intermediates
of the previously described catabolic events. The relationships between catabolism and
anabolism occur, however, not only at the common intermediates, as certain catabolic
pathways play a dual role and thus function in both anabolism and catabolism. These
pathways are referred to as amphibolic pathways, eg the tricarboxylic and glyoxylate
cycles. Hereby should be mentioned that, even where the same reactants are involved,
the enzymes acting in anabolic reactions are not necessarily the same and often are
different from those in catabolic reactions. This is a very important factor in regard to the
controls required for the relative rates of catabolism and anabolism.
2. Autotrophic carbon assimiIation
Most phototrophs and chemolithotrophs are strict autotrophs and perform their total
synthesis of cellular material from the C1-units carbon dioxide (CO
2
) or formaldehyde
(HCHO). Three of the pathways specifically employed for the conversion of these C1-units
into intermediates of central metabolism involve various sugar phosphate molecules,
whereas the fourth pathway involves part of the glyoxylate cycle. As can be visualized in
Figures 1-3, the schematic cycling mechanism of the first three pathways are very similar
in design. They differ mainly in the identity of the enzymes catalyzing the actual C1
assimilatory reactions. Further steps in all three cycles are catalyzed by the general
enzymes from the EMP, ED and the oxidative HMP pathways.

Figure 1: Calvin-Benson cycle of autotrophic carbon assimilation
1 - ribulose bisphosphate carboxylase; 2 - phosphoglycerate kinase; 3- triosephosphate
dehydrogenase; 4 - triosephosphate isomerase; 5 - fructose bisphosphate aldolase; 6 -
transketolase; 7 - transaldolase; 8 - ribose 5-P isomerase; 9 - ribulosephosphate
epimerase; 10 -phosphoribulokinase.PGA - 3-phosphoglycerate; 1,3-diPGA - 1,3-
bisphospho-glycerate; GA 3-P - glyceraldehyde 3-P; DHAP - dihydroxyacetonephosphate;
F-1,6-bisP - fructose 1,6-bisphosphate; E 4-P - erythrose 4-P; Xu 5-P - xylulose 5-P;
R 5-P - ribulose 5-P; Ru-bisP - ribulose 1,5-bisphosphate; S 7-P - sedoheptulose 7-P;
F 6-P - fructose 6-phosphate

The reduction of carbon dioxide to cell material is representative for most of the
phototrophs, which includes plants and algae, and is often referred to as the Calvin cycle
or Ribulose 5-phosphate pathway (Figure 1). n this cyclic mechanism, carbon dioxide is
incorporated with the help of the enzyme ribulose-1,5-bisphosphate carboxylase into
ribulose 1,5-bisphosphate forming 2 moles of 3-phosphoglycerate. This compound follws
the gluconeogenic pathway via glyceraldehyde, fructose 6-phosphate and the HMP
pathway.
n the case of aerobic and anaerobic C1-unit utilizers such as methanogens, methane
oxidizers etc, carbon dioxide serves as carbon and energy source and thus carbon dioxide
is first oxidized to formaldehyde, before either assimilation or energy production occurs. All
these microorganisms incorporate formaldehyde in either of two pathways, the ribulose
monophosphate cycle (Fig. 2) or the xylulose monophosphate cycle.

Figure 2: Ribulose monophosphate Pathway of Carbon Assimilation
Most of the methyIotrophs possess the enzyme hexulose 6-phosphate synthase forming
3-hexulose 6-phosphate and enter the gluconeogenic pathway at the fructose 6-phosphate
level. Those methylotrophs or methanogens, who substitute the hexulose 6- phosphate
synthase with another enzyme, the dihydroxyacetone synthase, incorporate their HCHO
into xylulose monophosphate thereby producing 2 moles of glyceraldehyde 3-phosphate.
Most methanogens and some methylotrophs have neither of these enzymes and possibly
use the serine pathway (Figure 3) with the specific enzymes serine
transhydroxymethylase, serine:glyoxylate aminotransferase and hydroxypyruvate
reductase. n this particular pathway, the C1-units combine with the amino acid glycine to
form serine to enter gluconeogenesis via the TCA and glyoxylate cycles.

Figure 3: Serine Pathway of Carbon Assimilation

3. Heterotrophic Biosynthesis
The microbial cell consists of five major types of macromolecules: proteins,
poIysaccharides, Iipids, RNA and DNA. These macromolecules consist of the respective
monomers, amino acids, sugar phosphates, fatty acids, ribonucleotides and
desoxyribonucleotides. All of these monomers the cell has to provide or to synthesise.
The enzymatic reactions involved in biosynthesis or anabolism are often called
biosynthetic reactions. Biosynthetic reactions are often energy-requiring, and ATP formed
in catabolic reactions is sued for this purpose. Although energy is required in certain key
biosynthetic reactions, the focus of biosynthesis is not on energy, but on carbon and the
pathways involved in the build-up of the macromolecules mentioned from simple starting
materials. All of these simple starting materials are organic compounds and intermediates
of the previously described catabolic events. The relationships between catabolism and
anabolism occur, however, not only at the common intermediates, as certain catabolic
pathways play a dual role and thus function in both anabolism and catabolism. These
pathways are referred to as amphibolic pathways, eg the tricarboxylic and glyoxylate
cycles. Hereby should be mentioned that, even where the same reactants are involved,
the enzymes acting in anabolic reactions are not necessarily the same and often are
different from those in catabolic reactions. This is a very important factor in regard to the
controls required for the relative rates of catabolism and anabolism.
3.1 Formation of Proteins
There are 20 amino acids common to proteins, whereby all of these amino acids are
amino acids in L-configuration. The synthesis of the carbon skeleton is relatively
straightforward, since there exist only a few organic compounds which are important
precursors:
Erythrose 4-P tyrosine, tryptophan
PhosphoenoIpyruvate phenylalanine
Ribose-5-P histidine
3-phosphogIycerate serine, glycine, cysteine
pyruvate alanine, valine, leucine
2-oxogIutarate glutamate, glutamine, arginine, proline
oxaIacetate aspartate, asparagine, methionine, lysine,
threonine, isoleucine

The attachment of the amino group is a crucial step. The two general enzymatic methods
of incorporating the amino group are transamination and amino acid dehydrogenations.
The former requires the coenzyme pyridoxal phosphate and the latter NAD(P)H. There is
no doubt that the two most important amino acids are glutamate and glutamine, which are
used frequently for transamination reactions.
The amino acids are then used for protein biosynthesis. t is the genetic code which
determines the sequence of amino acids in a protein and the point of formation is the
ribosome.
Protein biosynthesis can be thought of occurring in a number of steps: initiation,
elongation, termination-release and polypeptide folding. These steps occur at the
ribosomes, which represent the protein-synthesizing site. From the genetic code of the
DNA, the message of the right amino acid sequence is transcribed via the mRNA to the
ribosome. The translation of this message occurs via the tRNA, which brings the
corresponding amino acid to the ribosome, where the sequencing to a specific protein
occurs. Many proteins are used outside the cell as enzymes. A mechanism must therefore
exist to selectivle transfer some proteins across the cell membrane. This could be
explained by the so-called signaI hypothesis, which proposes the formation or
attachment of an extra N-terminal peptide sequence (some 15 amino acids), which permits
the enzyme to wind itself through the hydrophobic lipid membrane.
3.2 Formation of RibonucIeic acid [RNA] and DeoxyribonucIeic acid [DNA]
Ribonucleotides consist of a purine or pyrimidine base, ribose and phosphate groups. f a
purine or pyrimidine base is attached to a ribose, it is referred to as a ribonucleoside and if
a phosphate group is attached to this ribonucleoside, it is called a ribonucleotide.
Purines, such as adenine and guanine, have as their original intermediate ribose 5-
phosphate formed in the HMP pathway. n a sequence of reactions, the purine ring is built
up almost atom by atom using carbons and nitrogens derived from amino acids, carbon
dioxide and formyl groups (Figure 4). These various groups are built onto the ribose 5-
phosphate step by step until the key intermediate inosinic acid is formed. This first
compound with a purine ring serves as the branching point for the formation of adenylic
and guanylic acid, which after sequential phosphorylations can also lead to AMP
[adenosine monophosphate], ADP [adenosine diphosphate], ATP [adenosine
triphosphate] and GMP, GDPand GTP, respectively.
n contrast, the pyrimidine ring is built up before the ribose 5-phosphate is added (Figure
5). The compound with a pyrimidine ring is orotic acid. After the addition of the ribose 5-
phosphate, the important pyrimidines for RNA and DNA biosynthesis, uridine, thymidine
and cytidine are produced.

Figure 4: Purine Formation

The 5 bases, adenine, guanine, uridine, thymine and cytidine, so formed are now available
for the formation of DNA and RNA.
The structure of the single DNA chain consists of alternating units of phosphate and
deoxyribose, to which one of the bases is attached. The phosphate linkage is a diester,
since it is connected with two sugar molecules. At one end of the DNA molecule the sugar
has a phosphate on the 5-hydroxyl whereas at the other end the sugar has a free hydroxyl
at the 3 position. Since the complete DNA molecule is a double helix of two long chains,
both strands are complimentary, but not identical. This complementarity of the DNA
molecules is achieved by specific pairing of the purine and pyrimidine bases. For example,
adenine always pairs with thymine and guanine always pairs with cytosine. The specific
pairing and complementarity allows a replication to occur by simple unwinding of the
existing helix and the addition of a new structure via the complimentary pairing. Since the
DNA replicatoin occurs by the addition of short fragments, it is the DNA polymerase which
is responsible for connecting the nucleotide bases to the short fragments and the DNA
ligase for joining the fragments to form a new strand.
RNA plays a number of important roles in the expressoin of genetic information in the cell.
According to their individual functions, one differentiates between mRNA [messenger
RNA], tRNA [transfer RNA] and rRNA [ribosomla RNA]. n comparing the structures of
DNA and RNA, the latter exhibits three major chemical differences:

1. the sugar ribose replaces deoxyribose
2. the base uracil replaces thymine
3. RNA is single stranded in general.

RNA biosynthesis is directed by the genetic code, that is, it requires the presence of DNA
acting as template. The direction given (transcription) from the template is carried out
through the action of the enzyme RNA polymerase, which catalyses the formation of
phosphodiester bonds between ribonucleotides. The site at which mRNA synthesis begins
is not random, since each gene or group of related genes (operon) has a specific promoter
region at which the RNA polymerase first binds. Transcription now occurs by selective
opening of the DNA double helix.

Figure 5: Pyrimidine formation

3.3 Formation of Lipids
Most of the fatty acids occurring in lipids contain 16 or 18 carbon atoms and are saturated
or unsaturated, straight or branched with one or more double bonds. There is only one
precursor for the biosynthesis of all the fatty acids, acetyl-CoA. The reaction sequence of
fatty acid synthesis is essentially the same in all organisms. n order to differentiate
between fatty acid catabolism and fatty acid biosynthesis, the organisms employ CoA-
derivatives in the former and acyl carrier protein [ACP] in the latter.
The acetyl-ACP is attached to the enzyme fatty acid synthetase throughout the series of
reactions, in which successive two-carbon units are added and reduced. t is of interest to
note that, although the fatty acid chain is increased two carbons at a time, the immediate
precursor is a three-carbon compound, malonyl-ACP. This compound is synthesised from
acetyl-CoA via malonyl-CoA. Once the required chain length is reached, ACP is
hydrolysed and the appropriate fatty acid is formed.
Unsaturated fatty acids contain one or more double bonds. n most aerobic
microorganisms the formation of a double bond requires molecular oxygen, whereas under
anaerobic conditions, these fatty acids are synthesised through dehydration of a hydroxy
acid during fatty acid synthesis.
Branched fatty acids are formed through the replacement of the initiating molecule acetyl-
CoA with a branched chain fatty acid such as isobutyryl-CoA.
Branched and unsaturated fatty acids play an important role in maintaining the fluidity of
the cell membrane.
If the individuaIIy formed fatty acids are attached to or esterified with glycerol or
gIyceroI phosphate, fats or Iipids are being formed.
3.4 CeII WaII Formation
One of the most important structural features of the cell s the cell wall. The rigid layer of
both gram-negative and gram-positive bacteria is very similar in chemical composition and
is called peptidoglycan. This peptidoglycan is a mixed polymer of amino acids and amino
sugars. The amino sugars N-acetylmuramic acid and N-acetylglucosamine are connected
in glycosidic linkage and form the basis of the polymer. The amino acids L-alanine, D-
alanine, D-glutamic acid and either lysine or diaminopimelic acid (DAP) are connected in a
peptide linkage and form the crosslinks. t can therefore be realised that the basic structure
of the cell wall consists of glycan chains (sugars) connected by peptide cross-links. The
glycosidic bonds of the glycan chain are very strong, but these chains alone cannot
provide the rigidity in all directions. Therefore, the full strength of structure is obtained
when these chains are joined by peptide cross-links. This cross-linking occurs to
characteristically different extents in different bacteria, with greater rigidity coming from
increased cross-linkages.
A bacterial cell can synthesize several thousand different kinds of proteins, each
containing, on the average, approx. 200 amino acid residues linked together in a definite
sequence. The information required to direct the synthesis of those proteins is encoded by
the sequence of nucleotides in the cell's complement of DNA, most of which is in the form
of a double-stranded circular molecule, the bacterial chromosome or plasmids. By the
process of replication, the chromosome is precisely duplicated, thus assuring that progeny
cells receive information enabling them to synthesize the same protein.
The process by which the encoded information of the chromosome directs the order of
polymerization of amino acids into proteins occurs in two steps: transcription and
translation.
4.1 Transcription
The information content of one of the strands of DNA is transcribed into RNA; i.e. the DNA
serves as a template upon which a single strand of RNA is polymerised, the length of
which corresponds to from one to several genes on the bacterial chromosome. One class
of these RNA molecules, termed messenger RNA (mRNA) carries the information
encoded in the DNA to the protein synthesis machinery.
4.2 TransIation
Protein synthesis takes place on ribonucleoprotein particles called ribosomes, which
attach themselves to the molecules of mRNA. The information carried by the mRNA
molecules is translated into protein molecules by the transferRNA (tRNA). These
molecules are multifunctional: they are able to bind to the ribosome, to be attached to
specific amino acids and to recognise specific nucleotide sequences of the mRNA. Each
molecular species of tRNA recognises a specific sequence of nucleotides (a codon) on the
mRNA molecule and can be attached to a specific amino acid. Thus, the various amino
acids are brought by their cognate tRNA molecule to the ribosome, where they are
polymerised into protein in the sequence encoded by the mRNA. The products of
transcription, mRNA, tRNA, all participate in the synthesis of proteins. Thus the rate and
specificity of transcription determines, in large measure, the rate and the relative
proportion of the various proteins that are synthesized. All control of transcription is
affected by the frequency of its initiation and termination. The synthesis of proteins
consists of the linking together of activated amino acids in an orderly way. The key
problem of protein synthesis is thus the placing of the proper amino acid at the proper
place in the polypeptide chain. The site of protein biosynthesis are the ribosomes. Each
ribosome consists of two subunits, which in bacteria have sedimentation constants of 30S
and 50S. Each large subunit consists of a number of individual proteins, 21 in the 30 S
ribosome and 34 in the 50 S ribosome.
4.3 Activation
The activated forms of amino acid that are synthesized to form proteins are aminoacyl-
tRNAs. They are synthesized in two steps by a group of enzymes, aminoacyl-tRNA
synthetases. Each of these 20 enzymes is specific for a particular amino acid, but some
react with several different tRNA molecules, that is, several different types of tRNA
molecules can accept the same amino acid. n the synthetase reaction the amino acid
reacts with ATP to form an enzyme bound intermediate, aminoacyl adenylic acid. The
aminoacyl group is then transferred to the hydroxyl group of the terminal AMP residue that
all tRNA molecules contain at their 3' end.
4.4 Initiation
The initiation is concerned with the formation of the initial ribosome-tRNA-mRNA complex.

4.5 EIongation
Elongation is concerned with the formation of successive peptide bonds leading to the
synthesis of a polypeptide.
4.6 Termination-ReIease
Termination and release forms the completion of the polypeptide and its release from the
ribosome. The growing chain arrives at a termination codon, UAA, UAG or UGA, which
does not code for any amino acid and are called nonsense codons
4.7 PoIypeptide FoIding and Formation of FunctionaI Protein
Firstly the formyl group is being removed from f-met. The subsequent folding of the
polypeptide to assume a secondary and tertiary conformation is determined by its amino
acid sequence. Finally, the polypeptide associates with other polypeptides to form the
quaternary structure of the active enzyme or protein.
As recently as 1987, it was stated that enzymes that catalyse the folding of proteins were
not known. Since then, at least three classes of enzymes have been shown to catalyse
folding or refolding. One class was found in a group of ?heat-shock proteins? or HSPs,
which are synthesised by many kinds of cells in response to heat stress. Such proteins are
also expressed in response to other forms of environmental stress, and appear to form
part of a generalised stress response. Dozens of these proteins do indeed enhance the
rate of the refolding of unfolded proteins at the expense of ATP, and thus catalyse a true,
energy-coupled reaction, rather than simply providing a template of nucleating the folding
process. These folding enzymes have been called molecular chaperones or
chaperonins. The most famous of these are HSP 70 and its helper HSP 20. There are
several families of chaperonins, distinguished by structural similarities. Not all chaperones
are stress-inducible; some are constitutive. t has been clearly demonstrated that
chaperonins can aid in the refolding of denatured proteins in vivo, and prevent
aggregation. They can confer heat stability to proteins and thermotolerance or
osmotolerance to organisms. Clearly, moIecuIar chaperones are ubiquitous and
essentiaI, not an inconsequentiaI biochemicaI curiosity.
The importance of chaperonins appears to be far-reaching; they also play a role in gene
expression and regulation. While only a few such cases have been demonstrated, and
these are generally related to expression of other stress response proteins, it seems
reasonable to believe that they may control the activity of many protein factors important in
gene expression, such as sigma factors, repressors, etc.
Chaperones generally do not bind native proteins, but associate with unfolded or partially
unfolded proteins, probably via the same hydrophobic interactions that would otherwise
cause non-specific aggregation. There is even evidence that some chaperonins
specifically recognise certain folding intermediates but not others.
Molecular chaperones also appear to be useful as a laboratory tool. They can be used to
refold denatured enzymes and even untangle aggregated proteins in vitro. In vivo,
coexpression (by molecular genetics) of a chaperone and an enzyme one wishes to study
can lead to enhanced recovery of the active enzyme and a reduction in unfolded or
aggregated product.
Folding enzymes of the second class, protein disulfide isomerase (PDs), establish the
formation of proper disulfide bonds. Although there is evidence that at least one
chaperonine is capable of rearranging mispaired disulfide bonds, the PDs form a separate
class of folding-catalysing enzymes. Although protein disulfide isomerases have been
known for at least 20 years, they were perhaps thought of more as maintenance enzymes
rather than as catalysts for proper folding.
The third class of enzymes known to be involved in protein folding or refolding are proline
isomerases, which interconvert the cis- and trans-forms, of proline peptides.
Certainly an additional group of enzymes that could be thought of as catalysing the
formation of an active enzyme structure includes posttransitional processing enzymes.
These enzymes include methylating enzymes, glycosylating enzymes, kinases, and
proteases, among others. These enzymes are so diverse that it is perhaps misleading to
place them together as a class and certainly misleading to say that they catalyse folding
per se. Nonetheless, they are required in order to obtain active enzyme. For instance,
proteases are often necessary to remove leader (targeting) sequences or to activate
enzymes synthesised in an inactive form.
Many proteins are used outside the cell and must somehow get from the site of synthesis
on cytoplasmic ribosomes through the cell membrane. n prokaryotes, periplasmic
enzymes and extracellular enzymes are secretory enzymes. How is it possible for the cell
selectivity to transfer some proteins across a membrane, while leaving most proteins in
place in the cytoplasm. This is explained by the signaI hypothesis, which states that
secretory proteins are synthesized with an extra N-terminal peptide sequence, some 15-20
amino acids in length, which is called the signaI sequence. n this signal sequence,
hydrophobic amino acids predominate and may permit the enzyme to be threaded through
the hydrophobic lipid membrane. n many cases, the ribosomes that synthesize secretory
proteins are bound directly to the cell membrane, so that the protein is formed and passes
through the membrane simultaneously. Once the protein has been secreted, the signal
sequence is removed by a peptidase enzyme.
Not all enzymes are synthesized by the cell in the same amounts, some enzymes being
present in far greater numbers of molecules than others. Clearly the cell is able to regulate
enzyme synthesis.
The microbial cell is a complex of catabolic and anabolic events with many interconnected
pathways. Each metabolic pathway consists of a series of enzyme-catalysed reactions that
convert a substrate into product(s). Although the enzymes, which constitute such
pathways are not physically organised, they are highly organised in a chemical sense. The
concentration of enzymes of the pathways are such that the catalytic activity of certain
enzymes can limit the overall flux through the pathway, while other reactions can occur
rapidly and are limited by the substrate (intermediate) or cofactor concentrations. This
chemical organisation is synonymous with metabolic regulation.
n living cells the rates of metabolic processes may be varied in response to environmental
conditions in at least two ways. There exists a rapid mechanism operating within second
or minutes for the regulation of enzyme activity and this depends upon changes in the
catalytic activity of individual enzyme molecules. There also exists a slower mechanism
operating within hours or days that is dependent upon an increase or decrease in the
number of enzyme molecules through modification of the rate of synthesis.
5.1 Enzyme Activity ReguIation
The following four factors could be responsible for controlling fluxes through metabolic
pathways: substrate availability, cofactor availability, product removal and feedback
regulation.
Substrate avaiIabiIity. Any metabolic pathway could, in theory, be regulated very simply
by the availability of the substrate. A reduction in substrate concentration below saturation
level will decrease the activity of an enzyme and thus reduce the flux through the pathway.

Cofactor avaiIabiIity. A regulatory mechanism based upon the availability of a cofactor is
somewhat similar to a control by substrate availability. The major difference is, of course,
that cofactor synthesis is determined by the cell's genetic code. A typical example of
control by cofactor availability is the regulation of electron transport and oxidative
phosphorylation.

Product removaI and Feedback inhibition. f the substrate of the pathway is converted
to a product by a series of reactions, the removal of the product could control the rate of its
formation from the substrate. However, since most pathways appear to be controlled at
non-equilibrium reactions, product removal will be unimportant unless the product
interferes with a catalytic reaction. Feedback regulation of enzyme activity is the most
flexible and biologically widespread mechanism of metabolic control. Feedback control in
metabolic systems operates solely through the regulation of the activity of enzymes that
catalyse non-equilibrium reactions. This type of control has three remarkable features:

1. it is specific, as only the endproduct is effective;
2. it acts mostly on a single enzyme early in the pathway;
3. its action is immediate and rapid, and completely independent of the other enzymes.

The most remarkable and important feature of this inhibition is the fact that the inhibitory
product has no steric relationship to the normal substrate of the inhibited enzyme, thus no
competition occurs. Detailed investigations on the characteristic kinetics of these particular
enzymes revealed that

1. the activity vs substrate curve did not exhibit normal hyperbola, but is sigmoidal instead;
2. the enzymes were inhibited or activated by other substances, whereby the kinetics of
activation resembles normal Michaelis-Menten kinetics;
3. these unusual kinetics were observed with enzymes that have more than one binding
site;
4. the sensitivity of native proteins to an activator or inhibitor can be modified resulting in
an alteration of the three-dimensional structure of the protein.

These observations lead to the general theory of enzyme regulation, which is known as
allosterism (Figure 6).

Figure 6: Allosteric enzyme reactions

Feedback inhibition occurs in both catabolic and anabolic pathways. The main products in
catabolic pathways are ATP and reduced NAD. Both nucleotides are strong inhibitors of
allosteric control enzymes. The relative concentrations of AMP, ADP and ATP in a cell can
be used to calculate the energy charge of adenylate of the cell using the expression:
0.5[ADP] + 2[ATP]
-------------------------------
[AMP] + [ADP] + [ATP]

n using this expression, values in the range of 0 - 1.0 have been observed. In vitro
experiments revealed that the activity of many enzymes associated with ATP generation,
eg isocitrate dehydrogenase, is inhibited if the value is greater than 08. Conversely,
anabolic pathway enzymes requiring ATP, eg aspartate kinase, are activated when the
energy charge is high. A very similar control system exists with enzymes requiring the
cofactor NAD
+
for their oxidative reactions and those requiring NADH+H
+
for their
reduction reactions. Any disturbance in the balance of these cofactors by
overproduction leads therefore to an inhibition and a shortage to a reduction of flux.
All biosynthetic pathways also produce endproducts. Depending on the number of
products, the complexity is either simple or intricate. n principle, feedback inhibition is the
effect of the endproduct on the first enzyme of the biosynthetic pathway. n the case of
more than one endproduct being formed in branched biosynthetic pathways, this would
mean that the overproduction of one product would inhibit the first enzyme in that
particular branch of the pathway to avoid influencing the formation of the other product(s)
necessary for growth:

n order to cope with this specific feedback regulation, a number of different mechanisms
are known of which only the major ones are mentioned here:

1. SequentiaI Feedback is a mechanism, whereby the first catalysed reactions uses a
single enzyme a , for which the effector is not an endproduct, but the intermediate C. f
either endproduct F or J accumulates, their respective first enzyme in the pathway d or g
will be inhibited leading to an accumulation of C, which in turn or sequence inhibits
enzyme a.

2. Concerted Feedback is a mechanism, whereby enzyme a possesses two different
allosteric sites, each of which binds one of the specific endproducts. When only one of
these sites is occupied by an effector, activity of the enzyme is not affected. However,,
when both effectors are bound to the enzyme, it becomes inhibited.

3. IsofunctionaI Enzymes are two enzymes (a and a') which have the same catalytic
activity but are subject to feedback inhibition by different endproducts. t is believed that
these isofunctional enzymes form a physical attachment with the respective first enzyme of
the branch pathway, eg a with d and a' with g. The accumulation of J therefore would
inhibit the a' enzyme which immediately reduces the activity of the enzyme g allowing the
continuation of a certain flux through to the formation of product F

4. Combined activation and inhibition occurs in cases where a biosynthetic intermediate
formed by a specific reaction enters two completely different independent pathways. The
product or intermediate can then allosterically inhibit the enzyme in one pathway and
allosterically activate the enzyme in the second pathway of use.

These are only a few representative examples of major regulatory pattern observed. It is
assumed that such enzymes have deveIoped because an efficient controI of the rate
of enzyme activity enabIes the organism to adapt to changing environments.
5.2 Enzyme Synthesis ReguIation
The DNA of the microbial cell dictates the detailed synthesis of the protein and thus
enzymatic machinery. Despite their constant genotype, microbes are amazingly flexible in
their ability to alter their composition and metabolism in response to environmental
changes. The environment does not change the genetic makeup of the cell, but markedly
affects the phenotypic expression of the genes. n other words, it can greatly influence the
synthesis of many enzymes. For example, if an amino acid is added to the growth
medium, the organism will not produce this amino acid and it can be observed that all
enzymes involved in the synthesis are not formed. The external amino acid represses the
synthesis of these enzymes. (Figure 7) The complementary phenomenon to enzyme
repression is enzyme induction, where the synthesis of an enzyme occurs only in the
presence of the particular substrate. This substance or chemical compound is referred to
as the inducer, whereas the substance or chemical compound that represses enzyme
production is called a corepressor.
The question now is, of course, how these effectors are able to affect transcription in such
specific manner.

Repression of enzyme synthesis. Every operon in the DNA molecule produces a
represser protein. During synthesis, this represser cannot combine with the operator gene.
t is the corepressor which has to combine with the represser protein thereby changing its
configuration which now will fit and combine with the operator gene. f this occurs, mRNA
synthesis is blocked and protein synthesis cannot occur.

Figure 7: Repression of enzyme synthesis at the operon

Induction of enzyme synthesis. n this case, the regulator gene produces a represser
specific for the operator of the particular operon. The represser therefore blocks the
operator from initiating transcription to the mRNA and thus disallows protein synthesis.
When the inducer is added to the medium, it combines with the represser and thus
inactivates the represser by withdrawing it from the operator (Figure 8). The operator is
released from repression and transcription to the mRNA can occur.

Constitutive enzymes. Not all enzymes of the cell are inducible or repressible. A large
number of enzymes are produced continuously in the presence or absence of their
substrates. These enzymes are referred to as constitutive enzymes.

Figure 8: nduction of enzyme synthesis

5.3 CataboIite repression
Catabolite repression is a different type of enzyme repression and occurs when an
organism is offered two or more different energy sources, of which can easily be
catabolised. Only after the readily catabolisable energy source is exhausted is this
repression abolished (Figure 9). The mechanism of this catabolite repression involves a
catabolic activation protein [CAP] and cyclic AMP. Catabolic repression is also expressed
in growth measurements, where it is referred to as diauxie growth.

Figure 9 : Diauxic growth

For the biotechnological evaluation of the usefulness of a microbial process, the
stoichiometry of microbial metabolism together with its regulatory mechanisms can be a
very useful tool, since stoichiometric equations describing the consumption of a substrate
and the formation of biomass and products are always required. These stoichiometric
equations can be used in a number of ways:

1. to obtain relationships between different yield coefficients based on elemental and
energy balances describing microbial activity;
2. to obtain the theoretical limits of the maximum yields of biomass and products formed
from a given substrate;
3. to estimate yield coefficients from experimentally determined quantities such as
substrate consumption, biomass production, oxygen consumption, carbon dioxide
production, heat evolution and nitrogen consumption;
4. to explore reasons for discrepancies between measured yield values and those
predicted from the stoichiometry of a given system to systematic errors in measurement
and an incorrect description, such as the presence of an unknown product or substrate;
5. to construct figures that allow quick estimates of yield coefficients and functions of
experimentally measurable variables.
t should also be realised that product formation involving any biosynthetic intermediate or
endproduct is only possible with a thorough knowledge of the regulatory mechanisms
involved in the cell. Metabolism is the intricate interplay between anabolism and
catabolism via the regulatory mechanisms to observe the thermodynamic laws of
nature.
6. BibIiography
Please refer to the references in Chapter 9.

Deputy-Director, MIRCEN- Biotechnology Brisbane and Pacific Regional Network;
Chapter 12
MICROBIAL BIOTECHNOLOGY IN INDUSTRY - Enzyme
Production
[The following two chapters are lectures given in the Department of ndustrial Biotechnology, Faculty of
Agro-ndustry, Prince of Songkla University, Hat Yai, Thailand during a 2-month teaching fellowship around
1992]
Content:
1. Introduction
2. ChemicaI Nature and CIassification
2.1 Oxidoreductases
2.2 Transferases
2.3 HydroIases
2.4 Lyases
2.5 Isomerases
2.6 Ligases
3. Enzyme Assays
4.1 Screening of enzyme producers
4.2 Strain seIection
4.4 Strain maintenance
4.5.1 InocuIum
4.5.2 Medium composition and preparation
4.5.3 Process conditions and equipment
4.5.4 GeneraI fermentation aspects
4.5.5 CuItivation conditions
4.6 Purification
4.6.1 Extraction
4.6.2 SaIting-out and precipitation
4.6.3 DesaIting
4.6.4 Ion exchange chromatography
4.6.5 Hydroxyapatite chromatography
4.6.6 Size excIusion chromatography
4.6.7 Affinity chromatography
4.6.8 ConcIusion
4.6.9 Assessment of purity

5. Enzyme ImmobiIisation
5.1 Methods of immobiIisation
5.2 Properties of immobiIised enzymes
6. Biosensors
7. BibIiography
1. Introduction
Enzymes are the protein biocatalysts produced by living cells. They are produced by the
cells to bring about and control the numerous biochemical reactions involved in the
metabolic processes of the cells. Fortunately, most enzymes can be separated readily
from the cells that produce them and can perform their catalytic activities entirely apart
from the cells. A considerable number of enzyme preparations have found important
applications both in research and in industry.
The first major advantage of microorganisms as the source for useful industrial enzymes is
the potentially unlimited supply. Fermentation production capacity can be expanded almost
without limitation to meet any level of demand.
The second principal advantage is the large number of enzymes which can be obtained
economically from microorganisms. The only major limits are those imposed by the limits
of possible enzymatic catalysis. A well-designed and intensive search among microbial
strains can usually find an appropriate organism to produce almost any enzyme.
On the other hand, the multiplicity of enzymes produced by a single organism is
sometimes a disadvantage. Often in an industrial process requiring only a specific
enzymatic conversion, the presence of contaminating enzymes which will cause
undesirable reactions is a distinct handicap. n such cases it is necessary to remove the
undesirable enzyme contaminants, which may be difficult or costly. Fortunately, enzyme
purification methods, such as differential inactivation, fractional precipitation, and column
chromatography, have become applicable on a large scale, making it possible for the
enzyme manufacturer to supply commercial enzyme products having the necessary
performance characteristics.
For an appreciation of microbial enzymes, an understanding of simple enzyme theory is
necessary: what enzymes are, how they are named and classified, how they work and the
factors which affect their action.
2. ChemicaI Nature and cIassification
An enzyme is usually defined as a protein biocatalyst produced by a living cell. Enzymes
are therefore rather special members of that broad class of substances known as
catalysts. Catalysts influence the rates of chemical reactions without being used up; they
take part in the reactions but reappear in their original form. Theoretically, a catalyst can
convert an unlimited amount of reacting substance.
Like all catalysts, enzymes affect the velocities of chemical reactions, but not the extent of
the chemical changes which occur. Enzymes only catalyse reactions which are
thermodynamically possible, that is, are attended by losses of free energy. n order for
most chemical reactions to occur, a certain amount of resistance has to be overcome, the
molecules must be activated by supplying energy. An enzyme lowers the amount of
activation energy required by the reaction. Hence, enzymes are able to bring about, under
mild conditions near room temperature, reactions which in their absence would require
drastic conditions of high temperature or another high-energy source.
The enzymes produced by living cells are, of course, for the purpose of accomplishing
specific metabolic needs. How many enzymes exist is unknown but certainly the number
runs into many thousands.
Enzymes differ from other catalysts in several respects, mainly because of their protein
nature. Being proteins, the enzymes are denatured and inactivated when subjected to
non-physiological conditions, such as heat or strong chemicals. Hence two distinctive
properties of enzymes are their thermal lability and their sensitivity toward acids and
bases. But the most important distinction of enzymes is their specificity of action. For
example, chemical hydrolytic catalysts such as acids will catalyse the hydrolysis of many
different kinds of substances including esters, acetals, glycosides (sugars), or peptides
(proteins), whereas individual different enzymes are required for hydrolysing each of these
types of compounds and even of specific members of each class. Thus carbohydrases act
specifically on glycosides and cannot hydrolyse ester or peptide bonds. ndividual
carbohydrases are necessary for each of the individual glycosides. Sucrose, lactose and
maltose are all disaccharides, but separate and distinct enzymes are necessary for the
hydrolysis of each.
With enzymes, two types of specificity are distinguished: substrate specificity and reaction
specificity. Examples of substrate specificity are the hydrolysis of sucrose by sucrase
and the oxidation of glucose by glucose oxidase. Examples of reaction specificity are the
actions of proteinases which are capable of splitting particular peptide bonds of proteins.
These peptide bonds are amide linkages between carboxyl and amino groups of the amino
acids. ndividual proteinases have narrow reaction specificity as to which peptide bonds
they can split. For example, the action of trypsin is the rapid hydrolysis of only those bonds
linking the carboxyl groups of the two basic amino acids, lysine and arginine, to other
amino acids. Trypsin has little or no effect on peptide bonds formed by other amino acids.
solation and purification of enzymes are based upon stability, solubility, size, and charge
of the enzyme molecules, involving sequences of procedures of selective inactivation,
solvent or salt precipitation, chromatography, and electrophoresis. The size of enzymes
ranges from about 13,000 to over a million daltons. The molecular weights and amino acid
compositions of many enzymes have been determined, as have been the amino acid
sequences of some of the smaller enzymes. There is nothing in the amino acid analyses
or sequences which clearly differentiates enzymes from other proteins. All proteins contain
chemically reactive groups such as free amino, carboxyl, hydroxyl, sulfhydryl, and
imidazole groups and frequently nonprotein prosthetic groups. The mere presence of
reactive groups in protein molecules does not account for enzyme activity. Special
structures in the molecules must be responsible for the specific catalytic functions of the
enzyme proteins. t is known that the long peptide chains of native protein molecules are
folded, arranged, and cross-linked into three-dimensional structures. n these
conformationaI arrangements of enzyme molecules the reactive groups or combining
sites are situated in such a manner as to make up active centers which fit the reactive
groups of the substrates to enable substrate binding and catalytic activity. The molecular
conformation and structure of the active centers for a few enzymes have been elucidated.
When enzymes are denatured and thus inactivated by heat or strong chemicals, it has
been shown that the necessary tertiary arrangements for activity are destroyed by
unfolding and rearrangement of the peptide chains.
An interesting development relating to enzyme composition and structure has been the
demonstration of multiple forms of an enzyme, designated isoenzymes or isozymes.
These isozymes catalyse the same reactions but have very slightly different physical
properties. soenzymes occurring together can be most readily differentiated by zone
electrophoresis. The different electrophoretic mobilities indicate differences in electric
charge associated with slightly different amino acid composition of the isoenzyme proteins.
t has been clearly shown that commercial glucoamylase of fungal origin contains two
isozymes, but they appear to have no separate importance in the commercial production
of dextrose from starch.
soenzymes have, however, important practical applications in clinical diagnosis. For
example, electrophoresis of normal human serum shows five isoenzymes of lactate
dehydrogenase. Particular tissues have different patterns of occurrence of the lactate
dehydrogenase isoenzymes. Heart has predominantly isoenzymes 1 and 2, whereas liver
has almost entirely isoenzyme 5. n diseases leading to leakage of enzyme from damaged
cells, the isoenzyme pattern of serum may change from the normal toward that of the
particular tissue involved. Therefore it is now possible to distinguish between myocardial
infarction and liver disease by electrophoresis of serum and determining the lactate
dehydrogenase isozyme profile.
Although many microbial enzymes may readily be separated in soluble form from the cells
or from the insoluble fragments of the cells after disintegration of the cells, some enzymes
are frequently firmly associated with particulate matter in the cells. Such bound enzymes
are localized in the cellular structure and undoubtedly play very important metabolic roles
in such functions as nucleic acid replication, protein synthesis, and oxidative
phosphorylation. These bound enzymes are of the greatest significance in cellular
activities, but they have no industrial importance at the present time.
Since enzymes are proteins and all proteins are formed during active growth of the cell,
enzyme production is greatest during the logarithmic phase. We have, however,
extracellular and intracellular enzymes, depending upon the substrates available to the
microbial cell. Since only monomeric organic compounds are able to be transported
through the cellular membrane, all hydrolases responsible for hydrolysing complex natural
substrates into monomers must be extruded by the cell. There highest synthetic capability
exists during the early stages of growth.
The systematic name for an enzyme is derived in accordance with definite rules to identify
the enzyme and indicate its action as precisely as possible. n general, the systematic
name consists of two parts. The first part names the substrate, and the second, ending in -
ase, indicates the nature of the process. The systematic rules for nomenclature are quite
extensive but they cannot be applied if the substrate composition or the enzyme reaction is
not fully understood, in which case only trivial names are possible. The Enzyme
Commission also adopted more convenient trivial names for the enzyme for general use.
n the majority of cases the trivial names are those already commonly employed. n
biochemical publications it is now customary to give the classification number, systematic
name, and trivial name. To illustrate, the familiar glucoamylase (trivial name) is EC 3.2.1.3
or alpha-1,4-glucan glucohydrolase. The name indicates that the substrate is a glucan
(starch), a glucose polymer having the glucose molecules joined by alpha(1-->4)linkages,
and the reaction is the hydrolytic splitting of glucose.
The Enzyme Commission system consists therefore of a numerical classification hierarchy
of the form EC i.j.k.l, in which I. represents the class of reaction catalysed, j. denotes the
sub-class, k. denotes the sub-sub-class. The criteria used to assign j and k depend on the
class and represent details useful to distinguish one activity from another. All enzymes are
placed into one of the following classes:

2.1 Oxidoreductases
Oxidoreductases catalyse oxidation-reduction reactions. Their systematic names have the
form ?donor:acceptor oxidoreductase?, where the donor is the molecule becoming
oxidised (donating a hydrogen or electron). Their recommended common names have the
form ?donor dehydrogenase?, unless O
2
is the acceptor, in which case 'donor oxidase'
is permitted. The subclass describes the chemical group on the donor that actually
becomes oxidised (e.g. an alcohol, keto, or aldo-group). Sub-sub classes generally, but
not always , distinguish among acceptors (e.g. NAD(P)H, cytochromes, O
2
, etc.).
2.2 Transferases
Transferases catalyse group transfers from one molecule to another. Systematic names
logically have the form 'donor:acceptor- or group transfer' Recommended common names
are 'donor group transferase' or 'acceptor group transferase', but 'Acceptor-kinase'
(e.g. hexokinase) is used for many phosphotransferases. The subclasses distinguish in
general way among the various groups that are transferred (e.g. one-carbon transfers, acyl
transfers, glycosyl transfers) and the sub-subclasses employ greater detail in
distinguishing among the groups transferred. t is noteworthy that transamination reactions
between an amine and a ketone are classified as group transfer reactions even though the
ketone becomes reduced to an amine and the amine becomes oxidised to a ketone.
2.3 HydroIases
Hydrolases catalyse hydrolytic cleavage of C-C, C-N, C-O, or O-P bonds. They are
essentially group transfer reactions but the acceptor is always water. For this reason, and
because of the ubiquity and importance of hydrolases, they are awarded their own class.
2.4 Lyases
Lyases catalyse elimination reactions resulting in the cleavage of C-C, C-O, C-N or a few
other bonds, or the addition that constitutes the reverse of these reactions. Examples from
this category include decarboxylases, aldolases and dehydratases. The systematic names
are written as 'substrate group-Iyase' in which the hyphen is not optional. f the reverse
(addition) reaction is more important than the elimination reaction, the name 'product
synthase' may be used. Subclasses contain enzymes that break different bonds and sub-
subclasses distinguish among enzymes on the basis of the identity of the group
eliminated.
2.5 Isomerases
somerases catalyse structural rearrangements. Their recommended names correspond to
the kind of isomerisations carried out by members of the different subclasses: racemases
and epimerases, cis-trans-isomerases, tautomerases, mutases and cyclo-isomerases. The
sub-subclasses depend upon the nature of the substrate.
2.6 Ligases
Ligases catalyse bond formation coupled with the hydrolysis of a high energy phosphate
bond. The systematic names are written 'A:B ligase', and may specify 'ADP forming', etc.,
depending on the coupled energy source. Common names often include the term
synthetase, which the Commission discourages because of confusion with the name
synthase ( which does not involve ATP hydrolysis).

AII enzymes possess both a systematic name and a number. But, like microbial
taxonomy, the Enzyme Commission system is fundamentally a cIassification system,
rather than a naming system. The categories, like taxa, are not unique names, but
categories that contain groups of elements which can be further distinguished from one
another using other criteria. Although the EC nomenclature system is useful for identifying
enzymes, it is not entirely complete. To determine whether two enzymes are really the
same requires at least some degree of structure determination.

3. Enzyme Assay
Whatever is being done to obtain a good enzyme catalysed pathway or to study enzyme
regulations under set environmental conditions, it is of great importance to know the
quantity of enzymes being synthesized.
The amount of enzyme is not determined on the base of its chemical constitution but on
the basis of its catalytic activity, the rate of substrate conversion serving as a measure of
the activity. You will here more in the next few lectures on enzyme kinetics and thus will
not go into further explanations.
However, in order to obtain comparable values for a given enzyme the Enzyme
Commission of the nternational Union of Biochemists defined the 'internationaI unit' (U)
as the amount of enzyme that catalyses the conversion of 1 uM substrate per min under
standardised conditions of substrate concentrations, optimum pH, absence of inhibitors,
presence of activators.
This definition does not apply to most commercial enzyme processes because the assays
used do not even resemble the proposed conditions of enzyme application. For this reason
enzyme producer and user sometimes agree on an assay that meets the requirement of
the user. For example, in the analyses of amylases, the use of dextrose equivalents (D.E.)
units are commonly used.
n order to understand the synthesis and regulation of enzymes, it may be of advantage to
quickly remember the basic principles of enzyme or protein biosynthesis.
4.1 Screening of enzyme producers.
The first step in the manufacture of an enzyme involves the selection of an organism
suitable to produce the desired enzyme in amounts as large as possible. The general
aspects of this procedure can be outlined as follows:
1. Extracellular enzymes are preferred, because difficult and costly methods of cell
disruption are not necessary. As compared with intracellular enzymes, they are
present in relatively pure form in the culture liquid. ntracellular enzymes are
industrially used to a lesser extent because of difficult procedures of cell disruption
and separation of contaminating cell components.
2. High yields of enzymes should be obtained with an economical time required for
culture production.
3. The strain must be stable with respect to productivity, requirement for culture
conditions, and sporulation.
4. The organism should be able to grow on cheap substrates.
5. Synthetic activity should be as far as possible in the direction of the desired
enzyme. Formation of interfering by-products should be low.
6. Clarification of the culture liquor or extract should be possible without difficulties.
7. The strain must not produce toxic substances and should be free of antibiotic
activities. t should not belong to related strains that synthesize toxins. Mostly,
enzymes with particular properties, e.g. with respect to stability and activity, are
desired. This requires special screening programs.
4.2 Strain SeIection
n devising an enzyme fermentation it is important to begin with the most active strain
available. Known enzyme-producing strains can often be easily obtained from workers in
the field or from culture collections. n the absence of such cultures, a screening program
must be devised in which cultures from nature or from culture collections are examined for
enzyme activity. The major requirement in screening is simplicity, so that rapid
examination of a large number of strains is feasible.
n such strain selection one has to realize that macromolecules such as nucleic acids and
proteins are specific for the species and that the phylogenetic development which has
given rise to the microbiological variation basically has been caused by variations in these
molecules. As a consequence, it is to be expected that enzyme types, such as proteases
or alpha-amylase, which are found in several species, will have properties which vary as
much as the other properties of the organism. One usually finds that closely related
organisms have enzymes with closely related properties, while unrelated organisms have
enzyme systems which differ widely. For example, the protease Subtilisin Carlsberg from
Bacillus licheniformis is closely related to the protease Subtilisin NOVO from Bacillus
amyloliquefaciens, just as the species are closely related. Yet the differences are distinct
and thus specific for the species. Many incorrect classifications of microorganisms the
biochemical literature makes the establishment of such comparisons or rules very difficult.
The structural genes coding for the production of many enzymes are normally inactive in
the absence of the enzyme's substrate. That is, enzyme production is normally repressed.
The addition of a substrate induces or derepresses the enzyme, a phenomenon very
common in catabolic enzymes. Some inducers are very potent, e.g. certain galactosides
increase beta-galactosidase of E.coli 1000-fold, yielding cells possessing several per cent
of their cellular protein as this particular enzyme. Often the most potent inducers are
analogues of the substrate which are poor or inactive substrates. One can find products
rather than substrates as inducers of enzymes. Enzyme induction also occurs sometimes
upon addition of a coenzyme to the growth medium. For example, thiamine increases the
production of pyruvate decarboxylase.
Mutation can be used to eliminate the dependence of enzyme formation on inducer
addition. Such a mutation is termed reguIatory mutation since its locus is not a structural
gene but a regulatory gene. Therefore, an inducer is not needed, if a mutation at the
operator gene eliminates the production of an active repressor or if a mutation at the
operator gene eliminates its ability to bind a repressor. These mutants that produce a
normally inducible enzyme without inducer are called 'constitutive mutants', and can be
selected by a variety of methods. One of these methods is to grow the organism in a
chemostat with a limiting concentration of substrate inducer. This, for example, led to the
selection of E.coli mutants that no longer require a beta-galactosidase inducer, yet
produce high levels of beta-galactosidase. One also can plate out a population of
mutagenised cells on an agar plate with a medium containing as sole carbon source a
substrate that is not an inducer. Only constitutive mutants can grow under these
conditions. For example, constitutive beta-galactosidase producers were obtained with 2-
nitrophenyl-alpha-arabinoside.
Some enzymes are normally produced during growth but become repressed when
pathway endproducts buildup in concentration or are added to the growth medium. Such
low molecular weight endproducts (corepressors) are thought to combine with an
intracellular protein (aporepressor) coded by the regulatory gene, to produce a repressor.
This repressor then shuts off the structural gene coding for the enzyme and are called
repressibIe enzymes. A number of catabolic enzymes are repressed by immediate or
distant products of their activity. Thus proteases of many bacilli form poorly in media
containing certain amino acids. f one eliminates these compounds from the growth
medium will often increase enzyme yields considerably. Similarly, the limitation of
ammonia in growth media is known to derepress many enzymes that catabolize nitrogen
compounds. Sulfur compounds and phosphate can function very similarly on certain
enzymes.
More often than not, biosynthetic enzymes are repressed by endproducts. By limiting the
internal accumulation of endproduct corepressors, substantial increases in enzyme
production can be realized. Naturally, the presence of the endproduct as a medium
constituent should be avoided. For example,, production of glutamate dehydrogenase may
be increased 20-fold by using glucose or malate as carbon source for Bacillus
licheniformis instead of glutamate or casein hydrolysate
One way to limit internal build-up of endproducts is to add an inhibitor of the pathway to
the medium, thus limiting endproduct accumulation. For example the addition of 2-thiazole-
alanine to a bacterial culture can increase the 10 enzymes of the histidine biosynthesis up
to 30-fold.
Another way to reduce endproduct accumulation is to limit the growth factor supply fed to
an auxotrophic mutant. Such partial starvation leads to high enzyme production. nstead of
limited feeding one can use a slowly utilized derivative of the required endproduct. For
example, growth of an uracil auxotroph on dihydroorotic acid derepresses aspartate
transcarbamylase 1000-fold, yielding cells containing 7% of their protein as this enzyme.
Another means to derepress biosynthetic enzymes is by growing a partial or 'leaky'
auxotroph, which is an auxotroph that can grow in minimal medium slowly but is stimulated
by its growth factor, in the absence of its stimulatory endproduct requirement. A 500-fold
increase in aspartate transcarbamylase was possible by growing a leaky pyrimidine
auxotroph in minimal medium.
Regulatory mutants can be obtained that are not repressed by endproducts. Like mutants
no longer requiring inducer, these are also called constitutive mutants. They are thought
to be genetically altered in the regulatory gene so an inactive aporepressor is produced
that cannot combine with the corepressor. Alternatively, they may be mutated in the
operator gene so repressor binding does not occur. Such constitutive mutants, which
produce enzyme in the presence of normally repressing levels of endproducts, may be
selected by several procedures. One of the most popular method is selection for
resistance to a toxic analogue of the endproduct. Formation of many enzymes can be
removed from ammonia repression control by mutation to methylammonium resistance.
Certain enzymes, mainly of the catabolic inducible type, are markedly repressed when
cells are growing on a readily utilizable carbon source. This causes a decrease in the
intracellular concentration of cyclic AMP and in the absence of sufficient cyclic AMP,
structural genes may be turned off. Catabolic repression is very important in commercial
practice since many enzymes of current or potential industrial importance are subject to
this type of regulation. Avoiding the use of repressing carbon sources in the medium will
greatly stimulate production of enzymes sensitive to catabolite repression. For example,
growth of Bacillus stearothermophilus on glycerol instead of fructose increases
extracellular alpha-amylase production over 25-fold. Use of mannose instead of galactose
for growing Pseudomonas fluorescens var. cellulosa results in cells producing 1500times
as much cellulase. f one must use a repressing carbon source because of economic
involved, it is often possible to derepress enzyme production by limiting the rate of growth
by various feeding procedures. Slow feeding of glucose to above Pseudomonas strain still
increases cellulase yields about 200-fold. Mutation can be used to obtain mutants resistant
to catabolite repression. Many such mutants are apparently modified in their glucose
catabolic pathway, resulting in slower glucose utilization and derepression of a whole
series of enzymes. However, other mutants have been obtained in which resistance
specifically applies to a single enzyme or a single pathway.
n the isolation of enzyme-producing organisms or selection of mutants, traditional
microbiological methods are most often used. Especially important is the use of
enrichment cultures and selective media. Agar diffusion tests are used whenever possible
for the detection of enzyme activity. These tests can, however, be misleading as they
really only test exoenzyme production. n the design of isolation methods, the imagination
of the researcher is essential for the success.
Microorganisms may be pathogenic or producers of toxic materials, and this must be
constantly born in mind when working with unknown microorganisms. Until they are
identified, cultures and products should be handled with the proper care and precaution.
After the strain selection comes the strain development. Having obtained a mutant strain
growing on a plate does not mean that it grows in a liquid. The organism should grow on
an inexpensive medium and give a constant, high yield of enzyme in a short time.
Secondary enzyme activities and the content of metabolites in the fermented broth should
be minimal. Furthermore recovery of the enzyme should be simple and inexpensive and
lead to a stable product which can be handled safely and which has an acceptable
appearance. Last, but not least, the process must be safe to the personnel in the
production plant, and the effluents from the plant should not disturb the environment.
The fulfilment of these objectives requires a combined optimization of the strain properties
and process parameters. Optimization of strain properties is very attractive because this
offers an inexpensive and permanent solution to the problem. An example is the
development of a constitutive mutant which eliminates the need for an expensive inducer.

4.4 Strain Maintenance
The highly developed production strain must be protected against degeneration,
contamination and loss of viability. The most convenient way to secure this is to store the
strains lyophilized or at the temperature of liquid nitrogen. t is possible to store the strains
almost indefinitely without any changes using these methods.
Traditionally, microbial enzymes were produced by surface cultures. This technique is still
used for a few products, primarily of fungal origin.
Submerged culture methods dominate today in the production of enzymes, because
handling costs and the risk of contamination or infection are reduced, and because
modern methods of control are more easily adapted to these processes.

4.5.1 InocuIum.
Highly mutated strains are increasingly used for enzyme production. An important
requirement for a propagation technique is therefore that the production capability of the
strain be preserved and the technique should minimize the risk of contamination. t is
generally possible to develop a method where the seed flask is inoculated directly from a
lyophilized culture and where only one seed tank is used. The medium in the seed tank
often resembles the production medium. Excessive heat sterilization of the medium retards
the growth of the inoculum. The volume of the seed tank usually constitutes 3-10% of the
volume of the production fermenter. The propagation time in the seed tank varies from 10-
80 hours.

4.5.2 Medium Composition and Preparation.
The medium should provide the energy source for the process. t should therefore include
nutrient providing carbon and nitrogen sources and also special growth factor
requirements. Stimulating factors may be added to reduce the lag time or to increase the
growth rate. However, enzyme production may be negligible in a medium designed for
good growth. The strongest repression is see in media containing glucose.
Economy is very important in medium formulation. Typically, raw materials account for 60-
80% of the variable running costs of an enzyme fermentation process. Much
developmental work is directed towards the replacement of costly ingredients with
components available in large quantities at low costs, e.g. fertilizers for mineral salt
requirements. Most media are still sterilised batchwise in the fermenter. Continuous
sterilization methods are gaining wider application, particularly for the feeding medium.
The continuous heat sterilization is performed as a high temperature/short time process,
which results in improved preservation of growth factors and less colour development.

4.5.3 Process Conditions and Equipment.
Some microbial enzymes are relatively low-volume products, it has been difficult to justify
the development and construction of specialized equipment for submerged fermentation.
Equipment and techniques are most often adapted from antibiotic fermentations. Thus, tall
cylindrical fermenters of stainless steel with capacities of 10-100 tonnes, furnished with
strong mechanical agitators and air spargers are typical. The advantage of this setup is
flexibility as it is easy to switch between products.
Enzyme fermentations are especially vulnerable to microbial contamination. Rich media
with a neutral pH value are typical, and the protection afforded by antibiotic activity is
normally lacking. Strict attention must therefore be given to this contamination risk in the
design and construction of fermenters, pipes, and auxiliary equipment. A fully welded
system is recommended. Ports and valves should be steam-sealed, and all transfer of
cultures and media should be done by compressed sterile air. A positive pressure must be
maintained in the aseptic system.
As a rule, extracellular enzymes are produced by batch processes which last between 30
and 150 hours. The optimal harvesting time falls between the point of maximum
productivity and the point of maximum enzyme activity. Relative costs of raw material,
utilities, and recovery, as well as utilization of the plant capacity, determine the optimum.
While continuous methods are rarely applied, the batch process is often extended, and the
enzyme production favoured by continuous feeding of carbohydrate or protein. One
feeding strategy is to maintain a low reducing sugar level. One reason for the limited
application of continuous culture by the industry is the instability of the production strains.
Most enzyme fermentations have a high oxygen demand, requiring aeration and agitation
rates similar to those in antibiotic fermentations.
During process development, much attention must be given to the properties of the broth
in product recovery and to the quality of the final product. Since enzymes used for
technical purposes are marketed as rather crude protein solutions or precipitates, the
quality is strongly influenced by the fermentation method. Choice of raw materials,
sterilization method, foam control, aeration and agitation rates, and fermentation times
affect not only the product recovery yield and cost but also colour, smell, stability, powder
properties and similar quality parameters.
A general kinetic model for enzyme production has not been developed as yet (at least to
my knowledge). A few enzymes used commercially are formed during exponential growth,
but the vast majority are formed in the postexponential growth phase. A stoichiometric
relationship of the process cannot be expressed. The yield of useful enzyme protein may
reach 1-5% of the initial medium dry substance. The cell yield in a typical enzyme
fermentation may be 2-10% on a similar basis. Residual nutrients and metabolites usually
constitutes 5-10% of the broth at the end of fermentation.

4.5.4 GeneraI Fermentation aspects
As with other fermentation processes, for enzyme production the microorganisms are
cultivated by inoculating pure cultures into a sterile medium of suitable nutrient
composition, followed by incubation at controlled temperatures with the necessary
presence or absence of oxygen. Most commercial enzymes are derived from aerobic
organisms.
Growth CycIe. Everybody should be well aware that microorganisms multiply by cell
division and that there are five well defined phases - lag, logarithmic, stationary, declining
and survival phase - during incubation of an organism inoculated into a favourable growth
medium.
During the initial lag phase the culture becomes established and begins to multiply. This
lag phase may be very long or short, depending upon the organism, the medium and the
conditions of incubation. Extended lag phases are usually experienced when the growth
medium differs considerably in composition from that used in growing the culture as seed
culture or pre-culture or inoculum culture; when the culture is past the actively growing
stage, or when spore inoculation rather than vegetative cell inoculation is used. This initial
lag is usually of minor significance in the laboratory since maintenance of sterility in flasks
presents no problems, and time of fermentation is not important. n the plant, however,
minimizing or eliminating the lag is desirable since preventing contamination in large
culture vessels is a constant problem, and time of fermentation is very important in the
efficient utilization of plant equipment. Time aIways costs money ! By using inoculum
media of the same composition as used in the production fermenter and employing large
inocula of actively growing seed cultures, lag in plant fermenters may be almost
completely eliminated. Note that the pre-cuIture or seed cuIture or inocuIum decides
very often the economics of your production efforts.
The logarithmic or exponential growth phase is one of accelerating multiplication. During
this phase it can be assumed that the organisms are fully viable and all of equal vigour.
This is the stage where most of the ribosomes are formed for protein biosynthesis, which
is reflected in the growth of the organism. However, restrictive mechanisms, such as
accumulation of inhibitory products or exhaustion of essential nutrients, begin to come into
play well before maximum population density is reached. n an ordinary batch fermentation
the limit of growth is determined by the volume and the composition of the medium.
However, if fresh medium is continuously added to the culture, it is possible to prolong the
growth almost indefinitely in nearly logarithmic phase. This is, of course the basis of
continuous fermentation procedures.
Following the logarithmic phase is the stationary phase during which the cell population
remains almost constant. Some cells may continue to replicate, some remain static though
viable, and others may be dying. There is considerable variation in the vigour of individual
cells and in the type and rate of their metabolism.
n the declining phase, cell division still continues to occur but at an ever-decreasing rate,
and the number of new cells being formed is far outnumbered by the number of cells dying
off.
The last stage is the survival phase. Cell division ceases completely so that no new cells
are being formed. Only those cells which were formed previously continue to survive and
eventually die. Thus the curve of cell numbers versus time levels off and finally returns to
the horizontal axis.
There is great variability in the growth phase during which enzyme accumulation occurs,
depending upon the particular organism and enzyme. A desired enzyme may appear
mainly during any phase except the lag phase. For example, certain bacterial proteinases
are elaborated rapidly and almost entirely during the logarithmic phase, whereas
production of some bacterial amylases occurs mostly during the stationary phase. n some
cases an extracellular enzyme may begin to appear during the early part of the logarithmic
phase and continue to increase in amount during the later stages. ntracellular enzymes
are probably produced mainly during the logarithmic phase but are released into the
medium only as the cells undergo lysis during the declining phase. Therefore, recovery of
intracellular enzymes is usually best accomplished by harvesting the cells at the end of the
logarithmic phase, then releasing the enzyme into aqueous solution by lysis, mechanical
grinding, or ultrasonication.
During fermentation the concentration of a desired enzyme being produced will increase to
a maximum. Then, depending upon the particular enzyme, the concentration may remain
constant for a considerable time or it may decrease either slowly or rapidly. When the
latter occurs in a fermentation, there may be serious recovery problems involved in
obtaining good enzyme yields.
The problems of optimum time of harvesting and effective enzyme recovery methods, both
in the laboratory and in the plant, are affected by various factors of microbial growth and of
enzyme growth and stability. n the laboratory usually somewhat longer fermentation times
are required than in large-scale fermentations. Depending upon the organism and enzyme
desired, laboratory fermentations of 2-14 days are common, whereas similar fermentations
in the plant may require only 12 h to 6 days. n the laboratory small volumes are involved
so that when maximum enzyme production is reached the cultures can be harvested and
the enzyme recovered by simple methods, involving short times of minutes or a few hours
at most. n the plant, processing a large batch may require many hours in each of the
operations of filtering, concentrating, precipitating, recovering and drying. n fact, the most
difficult problems encountered in scaling up a process for producing a microbial enzyme
are usually not in the fermentation but rather in the subsequent recovery steps.

SteriIization. The only satisfactory means for sterilizing plant fermentation media prior to
inoculation with the desired cultures is by means of heat under pressure., and this is most
commonly employed for laboratory media also. However, there are major differences in
sterilizing conditions for laboratory and for plant fermenter media. n fact, sterilization is a
major problem in plant scale-up from laboratory fermentations.
n the laboratory, media in test tubes, flasks, and fermenter jars are sterilized in steam
autoclaves. Since small quantities of medium in tubes and flasks follow the temperature
cycle closely, heating and cooling periods are quite brief so that commonly 20 min at
120
o
C ensures sterility. When the volumes of medium and sizes of containers are
increased, there is a greater time lag of the temperature reached by the medium as
compared with that of the steam-space temperature. A 30 l vessel of aqueous sugar
solution requires about 2 hrs in an autoclave at an external steam temperature of 120
o
C
for complete sterilization. Media in which heat penetration is poor, such as those
containing undissolved solids or of viscous nature, require still longer times for sterilization.
Heat-sensitive medium components further complicate sterilization. f all ingredients are
soluble, in the laboratory small volumes of the complex media may be filter sterilized, or
the heat-sensitive material in solution may be separately filter sterilized and added
aseptically to the rest of the medium which has been heat sterilized.
n plant fermenters sterilization is often accomplished by heating the media in the
fermenters by means of steam jackets or steam coils, then cooling by circulating cold
water through the jackets or coils. Even with good agitation of the liquid in the fermenter, it
takes many minutes or hours, depending upon volume, to reach the sterilizing
temperature, say 120
o
C, and an equal or longer time to cool to inoculation temperature.
The long heating cycles are effective in sterilization but at the same time can cause
changes of destruction of essential nutrients as well as reactions between medium
constituents (caramelisation !!). Often such medium changes adversely affect growth of
the fermentation organism, its production of enzymes, or recovery of the enzyme from the
culture liquid. Sometimes it is possible to sterilize certain medium ingredients separately
from the main batch and add these aseptically at fermentation temperature to minimize
undesirable changes. Because of the volumes involved, filter sterilization of media or
medium components is not usually possible on the plant scale.
To avoid undesirable heat-caused changes in fermentation media, to approximate more
closely laboratory sterilizing processes, and to make more efficient use of fermenter
capacity, continuous high temperature, short-time sterilization methods are commonly
used for the media for large fermenters. Suitable continuous processing of free-flowing
liquids permits very fast heating, close control of the holding time, and rapid cooling. The
cool sterile medium flows continuously into the fermenter which has been previously
sterilised by steam under pressure. Usually the culture is introduced into the partially filled
fermenter, and growth begins during filling of the fermenter
The problem of contamination of fermentations by undesired organisms is much greater in
large-scale fermenters than in the laboratory. Contamination in laboratory flask
fermentations is not a serious problem, assuming good laboratory procedures are
followed. Preventing contamination in an industrial process is a constant battle.
Fermentations can be contaminated through such routes as faulty agitator seals, leaks in
cooling coils, inadequately protected inoculum, antifoam and sampling lines and valves,
and failure of air-sterilizing filters

Agitation and Aeration. Since most commercial enzymes are produced by aerobic
organisms, aeration and agitation must be continuous during the course of the
fermentation in submerged culture. n the laboratory the most common practice is to
employ shaken cultures in flasks. Aeration in tubes or cylinders employing porous air-
dispersing devices also may be used. Small jar fermenters provided with mechanical
agitators and air spargers, similar in arrangement to industrial deep tank fermenters, have
become common in well-equipped laboratories.
Plant fermenters for aerobic submerged culture fermentations were originally developed
for the production of antibiotics and now are widely used. For enzyme production they may
vary between 1000 to 30,000 gallon or 4000 to 120,000 litres capacity. To provide
agitation, a shaft, rotated by a powerful electric motor, runs from the top to the bottom of
the fermenter, with an efficient seal at the top and a steady bearing at the bottom. Near its
lower end the shaft carries a flat turbine impeller about two-fifths of the tank diameter with
either straight or slightly curved blades. Sterile air is introduced by a sparger immediately
below this impeller so that the air is intimately and continuously mixed with the fermenter
contents. There may be one to four additional impellers higher up the shaft. Most
fermenters have four vertical baffles inside the tank, about one-tenth of the tank diameter
in width. For temperature control, cold water is circulated as necessary through jackets or
coils. Foaming is controlled by introduction of sterile antifoam agents.
Agitation and aeration in laboratory shaken flasks are quite different from plant fermenters.
Laboratory jar fermenters simulate plant fermenters and are useful in developing
successful fermentations. However, they provide little useful information about optimum
plant fermenter operating conditions. To obtain maximum enzyme yields from any
individual plant fermentation, optimum conditions of aeration, agitation, and power input
must be determined rather empirically. When once established for a particular
fermentation and vessel, these conditions are adhered to rigidly in plant operations.

4.5.5 CuItivation Conditions
Let us now have a look at the various cultivation techniques used for the production of
enzymes and the equipment used.
Surface or SoIid Substrate CuItivation. This method plays an important role in
commercial enzyme production from fungal sources, especially in Japan and ndia.
Advantages and disadvantages of this method, as compared with submerged culture
technique, have often been analysed. Considering modern deep bed processes, the
following advantages can be stated as a matter of fact:
1. enzyme yield per unit volume of incubator is high;
2. power requirement is low;
3. minimum control is necessary;
4. extraction yields highly concentrated enzyme solutions;
5. only small equipment for enzyme recovery is required due to small
6. amounts of extracts obtained;
7. scaling-up is easy.

Problems which can be solved, if need be, may be listed as follows:
1. continuous operation is possible;
2. feeding substrates during cultivation is possible;
3. defined media can be applied by using suitable inert carriers
Caused by the nature of the complex media used, the extracts contain considerable
amounts of fungal pigments, the removal of which is difficult and costly. Their formation
might be avoided by using a 'solidified' synthetic medium.
The methods of solid substrate cultivation can be divided into two groups: thin layer and
deep bed process.
The thin Iayer techniques, also called tray process, work with substrate layers of 2-4 cm
height spread on wooden or metallic trays. These are incubated in air-conditioned rooms
or cabinets. Usually the heat produced by the growing culture is removed by moistened
cool air which passes over the surface of the trays or is pressed through the bran mass.
The deep bed process developed by Terui and coworkers in order to meet the enormous
demand for enzymes needed for the traditional soybean fermentations, uses substrate
layers usually as deep as 0.6 m, but beds as deep as 1.5-1.8 m have also been reported.
The dimensions of the rectangular beds is of the order of 5.5x61 m. Circular beds are also
operated. n general, the equipment used in deep bed processes is quite similar to that
known in the malting industry. Whereas automation of the tray process has not been
successful sofar, the deep bed process plants are fully automated. Continuously operated
deep bed techniques may, for example, guide the culture mass through air-conditioned
tunnels by means of conveyer belts.
The use of rotating drums for the production of mould bran on an industrial scale was only
of temporary importance. With this method manufacturers experienced many difficulties
and the desired results were not always obtained. Some workers attributed this to damage
of the mould mycelium by mechanical action, but it is not really clear why this method does
not work successfully. believe that this method is still under investigation in ndia.
Media used in solid substrate fermentations are mainly based on wheat bran. This material
is particularly suitable because of its high content of nutrients and its large surface. Other
basic substrates are rice bran and soybean or sweet potato flakes, as well as grains or
soybeans, etc. Kernels should be selected for optimal size or cracked to give particles of
the desired size. On bran, superficial growth of the fungus is sufficient for utilization of
nutrients. Kernels, however, must permit the inoculum to penetrate. This is not difficult with
polished rice, but corn or soybeans must be cracked or freed of the hulls. The amount of
water needed for moistening the substrates is in the range of 40 to 70%. n the case of
grains and soybeans the optimum content of moisture is about 30%.
Pressure sterilization of large masses of wet bran in bulk presents serious problems. A
convenient method to ensure thorough sterilization is by direct steam injection into the
bran. During this process the mass is agitated so that each particle of the moist bran is in
constant direct contact with the steam. Experience, however, has shown that when acidic
solutions are employed in place of water for moistening the bran it was quite sufficient to
sterilize the bran at 95
o
C for 15 to 30 minutes. n some cases decontamination of the bran
was achieved by means of bactericides, e.g. formaldehyde or beta-propiolactone.
noculation of the sterilized medium is carried out by use of spores in a dry or suspended
form. The amount of inoculum varies from process to process depending on a number of
factors. The actual amount must be determined empirically. t was found that even as low
an inoculum ratio as 0.04% of a dry spore culture was satisfactory in fungal amylase
production. Attempts had been undertaken to use an inoculum in the mycelial form which
can be easily produced in large quantities by submerged culture. However, this method is
not convenient, resulting in non-uniform growth of the fungus throughout the bran mass.
Conidia can be produced in large quantities in special cultures, whereby the method of
fermentation is quite similar to that applied in enzyme production. n order to promote
spore formation it may be beneficial to add a balanced solution of trace elements (Fe
3+
,
Zn
2+
, Ca
2+
, Mn
2+
).
Among the conditions of incubation, moisture and temperature present the most serious
problems. t was soon recognised that these two factors are dependent variables. Growing
cultures produce heat which tends to evaporate the water of the substrate. This effect is
intensified by warming the air when it passes the culture and thereby enlarging its capacity
to dissolve water vapour. These processes inevitably cause a drying out of the culture.
However, the water loss is partially replaced by water that is formed by the metabolic
activity of the organism. The difference must be made up by application of sprayed water.
The microbiological approach to this problem is the selection of slowly growing strains with
high productivity. Short-time fermentations may also be helpful.
Heat production is indeed considerable. ts magnitude can be readily appraised by the loss
in dry weight of the culture. t has been demonstrated that almost all this loss is due to the
oxidation of organic compounds to CO
2
. n many cases it was found that approximately
half of the dry weight of the bran disappears. t was for example observed with Aspergillus
niger that under industrial conditions up to 380 J/h/kg of fermented bran were liberated at
maximum heat production. Removal of this amount of heat required 20 m
3
air with 20-28
o
C
temperature and 100% moisture.
Regarding the moisture content of the medium, it has been found in many cultures that
careful observation of the tolerated limits has a decisive influence on the production of the
enzyme. n this respect it is very important to keep in mind the previously mentioned
formation of water by metabolic processes. nitial pH and the course of the pH during the
development of the culture also play a great role. Several means are known to influence
the pH development. For instance, this can be done by incorporation of suitable inorganic
or organic salts in the medium.
The semisolid surface culture method is now used extensively in producing such
commercial enzyme products as fungal amylase (Aspergillus oryzae), fungal protease
(A.oryzae), fungal glucoamylase (Rhizopus species), fungal cellulase (A.niger,
Trichoderma viride), and fungal milk clotting enzyme (Mucor pusillus).
4.6 Purification
t is nearly impossible for the inexperienced researcher to find detailed guidelines to
protein purification procedures in the literature. Numerous methods of protein purification
are available, and the successful purification process will require the use of several of
these methods in combination. The growth of biotechnology as an industry and the
confidentiality of methods that coincides with such development have made it difficult to
find published information on new purification development. Manufacturers of purification
equipment and materials provide little application data because of confidentiality
agreements they have with their clients.
As one sets out to purify proteins as fine chemicals there is much to consider. From the
practical view point the budgetary constraints and required yield will have a major impact
on the plan. Will the purification costs be supported by the final use ? s a protein purified
to homogeneity desired or may the protein be functionally pure ? Will the final use of the
product be regulated by the government authorities (e.g. FDA in USA) ? The ultimate scale
of the purification process plays a major role in the purification scheme developed. The
ultimate process should contain as few steps as possible in order to maximise yield and to
minimise the required investment of labour, materials and equipment. n general, one can
expect a 20% loss of yield with each critical purification step.
Due to all of the parameters involved in the process, such as starting material,
fermentation processes, cell lysis techniques, clarification techniques and the wide range
of purification options, developing a purification design that produces the optimal yield of
the purest protein in the least amount of time for the lowest cost is likely to take a great
deal of effort. t is therefore only possible to describe a few purification options for enzyme
purification, which should not be seen or used as a purification design.
The purification scheme can be significantly different depending on whether the protein is
expressed intracellularly or extracellularly. f the protein is expressed extracellularly,
several advantages exist, since it will not be necessary to rupture the cells, but a
concentration step is eventually necessary.
4.6.1 Extraction.
The first step in an enzyme purification is nearly always the extraction of proteins from
cells, tissues or the fermenter solution. Techniques to liberate enzymes from cells include
disruption using a French pressure cell, sonication or tissue homogenisation, or milling
with glass beads. The method used depends to some extent on the nature of the material
from which the enzymes are to be extracted. n the case of extracellular proteins, it is
advisable to combine the concentration of the protein with a purification step. This can be
achieved in a number of ways, such as ammonium sulfate precipitation, adsorption onto a
resin or by ultrafiltration
n general, cell disruption is performed at 4
0
C to prevent denaturation and to decrease
protease activity. A few enzymes are cold-labile, and purifications of these enzymes are
best carried out at room temperature. Cell disruption also liberates proteases. Nucleophilic
proteases such as serine proteases can be inhibited by the addition of inactivating agents
such as diisopropylfluorophosphate (DFP) or phenylmethylsulfonyl fluoride (PMSF). The
latter is easier to handle as DFP is rather volatile. Both of these reagents become
covalently attached to the active site of the nucleophilic group and cause permanent
inactivation. Because of the lability of freshly extracted enzymes, enzymes in various
states of purification are generally stored frozen (-20
o
C or even -70
o
C) and quickly thawed
by placing the tube in room-temperature water. Care is also taken to prevent foaming
during extraction. Furthermore, unpaired sulfhydryl groups oxidise easily in air-saturated
solutions. Consequently, reducing agents such as 2-mercaptoethanol or dithiothreitol may
be included.
Buffers for enzyme extraction. Enzymes are usually extracted into pH buffered solutions.
Sodium or potassium phosphate or acetate buffers have always been very popular for
buffering near neutrality. However, where phosphate ion may affect enzyme activity or
interfere with assays, or when other pH values are required, an array of organic, usually
zwitterionic, buffers are available for buffering within most pH ranges.
Tris(hydroxymethyl)aminomethane (Tris), commonly used to buffer near pH 8, HEPES,
near pH 7, and MES near pH 6 are common choices.
Membrane proteins present their own special set of problems as they are not water-
soluble. A useful approach has been to add chaotropes, non-ionic detergents (e.g. Triton
X-100) and emulsifiers (e.g. bile salts such as deoxycholate) to the extraction buffers

4.6.2 SaIting-out and precipitation
One of the oldest methods of purification, salting out, is still a useful procedure, either as a
crude purification step or as a method to concentrate the extracted proteins before the
next purification step. Ammonium sulfate fractionation is a popular method. The addition of
ammonium sulfate to a protein solution lowers the solubility of the protein and it lowers the
solubility of different proteins to different extents. Solid enzyme-grade ammonium sulfate is
pulverised and added slowly, with stirring, in the cold, to a crude extract of protein to a
particular final concentration which is usually expressed as a percent of saturation. The
precipitate is allowed to flocculate and is removed by centrifugation. The supernatant
solutions and resuspended pellets are assayed for total activity and total protein. f
necessary, more ammonium sulfate is added and with luck, one of the fractions (e.g. the
precipitate obtained between 40-60% saturation) may contain the bulk of the activity of
interest or may be enriched in the activity. This fraction is then used for further purification.

4.6.3 DesaIting
Before an ion-exchange chromatographic step or after an ammonium sulfate fractionation,
it is usually necessary to remove the salt from the solution of protein. Desalting can be
carried out either by dialysis or via gel filtration.
Dialysis. Dialysis is performed by filling a section of dialysis tubing [semipermeable
membrane] having a sufficiently small molecular weight 'cut-off' , with the protein solution,
and placing the filled tubing in a large volume of buffer. The decrease in salt concentration
can be calculated easily from the ratio of the volume inside and outside of the bag. Dialysis
requires a few hours, after which the bag may be transferred to fresh buffer.
During dialysis all small molecules, including salt ions, metal ions and cofactors, pass
through the membrane, which retains only macromolecules. Tightly bound metal ions and
cofactors are not effectively removed.
Since the initial solution in the bag is of much greater osmotic strength than the
surrounding buffer, the bag generally increases in volume. The volume of the contents of
the bag must be measured after dialysis if either protein or total enzyme units are to be
calculated.

Gel or size-exclusion chromatography. Gel filtration is a form of chromatography in which
solutes are separated from one another based upon the degree to which the solutes fit into
pores on the stationary phase. Gel filtration media can be obtained with many different
pore sizes, so that the technique may be tailored to the separation needed. For desalting,
usually a gel of rather small pore size is employed so that only very small solutes (MW
<5,000) fit easily into the pores, and enzymes are totally excluded. For bulk desalting of
protein solutions, usually a porous polysaccharide such as Sephadex G-25 or BioGel P10
is selected. The elution behaviour in gel filtration follows the standard rules of partition
chromatography:
Ve = Vo + KavVs
in which Ve is the elution volume, Vo is the volume of the mobile phase between particles
(= void volume), Kav is a partition coefficient, and Vs is the volume of the stationary phase.
t should be clear that Kav for salts is essentially 1, while that for proteins is essentially
zero, hence separation is maximally efficient for desalting. Because of the unusually large
separation, a desalting column can be loaded with a volume of sample that is up to 30% of
the bed volume to maximise throughput.
Gel filtration is not without artifacts. Some aromatic compounds and strongly charged
proteins adsorb to the packing itself and are retained by this mechanism. This problem can
usually be resolved by using a buffer (=mobile phase) of rather high ionic strength (>50
mM). Efficiency is maximised by using low flow rates and small diameter media. For
desalting, high throughput is more valuable than high resolution, so larger media (coarse)
are used, but flow rates should be kept slow.
4.6.4 Ion exchange chromatography
Since proteins have different net charge and charge distribution, ion exchange
chromatography can be an effective purification tool. For bench-top preparations, usually
gravity-flow columns are employed, but HPLC and automated HPLC-like systems have
grown on popularity. For gravity flow or for use with low pressure peristaltic pumps, ion
exchange media are usually carbohydrate-based. Charged groups are attached to solid
supports (inert phase) such as sepharose, Sephadex and cellulose. Since these
carbohydrates are compressible, they are not used in high pressure systems, and more
rigid inert phases such as TSK (a polyether-coated gel) are used.
The charged groups used with the solid supports depend to some extent on the chemistry
of the support material itself, but are remarkably similar. Groups containing charged
nitrogen atoms are almost universally used for anion exchange media. These include,
from strong to weak, quaternary amino methyl or ethyl (QAE), tertiary
amino(diethylaminoethyl, DEAE, or diethylaminomethyl) and secondary plus tertiary
nitrogens (polyethylenimine, PE). The conjugate base of the strongly acidic sulfonic acid
(i.e. alkyl, or aryl sulfonate) and the weakly acidic carboxylic acids (e.g. carboxymethyl,
CM) are the most common charged groups employed in cation exchangers. The
carboxymethyl packings must be used at a pH above their pKa (~4). Methods for
determining the optimal pH for separation of proteins depends, of course, on the proteins.
Since most proteins are acidic, they are negatively charged at pH 7-8. They therefore
adsorb to a positively charged stationary phase to which they act as counterions, providing
that other anions are not available to play the role of counterion. The cationic stationary
phase is known as an anionic exchanger because it functions by exchanging one anionic
counterion for another.
At pH values below the isoelectric point of a protein, the net charge is positive, so
negatively charged stationary phases (cation exchange phases) are used. f a protein has
an isoelectric point near neutrality, either a cation exchange or an anion exchange system
can be used, depending on the pH being used.
soelectric point titration curve analysis will provide information about the protein solution
that will aid in the optimisation of an ion exchange step. Pharmacia's PhastSystemTM will
generate a titration curve in approximately 100 minutes. This electrophoretic step will
indicate the buffer pH that will create the greatest difference in charged properties of the
proteins in the solution and whether the proteins will bind better to an anion or cation
exchange resin at that pH. When performing an EF titration curve analysis, running an EF
standard marker is recommended, as the pH gradient created by the ampholines may not
be linear. nformation generated from titration curve analysis should be used to define
optimal chromatography conditions which are best employed on a strong ion exchanger,
which can be utilised over a wider pH range than weak exchangers
Protein solutions are generally desalted, the loaded onto a column packed with a
stationary phase having the appropriate charge. Loading can often be done as rapidly as
the columns will flow without undue pressure; proteins that adsorb are retained at the top
of the column. As long as the capacity of the column is not exceeded, litres of a (desalted,
buffered) crude extract can be loaded onto a column of modest size, so that a pre-
chromatography concentration step is not required. After loading the column is washed
with the loading buffer to remove unadsorbed and weakly adsorbed proteins. The
adsorbed proteins are then eluted by washing the column with buffers of increasing salt
concentration (e.g. NaCl), which corresponds to increasing solvent strength. This method
of elution using a series of isocratic (constant strength) elutions of progressively increasing
strength is known as batch elution.
A simple and common solution to elution is to employ a linear concentration gradient of
salt using an easily constructed siphon apparatus. Such a gradient can cover a range from
0 to 1 M NaCl over the volume of a few hundred millilitres to a few litres, depending on the
dimensions of the column and the steepness of the gradient desired. A major advantage of
gradient elution is that proteins having a wide range of affinities for the column can be
eluted in a single run.
To exercise maximum control over the system, it is useful to separate the effects of pH
from those of ionic strength during ion exchange chromatography. One of the ions involved
in the buffering system bears the same charge as the protein and therefore act as a
displacing ion. Buffering ions seIected for use in ion exchange chromatography
shouId have the same charge as the coIumn, i.e. cations for an anion exchange
coIumn, anions for cation exchange coIumns. Hence phosphate buffers are used for
cation exchange chromatography, and Tris buffers are used for anion exchange.
For the sake of performance and reproducibility, it is necessary for the column to be
completely equilibrated with the starting solvent. Equilibration can be checked by
measurement of both pH and ionic strength (e.g. conductivity) prior to loading the column.
When performing preparative work, a column should be loaded to only 10% of capacity.
While a conventional column may be able to handle the pressures generated by a high
flow rate, the higher flow rate may result in proteins not binding to the column that would
normally bind at lower flow rates. Pharmacia has designed the FPLCTM system to
manage high performance ion exchange chromatography with Mono S and Mono Q high
performance resins. FPLC operates under moderate pressure, giving higher speed and
resolution than conventional chromatography. Mono S and Mono Q are composed of
smaller, more rigid beads than that of their Sepharose counterparts. These monobeads
are of plastic composition, which make them more hydrophilic than their Sepharose
counterparts. The smaller and more rigid the bead size, the better and faster the resulting
separation. The FPLC has been designed to deliver and withstand the pressures required
to deliver flow through columns composed of small beads. The rapid processing provided
by this system, along with titration curve analysis, will allow for efficient optimisation of an
ion exchange purification. A salt gradient providing high resolution can be applied to the
column in as little as 10 min. With automation, several pH conditions and gradient slopes
can be tried within a few hours.
Elution from an ion-exchange column could also be accomplished using a change in pH.
Stepwise pH changes are sometimes employed, but do not generally produce high
resolution of complex mixtures. A workable system along these lines has been devised
using a proprietary mixed-bed packing and a multi-component buffer system to elute
proteins at their isoelectric pH. This process is called chromato-focusing because of a
loose analogy to isoelectric focusing gel electrophoresis.
4.6.5 Hydroxyapatite Chromatography
Although size exclusion and ion exchange chromatography have been used often since
the 1960s, a few other chromatographic techniques are occasionally employed. A useful
stationary phase has been hydroxyapatite. Like other calcium phosphate gels employed
for chromatography, the adsorbent has had a checkered and mystical past; it always
seemed necessary to indicate the age of the adsorbent and how it had been aged.
Nevertheless, in order to explain the elution of proteins from hydroxyapatite, proteins are
classified into three loose groups: (1) basic proteins (p >8), which are eluted by 1-3 nM
Mg
2+
; (2) neutral proteins (5>p.8), which are eluted by 1-2 mM Mg
2+
; and (3) acidic
proteins which are not eluted by Mg
2+
, even at 3 M.
4.6.6 Size excIusion chromatography
Size exclusion chromatography (SEC) or gel filtration, separates proteins based on
molecular size. This method is also often referred to as gel permeation chromatography.
Gel filtration matrices are composed of porous beads that are typically cross-linked
dextran (Sephadex, Pharmacia) or cross-linked acrylamide (BioGel, BioRad). The major
differences between the wide range of gel filtration matrices is the pore size of the bead.
The size of the pores in each resin bead is influenced by the degree of cross-linking. A
range of matrices are available that can separate compounds with molecular weights
within the nominal range of 700 - 800,000 daltons. When these beads are packed into a
column, a volume external to the beads and a volume internal comprising the internal
volume of the beads is created. Molecules larger than the pore size of the beads will be
excluded from the internal volume, while proteins that will fit into the pores will travel
through both volumes. When flow is applied to the column, the distance through the
column is longer, then, for molecules able to fit into the pores than it is for molecules
excluded from the pores. Therefore, larger molecules will elute from the column first.
Proteins will not elute solely based on the molecular weight, but also based on the
molecular size.
Elution is performed isocratically since there is no binding to the column. A measurement
of elution is made in volume, referred to as elution volume, and not in concentration of
eluent. The selectivity of the resin is not adjustable by changing the composition of the
mobile phase and is only dependent on the dimensions of the resin itself. However, the
resolution provided by gel filtration is limited. Typically, fewer than 10 proteins can be
resolved from one elution. This technology is most useful when purifying a large protein
from a small one.
The size of the column load should be no more than 5-10% of the total volume of the
column [ protein concentration < 50 mg.ml]. A concentration step prior to gel filtration is
usually necessary and can be done by ultrafiltration, ammonium sulfate precipitation, or
ion exchange chromatography. Another disadvantage of size exclusion chromatography is
that the allowable flow rates are very low. These two parameters of SEC make this method
rather inefficient. No method is so inefficient, however, if it provides the required
purification, especially if other methods have failed.
4.6.7 Affinity Chromatography
Affinity chromatography is considered to be the simplest, yet most powerful
chromatographic method. Affinity chromatography is simply a method that takes
advantage of a protein?s natural interaction with other biomolecules. The molecule to
which the protein binds is referred to as the ligand. A good ligand choice will be one that is
involved with the target protein in either an enzyme-substrate interaction, an enzyme-
cofactor interaction or an enzyme-inhibitor complex. Affinity chromatography is a high
capacity, high-resolution purification step that is fast. Affinity purifications generally are
very fast to perform because no gradient may be necessary. All that is needed is
adsorption, washing and desorption. Affinity resins are typically expensive and may best
be used late in the purification stage, if multiple purification steps are necessary. t may be
disadvantageous to use affinity columns as the last step of purification, as the ligand
typically leaches from the column.
Affinity purification is particularly powerful in instances when the target protein is a small
percentage of the total cellular protein. This method is useful for concentrating target
proteins in dilute solution. These biological interactions often rely on the biological activity
of the target protein. Therefore affinity chromatography often serves as a means to
separate functionally active from inactive molecules. Proteins are stabilised when they are
absorbed to the ligands. The matrix providing the best support for affinity chromatography
is a cross-linked agarose. Good choices are CNBr-activated Sepharose 4B (Pharmacia)
and Affi-Gel (BioRad).
Due to the power of an affinity purification, this technique may be the only one necessary
to purify the target protein. n some instances, however, it is recommended to precede the
affinity column with a precipitation or ion exchange step to remove particulates, lipids,
major contaminants and to reduce the volume of the material to be processed.
The affinity resin should be prepared to contain 1-10 ug ligand/ml of resin with a low
molecular weight ligand or 1-10 mg protein ligand/ml of resin. Because the dimensions of
the column are not critical to the performance of the matrix, the column can be packed
short and squat to allow rapid flow rates. The size of the column is dependent on the
capacity of the resin. f the target protein is only loosely associated by the ligand, a longer
column will improve fractionation. Flow rates can be reduced or stopped or batch
adsorption can be used to maximise binding efficiency. Proteins typically elute from affinity
columns in broad peaks.
As with any chromatographic step, it is important to wash the column after the protein load,
at least until the A280 absorbance has established a minimum. This typically requires
approximately 3 column volumes of wash.
Antibody affinity methods. Probably no other chromatographic step can be as selective
as antibody affinity chromatography (also referred to as immunoaffinity chromatography). t
can be difficult, however, to prepare poly- or monoclonal antibodies to the target protein.
As a result, this mode of purification is very expensive. However, the resulting savings in
labour and additional equipment needs may compensate for the expense of this step. This
one purification step can increase the specific activity of a protein preparation several
hundred-fold to two thousand-fold
Dye ligand chromatography. Dye molecules often mimic biological compounds, such as
coenzymes, nucleotides and polynucleotides, to which the target protein may naturally
bind. These reactive dyes can bind proteins by either specific interactions at the protein's
active site or by a range of non-specific interactions. Dye resins have been prepared to
take advantage of this phenomenon. Dye chromatography is well suited for proteins that
have an affinity for aromatic compounds, such as those that interact with nucleotides and
cofactors. These dye ligand resins are more likely to withstand wear than are the affinity
resins prepared with antibodies or enzymes. Three examples of such dye-ligand resins are
Matrex Red (Amicon), Blue Sepharose CL-6B (Pharmacia), and Affi-Gel Blue (BioRad).
Blue dye, or Cibacron Blue F3G-A, is an analog of adenylyl-containing cofactors. The red
dye is Procion Red HE-3B.
Dyes typically used for this method of affinity purification are often of crude purity;
therefore care should be taken in choosing the source of dye, as dyes will typically leach
from the resin.
The binding capacity of these resins ranges from 1 to 15 mg protein.ml. An immobilised
dye can simultaneously bind 5-60% of the total protein in a crude cell free extract. As with
ion exchange resins, the bed should be prepared to accommodate 10-fold more protein
than is present in the solution to be purified. Proteins can be eluted from a dye ligand
column with high salt or with a competing ligand, such as ATP, a gradient is recommended
for fractionation. The bonding of some proteins to these affinity dyes can be enhanced by
the presence of a low concentration of a metallic cation such as Zn
2+
etc in the buffer.

Perfusion chromatography is one of the newest developments in protein purification by
liquid chromatography. Perfusion matrices are beads containing channels or pores. The
design allows the material to flow through the pores and interact with the active sites that
line the pores. Therefore, much more of the surface is readily available to the protein
sample. Because there is more contact with active sites, higher flow rates can be used
without sacrificing resolution and binding efficiency. The manufacturers of these columns
state that separations of acceptable resolution can be performed in minutes using these
columns; separations can be performed 100times faster than with conventional low
pressure chromatography.
4.6.8 ConcIusion
The purification methods indicate that liquid chromatography is generally recognised as
the technique that allows the highest degree of purification of biomolecules. n contrast to
precipitation, electrophoresis and ultrafiltration, chromatography does not involve heat
generation or major shear forces. Conditions close to physiological can be maintained.
The first step in designing a chromatographic purification strategy is to characterise as
much about the protein sample as possible. This includes characterisation of both the
protein of interest and of contaminating proteins. Knowledge of the molecular weight (both
native and denatured); amino acid sequence, isoelectric point, solubilities in the presence
of different salts, organic solvents and temperature; hydrophobicity, location in the cell and
binding characteristics to the chromatographic media as a function of pH will maximise the
successful design of the chromatography step. Taking advantage of the biochemicaI
differences between the target protein and the contaminants is the key to
successfuI purification. Purification of the target protein from a functional contaminant is
a challenge. A functional contaminant is one that interferes with the biological activity of
the target protein; often the proteins have similar binding sites and are therefore very
similar biochemically. For example, purifying a DNA binding protein such as DNA
polymerase from other DNA binding proteins that would interfere with the successful use
of a polymerase for molecular research is an arduous undertaking.
n general it is easy to remove the first 95% of the contaminants, but it is difficult to remove
the remaining 5%. However, when purifying proteins from wild-type organisms, the
pertinent protein can be 0.00001% or less of the total cell mass. This high degree of
required purity demands choosing reagents of high purity.
During the design of a purification process it is important to keep in mind the stability of the
protein both during the purification and as a purified product, the required yield, the
ultimate scale, and capital expense of the process. Other factors to be considered when
designing a purification procedure include:
a) the initial step should be of high capacity and low cost;
b) the lower capacity, higher cost steps should be used for the final steps of the
purification;
c) since the scale of a purification usually decreases as the process proceeds,
the most difficult and expensive steps should be used last;
d) choose purification steps that are very different from each other;
e) enzymes can loose activity when exchanged from one buffer into another.
The organisation of columns to minimise buffer exchanges is recommended;
f) the most plentiful impurity should be removed early and the most selective
purification steps should be used first.
g) the ideal chromatography step is designed such that the target protein does
not bind to the column and the contaminant does, or to have the target
protein be the first or last protein to be eluted, presenting the need to pool
away from contaminants on only one side of the peak;an inverse relationship
exists between the number of purification steps and the final yield of the
protein. A typical yield after each step is approximately 80% of that which
went into the step. Therefore it is necessary to limit the number of purification
steps.
Manufacturers of chromatographic resins have designed resins to bind proteins using
therefore on of several of the following interactions: electrostatic, hydrophobic, hydrogen
bonding or van der Waals. t was found that the best matrix for protein purification is a
cross-linked agarose. Several cellulose-based resins are available, but these fibrous
particles present flow rate challenges. Cross-linked acrylamides or styrenes tend to result
in non-specific binding to the resin itself, rather than to the functional groups.
The size of the column should be 5-20times the binding capacity of the protein preparation
as determined by batch experimentation. The dimensions of the coIumn can help or
hinder the purification and should be seriously considered. When increasing the scaIe of a
coIumn, increase the radius of the column but maintain the height and linear flow rate.
The linear flow rate is calculated by dividing the volumetric flow rate by the column cross-
sectional area.
Example: A Pharmacia XK-50 column has a cross-sectional area of 19.6 cm
2
. f a flow
rate of 5 ml/min was being applied to the column, the linear flow rate would be:
5 mI/min divided by 19.6 cm
2
= 0.25 mI/cm
2
/min
f the column is going to be scaled up into a Pharmacia BP 113, with a cross-sectional
area of 100 cm
2
, the flow rate needed for proper scale-up would be:
0.25 mI/cm
2
/min = voIumetric fIow rate divided by 100 cm
2

= 25 mI/min
The resolution of the purification is primarily determined by the elution conditions and not
by the length of the column. Columns should be wide and short, although long columns
are useful for proteins with weak binding characteristics as the long contact time will
increase binding.
Whatever type of liquid chromatography is performed, the column should be designed to
minimise pressure drops, mixing and loss of resolution. The dead space between the end
of the column and the fraction should be minimised to prevent mixing and loss of
resolution. This requires minimising the length of tubing between the column and UV-
detector and between the detector and the fraction.
The packing of a chromatographic column is critical to the performance of the column. An
unevenly packed chromatography bed and entrapped air bubbles will lead to channelling,
zone broadening and loss of resolution. Cross-linked resins are simple to pack. t is
recommended that any alcohol the resin is shipped in is first rinsed from resin with water.
This can easily be performed on a sintered glass funnel. Rinsing the alcohol from the resin
before it is poured into the column will prevent bubble formation from the degassing due to
the heat generated from the alcohol mixing with the aqueous buffer.
The resins can be used to purify proteins in one of three ways. The batch method does
not utilise a column and is the quickest and most amenable to scale up. The protein is
mixed into the resin and the resin is washed by filtration or centrifugation. For elution, the
protein-resin complex can be either poured into a column and eluted or batch eluted. The
batch method is best if the load has a high degree of particulates, which will impede
column flow during the load, or when the target protein is at low concentration and binding
will increase if the protein is kept in contact with the resin for a longer period of time. The
isocratic method elutes a protein, usually weakly bound to the resin, by continually
pumping the same buffer, without changing ionic strength or pH, onto the column. This can
be a powerful means of purification, but the protein can take a long time to elute, leading to
a large volume of a very dilute protein. The third method of elution, gradient elution, is not
amenable to scale up, but provides some of the best resolutions. n order to scale up, the
gradient needs to reach the same height over the same fluid length. For example, when
increasing the scale of a column from 100 ml to 1000ml with a 0-1 M NaCl gradient, the
total gradient volume must also be increased 10-fold.
While most modern liquid chromatographic resins are amenable to multiple uses,
frequently a cleaning procedure more complex than a simple salt wash is needed to
maintain acceptable performance. Before deciding to regenerate columns for subsequent
use, the cost of the labour required to properly clean and store resins should be compared
to that of purchasing new resin for future needs.

4.6.9 Assessment of purity
Specific activity. The ability to assess the purity of an enzyme is essential to detailed
enzymological studies. Probably the most fundamental measure of purity, though certainly
not the most sensitive to contaminants, is the specific activity. The specific activity of an
enzyme is the number of enzyme activity units divided by the amount protein,
usually expressed in mg protein.
For specific activity measurements, the rate of enzymatic reaction is measured at
saturating levels of substrate, so that it is V
max
that is determined. Activity units are
calculated in moles/time rather than in concentration/time, because a fixed amount of
enzyme turns over the same number of moles of substrate irrespective of the volume. The
standard activity unit is the InternationaI Unit of enzyme activity (IU), which is the
number of moles of substrate (or product) removed (or formed) per minute. The S unit of
activity, the katal or kat expresses the rate in mol.s
-1
, so
1 kat = 1 IU.
Coupled enzyme assays. t is often difficult to monitor the rate of an enzymatic reaction
because neither the substrate nor product has a measurable property (e.g. absorbance,
fluorescence, optical rotation) that would allow the enzymologist to distinguish between
them. One popular solution is to include in the assay another enzyme that is capable of
converting the product into a second product that has a distinguishable property. A very
common system involves the addition of pyridine-nucleotide-linked dehydrogenase and the
appropriate form of the coenzyme [NAD(P)H or NAD(P)
+
]. NAD(P)H has a strong
absorbance at 340 nm with an extinction coefficient of 6.22 x 10
3
M
-1
, whereas the oxidised
form of the coenzyme absorbs negligibly at 340 nm. Thus the production of glucose 6-
phosphate by hexokinase could be monitored by observing the reduction of NAD
+
to
NADH coupled to the oxidation of glucose 6-phosphate to phosphogluconate:
hexokinase dehydrogenase
gIucose gIucose 6-P + NAD
+
6-phosphogIuconate + NADH+H
+

Such an assay requires that the coupling enzyme and its substrate be present in sufficient
levels so that the second reaction is not rate-limiting.

Measurement of protein concentration. The other element of the specific activity is the
protein concentration. There are about a dozen methods for measuring protein, each of
which responds to a different property of proteins. For this reason it should not be
surprising that different methods sometimes give different answers.
Because of the UV absorbance of tryptophan, and to a lesser degree cysteine, proteins
absorb UV light near 280 nm. On average, a 1 mg/ml solution of protein has an
absorbance of 1 absorbance unit. The total concentration of protein in a solution
containing many proteins can therefore be measured quite accurately from a simple
absorbance measurement. However, the amino acid composition of individual proteins
varies sufficiently that their extinction coefficients range between about 0.4 and 2.0
absorbance units per mg/ml.
n addition to variability in extinction coefficients, UV absorbance measurements are highly
susceptible to interference, especially by nucleic acids. Thus crude extracts that may
contain DNA and RNA may give inappropriately high readings. A traditional solution to the
problem is to measure absorbance at two wavelengths, 260 and 280 nm.
Another problem associated with UV absorbance at 280 nm is that it is not very sensitive.
Colorimetric reactions have therefore been devised to enhance sensitivity. The Biuret
method is much more sensitive and depends upon the number of peptide bonds, so that it
gives relatively uniform detection of different proteins. Peptides with more than two peptide
bonds form a blue-coloured complex with Cu2+ salts (citrate or tartrate) in alkaline solution
which can conveniently be detected at 540 nm. This method is quite reliable, and protein
determinations used for commercial enzyme preparations are usually performed using the
biuret procedure.
The biuret procedure is not sensitive enough to be used in all cases, and a procedure
>100times more sensitive was developed by Lowry. This method depends on the Cu
2+
-
catalysed oxidation of aromatic amino acids by phosphotungstic and phosphomolybdic
acid that produces a blue colour which is measured at 500, 560, 650 or 700 nm (the
absorbance is greater at 700 nm). Like the biuret method, standard curves are generally
run along with the unknowns. The method has two drawbacks: it relies on the presence of
aromatic residues in a protein and it is subject to interference by a few common
components of enzyme solutions, including reducing agents (e.g. 2-mercaptoethanol) and
the common buffer, Tris.
To obtain uniform colour yields for different proteins and avoid interference from reducing
agents, a protein assay was developed utilising Coomassie brilliant blue G-250. This
'Bradford' reagent is dissolved in acidic ethanol solution. The blue colour is measured at
595 nm and compared to a standard curve. The method is about 10times more sensitive
than Lowry's method.
The newest popular method of protein determination is marketed by Pierce Chemical
Company and is termed the Pierce or BCA method. Like the Lowry method, it depends
on the reduction of Cu
2+
to Cu
+
, which is then complexed by bicinchoninic acid (BCA). This
complex has a high absorbance at 562 nm. Reducing agents and chelating agents
interfere with the BCA assay, but the colour yield is relatively constant.
The old Kjeldahl method for the determination of protein is inconvenient and too
insensitive.

PoIyacryIamide geI eIectrophoresis (PAGE) has been widely used to assess the purity
of a protein. Electrophoresis is a method for separation of proteins, thus the appearance of
a single protein band on a gel is evidence for, though not proof of purity. There are a
number of variations commonly employed for gel electrophoresis.

Native gels. The simplest variety of PAGE is known as a native gel electrophoresis,
referring to the state of the protein. n this variation a polyacrylamide is formed from a
mixture of acrylamide and N,N,N',N'-tetramethylethylenediamine (TEMED). The reaction is
actually initiated by ammonium persulfate, which removes a hydrogen atom from TEMED.
The total acrylamide known as %T determines the porosity of the gel. The fraction of
bisacrylamide, known as %C, determines the amount of crosslinking, which alters the
physical properties of the gel and is usually held constant between 2 and 5%. The
solutions containing the monomers are buffered at the desired pH and can be polymerised
in tubes of ~5mm diameter. After polymerisation, the top and bottom of the gel are
immersed in buffered electrolyte solutions and samples of buffered protein solution,
usually containing a small charged dye to report the progress of the electrophoretic
motion, are layered into the wells or onto a gel in a tube. Since most proteins are acidic,
buffers are used to hold the pH above neutrality, so that all of the proteins in the solution
have a negative charge. The anode (positive electrode) is placed in the bottom of the
reservoir and the cathode is placed in the top. When a DC potential of ~100 volts is
applied to the system, ion currents flow in the gel (usually a few milliamps/cm
2
). As a
protein moves through the gel, it experiences a decelerating force, or frictional drag, that is
proportional to its velocity. After electrophoresis, the gel is removed and stained for
protein. The relative mobility Rm is calculated as the ratio between the distance travelled
by the protein to that travelled by the tracking dye, which moves with the ion front.
The mobility and separation in native PAGE depends upon both the charge and size of the
protein.

SDS gels. n native PAGE, two proteins of very different size may co-electrophorese if
their charge densities are such that they produce the same steady-state velocity. A
common method to separate proteins solely on the basis of their molecular weight is to
treat them with the ionic detergent sodium dodecyI suIfate (SDS) and heat, then submit
the denatured samples to electrophoresis. The protein becomes unfolded and coated with
the surfactant, which has a negative charge. The total charge on a protein is proportional
to the number of detergent molecules bound, which is proportional to molecular weight.
Thus the charge to mass ratio for all proteins is approximately identical. Multimeric
proteins are converted to denatured monomers, so SDSPAGE can be used to detect the
presence of non-identical subunits.

Isoelectric Focusing gels. Another major modification of PAGE is gel electrofocusing
(EF), which is a direct adaptation of a preparative technique developed for use in density
gradient solutions. n gels, the technique involves the use of buffers of different pH in the
two buffer reservoirs. f the positive electrode (cathode) is placed in the lower reservoir and
the buffer is more basic than that in the upper reservoir, amphoteric anionic molecules will
move towards the cathode until they encounter more alkaline pH and lose their charge. f a
number of amphoteric molecules of different isoelectric point (called ampholytes) are
included in the gel when it is poured, they lose charge (and mobility) at different levels in
the gel. Thus a stable pH gradient is created within the gel during electrophoresis. Proteins
loaded onto such gels travel until they reach their own isoelectric point, then stop and
focus to a width that depends on the steepness of the gradient. The pH gradient can be
measured using a needle-tipped pH electrode.

HPLC. Because of the development of high efficiency ion exchange, hydrophobic
interaction and reverse phase columns, HPLC has become a good method for analysis of
proteins. The reverse phase columns are generally hydrocarbons covalently attached to
silica particles of diameters of 10, 5 or 3 m. Octyl silane (C8) is probably the most
common, although octadecyl silane (ODS, a variety of C18) is more common for small
molecules.
Since proteins have a reasonable absorbance at 280 nm and a strong absorbance below
210 nm, detectors using UV absorbance are the most common. For proteins the mobile
phase is generally a binary mixture of water and one or more of the following: acetonitrile,
methanol, or tetrahydrofuran, though many other systems exist.
5. Enzyme immobiIisation.
The disadvantage for the enzyme industry is, however, that they are soluble and unstable.
n order to overcome this disadvantage, a new enzyme technology developed, that of cell
immobilisation. An immobilised enzyme is one whose movement in space has been
restricted either completely or to small limited region, as is the case in microorganisms.
This immobilisation normally results in a water-insoluble form of the enzyme that is of
interest for several reasons:

1. t makes the recovery of the enzyme from a reaction liquor easier, and this is obviously
important in enzyme reactor economics;
2. t is useful to the biochemist on a model system for enzymes normally associated with
membranes in the living cell.

Many techniques for the preparation of immobilised forms of enzymes and other proteins
have been reported. During the last decade, these materials have been referred to, for
example, as water-insoluble enzymes, insoluble enzymes, matrix-bound enzymes and gel-
entrapped enzymes. The term immobilised enzyme has been recommended to cover all
these forms.
The methods for immobilisation have been subdivided into four main groups. The first
division is based on whether immobilisation has been achieved primarily by entrapment in
a limited space or binding to a support or carrier material. Entrapped enzymes are further
subdivided according to the structure of the entrapping system - single-enclosed space as
in encapsulation, or many small spaces, which may be linked, as in matrix entrapment.
The binding of enzymes to support materials may be by adsorption or by covaIent
binding. Covalently bound immobilised enzymes may be further subdivided according to
whether the covalent bond is between the enzyme and support or between enzyme
molecules. For the latter, the term cross-linked is used. We will consider each individually.
Although claims have been made for particular techniques, it is important to realise that
the choice of technique depends on the objective. For instance, an immobilised enzyme
prepared for model studies of the kinetic behaviour of enzymes present in biological
membranes is likely to have very different properties from an immobilised enzyme
prepared for use in a large-scale bioreactor.
n general, the advantages of immobilised enzymes are

1. Multiple or repetitive use of a single batch of enzymes;
2. Ability to stop reaction rapidly by removing the enzyme from the reaction solution;
3. n many cases, the enzyme is stabilised by bonding (maintenance of tertiary structure);
4. The processed solution is not contaminated with the enzyme;
5. Analytical purposes - long half-life, predictable decay rate, elimination of reagent
preparation.

Each one of these five (5) points contribute to the total utilisation of enzyme technology.
Enzymes on a milligram basis of pure enzyme protein are perhaps the most expensive
and difficult materials to obtain in reasonable quantities. Therefore, any procedure that can
economically extend the life time of these biologically active molecules should be
considered. f it is possible to immobilise an enzyme without appreciable loss of activity
during the immobilisation procedure, than not only reuse of the enzyme is gained, but also
the ability to process on a continuous basis, is achieved. Beyond the economic
advantages that may be achieved by the immobilisation of an enzyme, an additional
control can be exercised upon the extent of conversion of substrate to products. Thus, it is
apparent that the immobilised enzyme may be more precisely and more rapidly controlled
than the enzyme in solution.
t is very important to understand the changes in physical and chemical properties which
an enzyme would be expected to undergo immobilisation, if the best use is to be made of
the various immobilisation techniques available. Changes have been observed in the
stability of enzymes and in their kinetic properties due to the micro environment imposed
upon them by the supporting matrix and by the products of their own reaction. The stability
of enzymes might be expected either to increase or decrease on solubilisation, depending
on whether the carrier provides a micro environment capable of denaturing or of stabilising
the enzyme protein. The thermal stability of immobilised enzymes has been found to be
greater than that of the native enzyme in a number of cases.
Once an enzyme has been immobilised, it finds itself in a micro environment which may be
drastically different from that existing in free solution. The new micro environment may be
a result of the physical and chemical character of the support matrix alone, or it may be
due to interactions of the matrix with the substrates or products involved in the enzyme
reaction. The effect of the molecular weight of the substrate can be very pronounced.
Diffusion of large molecules will obviously be limited by steric interactions with the matrix,
and this is reflected in the fact that the relative activity of bound enzymes towards high
molecular weight substrates has been generally found to be lower than towards low
molecular weight substrates. The rationale for the replacement of existing applications of
soluble enzymes by immobilised enzymes is based on the following advantages:

1. An immobilised enzyme can be used to catalyse a continuously operated process and
hence will eventually replace all those processes currently operating batchwise;
2. An immobilised enzyme of the type where it is rendered insoluble in the substrate phase
is more easy to control than that operated where the enzyme is soluble. Thus, if a packed
bed enzyme reactor is used, action on the substrate ceases immediately it is out of contact
with the catalyst. Hence the cost of heat inactivation of the catalyst is saved and the
catalyst can be applied to those processes where heat inactivation is most desirable, e.g.
treatment of beer or a food that is heat-labile;
3. Two enzymes that are normally incompatible with respect to pH optima can be replaced
with two enzymes that have their own micro environment, either both immobilised on
solids or on opposite sides of a membrane. Alternatively, one enzyme can operate in
solution and the other bound to a surface. Hence the time given sequence of two enzyme
processes, e.g. starch ---> glucose, and glucose ---> fructose, can be much shortened;
4. mmobilised enzymes offer opportunities to cheapen the catalyst component of the
process cost, since when immobilised, their effective life time of use is considerably
extended. As a consequence or sequel of this, it often becomes possible to use more
enzyme catalyst and, if required, a purer enzyme catalyst. This again can result in a further
time shortening of the process and/or purer products;
5. mmobilised enzymes offer greater opportunities to carry out separation of the product
and substrate simultaneously which can result in the displacement of unfavourable
equilibria or succeed in overcoming any endproduct inhibition;
6. mmobilised enzymes in a modular form offer greater opportunities for a food
manufacturer to carry out his own operations in a more flexible manner as consumer
demand dictates.

Enzymes used in clinical assays and other assays can be replaced with enzymes bound to
tubes, packings or membranes. The first and last of these can be easily incorporated in an
autoanalyser with a consequent increase in its efficiency as related to the number of
assays simultaneously performable. The number of assays obtainable from a given
quantity of enzyme is also increased. This use of immobilised enzymes in analysis
systems allows not only continuous but also reagent less assays to be performed.
5.1 Methods of immobiIisation
The physicaI adsorption of an enzyme onto an insoluble matrix is probably the simplest
way of preparing immobilised enzymes. The method relies on non-specific physical
interaction between the enzyme protein and the surface of the matrix brought by mixing a
concentrated solution of enzyme with the solid. A wide variety of solids have been used,
e.g. charcoal, alumina, cellulose, kaolin, silica etc. A major advantage of adsorption is that
usually no reagents and only a minimum of activation steps are required. As a result,
adsorption is cheap and easily carried out, and tends to be less disruptive to the enzymic
protein than chemical means of attachment. The binding forces involved being mainly due
to hydrogen bonds, multiple salt linkages and van der Waal's forces. The disadvantage of
this method is, of course, the easy desorption of the protein due to changes in
temperature, pH, ionic strength or even the mere presence of the substrate. Another
disadvantage is that the adsorption surface is non-specific, and will therefore adsorb
further protein or other substances. This may or may not alter the properties of the
immobilised enzyme.
IncIusions. Enzymes have been immobilised by confining them within the lattice of
polymerised gels, which allow the free diffusion of low molecular weight substrates and
reaction products, but have a small enough pore size to prevent leakage of the high
molecular weight enzyme. The usual method is to polymerise the hydrophilic matrix in an
aqueous solution of the enzyme, and break up the polymeric mass to the desired particle
size. n most of the cases of immobilisation by inclusion, the monomer used was
acrylamide, and the cross-linking was affected by N,N-methylene bis-(acrylamide). As
there is no bond formation between the enzyme and the polymer matrix, inclusions provide
a method which is generally applicable to any enzyme and, in theory, involves no
disruption of the protein molecule. The main disadvantage is that only low molecular
weight substrates can diffuse into the vicinity of the enzyme, thus making the method
unsuitable for enzymes, which act on macromolecular substrates such as trypsin etc.
Cross-Iinking. mmobilisation of enzyme has been achieved by intermolecular cross-
linking of the protein, either to other protein molecules or to functional groups on an
insoluble support matrix.
CovaIent binding. The most intensely studies of the immobilisation techniques is the
formation of covalent bonds between the enzyme and the support matrix. Obviously the
choice is limited by the fact that the binding reaction must be performed under conditions
which do not cause loss of enzymic activity, and the active site of the enzyme must be
unaffected by the reagents used.
5.2 Properties of ImmobiIised Enzymes.
t is very important to understand the changes in physical and chemical properties which
an enzyme would be expected to undergo upon immobilisation, if the best use is to be
made of the various immobilisation techniques available. Changes have been observed in
the stability of the enzymes and in their kinetic properties due to the micro environment
imposed upon them by the supporting matrix and by the products of their own reaction.
The thermal stability of immobilised enzymes has been found to be greater than that of the
native enzyme in a number of cases.
Once an enzyme has been immobilised, it finds itself in a micro environment which may be
drastically different from that existing in free solution. The new micro environment may be
a result of the physical and chemical character of the support matrix alone, or it may be
due to interactions of the matrix with the substrates or products involved in the enzymic
reaction. A wealth of opportunities await the application of immobilised enzymes and to
this effect of immobilised microbial cells.
6. Biosensors
Since life itself depends upon almost incomprehensibly balanced enzyme-mediated
substrate-specific transfer of electrons, it may not be surprising that means to measure the
vital biochemical cellular processes would involve sensors composed of the same
substances. By looking at enzymes as specific chemical transducers, translating an
analyte into a substance capable of being detected by a chemically or physically sensitive
detector, a new class of sensors, intrinsically responsive to biological compounds, has
been developed. Combinations of enzymes, such as esterases, dehydrogenases, and
oxidases, and of detectors, such polarographic, conductimetric, potentiometric, acoustic,
and optical, offer promise to expand the selectivity, sensitivity and versatility of these
detectors. The first enzyme eIectrodes relied on enzymes physically entrapped on or
very near the surface of the sensor. Later on, chemical immobilisation, insolubilisation, or
fixation techniques were adopted.
Glucose and lactate electro-enzymatic systems are being widely used in biomedicine,
especially where rapid on-the-spot analysis of small samples of blood are desired.
Biosensors, meaning sensors which incorporate biological material in their structure were
first described in 1962 at a New York Academy of Sciences Symposium by Clark. He
described the combination of glucose oxidase with a Clark pO
2
electrode to measure
glucose by detecting the drop in oxygen when glucose was converted to gluconic acid and
hydrogen peroxide.
There are basically two kinds of polarographic enzyme electrode sensors. n one, the
analyte consumes oxygen in the presence of an enzyme and the measurement depends
upon a change in oxygen tension. n the other, the enzyme converts the analyte to a
substance to which the sensor is sensitive. The purpose of the enzyme is to transduce the
substance being measured, from one to which the sensor is not responsive, to one which it
is responsive. For this reason the enzyme layer was originally referred to as a transducer.
Biosensors with enzyme membrane are a common sight in medical or pathological
laboratories. Enzyme biosensors have been developed to detect a great number of
illnesses such as cardiac, prostate cancer, other cancers, diabetes, leukaemia etc etc.
Biosensors with enzyme membranes have a great future.
Apart from enzyme sensors, microbiaI eIectrodes have also been developed. The main
developmental work on microbial electrodes have been and still are carried out in Japan.
Microbial sensors are composed of immobilised microorganisms and an electrochemical
device and are suitable for the on-line control of biochemical processes.
A microbial sensor consisting of immobilised whole cells of Pseudomonas fluorescens and
an oxygen electrode was developed for the determination of glucose. The microbial sensor
was inserted into a sample solution and the sample solution was saturated with dissolved
oxygen and stirred magnetically while measurements were taken. The steady-state current
was attained within 10 min at 30C. The exact time depended on the concentration of
glucose added. When applied to molasses broth, an average error of about 10% were
observed for glucose readings compared to the enzymatic method.
Many microbial sensors have now been developed for acetic acid, aIcohoI, formic acid,
methane, gIutamic acid etc Even a BOD sensor has been developed using Trichospora
cutaneum. A linear relationship was observed between the current difference and the five-
day BOD assay of the standard solution up to 60 mgl
-1
. The minimum measurable BOD
was 3 mgl
-1

7. BibIiography

Bergmeyer, H.U. 1978 Principles of Enzymatic Analysis. Verlag Chemie, Weinheim

Enzyme NomencIature 1978 Recommendations of the Nomenclature Committee of the
nternational Union of Biochemistry on the Nomenclature and Classification of Enzymes.
Academic Press 1979

Foster,K.A., S.Frackman and J.F.JoIIy 1995 - Production of Enzymes as Fine
Chemicals. n Biotechnology (H.J.Rehm, G.Reed, eds.) Vol 9,73-120. VCH
Verlagsgesellschaft mbH, Weinheim, Germany

Lowe,C.R. and Dean,P.D.G. 1974 Affinity Chromatography. John Wiley & Sons, London

Scopes,R. 1982 Protein Purification Principles and Practice. Springer Verlag, Heidelberg

Smith,G. 1995: The Nature of Enzymes. n 'Biotechnology', 2nd ed. [H.J.Rehm, G.Reed,
eds.] Vol. 9: 5-72, VCH Verlagsgesellschaft mbH, Weinheim

Turner,A.P.F., I.Karube and G.S.WiIson 1989: Biosensors Fundamentals and
Applications. Oxford Science Publications, Oxford

Wang,D.I.C., Cooney,C.L., Demain,A.L., DunniII,P., Humphrey,A.E., LiIIy,M.D. 1979
Fermentation and Enzyme Technology. John Wiley & Sons, New York

Wong, J.Tze-Fei 1975. Kinetics of Enzyme Mechanisms Academic Press, London

Deputy-Director,MIRCEN-Biotechnology Brisbane and Pacific Regional Network;
Chapter 13

MICROBIAL TECHNOLOGY IN INDUSTRY - IndustriaI
AppIications of Enzymes
[This chapter is the continuation from chapter 12]

Content:
1. Pectinases
1.1 Pectin MethyIesterases
2. Lipases
2.1 Pancreatic Lipases
3. Proteases
3.2 MetaIIoproteinases
4. GIucose Oxidases
5. CataIase
6. GIucose Isomerase
7. IndustriaI Use of ProteoIytic Enzymes
8. Enzymes of the Detergent Industry
9. Enzymes of the Leather Industry
11. Enzymes in the Meat Industry
12. Enzymes in the Dairy Industry
12.1 Enzymes from rennet and rennet substitutes
12.2 Beta-1,4-GaIactosidases
12.3 Lysozyme
13.1 Syrups and Sweeteners
13.2 FueI AIcohoI
12.3 Baking
13.4 GIucose Isomerization
14. AnaIysis
15. BibIiography
1. Pectinases
Pectines are polymers of galacturonic acid and its methylesters and occur, together with
the heteropolymeric carbohydrates, in plants. n fruit and vegetables that are used to
produce juices, 60-80% of the pectin's carboxyl groups are esterified with methanol.
Carbohydrates, such as arabans, arabinoxylans, and galactans are associated with
pectins. Water-soluble pectines, having a high capacity to bind water, fill the intracellular
spaces of fruit. nsoluble pectines, so-called protopectins, are localised in the matrix and
are attached to heteropolymeric carbohydrates associated with the cellulose of the cell
wall.
Enzyme preparations with pectinase activity have been used since 1930 in the production
of fruit juices. Three groups of enzymes are involved in the cleavage of pectins:

1. pectin methylesterases;
2. depolymerizing enzymes, which attack the interior bonds of pectin chains
(endoenzyme);
3. exoenzymes, which liberate galacturonic acid units from the ends of the pectin
molecule.

Occurrence of pectolytic enzymes has been reported in a large number of bacteria and
fungi. Commercial enzymes are generally obtained from fungal sources since the pH
optima of these enzymes are in the range found naturally in materials to be processed.
Most potent strains are selected from Aspergillus niger. Japanese enzyme manufacturers
also use Sclerotinia libertiana and Coniothyrium diplodiella as producers of pectolytic
enzymes.
1.1 Pectin MethyIesterases (EC 3.1.1.11)
These enzymes attack pectins to yield pectic acid and methanol. Pectin methylesterase,
also called pectinesterase (PE) is a very specific enzyme, effecting successive removal of
methanol units from the reducing ends of pectin. Hydrolysis, catalysed by various
pectinesterases, proceeds with a yield of 98%. The pectic acid thus formed gelatinizes in
the presence of Ca
2+
ions. This process is used in France in the depectinization of
fermenting apple juice to produce cider. The activity of pectinesterase is determined either
by titrating the carboxyl groups released or by measuring the methanol liberated.
Pectinesterase occurs in fruit and vegetables, especially citrus fruit and tomatoes. The
properties of this enzyme, however, differ from those of pectinesterase obtained from
molds or bacteria.
Mold pectinesterase is active between pH 3 and 5; the bacterial enzyme, between pH 7
and 8. Commercial pectinesterase is a stabilized liquid containing 50-100 U/g. t is
employed in the production of apple cider and must be free from depolymerizing
pectinases.
Pectin depolymerases occur in plants, but industrially, they are isolated from cultures of
molds, and occasionally, bacteria. Depolymerases or endopectinases split the alpha-1,4-
bonds of the pectin chain. n fact, two mechanisms for bond cleavage have been
postulated: hydrolytic splitting of the glycosidic bond and transeliminative cleavage:
Depolymerases are classified on the basis of these mechanisms of cleavage and
substrate specificity as follows:

1) endo-pectin transeliminas [EC4.2.2.3], PTE, pectin lyase;
2) endo-pectic acid transeliminase [EC4.2.2.1], PATE
3) endo-polygalacturonase [EC 3.2.1.15]
4) endo-polymethylgalacturonase (PMG)

The activity of these enzymes can be followed by the rapid decrease in the viscosity of
pectin solutions. n fact, the viscosity falls below 50% of the starting value afgter 2-3% of
the glycosidic bonds have been cleaved. n addition the number of reducing groups
formed, determined later-on in the reaction, is an index of enzyme activity. The alcohol test
can be used to determine the last stage of the reaction, complete depectinization, when no
flocculation occurs on addition of 50% ethanol. The rate of transelimination can be
followed photometrically.
Liquid industriaI preparations of pectinase are stabiIized with saIts, sugars, or
gIyceroI. Pectinase in powdered form is standardized with lactose, sucrose, or
maltodextrin to give a definite activity. nternational units do not exist. Therefore, the
activities of preparations supplied by different producers cannot be compared with each
other.
The enzyme supplied as a liquid preparation Ioses approximateIy 10% of its activity
per year at storage temperatures up to 15
o
C . Apart from a mixture of the various
pectinases, commercial preparations contain small amounts of other important enzymes,
such as hemicellulases, cellulases including arabanase, proteinases, and amylases.
Biosynthesis of pectolytic enzymes is constitutive or controlled by the mechanisms of
induction or catabolite repression. No uniformity exists among the various organisms and
the various components of the enzyme complex.
On an industrial scale pectolytic enzymes are produced by the solid substrate cultivation
method as well as by submerged culture. Wheat bran or defatted rice bran have been
recognised as satisfatory basic substrates in solid substrate cultivation. t is also well
known that some by-products of the food industry, such as beet pulp, apple pulp, or grape
pulp, exert a promoting effect on enzyme formation. Other ingredients, e.g. nutrient salts,
acid or buffers are also incorporated to regulate the pH during the growth of fungi. The
time of cultivation can extend to 7 days, but when Aspergillis niger is used, the desired
enzyme level is normally reached within 36 and 72 hrs. After fermentation the mold bran is
dried and can be used as such. For obtaining concentrates the dried mold bran is
extracted with suitable aqueous solutions and concentrated under vacuum or by
ultrafiltration.
Submerged cultures, in contrast to solid substrate cultures, seem to have the
disadvantages of poor yields and undesirable composition.
Extensive use of pectolytic enzymes is made in processing fruit juices for increasing juice
yields on pressing, as aid in clarification of juices, and for depectinizing in order to obtain
high density fruit juice concentrates.
n the production of coffee beans the residual mucilaginous coating surrounding the bean
can be liquefied by commercial pectolytic enzyme preparations, thus offering an alternative
to the usually used fermentation process. The curing or fermentation of cocoa, tea and
tobacco also can involve pectolytic enzymes. One of the oldest applications of these
enzymes is the process of retting, in which textile fibers, such as flax, hemp, and jute, are
loosened from their plant stems. The enzyme system of Clostridium felsineum, an
orghanism that is involved in aerobic retting, contains endopolygalacturonate trans-
eliminase, but not pectinesterase. Recently, pectolytic enzymes have been proposed as a
means to make commercial softwoods, such as Sitka and Norway spruce, more
permeable to preservatives. t has been demonstrated that treatment with enzyme
preparations as well as with the specific bacteria that produce them is possible.
.
2. Lipases
Lipases [EC 3.1.1.3] are carboxylesterases that hydrolyse glycerides present as aqueous
emulsions:
triacyIgIyceroI-H
2
O diacyIgIyceroI + fatty acid anion
n that respect, they differ from other carboxylesterases, such as proteases and
pectinesterase, which act on substrates in aqueous solution. Therefore, lipases can be
regarded as enzymes that hydrolyse esters only at the interface between lipid and water in
a heterogeneous system.
A distinction is customarily made between enzymes that cleave esters of fatty acids and
lower alcohols (aliesterases), and 'real' lipases that hydrolyse glycerides. The specificty of
lipases varies considerably, and depends on the substrate chain length and on certain
positions in the substrate molecule.
2.1 Pancreatic Lipases (Pancreatin)
Lipase preparations are obtained from porcine pancreas, but these also contain esterases,
proteases or their zymogens, and amylases.
Apart from naturally occurring triglycerides, oils and fats, simple fatty acid esters and aryl
esters are also cleaved by this enzyme. Both tributyrin and triacetin are rapidly hydrolysed
and, therefore, are foten employed as analytical substrates.
Since lipases act only at the interface between fat droplets and aqueous phase, the
reaction rate depends on the degree of emulsification. Gum arabic, poly(vinyl alcohol), or
sodium deoxycholate promote emulsification. Calcium ions influence both enzyme activity
and stability, which is also dependent on the sodium chloride concentration (optimum at
about 0.5 g/l).
The conversion of natural fats and oils into those with specific characteristics has been
carried out by chemical and physical methods. Because lipases do not only hydrolyse fats
but also synthesize fats, new applications have been developed: hydrolysis of fats,
transesterification, stereospecific hydrolysis of racemic esters, and the use of lipases in
detergents. Commercial preparations of microbial lipases are produced by fermentation of
different fungi and bacteria. The industrial products are mixtures of lipases and esterases.
The chain length of the fatty acid and its position in the glycerol molecule significantly
affect the specificity of these enzymes.

Lipases from Aspergillus species. Different lipases from Aspergillus niger or Aspergillus
oryzae with varying specificity towards long-chain and short-chain fatty acids have been
described. The molecular masses are between 20,000 and 25,000; the pH optimum is
between 4.5 and 6.5. The lipase acts on coconut oil, and olive oil with yields of 48-93%.
Special types of such lipases are also used in cheese ripening.

Lipases from Candida cylindracea. These lipases have molecular masses of 120,000;
their isoelectric point is at pH 4.2 and their optimum of activity is between pH 5.2 and 7.2.
They hydrolyse olive oil to 95-97%.

Lipases from Rhizopus. Different strains of Rhizopus are used for the enzyme
production, such as R.arrhizus, R.javanicus, R.niveus, R.delemar. Most data are available
for the enzyme from R.arrhizus. ts molecular mass is 43,000, the isoelectric point at pH
6.3. The enzyme is a glycopeptide with 13-14% mannose per molecule. Rhizopus lipases
show a 1,3-regiospecificity, the optimum activity is at pH 5.0-7.0; the temperature optimum
is at 30-45
o
C.

Lipases from Mucor species. This lipase is a good catalyst for the transesterification in
the 1,3-position of glycerol. Different types are specific for short- or long-chain fatty acids.

Lipases from Pseudomonas. This enzyme has a molecular mass of 29,000 and an
isoelectric point at pH 5.8. The enzyme is active and stable at alkaline pH and therefore
interesting for the use in detergents.
t becomes very obvious from these details that many, perhaps most, bacteria and fungi
produce lipase. Potent producers are among the fat-producing microorganisms. However,
no distinct relationship between capacity of fat production and lipase production has been
found. The enzyme from Candida cylindracea is commercially available.
The production of lipase is markedly affected by many factors. Generally, synthetic media
produce lower yields of lipase than complex media. Lipase formation is highly dependent
on nutrient and physical conditions. n a number of cases, addition of lipid material or fatty
acids to the culture medium was found to enhance lipase production. Glucose is
unsuitable as the C source and ammonium ions seem to be unsuitable as N source.
3. Proteases
The microbial proteases which are of interest for application in the food industry are all of
the endopeptidase type and are all extracellular enzymes. There are many different types
of proteases produced by an extraordinarily large number of microorganisms, but in actual
practice the enzymes prepared commercially are of a very limited number of types and
they are derived from very few organisms.
The proteolytic enzymes from microorganisms are classified into 4 main groups, based on
their mechanism of action:
1. some proteinases contain mercapto groups at their active centres, these groups
being essential for enzymatic activity, e.g. papain, bromelain, ficin. These enzymes
are inhibited by oxidizing agents and heavy metals.
2. metalloproteinases require such metal ions as zinc, magnesium, or cobalt for
activity. The 'neutral' bacterial proteinases belong to this class of enzymes; they are
inhibited by chelating agents, e.g. EDTA.
3. proteinases that contain histidine residues at their active centers are inactivated by
alkylating agents such as DFP (diisopropyl fluorophosphate which attacks the
serine residue). Examples are trypsin, chymotrypsin, and proteinases from bacteria
and molds which are active under alkaline conditions.
4. acid proteinases of the pepsin group have asparagyl or glutamyl residues at their
active centers. Apart from pepsin, a number of proteinases from molds, which are
active under acidic conditions, belong to this category.
These proteinases are also referred to as thioI proteinases, metaIIoproteinases, serine
proteinases and acid proteinases.
ndustrial production of microbial proteases is carried out on a large scale by a number of
companies in Europe, Japan, and the United States. For cultivation of the microorganisms
the submerged fermentation is the preferred method; with bacteria it is the exclusively
used process. However, fungi usually give higher yields when cultured on solid media so
this method continuous to play a role. As in most fermentations there is a trend to use
highly concentrated media. The reason for this is that one can expect higher enzyme
yields per unit volume with a larger cell population, although there is no direct correlation
between growth and protease production. With regard to serine and metalloproteinases it
seems that low concentrations of purely carbonaceous substrates and high concentrations
of proteinaceous N sources stimulate production.
Many of the organisms excrete more than one kind of protease. The type of proteolytic
enzyme formed may depend on the composition of the medium. For example, Bacillus
NRRL B-3411 produces the preferable neutral protease when grown on a grain medium,
but mainly alkaline protease when cultured on a fishmeal-enzose-cerelose medium. The
biosynthesis of proteases is often correlated with particular growth phases of the microbial
culture. Under most growth conditions, Bacillus species produce extracellular protease
during the postexponential growth phase. Other bacilli synthesize proteases during the
exponential growth phase. However, these kinetics depend on the composition of the
medium.
For all protease preparations the degree of purification depends on the intended use. A
number of purification procedures are in existence, which follow the general description
given to you in earlier lectures. Particular care is necessary during the drying process in
order to avoid the formation of dust. For this reason proteas preparations are pelleted or
coated with some suitable material.
Bacterial proteases are used on a large-scale in enzyme-containing washing powders, but
they are not widely used in food processing. Minor uses are in the chillproofing of beer, in
the production of protein hydrolysates, in the production of condensed fish solubles, and
as feed supplement. n contrast to bacterial preparations, fungal proteases are the more
interesting group for the food industry. They are used, for example, for the modification of
wheat proteins in bread doughs, in meat tenderizing, and in several less important
applications.
These proteases are widespread in bacteria and fungi. They show maximum activity at
neutral to alkaline pH and are inhibited by diisopropyl fluorophosphate (DFP) or
phenylmethane sulfonylfluoride (PMSF). They can be classified into at least 5 groups:

1. trypsin-like proteinases
2. alkaline proteinases
3. Myxobacter alpha-lytic proteinase
4. staphylococcal proteinases
5. serine neutral proteinases.
Because of their superior economic importance, only the serine alkaline proteinase will be
considered.
Proteases of this type ae most active at pH 9.5-10.5; they are sensitive to DFP and potato
inhibitor, but not to tosyl-L-lysine chloromethyl ketone. All alkaline serine proteases show
specificity toward aromatic or hydrophobic amino acid residues, such as tyrosine,
phenylalanine, or leucine, at the carboxyl side of the cleavage point. The molecular weihts
are 26-34,000, slightly below the range of neutral metalloproteases. Most of the alkaline
proteases are stable from pH 5-10 at low temperatures, but show rapid loss of activity at
65
o
C.
Serine alkaline proteinase is produced by numerous species of bacteria and fungi. The
best known representatives of this type are the subtiIisins, which are produced by
Bacillus subtilis and related species. Alkaline serin proteinases produced by different bacilli
or different strains of Bacillus subtilis can be divided into two groups, subtiIisin CarIsberg
and subtiIisin Novo. These enzymes are quite distinct from each other, but possess
many similar properties.
The synthesis of these enzymes is linked to particular phases of development of the
microbial culture. Some strains, e.g., those of Bacillus megaterium, produce the protease
during the log phase of growth, while others, like those of Bacillus subtilis and Bacillus
cereus, produce it in the stationary phase. However, as mentioned before, the relationship
between growth cycle and enzyme formation depends on the ingredients of the substrate.
t is generally valid that the time of biosynthesis is genetically determined and can be
changed or extended by selecting proper mutants.
n most species production can be inhibited by certain components in the growth media,
such as free ferric ions, amino acids, carbon sources, or several of these. Catabolite
repression and availability of nucleic acid precursors are also thought to play a role in
alkaline protease synthesis.
Bacterial alkaline proteases are produced exclusively by the submerged culture method.
Amounts of more than 1 g protease per litre culture liquid are quite usual. With specially
selected strains markedly higher yields are possible, e.g. Bacillus subtilis strain AJ 3266
can produce more than 10 g enzyme per litre.
Fungal alkaline proteases are mainly produced from Aspergillus species, in both solid
substrate and deep tank fermentations. Solid substrate cultivations extensively used in
Japan, are carried out with wheat or rice bran or whole grains as the basic substrate. t has
been shown that ammonium ions strongly inhibit production of the enzyme, while nitrates
and Na salts of aspartic and glutamic acids promote its formation.
Serine alkaline proteases of bacterial origin are used in large amounts in laundering and to
a lesser extent in leather tanning and the food industry.
3.2 MetaIIoproteinases.
These types of enzymes play much lesser role in commercial applications than the serine
and acid proteinases. This is mainly due to their relatively poor stability.
Metalloproteinases exhibit maximum activity at pH 7 to 8. n the majority of cases they
contain a Zn atom in their active center. They are inhibited by metal chelating agents such
as EDTA, but not by DFP or thiol reagents.
Regarding their pH activity metalloproteinases are divided into neutral and alkaline types.
The neutral enzymes all have pH optima around 7.0 and a molecular weight of 35-45,000.
They are widespread in microorganisms, both fungi and bacteria. Strains used for
industrial production belong to the genera Aspergillus, Bacillus, Streptomyces. Only few
strains have been found which synthesize neutral protease free of accompanying serine
and acid proteases. Such strains are, for example, Bacillus cereus ATCC 14579 and
NCTC 945, Bacillus megaterium ATCC 14581 and MA, and Bacillus polymyxa ATCC 842.
The formation of neutral proteases by bacyeria does not seem to be correlated with
sporulation. n Bacillus subtilis enzyme synthesis is subject to catabolite repression.
Without pH adjustment during the fermentation, the culture produced the neutral protease
parallel to growth, and enzyme formation reached its maximu towards the end of the log
phase. The yield was 15times that obtained when the pH was controlled at 6.8.
Due to their high instability, processing of metalloproteinases may lead to high activity
losses. Therefore, the main problem in the concentration and purification of the enzyme is
its stabilization. This can be achieved by strictly observing the tolerated range of pH, by
the presence of metal ions (Zn
2+
for activity, Ca
2+
for stability) and by elimination of alkaline
protease activity.
The application of neutral metalloproteinases is very limited because of the mentioned
instability. Actual and potential uses are: treatment of beer, application in bakeries, and
reduction of dental plaque in humans.
These enzymes are without doubt the most interesting group of proteases with respect to
use in the food industry. They are characterized by maximum activity and stability at pH
2.0-5.0 with a molecular weight around 35,000. Acid proteinases are low in basic amino
acid content and have a low isoelectri point. They are sensitive to SH-reagents, metal
chelators, heavy metals, and DFP and are generally stable in the acid range, but are
rapidly inactivated at higher pH values.
Acid proteinases of commercial importance are prepared exclusively from fungal sources
and are tentatively divided into two subgroups by their physiological characteristcs:
pepsin-like acid proteinases and rennin-like proteinases.
Pepsin-like proteinases have usually been reported in the group of black aspergilli, such
as Aspergillus niger, Aspergillus awamori, Aspergillus usamii, Aspergillus saitoi, but also
occur in species of Penicillium, Rhizopus, and others. To a large extent they are produced
in solid substrate cultures. Biosynthesis of these enzymes is favoured by high C/N ratios .
Acid proteinases of the pepsin type play an important role in the production of fermented
foods by molds from soybeans, rice, and other cereals. They are further used in the baking
industry for the modification of wheat proteins in bread doughs.
Rennin-like acid proteinases [EC 3.4.4.3] are produced by strains of Mucor miehei, Mucor
pusillus, Endothia parasitica, Trametes sanguinea. The enzyme from the Mucor species
has been and is still now produced by the solid substrate culture method.
Microbial rennet substitutes must be free of lipase to avoid rancidity of the cheese. This
can be achieved by controlled heating or by adjusting to a low pH. Unspecific proteolytic
enzymes, which may cause a bitter taste of the cheese, must also be removed. Their
separation is obtained by adsorption on aluminosilicates. These adsorbents are
particularly advantageous because they can be mixed in to the culture liquid at the end of
fermentation, even in the presence of the medium., with a good separation effect and
without any loss of milk clotting activity. Bentonite, permutite, and attapulgite are also
suitable.
Because of their particular properties, rennet-like microbial proteases are used for clotting
milk in cheese manufacture. The process is based on the coagulation of casein under the
influence of the rennet-like protease. t is known that the casein in milk is mainly composed
of alphas-, beta-, and kappa-casein. n particular, kappa-casein plays an important role in
te coagulation process, because it keeps the casein micelles present in milk in solution
and protects them against flocculation by calcium ions. The clotting effect of rennins
consists of the destabilization of the casein complex. Two phases can be distinguished:

1. the primary or enzymatic phase, in which the protective colloid (K-casein) of the casein
micelle is broken down and a glycomacropeptide is splitt off as follows:

K-casein para-casein-gIycomacropeptide
nsoluble soluble

2 the secondary or nonenzymatic phase, in which the coagulum is formed under the
influence of calcium ions.
t would therefore be reasonable to develop a system for continuous clotting of milk
employing immobilised enzymes.
4. GIucose oxidase
Glucose oxidase (beta-D-glucose:oxygen oxidoreductase, EC 1.1.3.4) also known as
notatin, acts in the presence of molecular oxygen to convert glucose to gluconic acid and
hydrogen peroxide:
C
6
H
12
O
6
+ O
2
+ H
2
------> C
6
H
12
O
7
+ H
2
O
2

t is highly specific for beta-D-glucose, alyhough slight activity is found with 2-
deoxyglucose.
At present, glucose oxidase is commercially prepared from Aspergillus niger and
Penicillium amagasakiense in submerged culture. t has also been reported that
Penicillium notatum and Penicillium chrysogenum synthesize glucose oxidase on liquid
media in surface culture, but not in submerged culture.
During the growth of the fungal culture, the enzyme occurs in the phase following the lag
phase. By feeding glucose this phase can be extended and thus the enzyme yield
enhanced. The special culture conditions, however, depend markedly on microbial strains
used. For example, beet molasses has proved to be a suitable carbon source in
Penicillium purpurogenum, but not suitable in Penicillium chrysogenum; high aeration rates
supported enzyme synthesis in Penicillium purpurogenum, but not in Penicillium
chrysogenum
For the purpose of concentration and purification, glucose oxidase must be separated from
cells by extraction. The crude solutions also contain catalase which may interfere with
glucose oxidase in some applications. n these cases. separation is conducted by
adsorption of the catalase on alumina or kaolin.
Glucose oxidase is a good glycoprotein. The enzyme from Aspergillus niger contains
10.5% carbohydrate, which is believed to contribute to the stability and not to affect the
overall structure. The molecular weight of the Aspergillus niger enzyme is 186,000, that of
Penicillium notatum is 152,000. The optimum pH of glucose oxidase is about 5.5. The
enzyme is fully stable between pH 4 and 6 at 40
o
C for 2 hr. Specially stabilized
preparations for use at pH 2.5 are available. Use above pH 8.0 may be possible, but
requires high glucose concentrations. Glucose oxidase is very unstable above 50
o
C.
According to the reaction equation, glucose oxidase can be used in order to remove
glucose or oxygen or to form hydrogen peroxide or gluconic acid. ndeed, in food
processing glucose oxidase finds application for removal of residual glucose prior to the
preparation of dried eggs or to remove it from other products in order to reduce
nonenzymatic browning. t is highly effective in removing residual oxygen from beer, wine,
fruit juices, high fat products (mayonnaise), or packed dehydrated foods.
5. CataIase.
Catalase [EC 1.11.1.6] splits hydrogen peroxide to water and oxygen:
2 H
2
O
2
----------> 2 H
2
O + O
2

The enzyme is widely distributed in microorganisms. ts biological role has been studied by
a number of investigators. n methanol-utilizing yeasts, it is generally acceptable that
catalase must be involved in the metabolism of methanol, since hydrogen peroxide is
liberated during methanol oxidation by alcohol dehydrogenase.
Commercially, catalase is prepared from Aspergillus niger, Penicillium vitale, Micrococcus
lysodeikticus. t is a hemo-protein containing 4 ferri-protoporphyrin prosthetic groups per
molecule of enzyme, with a molecular weight of 250,000. The optimum pH of Aspergillus
niger catalase is at pH 6.0. The enzyme is inactivated by cyanides, phenols, alkali, urea,
freezing, and by sunlight under aerobic conditions.
Production of catalase is conducted in deep tank cultivation. Biosynthesis occurs
simultaneously with glucose oxidase formation. The ratio of these enzymes is controlled by
quality and quantity of the inoculum, the composition of the medium and by aeration
conditions.
Catalase produced by the directed biosynthesis can be separated selectively from extracts
containing catalase and glucose oxidase. The recovery of catalase from Micrococcus
lysodeikticus starts with lysing the cells in a solution of sodium chloride (0.5 to 2%). The
next step is fractionation of the lysate by centrifugation of a mixture of the lysate, an
organic solvent (ethanol in an final concentration of 40-50% v/v), and a salt (sodium or
potassium chloride adjusted to a concentration of 1-2% either before or after addition of
the solvent). The dissolved catalase can then be precipitated from solution by adding
ethanol to a final concentration of 75%.
Catalase finds application wherever the removal of hydrogen peroxide is desired.
Therefore, in the food industry catalase is employed to remove the excess of hydrogen
peroxide used for cold sterilization in milk and cheese processing. Catalase may also be
employed in cake baking as well as in irradiated foods, in the process of which hydrogen
peroxide is formed.
Catalase in combination with glucose oxidase is being used in so-called glucose
analysers, whereby glucose is oxidised, the hydrogen peroxide formed catalysed by
catalase and the resulting oxygen measured with an oxygen probe. The glucose oxidised
is directly proportional to the oxygen formed.
6. GIucose isomerase
This enzyme converts D-glucose to D-fructose. The main substrate of this enzyme,
however, is xylose and, indeed, the glucose isomerizing enzyme is a D-
xyloseketoisomerase [EC 5.3.1.5] with side activities to D-glucose and D-ribose.
A large number of genera of bacteria and some yeasts have been found to produce a
glucose isomerizing enzyme. But the strains most widely used as sources for commercial
production are members of the genus Streptomyces. The isomerase in Streptomyces is an
inducible enzyme which requires the presence of D-xylose in the culture medium for its
production. Media for the commercial production of glucose isomerase therefore are based
on xylan or xylan-containing raw materials such as wheat bran, maize husks, sulfite liquor
etc. As the enzyme is of the intracellular type, it can be used in the form of whole cells.
Another way, of course is immobilization of the cells. Purified preparations with higher
activities can be obtained by application of the usual methods of cell rupture and
solubilizing the enzyme.
Principal producers and users of glucose isomerase are found in the corn wet milling
industry, where the enzyme can be used as a substrate for D-fructose production.
However, in industrial practice, high D.E. starch hydrolysates are a more economical
souirce of D-glucose. Such a hydrolysate can be prepared by the combined action of
bacterial alpha-amylase, amyloglucosidase, and isoamylase on starch to yield a glucose
syrup of about 95-98D.E.. Subsequent isomerization is carried out in agitated vessels at
65oC and pH 7 for 18 to 24 hrs using, for example immobilised cells. Following conversion
to D-fructose, the immobilised enzyme is removed and the liquor further treated to give an
invert sugar syrup of about 45% fructose and 55% glucose.
7. IndustriaI use of proteoIytic enzymes
Proteolytic enzymes, proteases, or proteinases, are enzymes which under appropriate
conditions, specifically hydrolyze the peptide bonds of proteins. All proteases have
characteristic properties with regard to pH and temperature, ion requirements, specificity,
activity, and stability. The specificity depends on the amino acids involved in the peptide
bond to be hydrolysed. These biochemical parameters determine the application of a
protease. However, its commercial feasibility also depends on the development and
production costs of the enzyme, its market, and the economics of application. The
attached table surveys the uses of industrial proteases. Let us firstly deal with the use of
proteases in detergents, leather treatment, the synthesis of human insulin and the
production of aspartame (Table 1).

TabIe 1: ndustrial use of proteolytic enzymes

The worldwide requirement of enzymes for individual applications varies considerably.
Some enzymes are produced in large quantities, up to thousands of tons, but are of
relatively little unit value, e.g. $10-20/kg. These proteases are referred to as industrial bulk
enzymes. Other proteases are used in organic synthesis in amounts up to tens of tons.
The market for medical proteases is small in terms of tonnage, but their price per unit
activity is very high.
8. Enzymes for the Detergent Industry
As early as 1913, enzymes were used in laundry detergents in Germany. The rapid growth
of enzyme detergents was temporarily set back in the early 1970s when workers in
detergent factories developed allergies to the enzyme preparations. Enzyme
manufacturers solved this problem by developing dust-free protease formulations. n 1985,
approximately 70% of the heavy-duty laundry detergents (for domestic use) in Europe
contained enzymes, compared to 15% in the U.S.
Presently the major enzyme suppliers produce a variety of differently formulated proteases
for use in all types of liquid and powder detergents, with or without bleach and phosphates.
n addition to proteases, other enzymes are also used in detergents (e.g. alpha-amylase),
and the use of enzymes such as cellulases and lipases is being developed.

Requirements. The compositions of the detergent and washing liquor, as well as the
washing characteristics, determine the functional enzyme properties of a laundry
detergent. n addition the formulated enzyme preparation must be stable on storage. The
shelf life of an enzyme is affected by temperature, pH, water activity, bleach and the
presence of denaturing agents such as nonionic and anionic surfactants. For use in
powder detergents, enzymes are formulated with fillers and binding agents into small
beads or granules, sometimes surrounded with a layer of inert material such as a wax. To
avoid segregation of enzyme particles in the powder detergent, e.g. during transport, the
particle size of the enzyme beads must be more or less identical to the size of the
detergent particles. Furthermore, the particles must dissolve rapidly when the wash liquor
is prepared.

Characteristics. Composition of the wash liquor and laundering practices differ from
country to country. n Europe, detergent concentrations of 4-10 g/l are used. The detergent
formulation contains bleach, and the washing process is characterised by a heating up
phase followed by a constant temperature phase. n the U.S. and Japan, the detergent
concentration is lower. Bleach and builders are often added separately or not at all.
Washing is done at constant temperatures, significantly lower than those in Europe.
Enzyme suppliers have developed proteolytic enzymes that fulfill all the requirements of
detergent formulation, stability, and activity under washing conditions. The types of stain
subjected to proteolytic attach are composed of protein and other soil. The protein acts as
a binder and fixes the other soil components to the fabric.
Powdered laundry detergents, which constitute the largest part of the detergent market,
functions at high pH (11). The relationship between temperature and activity is another
importance characteristic.

Activity measurement. Formulated commercial enzyme preparations contain only a small
percentage of enzyme protein (on a weight basis). They are sold on the basis of activity. n
general, the supplier will specify in the product sheets the analytical method used to
determine activity. n most cases the enzyme is incubated at specified pH and temperature
with large substrate molecules, e.g. casein or hemoglobin. At the end of incubation, the
amount of hydrolyzed and therefore solubilized protein is measured spectrophotometrically
by UV absorption.

Washing performance. The contribution of an enzyme to the washing performance of a
detergent is determined in the laboratory by test washings in specially developed washing
machines. For this laboratory testing, different types of artificial stains such as milk, blood,
cocoa, grease, egg, grass are used. The stains are applied to different fabrics such as
cotton or polyester.
Protein hydrolysis is required for the efficient removal of ink. The amount of ink left in the
cloth after washing is determined by measuring the remission of cloth. This is an indicator
of the washing performance contributed by the enzyme to the detergent
Future trends. The development of new detergent compositions will also have an
important impact on the development of new proteases and other types of detergent
enzymes. n several countries, environmental considerations led to the partial or complete
substitution of phosphates by other sequestrants. As a result, detergent compositions were
changed. The pH was increased to 10, and enzymes with altered pH activity profiles were
required.
The tendency to reduce the amount of energy required for washing, i.e. the substitution of
biochemical energy for thermal and mechanical energy, will affect the use of enzymes in
detergents. Washing at lower temperatures requires the development of new bleaching
systems. n washing without a heating-up phase, traditional enzymes may not befully
compatible with the low-temperature bleaching systems. n the past, this was not a
problem since the detergents used for constant temperature processes did not contain a
bleaching agent. At present, a strong tendency exists to incorporate a bleaching system
into detergents for these washing processes. n that case, bleach-tolerant enzymes must
be developed for specific markets.
A tendency can be noted toward the development of multifunctional detergents, i.e. one
product with the characteristics of several - the ideal being a product that contains presoak
function, a wash function, builders, a bleaching system, and a softener. One compatibility
problem inherent in this type of formulation is the presence of anionic and cationic
surfactants in one detergent. However, the cationic surfactants, which have a softening
function, may be replaced by an alkaline cellulase which also acts as a softener, especially
on cotton fabrics.
Protein engineering techniques are likely to become a powerful tool for the improvement of
existing enzymes.
9. Enymes in the Leather industry.
The processing of skins and hides for the production of leather is a traditional art. Several
different proteases can be used in the individual stages of the manufacturing process. The
rationale behind the use of proteases for processing hides and skins lies in the fact that
protein is the major building block of hair and skin. Hair is composed of alpha-keratin -
fibrous, insoluble protein molecules containing a large fraction of cysteine residues and
having an alpha-helix conformation. The alpha-keratin is arranged in bundles of fibrils.
Different skin layers are composed of collagen, alpha-keratin (epidermis), and some
elastin. n addition, albumin, globuline, glycoproteins and other globular proteins are
present. Collagen contains a large fraction of glycine, alanine, proline and hydroxyproline.
t is arranged in a triople-helix conformation. The use of enzymes with different specificities
has made possible selective hydrolysis of the noncollagenous constituents of the skin. The
stages of leather manufacturing are curing, soaking, dehairing, dewooIing, bating,
tanning.
The first time enzymes are used is during soaking. At the tannery, hides and skins are
washed and soaked in surfactants and antimicrobial compounds. Alkaline proteases are
used to remove nonfibrillar proteins such as albumins and globulins. This is important for
the next process step. Proteases used by the detergent industry are generally suitable for
this purpose since they are relatively resistant to the increased pH of about 10 used in the
soaking bath. Trypsin, a pancreatic protease, is also used. Small amounts of amylase and
lipase activity in trypsdin preparations are particularly advantageous for skins with a fatty
flesh side.
Unhairing and dewooling. Unhairing was traditionally done by soaking the skins for
several days in a strongly alkaline bath (10% lime). Now the use of alkaline protease more
than halves the amount of lime required. As a result, the final quality of the leather is
improved and significantly less wastewater is produced.
Dewooling is performed by applying so-called dewooling paint to the flesh side of the skin
and keeping the skin at 20-35
o
C for 10-20 h before the wool is pulled. Dewooling paint is
composed of hydrated lime, sodium chlorite, alkaline proteases and water. The soaking
and dewooling steps are sometimes combined. Fungal lipases improve the removal of
wool grease.
Bating. n the bating step, hides and skins are deswollen and treated with enzymes and
chemicals to make them soft and supple, and to prepare them for tanning. Today, trypsin
and small amounts of alkaline and neutral proteases are used primarily. Bating conditions
such as pH, temperature, time, amount of enzyme and composition of the enzyme
preparation are determined by the type of leather to be produced.
Tanning. After treatment with an acid solution for deliming, skins and hides are coloured
by chrome tanning. Although enzymes are not directly involved in this step, the enzymatic
treatments of earlier steps influence the quality of tanning. n the near future, the
application of enzyme systems will increase.
The synthesis of aspartame, a low-calorie sweetener, has attracted much attention.
Aspartame is a dipeptide consisting of L-aspartic acid and the methylester of L-
phenylalanine. ts sweet taste depends on the L-conformation of the two amino acids, the
presence of the methylester and the correct coupling of the amino acids. Whereas alpha-
aspartame is 150-200-times sweeter than sucrose, beta-aspartame has a bitter taste.
The enzyme thermoIysin (EC 3.4.24.4), a neutral metalloprotease from Bacillus
thermoproteolyticus, is particularly suited for the synthesis of aspartame. Under
appropriate conditions, it catalyses the condensation of N-protected L-aspartic acid and
D,L-phenylalanine methylester. Reactions occur exclusievly at the alpha-carboxyl group of
aspartic acid and with the L-isomer of phenylalanine methyl ester.
For economic reasons, the enzyme must be recycled, e.h. precipitation, immobilization, or
use of a membrane reactor. Therefore three different production methods can be
distinguished

1. Synthesis in an aqueous system. Accumulation of the reaction product in the resin of an
immobilized enzyme or clogging of the reactor membrane would be disadvantageous.
2. Synthesis in a Two-Phase system. The reaction product is formed in the water phase
and then transferred immediately to an organic phase.
3. Synthesis in an Organic Solvent. This process seems particularly suitable for continuous
operation with an immobilised enzyme.

An industrial production unit based on the enzymatic synthesis of aspartame has been
developed and started in Japan and Holland.
11. Enzymes in the Meat Industry
Cooked meat is considered tender if it can be masticated easily and, at the same time,
retain the desired texture. Tenderness is influenced by a number of factors which are not
yet well understood. Numerous endogenous enzymes take part in the process including
endogenous proteinases, particularly the cathepsins. These enzymes change muscle
protein during maturation or ageing of meat.
Since 1940, attempts have been made to use exogenous enzyme preparations as meat
tenderizers. Proteinases capable of digesting connective tissue and muscle protein have
been chosen for this purpose.
Enzymes used on a commercial scale are papain [EC 3.4.22.2], bromelian [EC 3.4.22.5],
and ficin [EC 3.4.22.3]. The main problem associated with enzyme use is their even
distribution in the tissue. Factors influencing this distribution are diffusion, time, salt
content, and enzyme concentration.
f preparations are sprinkled only on the surface of the meat or if pieces of meat are dipped
into an enzyme solution, generally only the surface is tenderized and the interior remains
tough. n the kitchen, after enzyme application (e.g., 2% NaCl with 0.002% bromelain), the
meat is repeatedly poked to make it easier for the enzyme to penetrate. The main effect of
the proteinase is exerted only during cooking.
Commercial methods can be divided into premortem and postmortem procedures. n
postmortem treatment, a proteinase solution is spread into the carcass by repeated
injections, possibly under pressure. n the premortem method, the Swift technique
developed in 1960, a very pure, sterilized papain solution is injected intravenously 2-10
min before the animal is slaughtered.
Pancreatic proteinases are used in the maturation of fish. Bacterial proteinase is employed
to dissolve bone meat or segments of meat.
12. Enzymes in the Dairy Industry
The use of biocatalysts in food chemistry, especially in making dairy products, is one of the
oldest examples of biotechnology and goes back thousands of years. A typical case is the
use of either isolated biocatalysts (rennet) or cell cultures (lactobacilli, streptococci,
micrococci) in the production of cheese.
Biocatalysts are responsible for numerous reactions, such as the formation of aromatic
compounds, changes in matrix and structure and variation of pH.
A number of fermented products are made available today by use of purely biological
procedures; an example is the enormous variety of cheeses. The table in your notes
(Table 2) lists a number of isolated enzymes used in the production of dairy products.
12.1 Enzymes from rennet and rennet substitutes
The classical example of the use of a biocatalyst is in cheese making, where rennet is
added to gel the casein in milk. Today, other enzymes are employed in this step.

Rennet enzymes. Rennet, a mixture of chymosin [EC 3.4.4.3], , also called rennin, and
pepsin [EC 3.4.4.1], is obtained from the gastric mucosa of young mammals, e.g. calves
and lambs. The pepsin-to-chymosin ratio of different rennet preparations can vary
considerably because chymosin is present only in the stomachs of unweaned mammals
and is later replaced by pepsin. Only chymosin is able to convert specifically casein from
the sol to the gel state. Pepsin and other proteolytic enzymes are much less specific and
give rise to a number of degradation products which tend to taste bitter. For this reason,
pure chymosin and high-quality rennet are important
Chymosin is a highly specific endoproteinase. t destroys the protective colloidal function
of cappa-casein. As a result, the surface of the casein micelle becomes more hydrophobic,
leading to gel formation. Therefore, the proteolytic activity and specificity of rennet
preparations are of great importance, and both the chymosin and the pepsin contents of
rennet preparations must be determined accurately.

TabIe 2: Enzymes used in the processing of Dairy Products

Rennet substitutes. Because of the limited availability of pure chymosin, other proteolytic
substitutes have been employed in cheese making, e.g. enzymes formed by
microorganisms, which are considered to be safe according to food laws. Examples are
preparations from Mucor miehei, M.pusillus, Bacillus subtilis and Endothia parasitica. Apart
from microbial enzymes, proteolytic enzymes from other sources have also been tested for
their ability to coagulate milk, e.g. plant proteinases such as papaoin, ficin and bromelain.
Microbial enzymes are readily available and gradually replace chymosin in cheese making.
The search for new substitutes continues. Techniques of genetic engineering have also
been applied to the production of biologically active milk-coagulating enzymes. Escherichia
coli and Lactobacillus lactis have been manipulated genetically and made to produce large
amounts of chymosin in its zymogen form, prochymosin. Activation of the zymogen to give
chymosin and the action of this enzyme on milk in cheese making are currnetly being
under investigations. The specificity of other endopeptidases has been examined by
analysing the peptide fragments formed from casein or by measuring the ratio of digested
to undigested protein. A further parameter is the bitterness of the casein peptides
generated. The next step is to check the ability of the enzyme to coagulate the milk. Time
and temperature during gel formation are monitored and gel stability is analysed by use pf
rheological parameters.
Substantial changes in pH occur during the ripening of the cheese. Hence, the pH
dependence of the specificity of endoperoteinases used in chesse making must also be
tested. Large differences between nonspecific proteinases and highly specific chymosin
have been observed during ripening - a factor which can give rise to undesirable cheese
flavors.
Numerous tests are carried out to control the ripening and quality of cheeses produced
with rennet substitutes. n unripened cheese, the growth of microbial flora on the cheese is
encouraged after the initial processing. The ripeneing process, i.e. the growth and
metabolic activity of these microorganisms in turn is largely dependent on the peptide
spectrum formed during enzyme treatment.
Many difficulties can be overcome by using immobilised chymosin or rennet substitutes.
Because hydrophobic interactions play an important part in the formation of casein gel, gel
formation does not occur at low temperature (entropy effect). Hence, the cleavage of
cappa-casein (renneting) can be separated from gel formation. Cleavage is first carried out
in a reactor contain ing the immobilised enzyme at 4oC. After leaving the reactor, the
rennet-treated milk is slowly heated so that gel formation can take place and casein can be
separated in a continuous process. However, in practice, the enzyme reactor has been
found to have a relatively short life span because milk fat and proteins block the activity of
the immobilised enzymes. A further problem is, of course contamination, because milk is
an excellent substrate for contaminating organisms.
12.2 Beta-1,4-GaIactosidases
The use of beta-1,4-galactosidases [EC 3.2.1.23] in the cleavage of lactose is another
important application of biocatalysts. Beta-1,4-galactosidases are extensively distributed in
nature; they hydrolyse lactose to glucose and galactose. This improves the solubility and
increases the sweetness of the product.
The raw material for lactose is either whey having a pH of 4-6 or milk with a pH of 6.3-6.8.
The optimal pH for enzyme activity must correspond to the pH of the substrate. Yeast and
certain strains of Aspergillus and Bacillus produce beta-galactosidases with the required
properties. The beta-galactosidase from Escherichia coli, which possesses excellent
properties, has not been applied because it is not regarded as absolutely safe by the food
law.
The cleavage of lactose in milk and whey was first accomplished with soluble enzymes.
The consumer can add the enzyme to milk in the evening, and after 12 h in the
refrigerator, at least 80% of the lactose is hydrolysed. Soluble enzymes have also been
used in the preparation of a variety of dairy products such as cheese and yoghurt.
12.3 Lysozyme
The enzyme lysozyme [EC3.2.1.17] is a component of perishable foods such as eggs. t
protects the food from infection by favouring bacteriolysis. Attempts have been made to
replace the use of nitrate in cheese making by adding lysozyme from egg white; the
enzyme suppresses the growth of Clostridium thyrobutyricum.
The general application of lysozyme or other bacteriolytic enzymes offers a valuable, yet
still expensive, alternative to the undesirable use of nitrates.
The characteristic taste of heated milk is attributed to mercapto (sulfhydryl) compounds.
These compounds are released on heating, when the sulfhydryl oxidase contained in milk
is denatured simultaneously. Sulfhydryl oxidase can oxidise mercapto compounds and
banish the unpleasant taste associated with heated milk.
Enzymes and enzyme complexes are employed to improve the aroma and texture of dairy
products. The nature of the microorganisms used for fermentation is one of the factors
contributing to the enormous variety of cheeses, which differ from one another in aroma
and texture. For this purpose, commercially available lipases and proteinases have been
used. The metabolism of fat, protein, and lactose generates aroma and texture; therefore
enzyme complexes from product-specific cultures were then tested on suspensions of the
individual raw materials, and the formation of aroma compounds was found to be greatly
accelerated.
Starch and products derived from starch contribute to essential human nutrition and have
many industrial uses. The corn-processing industry, which began in the US in the 19th
century, is a major source of these products. The conversion of starch to dextrose and
oligosaccharides was based primarily on acid hydrolysis. Conversions at very low and very
high pH were known to result in undesirable byproducts and carbohydrate modification,
which limited the use of hydrolysis in foodstuffs. The application of enzymology to this
area, pioneered by Jokichi Takamine, the discoverer of malt diastase, introduced the use
of selective degradation and conversion under moderate conditions. Further work
indicated that the initial steps in the conversion of starch by acid could be followed by the
use of an enzyme to saccharify starch to oligosaccharides and dextrose.
The growth of the industry accelerated considerably with the discoveries

a) of dual enzyme conversion processes for improved conversion of starch to dextrose;
b) isomerizing enzymes for conversion of dextrose to fructose.
The commercial development of these processes provided an important, low-cost sugar
substitute derived from corn and other grains. Therefore, corn producers who, in 1970,
provided some 65x10
6
t of corn and processed 3x10
6
t corn to products, are now providing
nearly 250 x 10
6
t of corn and processing about 20 x 10
6
t to products. About 50% of these
products are directed to the manufacture of high-fructose corn syrup
A second major application for starch processing with enzymes is the production of fuel
alcohol. This became important in the 1970s and 1980s when alternatives and partial
substitutes for petroleum were sought from agricultural resources. This effort has also
aided in considering bioprocess alternatives for the production of other chemicals and
chemical intermediates.
A third application of starch-processing enzymes is their use in baking. This area is of
interest because it may lead to new apoplication of cell and enzymes in the processing of
foodstuffs in general.
13.1 Syrups and Sweeteners
Enzymes play an important role at various stages in this process. Corn kernels, treated
with sulfur dioxide and lactic acid bacteria, are softened so that fiber, protein and oil can be
separated from the starch by centrifugal force, based on density and size difference.
Enzymes are added only when the starch-water slurry has been prepared. However,
interest is increasing in the use of microbial and enzymatic augmentation, which involves
such enzymes as cellulases, glucanases, proteases, and pectinases, at this early stage of
the process. The objective is to improve stream quality and increase yield of protein or
starch. n addition, this could lower the cost of corn wet milling, aid environmental
protection and increase product saleability.
Many organic and inorganic impurities that remain in the starch milk can affect enzyme
performance. Examples include inhibitory cations and anions, factors that affect buffering
and pH adjustment, heat sensitive materials which would result in byproducts, and
microbial contamination.
The starch milk is adjusted to a pH of 6.0 and usually subjected to high-pressure steam in
a continuous cooker. A small amount of alpha-amylase is added at this point to augment
swelling, water absorption, and gelatinisation and subsequent thinning of the starch. More
amylase is added and liquefaction continues until the starch is degraded to polymer units
of 15-20 DE (dextrose equivalents, 1 DE has the same reducing power as a 1% aqueous
solution of pure dextrose). Total time to reach this level of conversion is up to 2 h in
continuous processes. Starches from different sources respond differently to this
treatment; some can be gelatinized and degraded more easily, based on gelatinization
temperature and the presence of fats, protein, and varying glycosidic linkages. The starch
must be broken down completely to limit the amount of dextrins, small oligosaccharide
polymers of glucose, and to prevent molecules from recombining into forms not as
susceptible to degradation.
The thermostable alpha-amylase currently in use commercially can withstand
temperatures near boiling point of water for a sufficient period of time to complete the
conversion. Present technology is based on alpha-amylase derived from bacteria (Bacillus
licheniformis) or fungi (Aspergillus niger).
An important development is the use of amylases that function at a pH slightly below 6.0.
Depending on process circumstances, lowering the pH results in improved quality, less
byproduct formation, and easier processing. Calcium is necessary to stabilise and activate
alpha-amylase. Compounds from corn such as phytin can competitively bind the calcium
and thus decrease enzyme performance.
The partially converted stream is the adjusted to pH 4.0 and cooled to about 60
0
C to
permit saccharification to occur. Amyloglucosidase, sometimes augmented with
pullulanase, is added to saccharification. The pullulanase allows more raoid degradation of
1,6-glycosidic bonds and higher conversion to glucose when the stream contains more
solids. The use of high solids lowers costs since less water must be removed in later
stages. The presence of an alpha-amylase component in the saccharification stage can
also benefit and augment amyloglucosidase in the disruption of 1,4- and 1,6-bonds.
Conversions that require too much time favour reversion (i.e. reformation of 1,6-bonds).
These reversion reactions yield byproducts such as isomaltose, which are considered
impurities, and reduce the yield of glucose. When transglucosidase is present as an
impurity in amyloglucosidase, it will also promote such undesirable reversion reactions.
The possible use of immobilised enzymes (amyloglucosidase and pullulanase) in
saccharification has been explored. To date, yields from such systems are lower than
those obtained in batch saccharification at comparative solid contents (30-35%) and
temperature levels (60
o
C). Equivalent yields can only be obtained at lower temperatures
(favouring microbial contamination) and higher dilution (greater costs). The corn wet-
milling industry would benefit from such a technology only if new methods were introduced
to improve process economics and aid in quality maintenance. Currently the immobilised
enzyme-assisted saccharification of the oligosaccharide recycle stream from furctose
enrichment is being investigated.
n a typical process, the effluent from saccharification is filtered to remove protein and fat,
and then purified by treatment with activated carbon and ion exchange. The result is a
solution of at least 95% dextrose. This solution can be either crystallised to yield pure
dextrose or sent on for isomerization-refining to fructose corn syrup.
Processes to prepare intermediate-level conversion syrups can be based on a
combination of acid and enzyme conversion. Alternatively, a series of enzymes, including
beta-amylase, amyloglucosidase, and debranching enzymes can be used in immobilised
form to yield these syrups.
The use of such syrups could increase considerably in future, but enzyme sales must be
large enough to creative incentives for such development. Current markets include
confectionery and fruit processing.
13.2 FueI AIcohoI
Starch conversion to produce fuel alcohol (ethanol) is analogous to the conversion to
syrups and sweeteners. A variety of grains and tubers are used worldwide for alcohol
production. For example, in the U.S. ground corn is the most common source, whole
cassava along with sugar cane is used in Brazil. When ground corn is used as raw
material, as opposed to starch, a large amount of undissolved solids must be handled.
As indicated in your notes, bacterial thermostable amylase and amyloglucosidase are
used to convert the feedstock to a fermentable substrate best suited for assimilation by
yeast or bacteria in ethanol production. The use of genetically modified yeast, which
synthesizes the amyloglucosidase as part of its growth and metabolism, has been
reported. This can be advantageous in a new facility to save capital cost' however, to
obtain the best combination of saccharification and fermentation time must be evaluated
on a case-by-case basis. A new bacterial fermentation using Zymomonas mobilis showed
great promise for advancement in production costs through simultaneous saccharification
and fermentation (see chapter 15).
Feedstock for this process can include recycled yeast and distiller's grains (backset),
mixed with ground corn and enzyme. The mixture is hydrated, slurried and subjected to
intensive liquefaction to ensure conversion of all the starch in the presence of undissolved
solids. This can involve several stages and temperatures. After secondary liquefaction, the
material is saccharified by treatment with amyloglucosidase. The saccharification time can
vary and is shorter than the time required for the analogous step in syrup manufactuire
since complete conversion to glucose prior to fermentation may be neither necessary nor
desirable, because excess glucose could cause excessive biomass formation at the
expense of ethanol yield or could cause substrate inhibition at the expense of ethanol
yield.
13.3 Baking
Cereal grains such as wheat or rye are used as sources of flour for baking (see also
chapter 17). Enzymes may either be endogenous to the grain or added as supplements for
several purposes. For example, proteases can be used to degrade gluten in a controlled
manner, and amylases can be used to obtain the desired profile of minimal dextrins,
fermentable sugars for yeast metabolism, and carbon dioxide formation. Free peptides and
amino acids resulting from the action of protease can combine with dextrins to yield
desired Maillard-type browning reactions; the beneficial result of this, if not excessive, is
brown crust, good colour and added flavour. Proteases can reduce the dough viscosity
caused by gluten and thus improve processing. The desired softening effect of proteases
can vary as greater softness is desirbale for biscuits and waffles.
Both types of amylases, alpha- and beta-amylase, are used in baking. Beta-amylase is
generally available in sufficient quantity in the grain, but alpha-amylase is oftyen deficient
and must be supplemented. The amount of additional enzyme must be sufficient for
desired gas production, volume control, and colour. t must not be added in excess
because this can result in excessive dextrin production, leading to loaf stickiness, dark
colour, and possibly insufficient product strength. The alpha-amylase supplement can be
derived from malted flour, however, fungal enzyme is commonly used because this alpha-
amylase and cereal beta-amylase are mesophilic and cease activity as the temperature
approaches and passes through the gelatinization point (70-80
o
C). This provides a means
to control dextrin formation. The malt amylases are thermally more stable and can lead to
excessive breakdown of starch. The enzymes derived from Aspergillus oryzae act in the
pH range 4.5-5.5. The pH of the dough can vary with the product, so a pH near 7.0 may
result and alter the enzyme dose. Levels of fungal amylase range from 5-250 SKB units
per 100 g flour depending on the application.
Proteases are usually derived from bacterial sources (neutral pH variety), but fungal
proteases can also be used in certain cases. The levels employed to aid in browning,
machining, and dough extension range from 2 g per 100 kg flour in crackers, through 15
g/100 kg flour in biscuits and up to 50-100 g/kg flour in waffles. The dose depends on the
pH of the dough. Lipoxygenases from soybean have been explored to improve whitening,
and phospholipase (animal source) has been studied as an antistaling agent.
Pentosanases (hemicellulases) have been used to cleave residual pentosans in flour in
certain cases to aid in antistaling and water absorption.
13.4 GIucose Isomerization
Enzymatic isomerization of glucose to fructose in starch processing is carried out on an
industrial scale worldwide. The commercial product obtained, HFCS typically contains 42
or 55% fructose based on dry substance.. t is used as an alternative sweetener to sucrose
or invert sugar in the food and beverage industry.
Glucose isomerase is produced by several microorganisms as an intracellular enzyme.
The commercially important varities show superior affinity to xylose and are classified as
xylose isomerase. The first patent on enzymatic isomerization of glucose was issued in
1960. The glucose isomerases used commercially today are all immobilised and
granulated to a particle size between 0.1 and 1.5mm. The enzymes are rather similar to
each other with respect to dependence on temperature, pH, and metal ion activation.
Typically, addition of Mg
2+
to the feed syrup is recommended, and Co
2+
and Fe
3+
are
potential activators, whereas Ca
2+
acts as an inhibitor by displacing Mg
2+
in the enzyme
molecule.
Glucose isomerization can only be made economically feasible by immobilising the
enzyme. A relatively high reaction temperature is necessary to obtain a reasonable
fructose yield. The pH must be ca. 7.0 or higher to secure satisfactory enzyme activity and
stability. Under these conditions, glucose and fructose are rather unstable and easily
decompose to organic acids and coloured byproducts (carbonyl compounds). To limit
byproduct formation, reaction time must be minimized, which can be done economically
only by using high concentrations of immobilised isomerase.
somerization of glucose to HFCS on an industrial scale is carried out almost exclusively in
continuous fixed-bed reactors, in which purified glucose syrup from the saccharification
stage of a starch processing plant is passed through a bed of granular, immobilised
glucose isomerase. The enzyme granules must be rigid enough to prevent bed
compaction during operation. At the temperature commonly used in industrial
isomerization, the equilibrium ratio of fructose to glucose is about 0.50, but to avoid
excessive reaction time, the conversion is normally limited to about 0.45.
The main criteria for selecting the feed syrup specifications are optimisation of enzyme
productivity and limitation of by-products. The use of immobilized enzymes requires highly
purified substrates to prevent rapid deactivation and clogging of the enzyme bed. nsoluble
impurities in the glucose feed syrup (fat, protein) are removed by filtration, and soluble
impurities (peptides, amino acids, salts) by treatment with activated carbon, followed by
ion exchange.
The dry-substance content of the feed syrup is adjusted to 40-50%. Higher syrup
concentration will result in a reduced isomerization rate due to diffusion resistance in the
pores of the immobilised enzyme. A de-aeration step removes dissolved oxygen that
would increase by-product formation. The pH is adjusted to the productivity optimum of the
enzyme.
The isomerization temperature is normally 55-60
o
C. Lower temperatures lead to increased
risk of microbial infection. Higher temperatures increase the isomerization rate but reduce
enzyme and monosaccharide stability.
During operation, the immobilised enzyme loses activity. When the feed syrup is carefully
purified, the most likely explanation for this activity decay is heat denaturation of the
enzyme. Typically, a reactor load of glucose isomerase is replaced after three half-lives,
i.e. when the activity has dropped to around `12.5% of the initial value. The most stable
glucose isomerases have half-lives of more than 100 days in industrial practice.
To maintain a constant fructose concentration in the product syrup, the feed flow rate is
adjusted according to the actual activity of the enzyme. With only one isomerization
reactor in operation, excessive variations in syrup production rate would result. To avoid
this, several reactors containing enzyme of different age are operated in combination. With
a system of eight reactors, the variation in total syrup flow can thus be limited to 13% of
the average. The isomerization reactors may be connected to operate in parallel or in
series.
n the U.S., reactor diameter is normally between 0.6 and 1.5 m. Typical bed height is 2-5
m. Minimum bed height - diameter ratio for one reactor is 3:1 to ensure good flow
distribution.
Around two-thirds of the HFCS produced worldwide is used in the chromatographically
enriched form containing 55% fructose for sweetening nonalcoholic beverages. The 42%
HFCS obtained directly by enzymatic isomerization is used mainly in the baking, canning,
and dairy industries. Because of the high hygroscopicity of the fructose component, HFCS
cannot replace sucrose in the manufacture of hard candy.
Both 42% and 55% HFCS are used almost exclusively as a reduced-cost replacement for
liquid sucrose and invert sugar; the price of HFCS is typically 10-20% lower than that of
sucrose, based on sweetening power. Pure fructose (100%) has a relative sweetnes of
1.5-1.7 (sucrose = 1.0).
14. AnaIysis
Product quality and consistency require sufficient analytical monitoring. n starch
processing, the solid content is measured by refractometry with proper temperature
control. The presence of protein as a contaminant can be detected by the Kjeldahl method.
f separation and identification of proteins are required, isoelectric focusing,
electrophoresis, and ion exchange chromatography are used. However, these latter
methods are not common as quality tests in industry. The dextrose equivalent (DE),
defined as the availability of free carbohydrate ends able to reduce CuSO
4
solutions is
measured to determine the degree of conversion of starch. More detailed knowledge of
carbohydrate composition requires the use of HPLC and gas chromatography. The
presence of undesirable colour can be measured spectrophotometrically at several
wavelengths. The relative amount of fructose present can be determined by optical
rotation with a polarimeter and by HPLA. Atomic absorption methods are used to
determine cations. The ionic purity of streams can be assessed by measuring electrical
conductivity.
15. BibIiography
Used, see previous chapter.
Chapter 14

MicrobiaI BiotechnoIogy in Industry - Production of
MicrobiaI Biomass for Food, Feed and FertiIiser
Content
1. Introduction
2. Microorganisms
2.1 Bacteria
2.2 Yeast
2.3 Fungi
2.4 AIgae
3. Production of microbiaI biomass as a nutritionaI protein source
3.1 Pruteen Process
3.2 Baker's yeast production
3.3 Fodder yeast production
3.4 PekiIo Process
3.5 Mushroom production
3.6 AIgaI biomass production
4. Production of microbiaI biomass as a protein-enrichment for animaI feed
4.1 Protein-enriched starch
4.2 Protein-enriched whey
4,3 Conversion of IignoceIIuIose into feed with white-rot fungi
5. SiIage - The use of microbiaI biomass for the production and preservation of
animaI feed
5.1 EnsiIing Process
5.2 SiIage microfIora
5.3 SiIage additives
5.4 SiIage quaIity
5.5 SiIage in tropicaI areas
5.6 SiIage from crop residues and by-products
6. Composting - The use of microbiaI biomass for the production of fertiIiser
6.1 PhysicaI factors affecting processing
6.2 ChemicaI factors affecting processing
6.3 MicrobioIogy of composting
6.4 HeaIth risks from pathogens
6.5 Odour sources
6.6 ConcIusions
7. Literature
1. Introduction
The industrial production of microbial biomass protein [MBP] was probably the first attempt
in the history of mankind to produce proteinaceous food and feedstuffs without the aid of
agriculture. Whereas agricultural production has its advantages through the use of free
solar energy and carbon dioxide from the atmosphere, the industrial production is
independent of climate and temperature, requires smaller space area, but a substrate or
carbon source for the microorganism to grow.
Furthermore, every microbial process used in industry and/or bio-integrated systems has
as a waste, microorganisms. Thus it is vital and absolutely necessary to select for any
process development beneficial microorganisms, which are neither pathogens nor
toxin producers of any kind. We cannot allow in future that microorganisms from any
product formation can be discarded and thus released into the environment in
concentrated form.
Microbial biomass proteins are potentially useful in supplementing the need for protein in
animal nutrition. The production of MBP from waste residues and surplus raw materials
could provide an economical control of some forms of environmental pollution from various
industrial and agricultural operations and concurrently alleviate some of the global
malnutrition. The cheapest microbial biomass protein is without any doubt that from
fermentation processes and product formation processes. Microbial biomass production
may become a very important factor in the battle against the 'greenhouse' effect and could
bring far reaching socio-economic benefit.
The characteristics of the microbiaI biomass protein depends on the substrates
used, the microorganisms empIoyed and the process aIternative chosen.
The application of microbial biomass protein for food and feed is inevitably connected with
strong considerations concerning the health of animal and man. The nutritional value of a
protein is dependent on its amino acid pattern and is judged to be better the more closely it
resembles the amino acid content of whole egg or the slight modification recommended as
a reference by the Food and Agricultural Organisation of the United Nations (FAO).
Special emphasis is given to the sulphur-containing amino acids such as cysteine,
methionine and cystine.
One of the first problems encountered with mass-produced microbial cells was its content
of nucleic acids. Whereas bacteria and in particular yeast have a very high nucleic acid
content, fungi and algae like plants are relatively low. Nucleic acids are undesirable at high
levels because their digestion leads to unacceptable high levels of uric acid, which may
lead to the precipitation of ureates in tissues and joints giving symptoms similar to those of
gout.
Toxic compounds, collectively known as mycotoxins are produced by many types of
filamentous fungi, such as Aspergillus niger and others. The requirements laid down by the
Food and Drug Administration [FDA] in the US not only demands laboratory testing for
traces of toxin, but also extensive palatability and feeding trials before any approval is
given.
The basic steps in MBP production consist of

a) the substrate or raw material, including possible treatment;
b) the growth of the microorganism;
c) the mechanical product separation
d) the thermal phase separation.

Raw material requirements for MBP production are governed by the requirements for
growth, which usually include carbon and energy source, nitrogen source, oxygen,
minerals and supplementary nutrients. Production media should be developed on the
basis of cell composition. The relationship between growth and substrate utilisation
together with the thermodynamics of the cell suggest a maximum value for a particular
type of substrate, whereas the minimum value depends very much on the skill of the
operator in regard as to how badly one grows the microorganism. Because of the great
influence of yield on the economics of MBP production, it is of great importance to
consider attempts to predict yields as a function of substrate or microorganism used.
Conceptually, the prediction of yield requires knowledge of

a) the pathway of dissimilation of the carbon source in sufficient detail for an estimate to be
made of ATP produced per unit of substrate oxidised;
b) the pathways by which the carbon source is assimilated, and the resulting cell
composition, so that an estimate can be made of ATP required per unit of cell synthesised;
c) the maintenance requirement, so that an estimate can be made of the ATP required for
maintaining the integrity of the cell and for all the other purposes considered as
maintenance;
d) the degree of uncoupling, so that one can estimate the ATP synthesised but broken
down in non-productive ways.

All these aspects have been dealt with in previous chapters and should be recuperated
from there.
A further consideration is, of course, that the costs of wastes that are suitable for
substrates are considerably lower than those of commercially available raw materials.
However, costs of collecting, transporting and pretreatment must be considered in
determining whether or not it is economically feasible to use these materials at a given
site. n general, carbon and energy costs may range from 14% for wastes to greater than
50% for highly purified substrates of total manufacturing costs. On the other hand it is
useless to develop an MBP process using wastes, if the demand and future availability
cannot be secured.
2. Microorganisms
Four types of microorganisms are used to produce microbial biomass: bacteria, yeasts,
fungi and algae. The choice of a microorganism depends on numerous criteria, the most
important of which is the nature of the substrate available. The other criteria are nutritional
[energy value, protein content, amino acid balance], technological [type of culture,
nutritional requirements, type of separation], and toxicological. The ideal microorganism
should possess the following characteristics:

1. high specific growth rate [] and biomass yield [Y
X/S
]
2. high affinity for the substrate
3. low nutritional requirements
4. ability to use complex substrates
5. ability to develop high density of cells
6. stability during multiplication
7. capacity for genetic modification
8. good tolerance to pH and temperature

n addition, it must have a low nucleic acid content, good digestibility and be non-
pathogenic and non-toxic.

TabIe 1: Microorganisms used according to carbon source

2.1 Bacteria
The specific growth rate and biomass yield of bacteria are greater than those of the other
categories of microorganisms. Total protein content may reach up to 80%. Their amino
acid profile is balanced and their sulfur containing amino acids and lysine concentrations
are high. n contrast, the nucleic acid of 10-16% is greater than that of yeast, fungi or
algae. Only a limited number of bacterial species can be used in foodstuffs as many are
pathogenic and their small size makes a separation from the liquid very difficult.
2.2 Yeast
Yeasts were probably the first microorganisms known and are generally best accepted by
the consumer. Yeasts are rarely toxic or pathogenic and can be used in human diets. They
have been used during World War and as protein extenders in sausages and other
food materials in Germany.
Although their protein content rarely exceeds 60%, their concentration in essential amino
acids such as lysine, tryptophan and threonine is satisfactory. They contain, however, only
small amounts of sulfur-containing amino acids such as methionine and cysteine. They are
also rich in vitamins of the B group and their nucleic acid content varies between 4 and
10%. Although their specific growth rate is relatively slow, they are larger than bacteria and
thus facilitating better separation.
2.3 Fungi
The use of fungi in food fermentation is as old as the human race exist. n particular in
Asia and SEAsia, fungi play an important role in foods such as tempeh, miso etc. As
microbial biomass producers, however, the use of fungi is relatively new. They are more
conventionally used for producing enzymes, organic acids and antibiotics. Their generation
times are significantly longer than bacteria and yeast. Their protein content is often smaller
with around 50% and they are deficient in sulfur amino acids. There are also problems with
wall digestibility, but there nucleic acid content is very low with 3-5%. The principal merit of
fungi are their ability to use a large number of complex growth substances such as
cellulose and starch and easy recovery by simple filtration of the mycelium, reducing
significantly the production costs.
The dominant use of fungi as food are in the form of the mushrooms.
2.4 AIgae
The enormous potential of algae is related to their ability to multiply with carbon dioxide as
the only carbon source , with some genera [Cyanophyta] using also atmospheric nitrogen.
Algae production takes place in natural ponds, lakes and lagoons. They are traditionally a
food complement for some populations in Mexico (Spirulina platensis) and Chad in Africa
(Spirulina maxima). Algae have a low sulfur-containing amino acid content, high vitamin
content and their nucleic acid content ranges between 4 to 6%. They are easy to recover,
but multiplication is relatively slow.

3. Production of microbiaI biomass as a nutritionaI protein source
Microbial biomass production processes fall into two main categories. Firstly there are
many processes employing diverse strains of microorganisms, metabolizable substrates
and fermentation equipment for the sole purpose of concentrated biomass production from
mostly waste, thereby reducing the environmental effects of the wastes, eg n-alkane, n-
paraffin [from the oil industry], sulfite waste liquor [from the paper industry], cellulosic
wastes [from the agricultural industry] and methanol [from the oil and gas industry]. The
second category of microbial biomass production is concerned with the enrichment of
renewable resources with protein for animal feed. Whereas the former is concentrated on
the total removal of the carbon and nitrogen sources, the latter has no such a concern.
The approaches and economics of both processes are therefore quite different.
Over the past century, numerous small and large scale industrial processes have been
operating, some are still operating, but the majority have ceased functioning.
3.1 Pruteen Process
The commercial production of microbial biomass [also referred to as 'single cell protein' or
SCP in earlier literature] from methanol was developed by C [mperial Chemical
ndustries] in UK. This process is using the bacterium Methylophilus methylotrophus. The
bacterium was isolated from soil and was chosen because it is safe, grows rapidly and
efficiently on methanol as its only carbon and energy source. t produces a product not
only rich in protein but also in the essential amino acids lysine and methionine. Dried
'Pruteen' contains 73.8% crude protein (64% true protein) and 3600 kcal/kg metabolisable
energy, making it a highly concentrated protein/energy feed ingredient. At the end of 1983,
granular 'Pruteen' was selling at more than US$ 600/ton.
The problems of the volatility and toxicity of the substrate were solved by the development
of a new type of fermenter, the 'pressure-cycle' fermenter. This air-lift fermenter is the
largest aerobic fermenter in operation in the world. ts capacity is nearly 1500 m
3
, the
height of the column is 42 m with a diameter of 7 m.
3.2 Baker's yeast production
The production of baker's yeast is probably the world's largest microbial biomass
production process. World production has been estimated to be around 2 million t/year of
pressed yeast per annum. Successive improvements to yeast strains and processes since
the 18th century have led to improved biomass yields from carbon sources. Most of the
manufacturers of compressed or dried yeast using molasses as their carbon and energy
source material. Most production processes today are using fed-batch cultivation systems,
whereby a typical production cycle lasts 5-7 days. A well organised fed batch system can
produce 48 tons of pressed yeast in 16 hours. This pressed yeast contains 30% dry matter
and can be dried to obtain dried yeast with a moisture content of only 6-8 percent.
3.3 Fodder yeast production
n the 1970s, a consortium of German companies developed a process for the production
of yeast biomass from n-paraffins. After screening about 500 yeasts, the yeast
Endomycopsis (Candida) lipolytica was selected from a soil dust sample at a petrol service
station.
A 4000 ltr loop fermenter with liquid jet propulsion with air being supplied through a two-
phase nozzle was used. The liquid jet effected dispersion of the liquid/gas mixture, thus
increasing the interfacial area for the oxygen transfer. At the same time, the medium was
distributed within the loop of the fermenter by means of internal circulation. The yield
obtained was around 0.95 g of cell dry weight per g alkane utilised with a productivity of of
2 g cell dry weight/litre/h. The resulting yeast paste was heat treated to kill the cells and
improve digestibility and spray dried to obtain a powder containing 3-5% moisture. The
dried yeast had a crude protein content of 60% and was suitable for animal feeding.
3.4 Pekilo Process
This process was developed at the Finnish Pulp and Paper nstitute using spent sulfite
liquor [SSL]. t is the first commercial continuously operating process, in which the product,
the filamentous microfungus Paecimolycus variotii is used as a feed ingredient. The
Jamsankoski-Pekilo mill has a production capacity of 100 m
3
/h of SSL producing about
15,000 t/year of Peliko-MBP product. The mycelial product has the advantage over the
bacterial and yeast cells in that it can be recovered on drum filters in a process that is less
costly than centrifugation. The dried MBP contains 55-60% crude protein, of which 87% is
digestible. t also contains vitamins and minerals and is used as an animal feed
supplement.
3.5 Mushroom production
Cereal straws and other plant by-products are either burnt in the field or utilised as feed for
low producing ruminants. More than 3.5 billion tons per annum of agricultural by-
products are produced in the world. A more efficient way of utilising lignocellulosics is in
the cultivation of edible fungi. By suitable treatment, lignocellulosics can be converted into
substrates for the cultivation of higher fungi. Fruiting bodies serve as delicious food, spent
substrate can be used as feed or as humus fertiliser.
The conversion of plant by-products by edible fungi has many remarkable advantages:

1. ndustrial plant residues and by-products (e.g. bagasse, sawdust, etc.) can efficiently be
extracted and reintegrated into the ecosystem through natural processes;
2. Solid and liquid waste (e.g. cereal straw, sawdust, sulfite liquor and other residues from
the paper industry) can be directly converted into fungal substrate;
3. Carbon sources of lignocellulosics, which have an unsuitable low digestibility can be
transformed into consumable biomass (i.e. fruit bodies);
4. Harvesting of flesh fruit bodies from the surface of the substrate (pure microbial
biomass) can be obtained without expensive separation;
5. Edible fungi represent a well characterised microbial biomass, which will be generally
accepted by the consumer. The presently cultivated species are presented in Table 2.

Edible fungi are cultivated worldwide under various climatic conditions. n Europe, North
America, Asia and Australia, Agaricus bisporus is traditionally grown. n the last decades,
the People?s Republic of China and Taiwan became the second or third largest producers
of mushrooms in the world.

3.5.1 EcoIogicaI background of mushroom production. A complex interdependence of
organisms in the terrestrial ecosystem determines the presence of single species of fungi
in the natural habitats of soil, wood, leaves and other plant residues. Fungal growth can be
facilitated according to the general or specific metabolic activities of competitors for
available nutrients. Easily available carbon and nitrogen sources are advantageous to
organisms with high metabolic turnover and growth rates. This raises the question of the
conditions necessary for Basidiomycetes and Ascomycetes to compete and survive in their
ecological niche.

TabIe 2: Edible Fungi [mushrooms]

n order to develop a reproducible method for domesticating and cultivating these fungi, it
is necessary to determine the conditions under which they colonise the substrate and grow
in nature. n comparison to pure (in vitro) cultures on artificially composed media, the
growth of fungi on natural, non-sterile substrates indicates a successful adaptation of
biotechnological processes to the ecological requirements of the fungus.
Colonisation of substrates by higher fungi is mainly due to their microbial 'saprophytic'
characteristics. Sterile substrates are colonised only in conjunction with free nutrients and
water availability. Optimal supplemented substrates are colonised faster than those with a
low level of the available nutrients.
There are distinct differences between fungal species and strains with respect to their
saprophytic colonisation ability. Pleurotus sp. And Stropharia rugosoannulata have
relatively high saprophytic colonisation ability, while Lentinus edodes, Flammulina
velutipis, Pholiota nameko, Kuchneromyces mutabilis and Agrocybe aegerita have low
saprophytic colonisation abilities. The latter species are able to colonise only sterile or well
'pasteurised' substrates.
The rate of growth of mycelia in solid substrates varies according to different physical,
chemical and biological parameters. Among the physical parameters affecting growth rate,
temperature changes have a relatively minor impact with variations tolerated over a broad
range. Optimal temperature is 25
o
C - 30
o
C, yet most of the mesophilic species, which are
adapted to tropical climates are able to adapt to temperatures above 35
o
C. Mycelial
growth and fruit body formation have different temperature optima. The fructification
usually requires lower temperatures.
The moisture content of the substrate and humidity of the environment are also very
important factors. An increase in the water content of the substrate corresponds to a
decrease in the content of air. Heat transfer capacity, water tension, gas exchange and
other factors are also affected by the moisture content. A substrate moisture content of 70-
80 % and an air relative humidity level of 80-95% generally favour the development of
mycelia and fruit bodies.
The pH of the organic substrate without inorganic additives is of less importance. For
example, Agrocybe aegerita is able to colonise substrate and to produce fruit bodies when
the pH ranges from pH 3.8 to 7.6. Some higher fungi are able to change the pH during
growth through solid substrate for their own advantage. Therefore it is necessary to
consider the buffer capacity of the natural habitat and artificially prepared substrates (e.g.
addition of CaCO
3
retards fructification of Pleurotus spp.).

The gas exchange between substrate and environment during fungal growth in solid
substrate is also very important. Many strictly aerobic fungi need only low oxygen
concentrations of 2-5% (v/v) in the atmosphere.
The influence of high CO
2
concentrations has been established for some cultivated
Pleurotus spp. An accumulation of CO
2
in solid substrates may inhibit the growth of
competitive microorganisms, yet it also stimulates the growth of mycelia. Consequently it is
possible to control mycelial growth and fruit body formation in mushroom cultivation by
means of carbon dioxide concentration.
Some species of fungi are able to excrete organic acids or antibiotics, which protect the
mycelial growth locally. n this particular case, thoroughly colonised substrates are often
unassailable for infected microorganisms.
Factors of fruit body initiation are characteristically different for each species. The
development stage of mycelium, substrate composition, climatic conditions [temperature,
air, humidity], light and composition of air are the most discussed factors of primordia and
fruit body formation. An exchange of air from the substrate and formation of carbon dioxide
gradients control the formation of primordia on the surface of the substrate. n closed or
insufficiently aerated containers primordia will be induced, but the developing fruit bodies
will later be deformed. Similar effects occur in harvesting rooms with insufficient air
exchange caused by an imperfect air conditioning system.
The yield of fruit bodies of one strain, when physical factors such as temperature, humidity
and air exchange are optimal, is limited by the nutritional value of the substrate. The yield
of Pleurotus sajor-caju on sterile substrate increased 50% with ammonium nitrate
supplementation, and around 300% with soya bean or alfa meal supplementation.

3.5.2 TechnicaI Background of Mushroom Production. The most important step in the
whole process of mushroom cultivation is the production of the spawn. This is obtained
under axenic conditions in specially equipped facilities of the mushroom spawn industry.
Different cereal grains such as rye, wheat, sorghum, millet etc. are generally used as
carriers for fungal mycelium. The utilisation of cereal grains takes into account the
following requirements: they allow fast mycelial development, easy handling and
steadiness during sterilisation. The epidermis and the endosperm of the grain contain
sufficient nutrients for mycelial growth. Additionally, the endosperm starch is an excellent
reservoir of water and acts against desiccation. The mycelium grows on the seed surface.
n the substrate, distributed grain spawn allows quick spreading of mycelium from a small
propagation centre. All the main cultivated mushrooms are grown on grain spawn:
Agaricus bisporus, Pleurotus spp., Agrocybe aegerita, Flammulina velutipes, Lentinus
edodes, Volvariella volvacea etc. n Asian countries, spawn for the production of wood-
decaying fungi is based on wood chips and sawdust-cereal bran mixtures.
The quality of the used cereal grain plays an important role in guaranteeing a consistent
production process. First, the stored grain is mechanically cleaned and sifted to achieve a
homogenous material. SecondIy, one of the most important factors influencing the
physical and chemical properties of the grain substrate, the fungal growth and the storage
life of the final product is the water content. A water content of 40-50% favours the
mycelial development and the storage life. Spawn with a higher water content accelerates
the fungal growth, but results in a shorter storage life.
For moistening, the grain is boiled in a pressure proof container using steam for heating.
At the end of boiling, the grain must be soft without the structure having collapsed.
Additives such as calcium carbonate and gypsum are mixed into the cooked seed to adjust
the pH to the required value between 6.5 and 7.5 and to prevent the grain particles from
clotting.
After filling the grain into plastic bottles, which are closed with a special filter plug or
special perforated and with membrane manufactured plastic bags, sterilisation is carried
out.
At the same time of the production of spawn substrate, the desired fungal strains have to
be stored. Many different methods are available for the preservation of strains, which do
not have to repeat here.
The inoculation of the sterilised spawn with the starter culture takes place in special rooms
with laminar flow hoods or on clean benches under axenic conditions. A high efficiency
of spawn production is achieved onIy if the transfer of the starter cuIture is carried
out under strictIy aseptic conditions [Figure 1]
noculation is followed by incubation, which is executed in rooms under controlled humidity
and temperature. Mechanical shaking of the container encourages homogenous growth of
mycelium throughout the substrate.
Due to the high sensitivity of the spawn during the production process (inocuIation
and incubation) to competitive microorganisms, steriIity and the quaIity of the
spawn have to be ensured.
Finished containers of Agaricus bisporus, Pleurotus spp., Flammulina velutipes, and
Lentinus edodes spawn are stored in the refrigerator at a constant temperature of 2-5
o
C.
During this time, the mycelium remains viable, but in a latent state. Spawn should be
transported in air-conditioned vehicles, because every fluctuation of the temperature
diminishes the quality.
n contrast to primary rot fungi, secondary rot species (Agaricus spp., Lepista nuda,
Coprinus spp. etc) prefer organic matter which has been more or less decomposed by
microorganisms in a rotting process over a long period of time. The 'composting' process
leads to an accumulation of organic nitrogen compounds in humic acids, which are
necessary for the colonisation of the substrate and for high yields of fruit bodies. High fruit
body yields can be achieved by mixing different carbon and nitrogen containing residues
(horse manure, straw, chicken manure etc.) And their microbial pretreatment.
The preparation of high quality substrates for white mushroom cultivation depends on a
composting process with an initial surplus of nitrogen (chicken manure, urea etc.) And the
presence of available carbon sources. There are three important metabolic factors which
influence the total amount of nitrogen incorporated into organic matter (ligno-protein
complex) of the mushroom complex:

1. Formation of microbial biomass in the compost;
2. Formation of a lignoprotein complex;
3. Formation of humic substances in compost.

Figure 1: Scheme for Spawn Production [acc. to Zadrazil et.al.1992]

Not only are chemical and biological changes of substrate important for the successful
production and high yields of white mushrooms, but also the physical structure of the
substrate, water holding capacity, porosity, composition and the quality of the casing soil
are critical. The yield of white mushroom fluctuates between 10 and 30 kg/m
2
production
cycle.
Mushroom cultivation involves various processes. n developing countries, most of the
work is done by hand due to low labour costs. n industrialised countries, the use of highly
efficient machinery results in a more economical production within a shorter period of time.
Mechanisation of outdoor composting is possible with special high-capacity turners with
stack, mix and water the compost two or three times a week

Figure 2: Mushroom Cultivation Cycle

The nature of biotechnological systems of mushroom farming are determined by the
economic constraints, climate and construction of production facilities. The annual per
square metre production rate of mushrooms in a building with 6 planar surfaces and 6
crops per year is calculated to be 720 kg. Mushroom cultivation may result in the highest
per square metre yield of all agricultural crops.

3.5.3 AIIergies caused by spores of edibIe fungi. Allergic diseases from the inhalation
of fungal spores by people involved in the cultivation of mushrooms have been
documented for different fungi. These allergic reactions are caused by substrate inhabiting
moulds, an allergy often compared to the well known "farmer's lung disease" . n the future
it will be necessary to ensure that workers in waste recycling plants are properly protected
against inhalation of fungal spores which may cause allergic reactions and other unknown
hazards. Careful handling and management of fungal substrates, ventilation of working
areas and wearing of protective respiration masks are suitable precautions to avoid
dangerous diseases.
Mushroom cultivation is a typical example of 'solid substrate cultivation'.

3.6 AIgaI Biomass production
The potential use of algae such as Spirulina (see blue-green algae, cyanobacteria] as a
source of protein for human consumption has been widely recognised. The history of
Spirulina as a staple food in human diet goes back for centuries. There is evidence from
the annals of the Spanish conquest of Mexico in the early sixteenth century that the Aztecs
harvested mats of algal biomass from Lake Texcoco. For many centuries, dried Spirulina
has also been used as a food by the people who live along the shores of Lake Chad in
Central Africa.
Spirulina has a high protein content of 60-70%, which is far more than other commonly
used vegetable sources. A special value of Spirulina is that it is readily digestible due to
the absence of cellulose in its cell walls and thus its protein can quickly be assimilated.
The composition of commercial Spirulina powder is 60% protein, 20% carbohydrates, 5%
fats, 7% minerals, and 3-6% moisture, making it a low-fat, low calorie, cholesterol-free
source of protein. Another important feature for the human diet is its vitamin content. t
contains high amounts of -carotene, vitamin B
12
, thiamin and riboflavin. t has been
frequently reported as an iron supplement as well as a source of essential fatty acid, as it
contains the all important -unsaturated fatty acids.
Apart from Spirulina, the algae Dunaliella, Porphyridium, Chlorella, Chlamydomonas,
Anabaena, Isochrisis and Tetraselmis are of commercial interest for health food,
aquaculture feed, soil conditioners, and biofertilisers.
The idea of producing algae for supplementing animal feed in conjunction with the
treatment of sewage in high rate algal ponds has mainly been studied in srael. The major
factor affecting algal productivity is the climate, ie solar radiation and temperature. The
experiments showed, however, that an annual algal production of 7 kg/m
2
pond area can
be achieved. Algae, in particular Spirulina, are absolute scavengers of nitrogen and
phosphorus
The second alga with great potential is Dunaliella. The remarkable feature of this alga is
that it produces intracellular glycerol in response to the osmotic stress imposed by
extracellular NaCl. The glycerol can be extracted and concentrated to a purity of 99%.
Azolla lives in symbiotic association with Anabaena azolla, a nitrogen-fixing blue-green
alga. The rate of nitrogen fixation in this association is almost equal to that of of the more
known Rhizobium-legume symbiosis. One hectare of Azolla can produce about 540-720
kg of protein per month. t can be fed to pigs, ducks, cattle and fish.
4. Production of microbiaI biomass as a protein-enrichment for animaI feed
n contrast to the industrial MBP production to reduce pollution problems, processes for
the enrichment of feed not necessarily require the complete utilisation of the raw material.
n contrary, the normal animal feed from renewable resources can be enriched with protein
through partial degradation of the renewable resource.
4.1 Protein-enriched starch
The rcha-Orstom process was proposed and developed by Senez and his coworkers
around 1980 in order to produce protein-enriched food from cassava (=manihot). An
Aspergillus niger strain is used to grow on a chopped and crushed cassava substrate. The
mycelium has a protein content of about 40%. The process is only carried out partially to
preserve some of the carbohydrates. The wetted cassava root is heated and wetted for a
production of around 30-35% dry weight. The cassava has to be complemented with other
nutrients in particular nitrogen. The protein content of the endproduct is about 20% after 30
hours of growth.
n Southeast Asia and the Pacific, rice, cassava, and sago are the main staple food crops.
Of these, cassava and sagopalm are inexpensive, making them obvious candidates for
further diversification and exploitation as starch sources. Sagopalm shows the best
prospects with a production capacity of 2-5 tons of dry starch/ha in the wild to 10-25
tons/ha in cultivated crops. The palm grows well in swampy areas which can only be
developed for other crops at high costs, and is a perennial. Clump densities of 590
palms/acre [= 1480 palms/ha] would allow a yearly harvest of 125-140 palms/year. Since a
well attended farm can produce 175 kg starch/palm, a total yield of 25 tons/ha can be
obtained (see also chapter 20).
n the case of cassava, 1 ha of land can produce up to 65 tons of cassava tuber. n pilot
plant experiments by the University of Queensland in Australia using Rhizopus
oligosporus, a filamentous fungus isolated from the Asian food tempeh, Sukara and Doelle
demonstrated in 1988 that 65 t cassava tuber can produce 3,500 kg microbial protein
together with a significant quantity of the highly active enzyme amyloglucosidase. Apart
from the approx. 45% protein content of the fungal protein, the enzyme in turn can be used
to convert more than 39,000 tons of cassava tuber into the monomeric glucose, which can
then be converted to 15.6 million litres of ethanol using the bacterium Zymomonas mobilis.
4.2 Protein enriched whey
Whey is the by-product of cheese, casein and butter-making. The composition of whey
varies depending of its origin, but it contains an average of 6-7% dry matter, of which
approximately 70% is lactose. The other constituents are protein, lipids, lactic acid and
minerals and some vitamins. Biomass can be produced from whey in three ways:

1. through a direct use of the lactose by microorganisms
2. conversion of lactose into glucose and galactose by an enzymatical process of chemical
hydrolysis and subsequent use of these two monomers as substrate source;
3. prior fermentation of lactose by lactic acid bacteria producing a mixture of lactic acid and
galactose.

Lactose can be used as carbon and energy substrate by many microorganisms, with the
most common species being Kluyveromyces marxianus
4.3 Conversion of IignoceIIuIose into feed with white-rot fungi.
The accumulation of lignin in plant materials used as feed results in a rapid decrease of
digestibility for rumen microorganisms. n order to increase the digestibility of
lignocellulose, physical, chemical and biological methods of delignification can be used.
The principle of these methods is the splitting of the cellulose-lignin complex by extraction
or decomposition of lignin. The main problems of biological upgrading of lignocellulosics
into feed are to find suitable microorganisms, with metabolic patterns different from those
of rumen flora and fauna and to develop cheap large scale processes.
deal microorganisms for upgrading of lignocellulosics into animal feed should have a
strong lignin metabolism with a low degradation of cellulose and hemicellulose. There are
three (3) major or principal biotechnological possibilities for upgrading plant wastes into
feed, which are outlined in the figure below. A distinction has to be made between the feed
for ruminants, which can contain cellulose and hemicellulose, and feed for monogastrics,
mainly containing microbial proteins and sugars. Either process must be cheap and simple
with a low cost technology. Similar processes are suitable to convert plant residues into
human food by the cultivation of edible fungi.

Figure 2: Different ways of microbial protein production for food and feed from
lignocellulosic waste products

5. SiIage - The use of microbiaI biomass for the production and
preservation of animaI feed
Silage is made of forages, crop residues or of agricultural or industrial by-products that
have been preserved by natural or artificial acidification, for use as animal feed.
The procedure to obtain silage is briefly as follows. Fresh forage is harvested, crop
residues or by-products are collected, stems may be squashed (conditioned), the material
wilted and/or chopped and additives may be added before it is stored with the exclusion of
air, so that facultative anaerobic lactic acid bacteria, present on the material, or added as
inoculants, can rapidly convert water-soluble carbohydrates (WSC) into acids. With the
appropriate fermentation the resulting pH becomes so low that all life processes come to a
halt and the material will be preserved for as long as it remains in airtight storage.
Silage is made in order to feed animals in periods when feed supply is inadequate, either
in terms of quantity or quality. n northern temperate climates this is usually the winter
period, but in other regions it may be an annual or incidental dry period. Silage may also
be made of materials of a higher feeding value than the normally available forage, e.g.
lucerne, maize, sorghum or other cereals and agricultural or industrial by-products, and
then used as a feed supplement.
Silage making is an addition to hay making, which is simply drying green material in the
sun. n climates with a low wet weather risk haymaking may be preferred, but where the
weather is variable silage making has largely replaced haymaking. n modern animal
husbandry the harvesting and storage techniques of both hay and silage making have
been developed to improve efficiency for both and the fermentation process and the
ensuing feeding value for silage making
5.1 The ensiIing process
The keywords for successful silage making are: adequate levels of WSC, exclusion of
oxygen, rapid reduction in pH, low buffering capacity of the crop, wilting of green material,
moderate temperatures and high nutritive value of the material to be ensiled.
Silage pH is not only determined by the fermentation, but also by the buffering capacity of
the forage. Buffering capacity of a crop is its ability to resist a change in pH upon the
addition of an acid or base. n the case of silage making it is expressed as milliequivalents
of acid needed per kg of dry matter to decrease the pH from 6 to 4. The buffering capacity
of forage depends mostly on its anion concentration (organic acids, orthophosphates,
sulfates, nitrates and chlorides) and to a lesser extent on CP concentration. Legumes have
a higher buffering capacity than grasses, although that in grasses can vary fourfold
between species. Wilting reduces buffering capacity.
The ensilage process can be divided into five phases.
5.1.1: The harvesting and storage phase Forage especially grown for silage or existing
grassland is cut and can be directly collected and stored or first wilted to between 30 and
50 percent dry matter. As a result of photosynthetic and respiration processes, the WSC
concentration of forage varies during the day and is higher in the afternoon than in early
morning. Therefore, cutting of forages for silage should be delayed until the afternoon to
maximize the amount of water-soluble carbohydrates.
n order to exclude as much air as possible before the fermentation process starts, the
material may be squashed (conditioned) and chopped before storage and must be
compressed before an airtight seal is affected. The wetter the material before ensilation,
the greater will be the risk of loss of WSC through respiration and for silage effluent to be
formed. Wilting increases the relative WSC concentration and thus the fermentability of the
forage and it eliminates effluent losses from the silo, reduces slurry production by the
cattle and the obnoxious odours of wet silage, which are serious environmental problems.
Wilting of green forage was not adopted in northwestern Europe to any extent until the
middle of the 20th Century, because agricultural advisors were of the opinion that the
weather in these parts was not suitable for wilting. t is a fact that slowly wilted grass,
particularly if rain occurs during the period between cutting and storing, is deleterious for
the quality of silage as rotting processes lead to large field losses. Only rapid wilting (< 24
hrs) offers great benefits for reduced losses and improved intake and animal performance.
Wilting can be improved by a cutting system developed in the UK by Vicon, which employs
a mower conditioner consisting of a disc mower with a twin roller, the top half of metal and
the bottom part a cylindrical nylon brush for squashing (conditioning) the grass. n addition,
the cut grass is spread into a swath over the full cutting width. Wilting above 50 percent
dry matter is not recommended because it is difficult to compress very dry material and
this may lead to poor fermentation and the development of mold. Compaction of the
material is of the utmost importance. With long stemmy material, chopping to about 6 mm
lengths is advisable to enhance compaction.
Good silo filling techniques, particularly compaction, will help to minimize the amount of
oxygen present between the plant particles in the silo. Good harvesting combined with
good silo filling techniques will thus minimize WSC losses due to respiration in the field
and in the silo, and in turn will leave more WSC available for lactic acid fermentation.
Silage can be stored in or above the ground in permanent or temporary silos that may be
vertical or horizontal or in bales or small receptacles, such as plastic bags or barrels,
depending on the size of operations. Modern large silos consist of a rectangular concrete
floor and three concrete sides with a height of about 2 meters. The most modern methods
of silage making entail wilted grass to be compressed into rectangular or round bales
covered with plastic, for which special machines have been developed, not only for large,
but also for small bales.
Although the principles of silage making apply to all methods, baled silage generally has
longer particle sizes and higher dry matter contents compared to forage ensiled in clamps,
which may restrict fermentation to some extent.
5.1.2 Aerobic Phase This phase normally only takes a few hours in which the
atmospheric oxygen present between the plant particles is reduced, due to the respiration
of the plant material and aerobic and facultative aerobic microorganisms such as yeasts
and enterobacteria. Furthermore, plant enzymes such as proteases and carbohydrases
are active during this phase, provided the pH is still within the normal range for fresh
forage juice (pH 6.5-6.0).

5.1.3. Fermentation Phase This phase starts when the silage becomes anaerobic, and it
continues for several days to several weeks, depending on the properties of the ensiled
forage crop and the ensiling conditions. f the fermentation proceeds successfully lactic
acid bacteria develop, and become the predominant population during this phase. Due to
the production of lactic and other acids the pH decreases to 3.8-5.0.
5.1.4 StabIe Phase As long as air is prevented from entering the silo, relatively little
occurs. Most microorganisms of phase 2 slowly decrease in numbers. Some acid tolerant
microorganisms survive this period in an almost inactive state, others such as clostridia
and bacilli survive as spores. Only some acid tolerant proteases and carbohydrases and
some specialized microorganisms, such as Lactobacillus buchneri continue to be active at
a low level.
5.1.5 Feed-out Phase This phase starts as soon as the silage gets exposed to air. During
feed-out this is unavoidable, but it can already start earlier due to damage of the silage
covering (e.g. by rodents or birds). The process of spoilage can be divided into two stages.
The onset of deterioration is due to the degradation of preserving organic acids by yeasts
and occasionally acetic acid bacteria. This will cause a rise in pH, and thus the second
spoilage stage is started, which is associated with increasing temperature, and activity of
spoilage microorganisms such as bacilli. The last stage also includes the activity of many
other (facultative) aerobic microorganisms such as moulds and enterobacteria. Aerobic
spoilage occurs in almost all silages that are opened and exposed to air. However the rate
of spoilage is highly dependent on the numbers and activity of the spoilage organisms in
the silage. Spoilage losses of 1.5-4.5 % dry matter loss/day can be observed in affected
areas. These losses are in the same range as losses that can occur in airtight silos during
several months of storage.
To avoid failures it is important to control and optimize each phase of the ensiling process.
n phase 2 good silo filling techniques will help to minimize the amount of oxygen present
between the plant particles in the silo. Good harvesting techniques combined with good
silo filling techniques will thus minimize WSC losses through aerobic respiration in the field
and in the silo, and in turn will leave more WSC available for lactic acid fermentation in
phase 3. During phases 3 and 4 the farmer cannot actively control the ensiling process.
Methods to optimize phases 3 and 4 are therefore based on the use of silage additives
that are already applied at the time of ensiling.
Phase 5 will start as soon as oxygen is available. To minimize spoilage losses during
storage an airtight silo is required, and any damage to the silo covering should be repaired
as soon as possible. During feed-out spoilage by air ingress can be minimized by a
sufficiently high feed-out rate. n addition, at the time of ensiling silage additives can be
applied that are able to decrease spoilage losses.
5.2 SiIage microfIora.
There are desirable (lactic acid bacteria) and undesirable microorganisms that can cause
anaerobic spoilage (yeasts, clostridia, enterobacteria) or aerobic spoilage (yeasts, bacilli,
listeria, molds). Many of the undesirable organisms also have a detrimental effect on
animal health and/or milk quality (e.g. listeria, clostridia, molds, bacilli).
n the initial fermentation phase desirable lactic acid bacteria belonging to the genera
Lactobacillus, Pediococcus, Leuconostoc, Enterococcus, Lactococcus and Streptococcus
as well as undesirable enterobacteria increase in numbers. The lactic acid bacteria are
facultative aerobes with a temperature tolerance range between 5 and 50
0
C and an
optimum between 25 and 40
0
C.
Some lactic acid bacteria cause a homofermentative and others a heterofermentative
fermentation. Heterofermentative bacteria and enterobacteria, which produce acetic acid,
are generally the first populations to develop. As the pH drops to below 5,
heterofermentative bacteria decrease in numbers, whilst homofermentative, lactic acid-
producing bacteria increase in numbers, causing a more rapid and efficient reduction in
pH.
Homofermentative lactic acid bacteria produce mostly lactic acid, whilst heterofermentative
lactic acid bacteria ferment sugars to lactic acid, acetic acid, ethanol and CO
2
.
Homofermentative fermentation is preferred, because lactic acid accelerates pH reduction,
and is a desirable nutrient and because ethanol production decreases the amount of WSC
available for lactic acid fermentation and it can also have a negative effect on milk taste.
Enterobacteria break down WSC and proteins, but they can also reduce NO
3
to NO
2
,
which can be further reduced to N
2
O and NH
3
. NO
2
can also be chemically transformed
into NO and NO
3
. n the presence of oxygen, NO is oxidized to various nitrogen oxides
amongst others NO
2
. Both NO and NO
2
are toxic to man and animals. However,
enterobacteria are not tolerant to low pH and therefore do not develop much when the
fermentation follows a normal course.
The most undesirable silage bacteria are Clostridium spp., because they convert WSC and
lactic acid into butyric acid and degrade amino acids to amines and ammonia. Ammonia
increases the buffering capacity, reducing the pH reduction.
Silage spoiled by clostridia has an obnoxious smell, lower feeding value and contains
clostridia spores, which survive even at low pH in the silage, they can pass through the
alimentary track of the animals and lead to milk infection via faeces and faecal
contamination of the udder and subsequently cause poor quality ("late blowing") cheese.
This can be prevented by adding NaNO
3
to clostridia-infected milk or by bacto-centrifuging
the milk to remove the spores. n Switzerland, farmers who produce milk for Emmental
cheese making are forbidden to feed silage. Under extreme conditions silage can be
infected by C. botulinum, usually caused by a dead small animal and this can cause death
(botulism) in cattle. However, C. botulinum has limited acid tolerance, and does not grow
in well-fermented silage.
Clostridia problems occur particularly when ensiled material is low in WSC and dry matter
contents at high temperatures; a rapid drop in silage pH can avoid these problems.
Therefore, wilting to a high dry matter content is advantageous to the quality of silage.
Acetic acid bacteria are obligate aerobes and acid-tolerant and they are undesirable,
because they can cause aerobic spoilage of, particularly, whole crop maize silages due to
their ability to oxidize lactate and acetate to CO
2
and H
2
O.
Bacilli are facultative aerobes and undesirable because they are inefficient lactic and
acetic acid producers and can lead to aerobic deterioration. Excessive bacillus growth can
be avoided by preventing soil or manure contamination of forage to be ensiled, high silage
storage temperatures and exposure to air.
Yeasts are undesirable because they convert WSC to CO
2
under anaerobic conditions and
lactic acid to CO
2
and H
2
O under aerobic conditions, which causes the pH to increase,
which may lead to the development of other spoilage organisms such as clostridia.
Molds are undesirable aerobic microorganisms that develop in air pockets in surface
layers of the silage and in the whole silage during the feed-out phase. They cause
reductions in feeding value and palatability and health problems in man and animals. Mold
spores can cause lung disease and allergenic reactions and mycotoxins can give rise to
digestive and fertility problems, reduced immune function, serious liver or kidney damage
and abortion.
Listeria are aerobic or facultatively anaerobic bacteria and can cause serious health
problems in man and animals. Especially pregnant and newly born sheep and goats are
susceptible to fatal listeriosis. Listeria can also lead to a contamination of raw milk. Listeria
problems are most prevalent in silages that are not perfectly anaerobic, which can occur
when the seal of a silage clamp or bale is perforated.
Additives can be used to reduce the activity of undesirable microorganisms
5.3 SiIage additives
n the past decade it has become increasingly common to use silage additives to improve
the ensiling process. The choice of additives appears to be sheer limitless if one looks at
the large number of chemical and biological silage additives that are commercially
available. Fortunately, the choice of a suitable additive is less complicated than it seems,
because the modes of action of most additives fall within a few categories (Table 3).

TabIe3: Categories of silage additives (adapted from McDonald et al. 1991)

Between products of one category differences exist in product properties such as general
effectiveness, suitability for certain crop type, and ease of handling and application. These
factors, together with the price and availability, will determine what product will be the most
adequate for a specific silage. A drawback of some of the chemical additives is that they
can be corrosive to the equipment used, and/or can be dangerous to handle.
The biological additives are non-corrosive and safe to handle, but they can be costly.
Furthermore, their effectiveness can be less reliable, since it is based on the activity of
living organisms.
Proper storage of these biological additives by the manufacturer, retailer and farmer is of
vital importance. Despite these disadvantages, in Europe and the USA bacterial inoculants
have nowadays become the most commonly used additives for corn, and grasses and
legumes that can be wilted to above 300 g dry matter kg
-1
. n the Netherlands the
absolute as well as the relative amount of silages treated with bacterial inoculants has
increased in the past 4 years. Last year 13.7 % of all grass silages in the Netherlands was
ensiled with an additive, of these treated silages 31 % was treated with an inoculant, 37%
with molasses and 29% with fermentation inhibitors.
The main reasons for using additives is to compensate for limitations in the desirable
silage microflora and/or the lack of WSC. When forage to be ensiled contains less than 35
percent dry matter the use of an appropriate additive is generally recommended.
Additives can be grouped into those that stimulate or inhibit fermentation or aerobic
deterioration and those that consist of nutrients or absorbents. Amongst the fermentation
stimulators are bacterial inoculants (lactic acid bacteria), enzymes and sugars (often
molasses). Fermentation inhibitors are commonly acids (formic) and salts (nitrite, sulfite or
NaCl) that inhibit clostridia activity. Nutrients added (urea, ammonia and minerals) are for
the benefit of feeding value. Dried sugar beet pulp and straw may be used to absorb silage
effluents.
A disadvantage of chemical additives is that they can be corrosive to equipment and
dangerous to handle. Biological additives are non-corrosive and safe, but they tend to be
expensive.
The amount of WSC for adequate fermentation depends on the dry matter content and the
buffering capacity of the forage. A fermentation coefficient (FC) was proposed by
Weissbach and Honig in 1996 which relates dry matter (DM) content, WSC and buffering
capacity (BC):
FC = DM (%) + 8 WSC/BC
FC < 35 indicates insufficient WSC or too low DM content. n this case sugars (e.g.
molasses) should be added. The critical sugar level for successful silage production is 25-
30 g kg
-1
fresh material. Most temperate grasses fulfill this requirement. However, tropical
grasses with a C
4
photosynthetic pathway have naturally low WSC concentrations and to
make good silage of these, sugars need to be added. Tropical forage grass crops (maize
and sorghum) are rich in starch and make excellent silage. Although silage bacteria cannot
ferment starch, hydrolysis of starch into sugars during wilting and prior to the onset of
anaerobic conditions in the silo or added enzymes that release extra sugars from starch
could boost the supply of sugars available for fermentation.
Fermentation inhibitors (formic acid, hexamethylene and nitrite) and molasses are
generally only used in wet forages with a low WSC concentration or high buffer capacity.
They inhibit butyric acid bacteria and can also reduce clostridial spore counts.
5.4 SiIage quaIity
Silage quality is judged by its feeding value (intake, digestibility and crude protein
concentration), pH, its chemical composition and the presence of harmful compounds.
Silages are considered stable when they have a sufficiently low pH and high lactic acid
concentration to prevent butyric acid formation. Silage should have a low pH, an ammonia
level below 10 percent, a high lactic acid and a low butyric acid concentration . Wilted
silages can have a higher pH than direct-cut silages for the qualification good.
Even silage with a very low pH and low average butyric acid concentration can harbour
spores of butyric acid bacteria in pockets where oxygen was present because of stemmy
material or where additives did not reach as a result of poor mixing. Therefore, before
forage is entered into the silo, it should be chopped, additives should be thoroughly mixed
and the material in the silo well compacted.
Protein degradation causes a reduction in silage nutritive value and gives rise to the
formation of toxic compounds such as amines, which reduce silage palatability
5.5 SiIage fermentation in tropicaI areas
Ensiling of forage crops or industry by-products could make an important contribution to
the optimization of tropical and sub-tropical animal production systems, but thus far it has
not yet been widely applied . This is not only due to the low prices for animal products, the
low levels of mechanization, and the high costs of silo sealing materials, but also due to
the lack of ensiling experience. More research is needed to address the specific problems
associated with tropical silages. Tropical grasses and legumes have for example a
relatively high concentration of cell wall components and the low level of fermentable
carbohydrates compared to temperate forage crops. Furthermore, on average storage
temperatures in tropical climates are higher than in temperate climates, which might give
bacilli a competitive advantage over lactic acid bacteria. n addition, it has to be taken into
account that some silo sealing materials cannot withstand intense sunlight, and thus might
impair the aerobic stability of the silage. Nevertheless, it seems likely that ensiling
technologies from temperate climates can be modified for tropical conditions.
5.6 SiIage from crop residues and by-products
Crop residues and agricultural and industrial by-products can be useful feed resources. n
industrialized countries there are well-developed technologies for recovering by-products
and converting them into protein-rich meals and/or energy-rich concentrates. Such
technologies are often lacking in developing countries, where large amounts of residues
and by-products from cereals, root crops, fruits and vegetables provide potential
supplements for fresh feeding, or conservation for later feeding of domestic animals. n
less developed societies by-products often become contaminating wastes that quickly go
sour and moldy, losing considerable quantities of soluble nutrients. Ensiling of these by-
products is the most suitable method of conservation.
Commonly ensiled crop residues and by-products are: rejected bananas, banana leaves
and pseudostems, roots and leaves of cassava and other starch crops (sweet potato, taro,
yams), citrus and pineapple pulps and leaves, tomato pulp, oil-crop residues (oil palm
fronds) and by-products of (olives, oil palm), seeds and pulp of grapes, brewers' extracted
malt and spent grain, fish by-products and poultry litter.
The basic principles of silage making apply equally to by-products as for forages. Crop
residues and by-products must be stored airtight and there must be sufficient WSC for
fermentation to acids to restrict the activities of undesirable bacteria. n order to eliminate
air the material must have a moisture content of no less than 50 percent and chopped into
small pieces to allow good compression.
6. Composting - The use of microbiaI biomass for the production of
fertiIiser
Composting is an ecosystem which self heats, i.e. temperature within the composting
mass rises because heat released metabolically accumulates faster than it is dissipated to
the surrounding environment. This self-heating tends to increase decomposition rates
unless inhibitively high temperatures are reached. Activity is therefore much more rapid
and less odorous under fully aerobic conditions.
Composting has become increasingly popular in the past decade as an alternative to
incineration or tipping of decomposable organic wastes. t can now be considered a useful
treatment process for almost every kind of biodegradable waste. Composting is
inexpensive, rapidly implemented, and a publicly acceptable treatment process.
Composting is a relatively simple process that offers small communities and large
organisations the means by which organic matter can be profitably stabilised for further
use as a biofertiliser and a soil quality enhancement material.
A definition of composting can be presented as follows: Composting is the bioIogicaI
decomposition of biodegradabIe organic constituents in a waste under controIIed
conditions:

Composting then is a microbiological process and depends on the activities of a large and
varied population of microorganisms to bring about a stabilisation of otherwise degradable
natural materials. Microorganisms attack therefore natural materials, but not materials
which could be regarded as the products of human ingenuity, such as plastics,
pesticides, herbicides and other products of the chemical industries.
Composting technologies may be categorised as
a) systems based on temperature of operation. These are either mesophiIic systems
[15
0
C to 40
0
C] or thermophiIic systems [45
0
C to 65
0
C]
b) systems based on oxygen availability. These are either systems using air (or
oxygen), i.e. aerobic composting or systems excluding oxygen, i.e. anaerobic
composting, eg the Ogden method of simply burying organic matter for at least 1
year.
c) systems based on mixing or non-mixing of the organic solids. These are classified
as either static piIes (or windows) of compostable organic matter or tumbIed
systems in which the organic material is continuously tumbled throughout the
breakdown process
Composting is a complex biological process. This complexity makes the process not easy
to understand, and when not well understood difficult to manage.
6.1 PhysicaI Factors Affecting Processing
6.1.1 PhysicaI structure. Unlike most other waste treatment processes which occur in an
aqueous phase, composting occurs within a physical matrix. The consequences of this
matrix structure are physical and chemical gradients, and site specificity of various factors
which influence microbial activity From an ecological perspective, composting systems are
similar to soil systems except that much more substrate is available. Composting matrices
are characteristically organic, with a high volumetric substrate density, and potentially high
rates of metabolic activity per unit volume. Also, as composting progresses, the structural
nature of the matrix changes, softening the texture and losing volume.
Unless mixed, the composting matrix limits the transfer of gases, heat, water nutrients and
microbial populations, causing distinct gradients. A large part of process management
consists of overcoming these gradients, and establish a uniformly favourable environment
for composting. An important part of process design is determining the compromise
between economics and material uniformity within the matrix.

6.1.2 Heat evoIution. The heat evolved during composting is almost exclusively derived
from biological reactions. Heat evolution and oxygen uptake are therefore proportionally
linked during aerobic metabolism, with approximately 14,000 kJ released per kg oxygen
consumed during the complete oxidation of organic matter. Thus, heat evolution from the
composting of different substrates can be either calculated based on the extent of
decomposition and the heat of combustion for the decomposed fractions. By combustion,
proteins and carbohydrates will yield around 15-25 kJ/g and lipids about 35-45 kJ.
Although heats of combustion are always higher than heats of metabolism [remember, the
microorganism is also conserving heat for its own growth], they are useful as
approximations. Heat outputs calculated on this bases were: sewage sludge 23 kJ/g;
refuse with high lipid content (14.7%) 22.1-28.5 kJ/g, rice hulls and rice flour 14.2 and 16.7
kJ/g. Storage of evolved heat within the mass leads to the elevated temperatures
characteristic for composting. Without intervention, the accumulating high temperatures
severely inhibit microbial activity.

6.1.3 Temperature as a seIective factor. Temperatures within composting materials are
a function of the rate of heat evolution and heat loss to the environment. n composting
systems, temperature is both a result of and a determinant of activity.
Enzyme activity rates generally double with each 10
o
C rise in temperature, until an
inactivation temperature is reached. Elevated temperatures during composting can
therefore promote rapid decomposition, but can also lead to thermal death. Thermal killing
eliminates pathogens but can also kill microbes required for decomposition. Temperature
selects for or against populations based on temperature tolerance. Most species can adapt
somewhat to varying temperatures, but the temperature variation common to composting
systems is much broader than the broadest capabilities of an individual species. Thermal
killing is very important during composting, in that if extensive pasteurisation occurs within
the composting mass, the rate of composting will be greatly retarded. So far it has not
been conclusively demonstrated that thermophiles have a significant role in composting at
temperatures higher than 70
o
C. Composting activities decrease at temperatures above
60
o
C, with optimal decomposition rates in the mid to upper 50s. Biologically, the maximum
temperature achievable through composting is approx. 82
o
C, at which point biological
activity and metabolic heat evolution ceases.
6.1.4 Heat fIow and controI. Heat flow is of critical importance in the control of
composting temperatures. Composting temperatures are determined by the rate of heat
evolution, but storage and the rate of heat loss. Heat loss is a function of conducting,
evaporation of water and sensible heating of air.
6.1.5 Water. Water is a critical factor in composting systems. Microbial cells have
physiological needs for water. Microbial cells can also be physically affected by the
solution of substrates and salts, the effect of per cent water content on gas exchange, and
the role of water as a medium for bacterial colonisation. The water content must be high
enough at the beginning of composting so that acceptable rates of activity can be realised,
without producing an excessively wet final product or interfering with materials handling.
For waste treatment, a dry final compost is generally desirable because it decreases
weight and bulk, and improves handling, storage and transport. As the water content
increases, the rate of gas transfer decreases. As the rate of oxygen transfer becomes
insufficient to meet the metabolic demand, the composting system will become restricted
in activity and becomes anaerobic.
Most waste composting is carried out at 50-70% water content, depending on the material
and the specific process.
6.2 ChemicaI Factors Affecting Processing
6.2.1 InterstitiaI oxygen concentration. The fact that 'anaerobic' and 'aerobic' are only
general regions on a gradient of redox potential values is often ignored. Like soils,
composting matrices can contain aerobic and anaerobic micro environments coexisting
within close proximity. Composting is greatly retarded in the absence of oxygen, which is a
common reason for composting failures. Oxygen supply rates are determined by diffusion
potential and process management. Diffusion rates are determined by the gas
concentration gradient and the resistance to flow. n turn, flow resistance is a function of
pore size, pore continuity and moisture content. Diffusion alone is far too close to supply a
large composting mass with sufficient oxygen.
f composting is allowed to become anaerobic, volatile organic acids, volatile sulfur and
nitrogen compounds and other compounds can be produced which can cause severe
odour problems. Under anaerobic conditions up to 15% of the total organic carbon content
can be converted into volatile organic acids. These organic acids are toxic to higher plants
and such composts can remain phytotoxic for years. Composted materials that have been
processed mostly anaerobically can be very difficult to dispose of in any manner because
of odours and poor handling characteristics, such as loss of structure.
6.2.2 pH . n most waste materials, the initial pH is rarely extreme enough to cause a
processing failure. Both fairly acidic and basic materials can be successfully composted
and lead to a product near neutrality. Rates of decomposition during composting increase
with increasing pH in the range of 6-9, and a low pH early in processing can retard
subsequent processing. With a rapid early activity, the pH can rise up to approximately 8.5
because of ammonification. At the completion of ammonification, the pH will stabilise
around 7.5-8.0. On oxygen limitation, pH will drop into the acidic range. Thus pH changes
can be used in many facilities as a rough gauge of composting process.

6.2.3 Ammonia. The effects of ammonia concentrations achieved during composting are
rarely considered in waste treatment composting. Ammonification is frequently a short
process in systems that promote high activity rates, completed in less than a week.
Ammonification is temperature dependent, with the quickest rate of completion between 40
and 50
0
C. Ammonia concentrations can peak at 1000 ppm or more in high protein
substrates. The ion equilibrium between ammonia and ammonium is determined by pH. At
pH 7 and below the ammonium ion is present, whereas at pH 9 and above free ammonia
dominates. Free ammonia can form stable products with organic matter by reacting with
sugars, carbohydrates, phenolic and many other carbons near their functional groups. This
conversion of ammonia into stable forms which resist further ammonification is very
important to the compost product and usage. Free ammonia is very reactive. High
ammonia concentrations can inhibit methanogenesis under anaerobic conditions, causing
volatile fatty acids to accumulate. Free ammonia can change population structures
because of its toxicity to many microbial populations.
6.3 MicrobioIogy of Composting
Populations of bacteria, actinomycetes and fungi have been investigated in various self-
heating systems, including mushroom compost, straw, wool, tree bark, sludges, and
refuse. Result of these investigations vary based on substrate, investigative interest,
microbial recovery techniques and means of expression, and especially on composting
process management. Trends show, however, that bacterial counts per substrate gram
can range from 10
8
to 10
12
at temperatures of 55-65
0
C. At mesophilic range, bacterial
counts can be a magnitude higher. Populations of thermophilic actinomycetes peak later
than those of bacteria, often achieving counts in the 10
7
- 10
9
range.
6.3.1 Actinomycetes The role of actinomycetes in composting was established a long
time ago. There are many thermophilic actinomycetes which can tolerate temperatures in
the 50s and a smaller number of species in the mid 60s. Actinomycetes prefer moist, but
aerobic conditions with neutral or slightly alkaline pH. Complex organic materials including
polymers are substrates utilised by these slower growing filamentous bacteria.
Actinomycetes tend to be common in the later stages of composting and can exhibit
extensive growth. Genera commonly found are Streptomyces, Thermoactinomyces and
Thermomonospora.

6.3.2 Bacteria Bacteria are by far the most important decomposers during the most active
stages of composting, partly because of their ability to grow rapidly on soluble proteins,
and other readily available substrates, and partly because they are the most tolerant of
high temperatures. Waste composting is usually managed to achieve processing
temperatures above 55
0
C to ensure pathogen destruction, restricting activity almost
exclusively to thermophilic bacteria. This bacterial decomposition can be rapid.
n controlled temperature waste composting a range of 50-65
0
C is commonly maintained.
Such high temperatures are selective for bacteria and specially for the genus Bacillus.
Species of this genus made p the majority of isolates from materials composting in the
upper 50s and low 60s, and that above 65
0
C sampling provided almost monocultures of
Bacillus stearothermophilus.
Bacterial genera reported commonly during composting include Bacillus, Clostridium and
Pseudomonas. Most commonly reported species would include B.coagulans,
B.licheniformis, B.spharicus, B.stearothermophilus and B.subtilis.

6.3.3 Fungi. Fungi have a limited role in waste composting except during curing, as they
are excluded by the temperature ranges common to waste composting. Most fungi are
eliminated above 50
0
C. Fungi are commonly recovered from composting materials late in
processing when the temperatures are more moderate, and remaining substrates are
predominantly cellulose and lignin.
Large numbers of different species of fungi have been reported from composted materials,
such as Mucor pusillus, Chaetomium thermophilum, Talaromyces dupontii and T.
Thermophilus, Thermoascus aurantiacus, Aspergillus fumigatus and Humicola grisea.

6.3.4 Interaction between popuIation and nutritionaI factors. Bacteria, actinomycetes
and fungi assimilate carbon and nitrogen differently. For mixed populations, 5-10% of the
substrate carbon is assimilated by bacteria, 15-30% by actinomycetes and 30-40% by
fungi. Both bacteria and actinomycetes have a protoplasmic C/N ratio of 2:1, while fungi
have a 10:1 ratio. As a result of assimilation of carbon and nitrogen, bacteria would need
1-2% nitrogen to degrade a unit of carbon while actinomycetes would need 3-6% and fungi
3-4%. The early attack of thermophilic bacteria on the initially high proteinaceous substrate
frees nitrogen through ammonification and makes it available for subsequent populations.
Differing nutrients available during composting will preferentially favour different
populations. Bacteria can utilise narrow carbon/nitrogen ratios of 10-20:1, while fungi can
utilise wide ratios of 150-200:1 or even much higher for wood decay fungi. Waste
substrates rich in protein would initially favour bacteria. After consumption of most of the
protein, fungi and actinomycetes would be better adapted to consume the remaining
complex carbohydrates.
6.3.5 PopuIation dynamics. Maximal specific growth rates and respiration rates of fungi
are generally about an order of magnitude lower than those of bacteria. This gives bacteria
an advantage over the fungi in the early utilization of composting substrates. Most fungi
are aerobes and are disadvantaged under anaerobic conditions. Such conditions can often
exist within aggregates of material, even when the air-filled pore space contains some
oxygen. Drier substrates favour fungi and actinomycetes which can bridge air gaps that
are effective barriers to colonisation by non-filamentous bacteria.
Population succession occurs rapidly during composting. The initial mesophilic population
is later followed by a thermophilic one as the temperature increases. A drop in heat output
can be observed in the 44-52
0
C range during the shift from mesophiles to thermophiles.
As soon as the temperature declines, mesophiles reappear again, especially fungi.
6.3.6 Interactions. ncreasingly important in the horticultural usage of compost is the
establishment of populations within the compost which can suppress the growth of plant
pathogens. The production of large populations of Trichoderma hamatum and T.harzianum
in composted hardwood bark is strongly suppressive against Rhizoctonia damping-off. On
the other hand, the presence of the thermophilic fungus Scytalidium thermophilum
stimulates the growth of mushrooms, in particular the common mushroom Agaricus
brunnescens.
6.4 HeaIth Risks from Pathogens
Composting has proven to be an effective process for the destruction of pathogens found
in solid waste materials. Two mechanisms of destruction are thermal killing and biological
antagonism.
6.5 Odour Sources
Compounds implicated in composting odours include organic acids, amines, aldehydes,
sulfides, thiols and ammonia. Of these, the organic acids, sulfides and thiols are normally
the worst offenders. Proteins are often associated with odour problems because they are
highly susceptible to bacterial decomposition, and the amine nitrogen can be a precursor
of odorous nitrogen compounds. Of the proteins, the sulphur containing cystine and
methionine are troublesome, with methionine by far the worst.
Odours can also be related to general material characteristics. For example, grass
clippings can cause odour problems because of their high moisture content, high nitrogen
levels and readily available organic matter.
Environmental conditions which lead to odour problems can be obvious, such as
anaerobic conditions.
Odours can be treated or prevented. Odour treatment consists of some means of
containing and treating the air after it has left the composting mass. Completely enclosed
composting systems are becoming more common-place because of the advantages of air
containment and treatment.
Odour prevention, or at least significant reduction, can be achieved through careful
process management. Lower temperature achievement and uniformly oxygenated
conditions that most favour decomposition also produce less odour. Optimising process
control can control odour and do it much more cost-effectively than any treatment system.

6.6 ConcIusion
Aerobic composting is the system most often used for rapid and successful composting. Of
considerable significance to the success of composting is the carbon to nitrogen ratio. At
one end of the scale are substances with low nitrogen and high carbon [eg cellulosic and
lignocellulosic] matter. Such material is not that good for composting. At the other end of
the scale with a C:N ratio of 5:1 are the animal manures, which again are not the oprimal
materials for processing. The best C:N ratio for composting is between 25:1 and 30:1.
Cellulosic and lignocellulosic materials can be best used for mushroom production (see
section 3.5), whereas the rich animal manure is ideal for anaerobic digestion and biogas
formation (see chapter 14). The residues of both are again good composting materials. For
composting, it is more usual to use a mixture of materials, eg animal manure mixed with
cellulosic materials.
Most economic is the production of mushrooms, biogas together or foIIowed by
composting.
7. BibIiography
Boze,H., MouIin,G. and GaIzy,P. 1995 - Production of Microbial Biomass, n
'Biotechnology', 2nd edition [Rehm,H.J. & G.Reed, eds], Vol. 9,167-220

Chang,S.T. and S.W.Chiu 1992 - Mushroom production an economic measure in
maintenance of food security. n 'Biotechnology: economic and social aspects'
[E.J.DaSilva, C.Ratledge & A.Sasson, eds], Cambridge University Press, pp 110-140

DoeIIe,H.W., Gumbira-Said,E., Sukara,E., and DoeIIe,M.B. 1993 - A socio-ecological
concept of microbial conversion for sustainable development and conservation of the
environment. Tropical Biodiversity 1(3): 195-200

EI-Nawawy,A.S. 1992 - Microbial Biomass Production from Organic Solid Substrates. n
Solid Substrate Cultivation (H.W.Doelle, D.A.Mitchell, C.E.Rolz, eds.), p. 247-268. Elsevier
Applied Science

GoIdberg,I. 1985 - Single Cell Protein, Springer-Verlag, Berlin

Hamdan,I.Y. and Senez,J.C. 1992 The economic viability of Single Cell Protein (SCP)
production in the twenty-first century. n: 'Biotechnology: economic and social aspects'
[E.J.DaSilva, C.Ratledge & A.Sasson, eds.], Cambridge University Press, pp 142-164

Manderson,G.J. 1997 Biosolids Processing with particular reference to Composting. n '
General Aspects of Available Biotechnological Systems for a Sustainable Development of
the Pacific Island Nations', ASM, Unesco, MRCEN sponsored Training course 1997

MiIIer,F.C. 1991 - Biodegradation of solid wastes by composting, n 'Biological
Degradation of Wastes' [A.M.Martin, ed.], p. 1-30, Elsevier Applied Science

Richmond,A. 1987 Spirulina . n 'Microalgal Biotechnology' [M.A.Borowitzka &
L.J.Borowitzka, eds.], Cambridge University Press, pp. 86-121

RingpfeiI,M. & Heinritz,B. 1986 Single-cell protein technology. n: 'Applied Microbiology'
[H.W.Doelle, C.G.Heden, eds.], D.Reidel Publishing Comp., Dordrecht and Unesco, Paris,
pp 111-134

Scrimshaw,N.S., Murray,E.B. 1995 Nutritional value and safety of Single Cell Protein.
n: Biotechnology', 2nd edition [H.J.Rehm & G.Reeds, eds], Vol 9,221-240

Senez,J.C. 1987 Single-cell Protein: past and present developments. n 'Microbial
Technology in the Developing World' [E.J.DaSilva, Y.R.Dommergues, E.J.Nyns,
C.Ratledge, eds], Oxford University Press, pp238-259

Stentiford,E.I. and C.M.Dodds 1992 - Composting. n Solid Substrate Cultivation
(H.W.Doelle, D.A.Mitchell, C.E.Rolz, eds.), p.211-245. Elsevier Applied Science

Suttie,J.M. 2000 Hay and Straw Conservation for small-scale farming and pastoral
conditions. FAO, Rome

Shaver,R.D. and Batajoo,K.K. 1995 - Fermented Feeds and Feed Products, n
'Biotechnology', 2nd edition [H.J.Rehm & G.Reed, eds.], Vol. 9,769-784, VCH
Verlagsgesellschaft Weinheim

'tMannetje, L.[ed.] 2000 Silage making in the tropics with particular emphasis on
smallholders. Proceedings of FAO Electronic Conference on tropical silage 1999, FAO
Plant Production and Protection Paper 161, FAO Rome

ZadraziI,F., Ostermann,D. and DaICompare,G. 1992 - Production of edible mushrooms,
n' Solid Substrate Cultivation' [H.W.Doelle, D.A.Mitchell & C.E.Rolz, eds], pp283-320,
Elsevier Applied Science

ZadraziI,F. 1992 - Conversion of lignocellulose into feed with white-rot fungi, n 'Solid
Substrate Cultivation' [H.W.Doelle, D.A.Mitchell & C.E.Rolz, eds.], pp321-340, Elsevier
Applied Science

Past Chairman, nternational Organisation of Biotechnology and Bioengineering.
Chapter 15
MicrobiaI BiotechnoIogy in Industry - BIOENERGY
PRODUCTION
Content
1. Introduction
2. BiofueI from soIids as eIectricity
2.1 Pretreatment of biomass
2.2 Direct combustion
2.3 Co-firing
2.4 Gasification
2.5 Small Modular Systems
3. BiofueI in the form of gas
3.1 Hydrogen
3.2 Methane or biogas
4. BiofueI in the form of Iiquid
4.1 Ethanol
4.2 Diesel
5. BiofueI from phytopIankton
6. References
1. Introduction
There exists an ever increasing awareness and concern to foster the development of
biotechnology into the direction of producing fuels and chemicals from renewable biomass
such as starch, sucrose, cellulose and lignocellulose at the expense of the petrochemical
and the coal conversion industries. All biomass or organic matter can in one way or
another be used as a fuel. All organic matter is ultimately derived from photosynthesis,
which indicates that all biomass transformations into fuel involving primarily photosynthesis
are strongly influenced by the geographical location and an optimal process of energy
conversion. The latter is a very important factor also if photosynthetic biomass is
secondary and primary sources range from agricultural wastes, such as straw, to wet
wastes, such as human and animal waste materials. Fuel energy can therefore be
physically recovered through incineration of sewage sludge, municipal refuge, solid waste
of animals and crops (e.g. bagasse) or chemically by pyrolysis or gasification, or by
microbial conversion to gasses (methane and hydrogen) or alcohols (ethanol, methanol,
butanol).
Biofuels are alcohols, ethers, esters, and other chemicals made from lignocellulosic
biomass such as herbaceous and woody plants, agricultural and forestry residues,
polymers [eg starch, sugar and plant oils], and a large portion of municipal solid and
industrial wastes. Biofuels can be in the form of solids, gas and liquids [Figure 1]. Biofuels
offer many benefits, since they are good for the environment and health because they add
fewer emissions to the atmosphere than petroleum and coal fuels and use wastes that
have currently no use. Biofuels, in contrast to petroleum and coal fuels, are a renewable,
inexhaustible source of fuel, reducing dependency on foreign oil as they grow domestically
and thus help in becoming self-efficient in energy supply.
Modern applications of biofuel generation (Figure 1) cover not only heat and power
generation from biomass, but also include domestic applications such as improved
cooking and heating stoves. Besides direct combustion of solid fuels, the application of
gaseous fuels [gasification and biogas] and liquid fuels for transportation [ethanol,
biodiesel] made from biomass are also considered as modern.

Figure 1: General outline of biofuel generation

f one compares fossil [non-renewable] fuels with biofuels [renewable], it should not be
forgotten that all fossil fuels originated from biomass. Fossil fuels have been subjected to
extremely cost-free processing operations through a combined action of climate and
geological forces. Biofuels, on the other hand, have the unmeasurable advantage of being
renewable and thus will always be available. Economic considerations hinge therefore on

1. how well will we be able to copy or replace the cost-free processing operation;
2. what is the economic price for non-renewable contra renewable resources;
3. can we ever put a price on medical treatments and ecological devastation caused
through the use of non-renewable resources.

From thermodynamics and bioenergetics, fuel can be any chemical substance that
generates heat during its reaction with a second substance. Thus, the combustion of
hydrogen is 141.9 GJ/t, carbon as coal 34.4 GJ/t, a typical oil 44 GJ/t, methane gas 55.7
GJ/t and ash-free biomass 20 GJ/t.
2. BiofueI from soIids as eIectricity
Biopower is the use of biomass to generate electricity. There exist four major types of
biopower sytems: direct combustion, cofiring, gasification, and small modular [see
also http://www.nrel.gov/clean_energy/biopower.html or/and
http://www.eren.doe.gov/biopower as well as http://members.tripod.de
Before biomass can be converted into other forms of energy, an intermediate step is often
necessary to facilitate the handling of biomass as well as improve the quality of the final
combustion process. Drying, sizing and briquetting and/or pelleting of the feedstocks are
among these pre-treatment activities.
2.1 Pre-Treatment of Biomass
Drying has the objective to decrease the moisture content of the fuel to a level suitable for
use in the subsequent conversion process. Mechanical drying [eg centrifuging or pressing]
can only be used for very wet materials to be dried to a moisture content of about 50%
(wet base). f a moisture content below 50% is to achieved, thermal drying is required.
A modern dryer uses about 5,000-10,000 kJ thermal energy for the evaporation of 1 kg
water, which means that decreasing the moisture content by approximately 10%
consumes about 4-7% of the heating value of the dry material.
Sizing simply means that the biomass and/or biomass residues are cut into smaller sizes
for easy handling and efficient combustion. Straw and stalk-type materials are chopped
into granular material for easy transportation. Similarly, wood is cut into chips for their
efficient use in boilers. Size reduction is an expensive operation and requires high
investment and operation costs.
Briquetting is a densification process of loose organic material, such as rice husk, saw
dust and coffee husk, and aims also to improve handling and combustion characteristics.
Briquetting is practised on a limited scale in Asia, mainly due to its high production cost.
2.2 Direct combustion
Most of the Biopower plants in the world use direct combustion as it deals mainly with
primary fuels in the form in which it is available in nature or after some form of processing
[briquetting, pelleting, heat, charcoal]. Briquetting and pelleting are densification processes
of loose organic materials such as rice husks, saw dust, coffee husks, municipal wastes
etc, aiming to improve handling and combustion characteristics for stoves, fireplaces, kiln
etc. Biomass-fired power plants have been installed in a number of countries in Asia and
Europe. They burn bioenergy feedstocks directly to produce steam. This steam is usually
captured by a turbine, and a generator then converts it into electricity. These plants have
the option to deliver electricity to the grid, so-called dendropower, utilise the electricity to
satisfy the power demand of a stand-alone production process or a combination of both.
Combined heat and power plants [CHP plants] , called cogeneration, often integrated with
a pellet-manufacturing process have been installed in Scandinavia and are becoming
increasingly popular also in Asia.
At a cost of SEK 216 million [approx. A$ 41 million], a Swedish company uses
unprocessed biomass residues producing 120 GWh electricity and 210 GWh heat or in an
integrated operation 170 GWh electricity, 230 GWh heat with 130,000 tonnes of pellets.
The electricity consumption in the pellet plant is around 100 kWh/tonne. Whereas the heat
is connected to a district housing system for heating houses and schools, the surplus
electricity goes into the grid and the pellets are transported to the market place.
The world's first straw-fired CHP plant was constructed in 1989 in Denmark. The plant
uses about 26,000 tonnes of straw annually and has a nominal production capacity of 5
MWe and 13 MJ/s heat. The annual electricity production is around 17 GWh,
corresponding to the consumption of around 3,000 households. Heating output in 1998
was around 228 TJ. Around DKK 102 million [approx A$ 22 million] has been invested in
the plant itself and around DKK 12 million in transmission pipes.
n the United States, direct combustion with more than 7,000 MW of installed capacity is
the second-most utilised renewable power generation resource.
Direct combustion plants are similar in concept to most existing fossil-fuel fired power
plants, replacing oil with biomass.
2.3 Co-firing
Co-generation of both heat and power is increasingly applied in various wood and
agroprocessing industries such as sugar, palm oil and rice mills in Asia, in particular in the
Philippines. Co-firing involves replacing a portion of the coal with biomass at an existing
power plant boiler. t is the most economic near-term option for introducing new biomass
power generation. Since much of the existing power plant equipment can be used without
major modifications, co-firing is far less expensive than building a new BioPower plant.
Compared to the coal, biomass reduces SO
2
[sulphur dioxide], nitrogen oxides as well as
other air emissions. After 'tuning' the boiler for peak performance, there is little or no loss
in efficiency from adding biomass. This allows the energy in biomass to be converted to
electricity with the high efficiency (33-37%) of a modern coal-fired power plant. Biomass
can replace up to 15% of coal in such a cofiring operation.
t is encouraging to learn that the Australian Government is at last introducing programmes
which will require the electricity industry to achieve a significant increase in the contribution
of renewable energy generators. Currently the dominant fuel in the Australian biomass
industry is sugarcane bagasse. About 60 years ago, mills started to generate electricity
mainly for their own needs. Bagasse currently fires 14% of Australians cogeneration
capacity with 302.8 MWe installed. Estimates made by the Sugar Research nstitute
suggests that there is enough waste bagasse currently produced in Australia to provide
fuel for an additional 3,000 MW [see also CADDET 1999].
2.4 Gasification
Gasification is a major and unique element in the development of improved BioPower
systems. t is a thermodynamical process that converts solid biomass raw materials to a
clean fuel gas form. The fuel gas form allows biomass to use a wide range of energy
conversion devices to produce power. Biomass gasifiers operate by heating biomass in an
environment where the solid biomass breaks down to form a flammable gas. This offers
advantages over directly burning the biomass. The biogas can be cleaned and filtered to
remove problem chemical compounds. The gas can be used in more efficient power
generation systems called combined-cycles, which combine gas turbines and steam
turbines to produce electricity. The efficiency of these systems can reach 60 %, which
means that this technology can be twice as efficient as conventional boilers producing
electricity. Gasification systems can be coupled in future with fuel cell systems. Fuel cells
convert hydrogen gas to electricity and heat. There are very little air emissions and the
primary exhaust is water vapour.
Gasification is essentially a two-step, endothermic [heat absorbing] process in which a
solid fuel [biomass] is thermodynamically converted into a gas.
The first step is the most important step, mostly referred to as 'pyrolysis' , which is the
thermal decomposition of biomass fuels in the absence of oxygen into three kinds of
products: solid, liquid and gases. The ratio of the products is influenced by the chemical
composition of biomass fuels and the operating conditions:

Figure 2 : Biomass Gasification Process Diagram
[see:http://www.eren.doe.gov/biopower/projects/ia_tech_gas1.htm]

n the first reaction, pyrolysis, the volatile components of the fuel are vaporised at
temperatures below 600
0
C by a set of complex reactions. ncluded in the volatile vapours
are hydrocarbon gases, hydrogen, carbon monoxide [CO], carbon dioxide [CO
2
], tar, and
water vapour. Char (fixed carbon) and ash are the pyrolysis by-products, which are not
vapourised.
n the second step, the char is gasified through reactions with oxygen, steam and
hydrogen. Some of the unburned char is combusted to release the heat needed for the
endothermic gasification reactions.
Large commercial-scale gasifiers will use about 1,500 tonnes of biomass per day to
generate up to 120 Megawatts of electricity, which is enough for about 120,000
households.
Biocrude oiIs can also be produced through similar chemical reactions (pyrolysis) as
used in biomass gasification. These processes produce a liquid fuel from biomass instead
of a gaseous one. n general, the reactions take place at different temperatures and at
different reaction rates compared to gasification.
2.5 SmaII ModuIar Systems
Small, modular BioPower systems have the potential to help supply electric power to the
more than 2.5 billion people in the world who currently live without electricity. The potential
exists because most of these people live in areas where large amounts of biomass are
available for fuel. Small systems, with rated capacities less than 5 MW could potentially
provide power at the village level to serve many of these people and their industrial
enterprises.
Up-to-date examples of electricity generation from biomass can be found on the website of
CADDET.
Not only biomass can be converted into electricity. The Fibrowatt Company in the UK is
using poultry litter for electricity generation. The UK poultry farming industry produces
more than 1.5 million tonnes per annum of such litter from broiler poultry farms. This litter
consists of a mixture of woodshavings and/or straw or other suitable bedding material and
poultry droppings, and is an excellen fuel for electricity generation with nearly half the
calorific value of coal [see http://www.fibrowatt.com/ourtech.html.
There are no waste products from this process. nstead, a valuable by-product is produced
in form of a nitrogen-free ash, rich in potash and phosphate, which is being marketed as
an environmentally friendly fertiliser.
3. BiofueI in the form of gas
3.1 Hydrogen
Hydrogen is produced by a large number of microorganisms, both photosynthetic and
chemosynthetic. Such spontaneous hydrogen evolution is connected with the physiological
role of regulation, enabling organisms to dissipate excess reducing power.
Quantitatively, solar energy is clearly the most important energy source and photosynthetic
systems derived from them may be used in various ways to trap solar energy. The
photosynthetic electron transport system can therefore be regarded as a solar cell which
generates a potential difference of approximately 1.2 Volt. Normally these light-potential
electrons can be used to reduce NADP
+
to NADP
+
+ H
+
via ferredoxin (see chapter 10),
but in the presence of the enzyme hydrogenase, they can be converted to molecular
hydrogen:
2 H
+
+ 2e
-
----------> H
2

Since the photochemically generated reducing power (NADPH) is mainly used for carbon
dioxide fixation, in order to obtain efficient whole organism hydrogen generating systems, it
may be necessary to interfere with the normal process of photosynthesis in order to
stimulate hydrogen production. An alternative is, of course, to use modern gene
technology to construct so-called 'bioIogicaI fueI ceIIs'.
Green algae species like Anabaena cylindrica have been cultured in a number of
experiments and were capable of producing hydrogen at a rate of 0.4% of the solar
conversion efficiency. ndications are that this conversion efficiency could be as high as 2
or 3 per cent. f such hydrogen production is possible, it means that up to 0.65 litres of
hydrogen could be produced per square meter of culture medium surface area. This figure
would be ten times higher if an assumed theoretical maximum solar energy conversion
efficiency of 20% could be approached.
A further thought is to use the direct conversion of the free energy of a chemical reaction
for electricity generation (biological fuel cells). n this case, oxidation occurs at the anode
and reduction at the cathode with both electrode compartments being separated by a
membrane. The advantage of such a system would be that the reactions in a fuel cell
involves charge transfer and not a heat transfer. Consequently, the theoretical maximum
efficiency of fuel cells is often 100%, with a not uncommon efficiency of 50-70%. The
limitations of such biological fuel cells is, of course, the slowness of electron transfer
reactions at the electrodes. The hydrogen/oxygen fuel cell is the most developed and the
use of hydrogenases and cytochrome oxidases from different sources as catalysts
requires further investigations.
n the case of chemosynthetic microorganisms, members of the genus Clostridium are well
known for their high hydrogen evolution during anaerobic glucose degradation:
C
6
H
12
O
6
+ H
2
O ---------> 6 CO
2
+ 12 H
2

Superficially, such a conversion looks very attractive as it enables more than 99% of the
thermal value of glucose to be conserved in molecular hydrogen. However, the bacteria
also require metabolic energy for growth, which would limit the production of hydrogen
from carbohydrates to about 4 mpl/mol of hexose and hence to around 33% of the
theoretical yield.
3.2 Methane or biogas
Biogas is a mixture of roughly 70% methane and 30% carbon dioxide, is colourless,
odourless and inflammable. n its crude form, it is largely used for cooking, lighting, and to
power stationary engines for the generation of electricity. When purified, biogas is
chemically identical to methane derived from any other source such as natural gas.
One thousand cubic feet of biogas has an energy equivalent of 600 cubic feet of natural
gas, 28.8 l of butane, 23.4 l of petrol or 20.7 l of diesel oil. Biogas systems constitute not
only a renewable source for energy, but also provides biofertiliser for the regeneration of
our soil fertility. These systems are of interest in respect to waste recycling, public health
and hygiene, pollution control and environmental management.
The conversion of waste materials into methane requires the complex interaction of mixed
populations of microorganisms. Based on their metabolic characteristics, four general
categories can be distinguished:

1. those microorganisms which are capable of producing extracellular enzymes
[proteases, lipases, amylases etc.] for the breakdown of polysaccharides or other
polymers into monomeric structures, such as sugars, amino and fatty acids ; this process
is referred to as Iiquefaction;
2. those microorganisms which are capable of fermenting the monomeric structures
produced in (1) to volatile fatty acids, e.g. fermentative bacteria [Escherichia coli, Bacillus
sp., Enterobacter spp. etc.]; a process referred to as fermentation;
3. those microorganisms which are capable of converting all the volatile fatty acids other
than acetic acid into acetic acid and carbon dioxide, e.g. acetogenic bacteria
[Syntrophobacter wolinii, S.wolfei, Syntrophonomas wolfei]; a process referred to as
acetogenesis;
4. those microorganisms which are capable of converting acetic acid, carbon dioxide and
hydrogen into methane plus carbon dioxide, e.g. methanogenic bacteria ; the process
referred to as methanogenesis.

Although most of the carbohydrates finish up in acetic acid and carbon dioxide, fat and
protein metabolism in general form higher fatty acids such as propionic, butyric, valeric
and other volatile fatty acids. n methanogenic environments it is therefore not unusual for
the methanogens, which can only utilise acetate and carbon dioxide, to co-exist in close
association with another metabolically specific bacterium. This close association is called
Syntrophism and is based upon closely integrated biochemical features of the two
bacteria in this anaerobic consortium. One member of this pair is called a 'methanogen'
because it generates methane, whereas the other member is called an acetogenic
hydrogen plus acetic acid producing bacterium or short 'acetogen'. The most important
feature in this syntrophism is the 'interspecies hydrogen transfer', without which no
methane can be formed.

Figure 3: Methane production from natural organic wastes.

Methanogens contain several cofactors not found in other bacteria. Three of them, e.g.
methanopterin, methanofuran and CoM are carriers of C-1 units during its reduction from
carbon dioxide to methane. Factor 420 probably functions as a hydrogen carrier in these
reductions, and factor 430, an unusual tetrapyrrol, is the prosthetic group of methyl-CoM
reductase, the last enzyme in the pathway [see also chapter 10].
The overall autotrophic reaction of methanogenesis
4 H
2
+ CO
2
CH
4
+ 2 H
2
O G = - 32 kcaI
shows that carbon dioxide reduction to methane is an eight-electron process, which means
it must contain at least 4 individual reactions, the sum of which gives a significant energy
release equivalent to about 3 ATP.
The acetoclastic reaction, on the other hand,
CH
3
COO
-
+ H
+
CH
4
+ CO
2
G = - 9 kcaI
involves a simple decarboxylation plus hydrogen transfer with a less energetically useful
reaction.
The loading of the digester [or anaerobic fermenter] is determined by the solids of the
influents, the retention time, digester size and temperature. During the digestion it is
important to control the process, which normally is done by pH and total volatile fatty acid
(VFA) determination.

Figure 4: Schematic diagram of biogas production

The most impressive installations of methane producing fermenters can be found without
any doubt in the Peoples Republic of China. Digesters up to 400 m
3
in series of 5-6 are a
common side on the outskirts of Shanghai or in rural areas. t is in the rural areas where
there exist some 4 million Gobar plants in China today.
The Maya Farm on the outskirts of Manila in the Philippines is also an excellent example
of the utilisation of methane produced from animal waste for electricity and power
generation.
Each person in the UK needs an average of about 5500 BTUs or 5830 KJ/day for cooking
purposes, which corresponds to about 225 litres of biogas. This amount of biogas can be
produced from two (2) pigs, allowing one-third of the gas to be returned for heating the
digester.
A farm of 1000 pigs therefore would generate enough energy to service about 500 people
(families) with cooking gas. Biogas can also heat water for central heating. Gas-fired
boilers have a similar heat conversion efficiency of about 75%. Since the normal range of
central heating systems require an energy input of 42,000-131,250 KJ/h or 1876-5825
litres/h, such a system could be serviced by the gas produced from the waste of 250-780
pigs.
Biogas digestion was introduced into developing countries as a low-cost alternative source
of energy to partially alleviate the problem of acute energy shortage for households and
eliminate health hazardeous animal and human manure. However, few farmers were using
this technology because of the high cost of the digesters, difficulty in installing them and
difficulty in getting spare parts [Bui Xuan An et al. 1997]. The biogas programme
developed quickly only in those countries, where substantial support from governments
and aid agencies was available (Gunnerson 1986, Karki 1996). The development of a new
polyethylene tubular film biodigester technology based on the bag digester digester model
(Pound et al. 1981) and later simplified by Preston and his coworkers in Colombia (Botero
& Preston 1987), Vietnam (Bui Xuan An et al. 1994) and now in Cambodia (see Figure
5+6 ) led to the installation of more than 4,000 polyethylene biodigesters in Vietnam alone,
all paid by the farmers. Whereas the price of a concrete digester plant installed for an
average family in Vietnam varied between US$ 180 to 340 (Thong 1989), the average
price for the polyethylene tubular digester was only US$ 5/m
3
. This new polyethylene
tubular film biodigester technology is a cheap and simple way to produce gas for small-
scale farms. t is appealing to rural people because of the low investment, fast payback,
simple technology, positive effects on the environment and women's lives in rural areas.
A typical example for biogas production from pig manure using different loading rates to
polyethylene tubular biodigesters has been reported by Bui Xuan An & Preston (1999).

Figure 5: Polyethylene biodigester tube

Biogas production has not only taken off in developing countries, but has also found a new
life in European countries. By the end of 1998, there were 24 biogas plants in Finland
(Leinonen & Kuittinen 1999). The biogas produced 112 GWh of energy from sewage
sludge, 19 GWh from industrial waste and 2.4 GWh from other installations. n addition
about 45 GWh of landfill gas was collected.
Growth and concentration of the livestock industry create opportunities for the proper
disposal of the large quantities of manures generated at dairy, swine, and poultry farms.
Pollutants from unmanaged livestock wastes can degrade the environment, and methane
emitted from decomposing manure may contribute to global climate change. Lusk (1998)
presents a number of case studies indicating that anaerobic digestion benefits farmers
monetarily and mitigates possible manure pollution problems, thereby sustaining
development while maintaining environmental quality.
P.Lusk (1998) comes to the conclusion that one management system not only provides
pollution prevention but also can convert a manure problem into a new profit center.
Economic evaluations and case studies of operating systems indicate that the anaerobic
digestion of livestock manues s a commercially available bioconversion technology with
considerable potential for providing profitable co-products, including cost-effective
renewable fuel for livestock production operations.
4. BiofueI in the form of Iiquid
4.1 BioethanoI
The production of ethanol by yeast fermentation of simple sugars has been known
throughout history as the means for producing alcoholic beverages and liquors. Ethanol
has also been produced as a chemical, surgical spirit and for solvent purposes for several
decades microbiologically from molasses or chemically from ethylene and thus from oil
refinery fractionation.
Although fueI aIcohoI has been around with us since ever the first Daimler-Benz car has
been produced, biofueI as such is a recent idea stemming from the 'energy crisis' in the
1970's, the increasing awareness of depletion of non-renewable resources and air
pollution problems caused by the exhaust gases of petrol driven automobiles, in particular
petrol with a high lead content added for higher octane levels.
f one considers ethanol production from biomass, it should be realised that it can only be
used for automotive fuel, because of its high octane rating, but also as a chemical.

Figure 6: Use of ethanol as a chemical

Any successful production of ethanol depends upon a means for producing sugar
monomers from the natural polymers starch, cellulose and sucrose.
n the case of yeast [Saccharomyces cerevisiae], the sugar is metabolised via the
Embden-Meyerhof-Parnas (EMP) pathway, also referred to as the glycolytic pathway,
whereas the bacterium [Zymomonas mobilis] uses the Entner-Doudoroff pathway. Both
microorganisms use the pyruvate decarboxyIase for the conversion of pyruvate into
acetaldehyde plus carbon dioxide followed by an alcohol dehydrogenase to convert all the
acetaldehyde to ethanol. The main difference in using different pathways could be
explained energetically. Whereas the glycolytic pathway produces a net 2 mol of ATP, the
ED pathway has only 1 mol ATP net gain.
The key difference between the use of Saccharomyces or Zymomonas is the relationship
between growth and product formation as well as substrate and endproduct inhibition. n
yeast there exists a very close relationship between growth and product formation, which
necessitates the addition of air to the fermentation vessel and high biomass concentrations
for high ethanol production. Kinetic models incorporating the fact that sugar uptake is
related to biomass growth rate and ethanol production rate by constant yield coefficients
predict that an ethanol concentration between 70 and 90 g/l inhibits totally cell growth and
between 80 and 110 g/l ethanol production. This phenomenon has been related to
membrane transport effects. Air is required for the synthesis of ergosterol, which in turn is
essential for the synthesis of unsaturated fatty acids in the cellular membrane structure.
These unsaturated fatty acids are responsible for ethanol tolerance allowing a greater
membrane fluidity and thus exit of ethanol from the inside to the outside of the cell.

Figure 7: Ethanol formation using yeast

n contrast, the bacterium Zymomonas is an anaerobic organism tolerating air and
contains the unsaturated fatty acids in its cellular membrane. These are two of the main
reasons why Zymomonas mobilis can tolerate substrate concentrations well above 20%
(w/v) sugar and is able to grow and produce up to 15% (v/v) ethanol.

Figure 8: Ethanol formation using Zymomonas mobilis

A further difference exists in the transport systems. Whereas yeasts require energy in the
form of ATP for its active transport system, Zymomonas has a facilitated diffusion system,
which relies on membrane carrier proteins and concentration gradients.
Whereas ethanol production in yeast is firmly growth-associated, this is not the case with
Zymomonas. This bacterium exhibits unbalanced growth and uncoupled product
formation, which is reflected in the carbon requirement for cellular biosynthesis.
Typical fermentation times for the production of 8-10% (v/v) ethanol are for yeasts 40-50
hrs, and for Zymomonas 18-24 hrs and lately this time was cut to 10-14 hrs.
ndustrial ethanol production suffers, however, like all low-value fermentation products in
its economic viability in our industrialised system. Furthermore, CO
2
is a by-product and
contributes to the so-called 'greenhouse effect'. t is therefore of vital importance for a
viable ethanol industry to seek value-added products as by-products for cross-
subsidisation and an educational effort that pollution also costs money in medical bills.
n the USA efforts have been made with great success to not only compress CO
2
to dry
ice, but also to use the residual or waste from grain to ethanol conversion plants as animal
feed. Such development in the microbial fermentation industry would make this industry
environmentally friendly and contribute to a waste-free industrial product formation.
Since it is very important to recognise differences between microorganisms in microbial
process development, let us have a closer look at the two different bioethanol producers
and the role of basic science in the improvement of industrial processes.
t was mentioned earlier that the underlying reason for favouring Zymomonas over yeast
lies in the different metabolic pathways used by both microorganisms. While yeasts use
the energy-requiring active transport system followed by an EMP pathway underlying
complex regulation mediated by a hexokinase and a phosphofructokinase, Zymomonas
transports sugars by a low-affinity, high-velocity facilitated diffusion process. The latter is
followed by differently regulated enzymes in the ED pathway. No hexokinase or
phosphofructokinase are present as energy regulators. These enzymes are replaced by
glucokinase and fructokinase (Doelle 1982). Consequently, fermentation is not limited by
substrate uptake at substrate concentrations around or above the Km of the uptake
process. Thus, Zymomonas in contrast to yeast are able to grow on high sugar
environments.The tight energy regulation in yeast cells is necessary, because yeasts are
essentially aerobic microorganisms, which require air, mitochondria and a full electron
transport chain for optimal growth. They do not grow in the absence of air and the ethanol
production yield depends on each and every single cell trying to survive under anaerobic
conditions. Only if the growth is reduced do yeast produce ethanol.
Although the production of ethanol by yeast is an anaerobic process, growth of new cells
requires oxygen and traces of oxygen (or air) are needed to support alcohol-producing
cells. At the metabolic level the regulation of ethanol production is very complex: the
concentration of glucose, oxygen and product all effect yeast metabolism, cell viability, cell
growth, division and ethanol production.
Where practicable, the level of fermentable solids used is in the range of 16-25% (w/v)
giving final concentrations of 6-12 % (v/v) ethanol. The preferred temperature is 25-33
o
C
and the pH between 4 and 5 to reduce contamination by bacteria. n batch fermentation
the substrate is fermented out by a growth of a freshly cultured inoculum initially under
aerobic conditions. Hence a new culture has to be used for each batch. This continual
propagation of cells is costly in terms of substrate. Most manufacturers of ethanol
purchase dried yeast from a separate company.
Furthermore, with yeast grown in a batch system, about 5% of the sugar may be used for
cell growth and maintenance energy or for the synthesis of other compounds such as
glycerol, lactic acid, acetic acid, acetaldehyde and also fusel oils [higher alcohols] from
proteins. The maximum weight yield of a top yeast is thus about 48% of feed stock, just to
name an example:
15% glucose forms theoretically 76.65 g or 9.7% (v/v) ethanol
In the case of yeast : 72 g or 9.1% (v/v) with a maximal conversion efficiency of 93%
In the case of Zymomonas: 75 g or 9.5% (v/v) with a maximal 98% conversion efficiency

Most yeast in industrial process reach only 86-88% conversion efficiency.
With yeast the productivity on a cell dry weight basis may vary between 1 and 2 g
ethanol/h/g cell. n comparison, Zymomonas has productivity of 5-10 g ethanol/h/g.
Since the productivity in the reactor reflects the mode of operation, yeast cell densities
have to be increased and often reach 10% of more compared to 1% of Zymomonas. A
normal 36 h fermentation yields 5% (v/v) ethanol at an average of 1.4 g ethanol/l/h, thus
yeast fermentations are mostly carried out over a 50-70 hrs period of time.
At the end of the fermentation, the ethanol concentration lies between 6-12% (v/v). A high
ethanol concentration is important, since the steam consumption for distillation increases
rapidly, e.g. from about 2.25 kg steam/l of 96% ethanol for a 10% beer to over 4 kg steam/l
for a 5% beer.
n contrast, Zymomonas has no such tight regulation and thus ethanol production does not
depend on the number of cells, as Zymomonas grows under anaerobic conditions. This
phenomenon of independent product formation is referred to as 'uncoupIed growth'.
There appear to be two general forms of uncoupled growth: firstIy, an energetic
uncoupling, where much of the energy generated is diverted away from growth caused by
high maintenance energy requirements. Maintenance energy is a non-growth associated
component of metabolism, e.g. maintaining a proton gradient etc. SecondIy, the diversion
of substrate carbon away from cellular macromolecule formation to endproduct formation.
The two forms of uncoupled growth thus occur independently and can therefore be
manipulated. This means it is possible to manipulate specific substrate uptake rates and
also improve ethanol yields.
High and rapid ethanol formation causes, of course, also a rapid formation of carbon
dioxide, which does lead to a supersaturation of the gas in the solution. Although the major
portion of the dissolved CO
2
is a function of the concentration of other nonpolar and ionic
species, in particular ethanol. One has to consider therefore the other molecular species of
CO
2
that are in equilibrium with carbon dioxide:
CO
2
+ H
2
O H
2
CO
3
HCO
3
-
+ H
+

because CO
2(aqueous)
is able to react with free amino groups or proteins and H
2
CO
3
to
associate with positively charged groups on proteins via a dipole-protein interaction, the
total amount of CO
2
in the solution can increase significantly causing supersaturation. The
gas exchange CO
2[aqueous]
<-------> CO
2[gas]
further depends on the rate of nucleation. The
resulting supersaturation suggests a nonideal behaviour of CO
2
in solution and the
presence of increasing amounts of HCO
3-
and CO
2[aqueous]
exerts its effect on the fluidity of
the hydrophobic fatty acid core, bicarbonate exerts its effects on the charged phospholipid
head groups and proteins at the surface of the membrane. This in turn means that the
bicarbonate could be responsible for the destabilisation of the membrane.
This effect of CO
2
can only be overcome by some protection of the cellular membrane.
The organism does this by forming a polymeric fructose layer around its cell, reducing the
conversion efficiency of glucose to ethanol. Such protection can be done through
immobilisation. The results of such immobilisation have shown a production of 18% (v/v)
ethanol within 6 hrs, whereas free cells produce only 11-12% (v/v) in 24 hours.
Sugarcane is the top raw material with an ethanol yield of 5,150 l/ha, followed closely by
artichokes (5,000 l/ha), sugar beet (4,755 l/ha), cassava (4,450 l/ha), sweet sorghum
(2,500 l/ha) and grain (2-3,000 l/ha).
The worldwide bioethanol production volume is about 33 x 109 litre/year and is estimated
to grow to 36-37 x 109 litre/year by the year 2005. The largest market for fuel ethanol can
be found in Brazil (14 x 109 l/year) followed by the USA. t is often forgotten that the first
cars ever built by Daimler-Benz run on ethanol, that Brazil has used ethanol from
sugarcane since 1903 adding 5% to petrol and reached a production volume of 650 million
litres in 1941. Petrol blended with 30-50% ethanol and named Latol had been used in cars
in Latvia before World War .
The price of petrol in the USA varies between US$ 0.11-0.17/l and for ethanol between
US$ 0.26 and 0.40, whereas in France the ethanol production costs from sugar beet varies
between US$ 0.56 and 0.64 /ltr and from wheat around US$ 0.50/l. Two decades ago it
was estimated that the costs of ethanol production in Australia would be around A$
0.50/ltr.
Similar to the electricity generation from solid fuels, bioethanol would have an enormous
impact on the rural economy. t has to be realised, however, that high ethanol yields can
only be achieved from high sugar or starch/glucose raw material. On the other hand,
biofuel production in contrast to sugar or grain production can still proceed profitably in
times of bad weather and low sugar contents in both sugarcane juice and grain grades. t
is totally wrong to compare prices of first grade sugar or grain with ethanol prices, since
lower grade standards are excellent raw materials for bioethanol. n a bioenergy
production unit, no grain or sugarcane comes to waste. Furthermore, the residuals of the
biodiesel and bioethanol production are excellent by-products for pharmaceutical
industries and cattle industries respectively.
n view of all these biofuel generation technologies readily available and proven in the US,
Europe, SEAsia and South America as well as in some countries of Africa, eg Malawi,
Zimbabwe, it is very hard to comprehend why rural industries and governments failed sofar
to see the benefits coming from these available technologies. t would relieve the external
world price pressure in favour of a stable domestic market and thus improve and settle the
rural economy. As mentioned earlier, all these technologies are still applicable during bad
weather seasons, as straw, bagasse and low sugar contents would still provide the
industry with a good income. The establishment of a biofuel industry would furthermore
create thousands of jobs and thus benefit the whole economy apart from becoming more
independent from overseas oil imports.
The US Department of Energy has reported that the bioethanol industry alone is
responsible for approximately 200,000 jobs in the USA and from 1996 to 2001 will add US
$ 51 billion to the US economy. t has also helped the rural corn industry to recover and
stopped the then increasing migration trends from the rural to the urban cities. Based on
this success, the President of the USA announced a tripling of the use of biomass
technologies by the year 2010 in order to produce fuels and materials thereby increasing
the income to farms, lessen oil imports and lessen the risk to global warming. Biofuel is
therefore going into direct competition with the petrochemical products.
4.2 BiodieseI
Biodiesel can be manufactured by adding transesterification equipment to existing oil seed
crushing and refining facilities (Louwrier 2003). ts use as a fuel is very comparable to its
conventional counterpart. The power generated by an engine using biodiesel is about the
same as conventional diesel (128,000 vs 130,500 BTUs, respectively). The result of this is
that the engine torque and effective horsepower do not change, despite a change in fuel.
Furthermore, this means that the fuel consumption of the average diesel engine running
on biodiesel will remain unchanged.
The final method of biodiesel manufacture is the transesterification of plant oil with
methanol or ethanol in the presence of a catalyst. Essentially, the triglyceride is split so
that the fatty acids are cut from the glycerol backbone. These fatty acids are
simultaneously converted to their methylesters during this process, which are compounds
that have chemical characteristics similar to those of conventional diesel fuel in terms of
combustion. Such oils include soybean, canola, rapeseed, tallow and other vegetable oil.
n addition, glycerol, a valuable byproduct of the process, can easily be isolated and sold,
off-setting some of the production costs. Glycerol is being used in over 1500 applications
such as drugs, polymers, paints, cosmetics and many others. The world produced and
used almost 700,000 tonnes of glycerol in 1995, and Europe alone produces currently
about 45,000 tonnes of glycerol per year from the biodiesel process. At the present time, a
biodiesel production capability of about 10 million gallons per year [ 1 gallon approx. 4.5 l]
exists in Austria alone.
Whereas Europe uses rapeseed oil, the US produces biodiesel mainly from soybean oil,
where approximately 7 lb of soybean oil are needed to make 1 gallon [about 3.8 l] of
biofuel together with lb of crude glycerin. At present, Procter & Gamble, the sole US
producer, has the capacity to supply up to 25 million gallons [ approx. 95 million litres] per
year. The price of 100% biofuel ranged from US$ 2.20 to US$ 2.90 per gallon [ US$ 0.58-
0.76 per litre], but in general, a 20/80 blend of biofuel and diesel are recommended. No
engine modification is required.
Every gallon of biodiesel displaces 0.95 gallons of petroleum-based diesel over its life
cycle. Biodiesel is non-toxic and biodegradable.
5. BiofueI from PhytopIankton
Phytoplankton lipids are highly reduced hydrocarbons produced by direct conversion of the
sun's energy to chemical energy via the process of photosynthesis. Phytoplankton lipids
are typically esters of glycerol and fatty acids having carbon numbers in the range of C
14
to
C
20
. Diatoms are unique in that linolenic acid [18:3] is only a minr constituent, whereas this
fatty acid is common in green algae (Chlorophyta) and higher plants. Blue-green algae
(Cyanophyta) tend to have large amounts of polyunsaturated fatty acids (25-60% of total),
while in eukaryotic algae, saturated and monounsaturated fatty acids predominate. Total
lipid fractions in healthy phytoplankton vary therefore substantially from less than 1% to
more than 40% dry weight. n order to propose a design for a mass culturing system for
lipid production, the trade-off between harvesting log-phase cells with low lipids vs nutrient
starved cells with high lipid fraction must be assessed. The most versatile schematic
outline of the process to produce Phytoplankton lipids on sewage would ideally include an
exponential growth phase, a nutrient stressed linear growth phase followed by a direct
liquid-liquid extraction of the culture to recover the oil product (Figure 9). The exponential
phase of growth would utilise current high-rate pond technology, whereas the nutrient
stress system might utilise continuous plug-flow hydraulics to improve nutrient limitation.
Alternative paths in the overall mass culturing process could include methane digestion of
the remaining biomass after liquid extraction or of the entire lipid-rich biomass. The energy
yields are based on a productivity of 15 g m
-2
d
-1
and a lipid fraction of 50%. The energy
yields of methane digestion were calculated according to Buswell's formula.

Figure 9: Bioenergy production from Phytoplankton

f sewage treatment is the economic key to a commercial mass culture process, a
knowledge of the upper limit of supply of this resource will provide a proper perspective for
development
6. REFERENCES and Iiterature for further reading
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Anaerobic Digestion at http://www.dmu.ac.uk/ln/itc/itc.htm

Anaerobic Digestion Network at http://www.ad-nett.org

BiodieseI at http://www.afdc.doe.gov/altfuel/bio_general.html

BiodieseI TechnoIogy at http://www.ott.doe.gov/biofuels/biodiesel.html

BioethanoI at http://journeytoforever.org/ethanol.html

BioethanoI TechnoIogy at http://www.ott.doe.gov/biofuels/bioethanol.html

BiofueIs at http://journeytoforever.org/biofuel.html

BiofueIs and the Economy at http://www.biofuels.nrel.gov/economics.html
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Rodriguez,L. & T.R.Preston 2001 Biodigester installation manual.
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ual.htm

RosiIIo-CaIIe,F., HaII,D.O., Arora,A.L. and Carioca,J.O.B. 1992 Bioethanol
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'Biotechnology: economic and social aspects' [E.J.DaSilva, C.Ratledge and A.Sasson,
eds], Cambridge University Press, pp 23-54

Ross,C.C., Drake,T.J., WaIsh,J.L. 1996 Handbook of Biogas Utilisatoin, 2nd ed., US
Department of Energy, Tennessee Valley Authority, Muscle Shoals, Alabama, USA

Horst W.DoeIIe
Chapter 16
MicrobiaI BiotechnoIogy in Industry - Production of Bio-
ChemicaIs

Content
1. Introduction
2.1 Acetic acid
2.2 Citric acid
2.3 Lactic acid
2.4 Amino acids
3.1 Polysaccharides
3.1.1 Dextran
3.1.2 Xanthan gum
3.1.3 Alginate
3.1.4 Approaches to the improvement of microbial polysaccharide production
3.1.5 Poly-beta-hydroxybutyrate
3.2 Antibiotics
3.2.1 Mode of action
3.2.2 Production
4. Bioinsecticides
4.1 Principles
4.2 Stages in the investigation
4.3 Presently used candidates for biological control agents
4.3.1 Bacteria
4.3.2 Viruses
4.3.3 Fungi
4.3.4 Protozoa
4.4 Production of biological insecticides
4.4.1 Submerged fermentation
4.4.2 Surface culture
4.4.3 in vivo culture
4.5 Bioassays
4.6 Formulation and use of bionsecticides
4.7 Safety testing of bioinsecticides
4.8 Future
5. References
1. Introduction
An organic substance with an industrial application can be produced either employing
microorganisms or chemical synthesis. The decision of choice is obviously an economic
one, whereby the key role is played by the cost of the raw materials. n the case of
microorganisms, the chief raw material is the growth substrate, e.g. molasses, starch
etc.(Okafor 1978), whereas in chemical synthesis the raw material is petroleum or a
derivative of it. Secondary considerations are conversion efficiency, recovery and waste
treatment costs.
Chemicals other than fuels can be divided into primary and secondary product formation
(Rose 1979)depending upon their participation in the metabolic functions of the microbial
cell (Doelle 1994).
2.1 Acetic acid
The most important chemicals produced so far microbiologically are ethanol (see chapter ;
Kosaric 1996), glycerol (Rehm 1996), acetic acid (vinegar)(Ebner et al. 1996; Ebner
1982), citric acid (Kapoor et al. 1982) and lactic acid (Kascak et al. 1996). Of these, acetic
acid is undoubtedly the most important organic acid. Apart from the use as vinegar, acetic
acid has also applications in the manufacture of rubber, plastics, acetate fibres,
pharmaceuticals, dyes and photographic materials. Acetic Acid can be formed by the
microbiological oxidation of ethanol, but except in the case of vinegar, the process is not
currently competitive with the chemical synthesis based on the carbonylation of methanol.
The organisms involved in acetic acid production are generally known under the name
'acetic acid bacteria', with the genera Acetobacter and Acetomonas being predominantly
involved. During acetification, oxidation of ethanol occurs in a twostage oxidation system
from ethanol via acetaldehyde to acetate:
CH
3
CH
2
OH + O.5 O
2
------- > CH
3
CHO + H
2
O

CH
3
CHO + O.5 O
2
-------> CH
3
COOH + H
2
O
This is a highly exothermic reaction, and all fermenters or 'acetators' have to be cooled.
The reaction can occur over the temperature range 20 - 44
o
C with an optimum at 30 -
32
o
C.
The second reaction involving acetaldehyde can, under certain environmental conditions,
undergo a dismutation reaction, whereby only 50% of the acetaldehyde is converted to
acetic acid, the remaining half returning to ethanol:
2 CH
3
CHO + H
2
O --------> CH
3
CH
2
OH + CH
3
COOH
Although this reaction is predominantly observed under anaerobic conditions, it can also
occur aerobically.
Vinegar production can be obtained in batch, semicontinuous and continuous
fermentation. High substrate and product concentration together with oxygen supply are
the main criteria. Most widespread today are probably the Frings generators, which are
semicontinuous operated vortex stirred tanks, for which minor modifications can make
them almost continuous, giving efficiencies of up to 0.5 with conversions of 96 - 98%.
Fully continuous processes have been introduced in the 1970's using tower fermenters.
There are no differences in the final product irrespective of the method used, but economic
refinement is dominant in the further development.
2.2 Citric acid
Citric acid is an intermediate metabolite and essential ingredient in foods. t is produced
most efficiently from molasses and other raw materials by Aspergillus niger (Roehm et al.
1996).
t was shown in 1916 that a number of black aspergilli made citric acid and that the highest
yield of citric acid occurred when the development of mycelium was restricted and not
when it was stimulated. n using ferrocyanidetreated beet molasses, the specific activity of
the condensing enzyme increases, but aconitate hydratase and isocitrate dehydrogenase
activities are lost. Since aconitate hydratase needs iron for its activity, the elimination of
this ion leads to citric acid accumulation. Therefore, the tricarboxylic acid cycle accounts
for the formation of citric acid from carbohydrates but not from acetate.
Miles Laboratories patented in the early 1950's the use of Aspergillus niger in deep
cultures and a medium in which the iron content was under one part per million. This low
concentration was obtained by passing a solution of pure sucrose or of invert molasses
through a cation-exchange resin. For submerged fermentation it is stated that the
fermenters must be made from, or lined with, a material which is resistant to citric acid and
will not contaminate the fermentation with iron. The fermentation usually requires 10 - 14
days to convert 120 - 150 g/l of sugar solution. The maximum yield quoted on the total
sugar was 65.4% as citric acid monohydrate.
n another patent, the addition of morpholine at 100 - 1,000 ppm is claimed to give
improved citric acid yields, 80.4% being the highest quoted.
Although citric acid production is a rather successful industry, a number of factors are very
critical. Firstly, the standard inoculum must be very carefully prepared making certain that
little filamentous growth is present. Secondly, a fairly strict control must be exerted on the
concentration of ferrocyanide, which needed values of about 20 ppm at the start of the
acid producing stage. The theoretical stoichiometric conversion of sucrose to the hydrated
acid is 122% and sugar has to be used both to provide carbon and energy for growth of
the fungal mycelium.
For a long time the production of citric acid has been based on the use of molasses and
various strains of Aspergillus niger and occasionally Asp. wenti. Although several reports
of citric acid production by Penicillium are available, in practice, organisms in this group
are not used because of low productivity. n recent times yeasts, especially Candida spp.
have been used to produce the acid from sugar.
Paraffins have also been reported as substrates around 1970. n the processes described
mainly by Japanese workers, bacteria and yeasts have been used. Among the bacteria
were Arthrobacter paraffineus and corynebacteria; the yeasts include Candida lipolytica
and Candida oleiphila.
Fermentation with molasses and other sugar sources can be either surface or submerged.

2.2.1 Surface fermentation. Surface fermentation (Doelle et al 1992) using Aspergillus
niger may be done on rice bran, as is the case in Japan, or in liquid solution in flat
aluminium or stainless steel pans. Special strains of the fungus are used, which can
produce citric acid despite the high content of trace metals in rice bran. The citric acid is
extracted from rice bran by leaching and is then precipitated from the solution as calcium
citrate. With surface liquid fermentation, a sugar base such as high-test molasses or beet
molasses is used. The temperature is 28-34
o
C and it lasts for about ten days.

2.2.2 Submerged fermentation As in all other processes where citric acid is made, the
fermentation vessel is made of acid-resistant materials such as stainless steel. The
carbohydrate sources are molasses decationized by ion exchange, sucrose or glucose.
MgSO
4
and KH
2
PO
4
at about 1% and 0.05-2% respectively are added. The pH is never
allowed higher than 3.5. Copper is used at up to 500 ppm as an antagonist of the enzyme
aconitase which requires iron. 1-5% of methanol, isopropanol or ethanol when added to
fermentations containing unpurified materials increase the yield; the yields are reduced in
media with purified materials. Because high aeration is deleterious to citric acid production,
mechanical agitation is not necessary and air may be bubbled through. Antifoam is added.
The fungus occurs as a uniform dispersal of pellets in the medium. The fermentation lasts
for five to fourteen days.

Extraction. The broth is filtered until clear. Calcium citrate is precipitated by the addition of
magnesium-free Ca(OH)
2
. Since magnesium is more soluble than calcium, some acid
may be lost in the solution as magnesium citrate if magnesium is added. Calcium citrate is
the filtered and the filter cake is treated with sulphuric acid to precipitate the calcium. The
dilute solution containing citric acid is purified by treatment with activated carbon and
passing through ion exchange beds. The purified dilute acid is evaporated to yield crystals
of citric acid. Further purification may be required for pharmaceutical standard.

Use. Citric acid is a major food additive, particularly in the manufacture of jellies, jams,
sweets and soft drinks. t is also used for flavouring various foods and soft drinks. Sodium
citrate is often used on processed cheese manufacture.
Sodium citrate is also used in blood transfusion for the prevention of blood clogging. The
acid is used in effervescent powders which depend for their effervescens on the CO
2

produced from the reaction between citric acid and sodium bicarbonate.
n the cosmetic industry, citric acid finds use in astringent lotions such as aftershave
lotions because of its low pH. Citric acid is also used in hair rinses and hair and wig setting
fluids.
Citric acid has recently been used in the detergent industry as replacement of phosphates,
as precursors of the latter give rise to eutrophication

2.3 Lactic Acid

Lactic acid, which is mainly sold as the optically inactive DL-mixture of isomers, has for a
long time been made by fermentation. Over the past decades, however, the fermentation
process has been under strong competition from a purely chemical process. Whereas the
chemical process is only able to produce inactive DL-mixtures, the fermentation process is
able to produce in addition each individual isomer through proper selection of the microbial
strain (Kascak et al 1996). n order to be able to meet a relatively small demand for lactic
acid, the process has to be flexible, which means that the manufacturer should be able to
produce all three different types of lactic acid [DL, D(-), L(+)] in the same plant.
For the selection of the proper strain, one has to be familiar with the biochemistry of lactic
acid producing microorganisms. There are numerous species of bacteria and fungi that are
capable of producing relatively large amounts of lactic acid from carbohydrates, but only a
few which would meet the criteria for process development. The latter are mainly confined
to the family of Lactobacillaceae.
One of the most important features of the lactic acid bacteria for microbial process
development is the differentiation of homofermentative and heterofermentative species.
Whereas the homofermenters virtually produce a single fermentation product, lactic acid,
the heterofermenters produce only half the amount of lactic acid plus a series of other by-
products. This differentiation is of vital importance for the selection of strains for a process

a) to produce lactic acid;
b) to produce dairy products or food fermentation.

t is nowadays very simple to find out which lactic acid bacterium is a homo- and which
one is a heterofermenter. The homofermenters use the EMP pathway for glucose
metabolism, the heterofermenters an entirely different pathway, the phosphoketolase
pathway. The latter is a modification of the hexosemonophosphate pathway, whereby the
transaldolase-transketolase reactions are replaced by the enzyme phosphoketolase which
splits xylulose-5-phosphate into glyceraldehyde 3-phosphate plus acetyl-phosphate.
Since both subgroups follow different pathways, simple enzyme fingerprinting will quickly
determine the subgroup of your organism.
Since the yields for the two subgroups are

1 glucose ---> 2 lactic acid [homofermenter]
1 glucose ---> 1 lactic acid + 1 ethanol + 1 CO
2
or other minor products [heterofermenter]

it becomes obvious that a bioprocess for lactic acid must require a pure homofermenter.
The next step of selection involves the type of lactic acid to be required. This again
requires biochemical knowledge of lactic acid formation from pyruvate. There exist three
enzymes which are responsible for the different types of lactic acid formation:

a) a stereospecific L(+)-lactate dehydrogenase
b) a stereospecific D(-)-lactate dehydrogenase
c) a lactate racemase

Figure 1: The actions of stereospecific lactate dehydrogenases
t is well known, for example, that L.leichmanii produces pure D(-)lactic acid, L.casei is a
pure L(+)lactic acid producer, whereas L.plantarum produces the mixture of both isomers.
Furthermore, there exist several types of heterofermentative mechanisms, e.g. the
Leuconostoc-type [acetate, ethanol, glycerol], the Peptostreptococcus-type [propionate]
and a third group producing butyric acid as byproducts of glucose metabolism.

TabIe 1: Homo- and heterofermentative lactic acid bacteria

After the selection of the strain has been carried out, the optimization of the process
depends very much on the substrate available and the downstream processing for the
purification of the lactic acid.
Lactic acid is generally produced from glucose, maltose, sucrose or lactose. Starches,
especially those from corn and potatoes, are hydrolysed first using amylases or sulphuric
acid before commencement of lactic acid production. Molasses or sugarcane syrup can
also be used. The use of whey or sulphite waste liquor produces a number of byproducts,
in particular sulphite waste waste liquor, as pentoses can only be utilized by the
heterofermentative group of lactic acid bacteria.

2.3.1 Process n order to start a process for lactic acid production, great care has to be
given to the inoculum as well as the removal of free lactic acid. Growth is significantly
affected at as low as 1-2% lactic acid in the medium. This inhibition is circumvented
through the addition of calcium carbonate, which neutralises the lactic acid in forming its
Ca-salt.
The sugar concentration in the fermentation tank medium is usually not higher than 12%.
At higher concentrations, calcium lactate may crystallise out in the fermenter with the
formation of a solid mass, which is hard to handle in downstream processing. The
inoculum is usually between 5 and 10% of the fermentation broth volume. The pH of the
culture broth is kept in the range 5.5-6.5 by neutralisation of the acid produced. A
continuous pH control is of advantage, as this increases the yield and rates. f the lactic
acid is not continuously neutralised, high acidity will develop and fermentation will not get
to completion. At present, plant fermentation time is commonly 5-10 days. A continuous
pH control at 6.3-6.5 with Ca(OH)
2
completes fermentation of 12-13% glucose in 72 hours.
Commercial fermentation yields are 93-95% of the glucose supplied.
The fermentation is essentially anaerobic, but strict anaerobic conditions are not required.
Gentle stirring is necessary to keep CaCO
3
in suspension. The fermentation temperature
required depends on the strain used, e.g. L.delbruckii 45
o
C, L.bulgaricus 45-50
o
C and
L.plantarum, L.casei at 30
o
C.

2.3.2 Product Recovery The problems in the lactic acid process lie almost exclusively in
the recovery. Both the D- and L-form of lactic acid can only be crystallised with great
difficulty and in low yields. n their purest form the acids are usually colourless syrups that
readily adsorb water.
The suspended solids and most of the microorganisms are removed from the solution by
conventional industrial precoat filters. For technical grade products, calcium present is
precipitated as calcium sulphate dihydrate, which can be filtered off, and then the filtrate is
concentrated to 35-40% lactic acid by evaporation. f more calcium sulphate precipitates, it
is removed by filtration.
Food-grade lactic acid is an aqueous solution of 60-65% acidity.
2.4 Amino acids
n order to obtain chemicals from biosynthetic events, a thorough knowledge of metabolic
pathways and their regulatory mechanisms are necessary. Amongst these intermediates,
the amino acids are undoubtedly the most important ones (Nakayama 1982;
Leuchtenberger 1996; Esaki et al. 1996), since of the 20 amino acids necessary for protein
formation, eight cannot be synthesized by man. Amongst these eight essential amino
acids, lysine and methionine are particularly important in nutrition as most cereal grains
are deficient in these two amino acids.
Following the increasing demand for monosodium glutamate as a flavouring agent, an
efficient L-glutamic acid producing microorganism, namely Corynebacterium glutamicum
(syn. Micrococcus glutamicus) was isolated in Japan. Since then, a lot of research has
gone into microbial amino acid production. The primary reason for these efforts was the
hope to improve the nutritional value of low-cost vegetable proteins by enrichments with
essential amino acids.
Today, the total world production of L-glutamic acid is considered to be well in excess of
150,000 x 10
3
kg/year. L-Lysine production is considered to be about 15,000 x 10
3
kg/year.
Once C.glutamicum was discovered by screening isolates from nature, similar efforts lead
to the isolation of bacteria producing DL-alanine or L-valine. However, it was found that
most wild-type strains isolated from nature could not produce industrially significant
amounts of other amino acids except a few. One of the main reasons for this fact is that
regulation of cellular metabolism avoids oversynthesis.
An auxotrophic mutant which cannot produce the regulatory effector or corepressor
overproduces and excretes the precursor or the related metabolite of a blocked reaction
when grown on a limiting supply of the required nutrient. This is the principle of the
application of an auxotrophic mutant to the microbial production of amino acids.
t is obviously useless to accumulate the endproduct of an unbranched pathway such as
arginine or histidine with an auxotrophic mutant. The production of such a metabolite
depends on the use of reguIatory mutants. A mutant which has lost some biosynthetic
regulation can be obtained by selecting for analogue resistance.
To improve the yield of an amino acid, mutants having multiple markers including
auxotrophy and analogue-resistance contributing to the production of the designated
amino acid are selected. Multiple markers also contribute to the yield by stabilising the
productivity against back mutation during fermentation.
With metabolic regulation, the permeability barrier is another mechanism which protects
the microorganism from leaking organic compounds to the environment. t allows cells to
retain intermediates and macromolecules necessary for life of the microorganism.
Production of L-glutamic acid by C.glutamicum was found to be due mainly to the
permeability change induced by limiting the supply of biotin required by the bacterium.
PermeabiIity is thus another important factor for amino acid production.
Although certain specific environmental conditions make it possible to exploit a single
enzymic process or a process using a precursor, most amino acids can now be produced
by the so-called 'direct fermentation' process, e.g. microbial production from a cheap
carbon source by a fermentation process.

2.4.1 GIutamic Acid The production of glutamic acid from carbohydrate in high yield is
carried out by a group of bacteria represented by Corynebacterium glutamicum [Figure 2].
These bacteria were classified species in different genera and include apart from
C.glutamicum, Brevibacterium flavum, Brev. lactofermentum, Brev. divaricatum, Brev.
thiogenitalis, Corynebacterium callunae, C.herculis, Microbacterium ammoniaphilum and
others. The GC content of these bacteria falls into a narrow range (51.2-54.4 mol%).
The special conditions which allow these bacteria to excrete large amounts of glutamic
acid are a nutritional requirement for biotin and the lack, or very low content of alpha-
ketoglutarate dehydrogenase. The biotin requirement is the major controlling factor in the
fermentation. When enough biotin is supplied for growth, the organism produces lactate.
Under conditions of suboptimal growth, glutamate is excreted.
Under optimal culture conditions, glutamic acid bacteria convert about 50% of the supplied
carbohydrate into L-glutamic acid with little formation of by-products. Various carbohydrate
materials can be used as the carbon source. Glucose and sucrose are particularly
suitable. For industrial purposes, hydrolysed starch solutions, cane molasses and beet
molasses are preferred. Other carbon sources such as acetic acid and ethanol are also
used. Carbon sources, such as cane molasses, with a high content of biotin, are used with
the addition of penicillin during logarithmic growth or of fatty acid derivatives such as
polyoxyethylene sorbitan mono-oleate (Tween 60) before or during logarithmic growth.
Ammonia and various ammonia salts including urea are being used as a nitrogen source.
Although ammonium ions are necessary, high concentrations can be inhibitory. The most
important factor in the medium is biotin, which is an essential growth factor. The optimal
concentration of biotin for these organisms depends on the strain, kind and concentration
of the carbon source, but is generally slightly below 5 ug/l of medium. To obtain glutamic
acid, the biotin concentration has to be below this optimum for growth. Certain iron-
chelating compounds are necessary sometimes.
The pH value optimal for growth and glutamic acid production is 7.0-8.0. Continuous
feeding of NH
4
+
can adjust the pH value and also supply ammonium ions to the medium.
Under conditions of insufficient oxygen, the production of glutamic acid is poor and large
amounts of lactic and succinic acid accumulate. Excess oxygen, on the other hand,
increases the amount lactic acid and oxoglutaric acid.
The optimal temperature is usually 30-35
o
C and could require 96 hours.
The availability of acetic acid at a reasonable price and the waste water problem
associated with the use of cane molasses prompted the search for a process using acetic
acid as carbon source. With Brevibacterium flavum, L-glutamic acid production reached 98
g/l (48% on the basis of acetic acid) in 48 h.

Figure 2: Metabolic pathways involved in the production of glutamic acid [adapted from
Kinoshita & Nakayama 1978]
2.4.2 Lysin Direct production of L-lysine from carbohydrate was developed first with a
homoserine or threonine- plus methionine-requiring auxotroph of C.glutamicum. The same
type process was reported with a homoserine-requiring auxotroph of Brevibacterium
flavum. The fermentation performance of the homoserine-requiring auxotroph could be
stabilised by the use of mutants having a double amino acid deficiency, one of which is
homoserine. Double auxotrophs, which require in addition to homoserine at least one of
the amino acids threonine, isoleucine or methionine for growth, have been found to be
highly stabilised showing little tendency to revert to homoserine independence.
Cane molasses is now generally used as a carbon source in the industrial production of L-
lysine, although other carbohydrate materials, acetic acid or ethanol can also be used. The
pH value of the medium is maintained near neutrality during the fermentation by feeding
ammonia or urea.
An example of a fermentation using cane molasses in a 2 K fermentor is as follows:
First Seed Culture: 2% glucose, 1% peptone, 0.5% meat extract and 0.25% NaCl in tap
water.
Second Seed Culture: 5% cane molasses, 2% (NH
4
)
2
SO
4
, 5% corn-steep liquor and 1%
CaCO
3
in tap water.
Fermentation Medium: 20% cane molasses (as glucose) and 1.8% soybean meal
hydrolysate (as weight of meal before hydrolysis with 6N H
2
SO
4
and neutralization with
ammonium water) in tap water. The fermentation was carried out at 28
o
C. C.glutamicum
No. 901 (homoserine-requiring auxotroph) produced 44 g L-lysine/l in 60 h. Foaming in the
aerated culture can be repressed by addition of proper antifoaming agents. The growth
factors are supplied in limited amounts suboptimal for growth. The biotin concentration in
the medium must generally be greater than 30 ug/l.

2.4.2.1 Amino acid production from biosynthetic precursors. The use of precursors in
amino acid production effectively bypasses the metabolic control exerted by feedback
inhibition and repression. L-Leucine is synthesized via alpha-ketobutyrate from L-
threonine. The first step in this pathway, a hydratase, is subject to feedback inhibition by L-
isoleucine in Serratia marcescens. The addition of D-threonine to the medium serves to
induce a D-threonine hydratase, which is not inhibited by L-isoleucine, and thus L-
isoleucine synthesis from D-threonine can continue without any effective metabolic control.
Similarly, L-serine can be produced from glycine precursors via the action of a
hydroxymethyltransferase, which requires sufficient quantitites of
methylenetetrahydrofolate. The C-1 supply can be satisfied by glycine, formaldehyde,
formate, sarcosine etc. A methionine auxotroph of Arthrobacter globiformis produces 5.2
g/l of serine when glycine, glucose and methanol are present.

2.4.2.2 Enzymatic synthesis of amino acids The use of enzymes in isolated form in
chemical applications can either be single or multi-step in nature and techniques used
range from utilization in situ in the intact but non-growing organism to immobilized
preparations Easaki et al. 1996). Five classes of enzymes have been evaluated in this
context:

a) HydroIytic enzymes (hydrolases) such as L-alpha-amino-eta-caprolactam lyase
(L-lysine productio) or 2-amino-thiazoline-4-carboxylate hydrolase (L-cysteine).
Surfactants are used to permeabilize whole organisms to allow use of the enzymatic
activity without purification. Mutants can then be constructed which do not further
metabolize the target product.

b) Lyases are often involved in deamination reactions. Aspartase can be used in
reverse reaction to promote the formation of L-aspartate from ammonium fumarate.
Alternatively, hydrazine or hydroxylamine can serve as the ammonium donor. Similarly,
phenylalanine ammonia lyase catalyzes the cleavage of L-phenylalanine to trans-cinnamic
acid and ammonia. Although the equilibrium lies in favour of the cleavage reaction,
synthesis is favoured by high ammonia concentrations.

c) PyridoxaI phosphate is a common coenzyme involved in amino acid metabolism and
pyridoxal phosphate linked enzymes are versatile, being involved in racemizations,
transaminations, decarboxylations, eliminations and replacement reactions. t is believed
that the function of this coenzyme is to activate the amino acid to facilitate its interaction
with the apoenzyme.
L-Tyrosine phenol-lyase (beta-tyrosinase) catalyzes a beta elimination reaction, in which
L-tyrosine is cleaved to produce pyruvate, phenol and ammonia. The enzyme is produced
in large amounts in Erwinia herbicola under optimal growth conditions. t can be used for
the synthesis of tyrosine and has been used in the immobilised form for the continuous
production of tyrosine.
ts substrate specificity is such that it will also catalyze the beta-replacement reaction
between DL-serine and pyrocatechol to form L-DOPA.
L-Tryptophan indole lyase (tryptophanase) is a deaminating enzyme, found extensively in
nature, which catalyzes the alpha,beta-elimination and beta-replacement reactions. t too
possesses a broad substrate specificity [L-tryptophan, L-cysteine, S-methyl-L-cysteine, L-
serine] and can be reversed to effect the synthesis of L-tryptophane from indole, pyruvate
and ammonia.
L-Methionine-gamma-lyase is an inducible enzyme that catalyzes elimination reactions
with various amino acids, including derivatives of L-methionine and L-cysteine. L-
Methionine is cleaved to form methanethiol, alpha-ketobutyrate and ammonia. n its
synthetic mode it can be used to produce new sulphur amino acids, by using alkanethiols
or arylthioalcohols.

c) Amino acid dehydrogenases, such as leucine and alanine dehydrogenases,
catalyze reversible deaminations and have been used in continuous amino acid production
from the corresponding keto analogue. The amino acid dehydrogenase is retained within
the membrane reactor by an ultrafiltration membrane and utilizes the same pool of NADH,
which is retained within the reactor by covalent coupling to polyethylene glycol and
continuously regenerated by the action of formate dehydrogenase (cofactor recycling).

d) ATP-dependent amination of glutamate, catalyzed by glutamine synthase, has
been coupled to a yeast sugar fermentation. The energy released during fermentation is
used to drive glutamine synthesis. Using cell-free extracts of baker's yeast and the
glutamine synthase of Gluconobacter suboxydans, a 92% molar yield of glutamine has
been achieved, using glucose, glutamate and ammonium ions as substrates.

2.4.2.3 Uses of Amino Acids Amino acids have a wide variety of applications in many
spheres of commerce:

1.They are used as nutritional supplements in foods, e.g. lysine, tryptophan and threonine
are used to enrich vegetable protein, methionine to enrich soybean meal;

2. n food processing, amino acids find application as flavour enhancers and additives. The
L-enantiomer of monosodium glutamate is extensively used for its meaty taste, whereas
glycine is used as a sweetener, bacteriostatic agent and antioxidant.

3. amino acids are used therapeutically in infusions and some analogous find use as
pseychotherapeutic agents

4. as precursors they are widely used in the chemical and pharmaceutical industries for
the manufacture of detergents, polyamoni acids, polyurethane and agricultural chemicals.

TabIe 2. : Amino acids produced from wild-type and mutant strains.

3.1 PoIysaccharides
Polysaccharides have been recognised and exploited by man for centuries. As far as we
know, they occur as energy reserves and structural materials in the tissues of living cells.
n animals, their structural roles is performed in connective tissue, in plants in the cell
walls, and in microorganisms as cell wall material and extracellular capsules. A large
number of polysaccharides obtained from plant tissues, including seeds, seaweeds, fruits
and trees, have been developed into commercially important products known collectively
as industrial gums. The commercial usefulness of gums is based upon their ability to alter
the rheological properties of water. They do this by performing two broadly interconnected
functions, namely by gelling aqueous solutions or by modifying their flow characteristics.
Other types of gelforming polysaccharides are pectins and alginates which are extracted
from citrus fruits and brown seaweeds respectively.
Due to their extremely diverse physical properties, polysaccharides have found many
applications in, amongst others, the food, pharmaceutical, cosmetic, paper, oil and textile
industries.
Let us concentrate at the microbial polysaccharides. The microorganisms involved include
bacteria, fungi and yeasts, many of which form polysaccharides as exocellular capsules
and slimes. These exopolysaccharides are therefore found in two different forms:

a) attached to the microbial cell: capsuIes
b) secreted from the cell into the surrounding environment: soIubIe sIime
The two forms can readily be distinguished through negative staining techniques, among
the most useful of which is the ndia nk procedure .
There are clearly two groups of exopolysaccharides (Sutherland 1996), those composed of
a single structural unit (homopolysaccharides) and those which are constructed from two
or more monomers (heteropolysaccharides).
Extracellular homopolysaccharides form two distinct groups, depending upon their site of
synthesis. Dextrans and levans are different from other homopolysaccharides in that they
are formed from a specific substrate, sucrose, by essentially extracellular biosynthetic
processes which probably also involve a suitable acceptor molecule. n contrast to levan,
dextrans are widely used in the pharmaceutical industry.
Most microbial exopolysaccharides are heteropolymers composed of neutral sugars and,
commonly, uronic acids. Some may contain amino sugars in place of uronic acids. The
other common component of bacterial heteropolysaccharides is pyruvate in the form of a
ketal.
All commercial scale polysaccharide formation processes are aerobic. As the viscosity of
the medium increases with polysaccharide formation, oxygen transfer to the cells becomes
increasingly more difficult. Temperature is often a critical factor. All microorganisms are
mesophilic. n order to increase oxygen transfer, usually hot air is sparged through the
medium with high degrees of aeration. Optimum pH for the synthesis of bacterial
polysaccharides is 6.0 7.5 and for fungi pH 4.0 5.5.

3.1.1 Dextrans Dextrans are polyglucans produced by a wide range of bacterial species,
including Klebsiella spp., Acetobacter spp., streptococci and Leuconostoc sp. They include
grampositive as well as gramnegative bacteria, aerobic as well as anaerobic. Most work
has, however, been carried out with Leuconostoc mesenteroides.
Dextrans are normally high molecular weight polysaccharides containing up to 90-95% of
alpha, 1,6 linked glucose residues, the remaining linkages being either alpha, 1-->4 or
alpha, 1-->3. The differentiation of dextrans into three groups, A, B, and C has been based
on the presence of certain proportions of the three types of linkages. The molecular weight
range might be from 50,000 to 3x10
8
daltons. Dextrans have found uses in two major
areas:

a) as blood extenders; and
b) as the basis for a wide range of adsorbents for use in the biochemical and
pharmaceutical industries as well as in the research laboratories.

Production is obtained in fermenters inoculated with 10% of a seed culture. The enzyme
dextransucrase catalyzing the reaction
n sucrose ----> (gIucose)
n
+ n fructose
is rapidly produced and the pH value in the culture falls. Adjustments with alkali have to be
done and further sucrose is added at intervals. As the production of dextran is rapid and
basically a process involving the use of bacterial extracellular enzymes present in the
culture fluid, contamination presents less of a problem than is found in more prolonged
fermentations. Cultures of Leuconostoc mesenteroides do not require vigorous aeration.
Low molecular weight dextrans are normally added to the culture fluid prior to inoculation
in order to provide receptor molecules for polysaccharide formation. The fermentation
process is therefore essentially the use of a cellfree enzyme system. The substrate does
not enter the microbial cell, consequently the complex regulatory processes associated
with such uptake mechanisms are not involved.
The product is fractionated with organic solvents prior to hydrolysis to yield the desired
average molecular weight. They may be modified by the introduction of crosslinking to
yield a threedimensional network of polysaccharide chains. As the polysaccharide still
contains a high proportion of free hydroxyl groups, it is strongly hydrophilic and can be
produced in a bead form capable of swelling in aqueous solutions.
Alternatively, alkylation can be introduced to produce lipophilic properties. Other
derivatives have been prepared to enable the coupling of molecules for affinity
chromatography. Derivatives such as carboxymethyl and diethylaminoethyl dextrans from
extremely useful ionexchange adsorbents for the purification of enzymes and other
proteins. Dextran derivatives may also be used to form the basic support for insolubilised
enzymes or whole cells.
3.1.2 Xanthan gum. The most interesting polysaccharide to be produced from microbial
sources in recent years has undoubtedly been the exopolysaccharide of Xanthomonas
campestris and related strains of Xanthomonas. t is composed of D-glucose, D-mannose
and D-glucuronic acid residues and was both acetylated and pyruvylated (Lawson &
Sutherland 1978). The monosaccharides were present in the approximate molar ratio
3:3:2. ndustrial production of xanthan has been by batch fermentation. nitial studies used
fermenters of up to 900 ltr capacity with a culture medium containing corn syrup, distiller's
solubles and mineral salts. Growth conditions are carefully monitored and controlled, the
important variables being temperature, pH value and fermentation time. Aeration and
mixing of the fermentation broth are critical variables, due to the extremely high viscosities
encountered, oxygen transfer rate is affected and will become limiting unless the fermenter
baffle and impeller geometry are carefully designed for optimum gas transfer.
When the fermentation is terminated, the broth has a pH value of about 6.0 and a viscosity
of greater than 30,000 cP (contraves viscometer at 25
o
C and a shear rate of 1/sec). The
production of xanthan occurred principally during the first 72 hours of culture. The products
are precipitated using methanol, isopropanol in the presence of potassium chloride. The
wet cake can then be recovered by filtration or centrifugation, and the product shredded
before drying on a moving band drier. The alcohol is normally recovered from the spent
liquor by distillation and from the cake by passing the hot gases from the drier into
charcoal recovery columns. The yield of product is such, that a fermentation can be
expected to be between 75 and 80%. The concentration of gum, however, is limited to less
than 5% in the fermenter, mainly due to the high broth viscosity encountered.
Continuous production of xanthan gum has been investigated in the laboratory, but there is
no evidence of industrial fermentations which employ this mode of cultivation. f continuous
culture of xanthan could be achieved on an industrial scale, production costs would be
greatly lowered as batch processes run to approx. 80 hours, whereas a continuous
process with a dilution rate of 0.05 h
-1
would give a 20 h fermentation.
Xanthan has been widely accepted for both food and nonfood uses. The value of xanthan
to the food industry is due to its unusual solution properties. The major food use is as
stabiliser and it is included in French dressing, fruitflavoured beverages, processed cheese
and other dairy products. A more recent opportunity for the gum stems from research
being undertaken by many oil companies throughout the world, namely enhanced oil
recovery. Enhanced oil recovery is the general technique of winning further oil from a well
after the natural pressure has ceased to force the oil out of the well.

3.1.3 AIginate. Alginic acid is another commercially important gum with gelling and
viscoelastic properties of use in the food, textile, pharmaceutical and paper industries.
Traditionally, alginates have been regarded as products found in the cells of Laminaria
spp. and other seaweeds, where they form important structural polymers. n these
seaweeds, the alginate content varies considerably, and the extracted polymer shows
variations in the ratio of mannuronic acid to guluronic acid residues. Recent developments
have indicated that it is possible to produce bacterial alginates using Azotobacter
vinelandii, in which the ratio of uronic acids can be controlled to some extent.
Of many fermentation parameters examined in batch culture, the phosphate concentration
was most critical. Because of the low buffering capacity of the lowphosphate medium, pH
control became necessary to maintain the level of polysaccharide production. These
improvements gave a 25% yield from the sucrose supplied. Further improvements were
achieved by continuous culture. The maximum efficiency was increased to 50%.
The major role of the polysaccharide is as a stabiliser for icecreams, instant deserts,
frozen custard, creams and cake mixes.

3.1.4 Approaches to the improvement of microbiaI poIysaccharide production.
Advances in the use of microorganisms to produce industrially useful polysaccharides may
be made by effecting the following improvements:

a) increasing the rate and extent of polysaccharide formation;
b) modifying the polysaccharide produced;
c) altering the surface properties of the producer microorganism to simplify cell
separation in subsequent downstream processing;
d) eliminating enzyme activities that may make unwanted modifications to the
polysaccharide;
e) transferring the genetic determinants of polysaccharide synthesis to more amenable
host process organisms.

ncrease in the rate or extent of conversion of the carbohydrate substrate to the polymer
product will require increase in the specific activities of the synthetic enzymes involved,
alteration of the control mechanisms of the synthetic process or increased availability of
polysaccharide precursors. The number of enzyme steps involved in the synthesis will
depend on the complexity of the particular polymer, but any attempt to increase polymer
production will require a detailed knowledge of the synthetic pathway and of its metabolic
control. At present, improvements in yield are brought about by random mutational events.
The rate of substrate uptake may be improved by the duplication of genes involved in
uptake mechanisms, but this may not be necessary if the organism possesses several
transport routes for each substrate.

Modification of the polysaccharide, for the purpose of enhancing one or more particular
characteristics, also requires some prior knowledge of the synthetic pathway. Xanthan
forms microgels in aqueous solution due to the interaction of the pyruvate ketals of the
polymer with cations. These groups may be removed chemically by treatment with oxalic
or trifluoroacetic acid. Alternatively, mutants can be isolated which produce non-pyruvated
or non-acetylated xanthans that are otherwise unaltered in carbohydrate structure and
have lower viscosity.

AIteration of the surface properties of the producer organism, e.g. by loss of surface
polymeric material such as lipopolysaccharides, eases polysaccharide harvesting. Such
mutant cultures will autoagglutinate, spontaneously flocculate and reduce the amount of
centrifugation required. However, care must be exercised to ensure that such mutants do
not 'leak', losing cell material, such as proteins from the periplasmic space, or lyse to
contaminate the final product. Other surface alterations involve the mutation of capsular
organisms to stable, slime-forming bacteria or the isolation of phage-resistant mutants, to
reduce the risk of phage contamination during the production run.

Some microorganisms produce exopolysaccharides that are subsequently degraded by
hydroIytic enzymes. Azotobacter vinelandii synthesizes alginate and an alginase.
Xanthan producers often secrete an active cellulase that may be the cause of unwanted
degradation if xanthan is subsequently added to cellulose-based products. Control of such
an unwanted enzymic activity may be achieved either by careful manipulation of culture
conditions or by the use of mutants unable to produce such hydrolytic enzymes.
Polysaccharide-producing strains also synthesize other polymeric products, such as other
polysaccharides, poly-beta-hydroxybutyrate or glycogen, which may represent a
considerable carbon drain from the desired synthetic process. Mutants defective in these
alternative synthetic pathways should be sought. However, prior knowledge of the
regulatory mechanisms of both pathways is required for this approach (glycogen !)

The transfer of genetic determinants of polysaccharide synthesis may be advantageous
under a number of circumstances. For example, transfer of alginate synthesis from a strain
of Pseudomonas aeruginosa, originally isolated from a patient with cystic fibrosis, to more
harmless, non-pathogenic Pseudomonas spp. would allow this synthetic capacity to be
employed commercially. A chromosomal locus has been postulated for the genes of
alginate synthesis. Transfer of the genes for xanthan synthesis to a host that is not
pathogenic to plants would also be advantageous. Alternatively, polysaccharide synthesis
could be introduced into bacterial strains that possess faster growth rates. t has been
found that strains unable to synthesize exopolysaccharides could be converted into
polymer producing strains by selection for carbenicllin resistance. This technique has been
successfully applied to a variety of Pseudomonas spp. to produce muc mutants, which
excrete polysaccharides.

3.1.5 PoIy-beta-hydroxybutyrate (PHB) PHB is a thermoplastic polyester consisting of
repeat units of the formula -CH(CH
3
)-CH
2
-CO-O. For over fifty years PHB has been
recognised as an energy reserve material accumulated by a wide variety of
microorganisms (e.g. Alcaligenes, Azotobacter, Bacillus, Nocardia, Pseudomonas,
Rhizobium). Under certain conditions, some species, such as Alcaligenes eutrophus and
Azotobacter beijerinckii can accumulate up to 70% of their dry weight as this polymeric
material (Steinbuechel 1996). Although many species are capable of PHB accumulation,
both the extent of that accumulation and the molecular weight of the particular polymer are
too low to render them useful for commercial polymer production. PHB content of the
biomass needs to be at least 35-40% of the dry weight and the molecular weight of the
polymer should be of the order of 20,000-300,000.
3.2 Antibiotics
Of the approximately 2000 antibiotics hitherto described the vast majority are of no
economic value. Toxicity or ineffectiveness in vivo has eliminated most from consideration
as agents in chemotherapy. Most of these were, of course, discovered as the result of
screening programs for new entities or as modifications of known antibiotics.
n treating infectious diseases, modern physicians have available to them numerous
antimicrobial agents from which to make a choice, including the antibiotics (Gutierrez et al.
2003). On the basis of their breadth of activity, these can be classified as having narrow,
intermediate or broad spectra of activity.
The narrow spectrum antibiotics are primarily limited to those active against gram-positive
organisms, although some are also active against neisseria and spirochaetes. Penicillin G,
for example, is highly effective in most infections caused by streptococci, pneumococci,
and sensitive staphylococci. Several of the newer penicillins, because they are unaffected
by penicillinase, are useful against resistant staphylococci (Queener & Swartz 1979).
n addition to gram-positive cocci, intermediate spectrum antibiotics are active against
some gram-negative bacteria and some mycobacteria. They are perhaps exemplified by
the aminoglycoside antibiotics (Claridge 1979), including gentamicin, kanamycin,
neomycin, and streptomycin. The cephalosporins may also be considered intermediate
spectrum antibiotics.
The broad spectrum antibiotics - the tetracyclines (Hostalek et al. 1979) and
chloramphenicol - are used in a variety of infections caused by both gram-positive and
gram-negative bacteria, although chloramphenicol must, in every case, be employed with
caution because of the occasional occurrence of aplastic anemia. The tetracyclines are
useful in trachoma.
There is currently an antibiotic available for each of the three general types of fungus
infections of human beings. Griseofulvin is effective in many superficial infections caused
by some species of Trichophyton, Microsporum, Epidermophyton. Amphotericin B, which
is a limited utility because of its toxicity, is employed in deep mycosis, particularly North
American blastomycosis, histoplasmosis, coccidioidomycosis, cryptococcosis and
candidiasis. Candida infections of the elementary tract or localized vaginal or skin
infections usually respond to nystatin.
n spite of the impressive advances in antimicrobial therapy there is a need for additional
agents to fill the gaps in the currently available drugs. For example, new agents are
needed to combat refractory gram-negative bacteira, especially strains of Pseudomonas,
Proteus, Aerobacter and Salmonella. Many mycobacteria, especially the atypical ones,
resist treatment with available drugs. There is also always the problem of the development
of resistant strains, some of which succumb to the newer antibiotics only to have other
strains appear in their stead.
No general discussion of antibiotics would be complete without mentioning the agricultural
and nonmedical uses of antibiotics:

a) disease therapy in livestock, poultry and plants;
b) prevention of disease in livestock, poultry and plants;
c) improvements in weight gains and feed conversion of livestock and poultry;
d) food preservation;
e) biochemical tools;
f) selective agents in culture media.

Many countries are now introducing legislation not allowing the use of antibiotics for meat
production destined for the human market.

3.2.1 Mode of Action. n general, antibiotics may either prevent the formation of DNA or
RNA, inhibit protein synthesis, interfere with the formation of the cell wall, or impair the
integrety of the cell membrane. For some antibiotics the site of action has been pinpointed
with considerable accuracy. For others the precise locus is unknown:

1. Interference with nucIeic acid

griseofulvin; novobiocin, rifampin

2. Impairment of the transIation of genetic information into protein synthesis

chloramphenicol ; neomycin; erythromycin; spectinomycin; gentamicin; streptomycin;
kanamycin; tetracyclines; lincomycin

3. Impairment of the synthesis and function of the ceII waII

bacitracin penicillins
cephalosporins vancomycin

4. Impairment of the function of the ceII membrane

amphotericin B; polymyxin
colistin; streptomycin
nystatin; tyrothricin

Novobiocin interferes with DNA polymerization and also inhibits DNA-dependent RNA
polymerase, as does rifampin. The locus of action of griseofulvin has not been precisely
worked out but the antibiotic may prevent the assembly of purine nucleotides.

3.2.2 Production Penicillin has been selected as the best known of the many antibiotics
produced at the present time. Penicillin is the name applied by Fleming in 1929 to the
bacteriostatic principle produced by Penicillium notatum. Since then it has been found that
penicillin is produced by a variety of moulds belonging to other species and genera, and
also that there is a series of closely related penicillins, all of which show approximately the
same antibiotic characteristics. The yields with Fleming's organism were poor and many
strains were tested to improve yields in submerged cultures. The penicillinproducing
moulds are characterised by unusual variability. n general one can say, the greater the
productivity of the strain, the less stable the strain becomes. Stock cultures can be
maintained in agar slants, in dry soil, in lyophilised form, or as spore or cell suspension
stored in liquid nitrogen.
Most of the recent work on the penicillin fermentation process has been concerned with
increasing production and decreasing manufacturing costs. Surface cultures of P.notatum
for penicillin production was first carried out in simple chemically defined media, which
later were improved through the addition of lactose, ammonium lactate, acetate and
phenylacetate, which is a precursor of benzylpenicillin. The addition of vegetable oils or
corn steep liquor significantly improved the penicillin production by P.chrysogenum. The
replacement of lactose with the continuous addition of glucose or sucrose did not change
antibiotic production, but resulted in a financial advantage. ncreased antibiotic production
was also attained through pH control between pH 6.8 and 7.4.
Aeration and agitation are fundamental for the penicillin process, and the theory of oxygen
transfer in largescale fermentation had to be investigated. t is agreed that high levels of
aeration and agitation are required to maintain the desired dissolved oxygen tension.
Temperature of incubation is very important and most operations are conducted at 25
o
C.
The production of penicillin G by fermentation is carried out in liquid culture. The culture
volume is typically 40 - 200,000 litres. The process is aerobic, having a volumetric oxygen
uptake rate in the range of 0.4 to 0.8 mmol/ltr/min. Oxygen is supplied by passing air
through the culture at a rate of 0.5 to 1.0 volumes of air/volume of fluid/min, and the air is
vigorously contacted with the fluid using turbine agitators of various designs. Power
introduced to the culture is generally of the order of 14 watts.ltr including that introduced by
the air stream. The vessels are fitted with coils, jackets or cascade systems for heat
removal, and control systems for the maintenance of the desired temperature, airflow
rates, agitator speed, pH value and various nutrient feed rates. Air filtration and aseptic
vessel design assures culture sterility.

The first step in the fermentation process is inoculation of vegetative cultures. The main
purpose of this and subsequent inoculum development steps is to increase the
concentration of fungal mycelium (biomass) to give a population which can be added to
the next step to assure that each step will be reasonably short and the largescale
equipment is used efficiently.
noculum development stages are typically conducted at around 25
o
C in shake cultures
and agitated vessels. A typical vegetative or seedstage medium contains an organic
nitrogen source, such as corn steep liquor, and 2%(w/v) sucrose or glucose. Calcium
carbonate is often added as a buffer at 0.51.0%(w/v). Log phase growth is usually
desirable in these stages, and a mass doubling time of about six hours is achieved.
The yield of penicillin per unit volume in a fermenter is the product of three parameters:

a) the concentration of cells X;
b) the specific rate of penicillin synthesis qpen;
c) the duration of the fermentation t.

The object is to achieve high X and qpen values as quick as possible.
n a typical penicillin fermentation, most of the cell mass necessary for high penicillin yields
is obtained during the first 40 hrs of the fermentation, starting from an inoculum consisting
of 10%(v/v) of a vegetative culture containing 20 g dry cell weight/litre. Once a cell
concentration sufficient to support a satisfactory volumetric yield of penicillin has been
obtained, growth must continue at a certain minimum rate if a high qpen value is to be
maintained. Hence, in the typical industrial penicillin fermentation, the concentration of
cells achieved at the end of a rapid growth phase is limited to allow for the additional cell
mass to be added during growth in the 'slow growth' or 'production' phase.
Before a high qpen value can be obtained in the fermentation, the content of glucose in the
cell must be minimally in excess over that required to maintain a desired growth rate. The
presence of excess glucose at this stage causes an accumulation of acid and excessive
biomass. Too little glucose can cause use of organic nitrogen as the carbon source and
excessive pH values, with correspondingly low qpen values. n modern facilities, hexose is
fed throughout the fermentation via closeloop computer control. Such systems allow
appropriate control over the rate of growth and carbohydrate combustion, both of which
affect qpen values.
Since the management of the oxidation of carbohydrate by P.chrysogenum so markedly
affects values for u and qpen, it is not surprising that the efficiency of most modern
penicillin fermentations can be judged on their yield of penicillin from carbohydrate,
designated Ypen/carbohydrate. A theoretical maximum value for the modern industrial
penicillin fermentation has been estimated to be around 0.12 g/g, which assumes a qpen
value of 5.1 mg/g cell/h for the modern process. The only real values available are 0.073
g/g and 1.8 mg/g cell/h.
Continuous countercurrent solvent extraction of the fermentation broth forms the basis for
the isolation and purification of penicillin.
At harvest, the culture has the consistency of dilute sludge and is light tan to dark brown in
colour. Mycelium is separated from the penicillincontaining broth and is washed on the
filter. The penicillincontaining filtrate is cooled in a heat exchanger to 0 - 4
o
C and the
filtrate is further clarified by a second filtration with 1.0 - 1.5% Hyflo, to precipitate
dissolved proteinaceous material. Penicillin is then extracted into amylacetate or
butylacetate by a continuous countercurrent process. The extraction solution is important
since penicillin degrades under acid conditions with first order degradation kinetics. This
rate is proportional to temperature and reciprocal to pH.
Depending on the final specification or end use, the penicillincontaining solvent may be
treated with carbon to remove pigments and other impurities. Penicillins may now be
extracted into water by addition of sufficient alkali or buffer at pH 5.0 - 7.5. The volume
ratio of water to solvent could be as low as 0.1 to 0.2. An excess of potassium or sodium is
added as the alkali or acetate to an aqueous or solvent stream containing a high
concentration of penicillin and the crystals are collected in a basket centrifuge or on a filter.
The crystals may be washed and predried with anhydrous isopropanol, butanol or other
volatile solvents which remove some impurities. Drying may be accomplished with warm
air, vacuum or radiant heat. The crystalline penicillin is sold as an intermediate or is further
processed to pharmaceutical grade. The latter are mainly referred to as semisynthetic
penicillins, which are acid stable, orally absorbed and very resistant to the enzyme
penicillinase inactivation.
All antibiotics require an extensive downstream processing process, which is the most
expensive part of the total production in contrast to the primary product formation.

4. Bio-insecticides
4.1 PrincipIes
The fact that certain microorganisms can inhibit the growth of others has been known
since the earliest days of microbiology and has stimulated much research, of which the
discovery and development of antibiotics for clinical use is probably the most important.
The possibility that a population of one microorganism might, by antagonistic or
competitive mechanism, be used to control a different microbial population, a plant
pathogen, for example, has also incited great interest but, regrettably, has led to few
developments of agricultural significance.
Biological control occurs naturally and helps to keep plant diseases in check but it is rarely
possible to explain how such control operates or how it might be manipulated to
agricultural advantage. Progress in this applied research area is slow, undoubtedly
because it must wait upon the accumulation of much fundamental knowledge on the
behaviour of mixed populations in soil and plant surfaces.
To use pathogens for pest control the basic requirements for successful microbial control
must be met. These requirements are
a) an adequate reservoir of pathogens in the pest population or in the environment;
b) good transmission of pathogens from reservoir to healthy pests;
c) rapid build-up of disease to prevent the pests becoming economically important or - with
insect vectors of human disease - to prevent the vector reaching significant numbers at the
stage when it infects man.

These requirements can be achieved in a number of ways:
1. a pathogen may be introduced into a new geographical area;
2. a strain more efficient than the native strain of a pathogen may be introduced;
3. the natural pathogen reservoir may be supplemented or the environment altered to
achieve disease earlier than usual;
4. a pathogen may be applied extensively to swamp the pest, i.e. used as a microbial
insecticide, which may have to be applied frequently during the pest season.
4.2 Stages in the investigation
The first step in the evaluation of potential microbial control is to obtain information about
the biology of the pest in question and its associated pathogens. Emphasis should be
placed on detecting the infective stages of the pathogens and the weak points in the pest's
life-style. This permits assessment of each pathogen to decide which causes the greatest
mortality and which - taking all factors into consideration - would be the most feasible
microbial control agents. Field tests should follow on an increasing scale. Data,
commensurate with the planned stage of investigation, should be obtained on the safety of
pathogens to man, domestic animals and wildlife.
The following are desirable in microorganisms to be used in the biological control of
insects:

1. the agent should be highly virulent for the target insect, but should kill no other insect;
2. the killing should be done quickly so that in the case of crops, damage is kept as low as
possible, and in the case of vectors of disease before extensive transmission of the
disease occurs;
3. the killing ability should be predictable;
4. it should not be harmful to man, animals or crops;
5. it should be technically amenable to cheap industrial production;
6. when produced, it should be stable under conditions of use such as under the high
temperature and UV-light of ordinary sunlight;
7. it should be viable over reasonably long periods to permit storage and transportation as
necessary;
8. it should ideally persist or recycle and/or be able to search for its host.
4.3 PresentIy used candidates for bioIogicaI controI agents
4.3.1 Bacteria.
A large number of bacteria are pathogenic to insects including Bacillus spp.,
Pseudomonas sp., Klebsiella sp., Serratia marcescens. n practice, spore formers have
been developed commercially because they survive more easily in the environment, but
especially because they are more easily mass produced. The four bacilli which are
currently being produced for control purposes are:
a) Bacillus thuringiensis: a complex of several organisms regarded by some as being
variants of B.cereus. There are fourteen serotypes based by some flagellar or H-antigens.
Bacillus thuringiensis produces at least three toxins, a phospholipase C, a water-soluble
heat stable B-entoxin, potentially toxic to mammals, and a crystalline, d-toxin or the
parasporal body which is enclosed within the sporangium. The crystalline d-toxin is the
active principle against most insects. The spores and crystals are released in the medium.
B.thuringiensis is the microbial control agent most used in the developed world. t is an
aerobic spore-forming species readily cultured in media and produced commercially by
convbentional, liquid, stirred-tank fermentation. At sporulation, each cell produces a
bipyramidal crystal of protein, a potent insect gut toxin that is the major component of
commercial products in killing most susceptible species.
First described in 1911 from flour-moth larvae in a European flour mill, it subsequently
proved common in Lepidoptera in dry protected habitats such as warehouses, food
factories, silkworm farms, and beehives. Although spores and crystals in dry larval
cadavers survive almost iundefinitely in food residues left when a store is unloaded, the
bacterium does not spread enough to give useful control. t must be applied regularly to
crops and forests as a microbial insecticide.
The crystal of protein, that is produced with every spore of B.thuringiensis, is an active
protoxin, composed of molecules of mass 130 kDa. t is activated by alkaline dissolution
and breakdown by gut proteases into active toxins, varying in molecular mass from 60 to
65 kDa. These toxins destroy the epithelium of the insect mid-gut, acting on the cell
membrane. n Lepidoptera, they probably bind with glycoproteins, causing cytolysis by
altering membrane permeability and destroying regulation of the passage of glucose and
ions such as K
+
.
Thus at first sight, the B.thuringiensis crystal appears to be an ideal insecticide. t is
specific to and highly potent in a wide range of pest insect larvae (Moazami 2003). t is
harmless to man, even if eaten in considerable quantities or injected into the body,
harmless to wildlife and biodegradable. However, its use is restricted by its being a
stomach poison that must be eaten by larvae to take effect, i.e. it has no contact action
and attacks no other developmental stage of the insect.
The disadvantages of B.thuringiensis can be alleviated by selection and genetic
manipulation. Extensive selection from natural strains in the developed world produced a
succession of better strains. These were adopted over two decades by industry, increasing
potency of products by over 100-fold, and improving host range. The genes controlling the
crystal toxins are borne on plasmids, which can be exchanged between strains with
relative ease. This enables strains to be genetically tailored to the needs of particular pest
complexes. What is probably the first patent of a genetically modified B.thuringiensis strain
was taken out in 1984 in the UK.
The developing world has contributed new strains and varieties of B.thuringiensis
particularly from countries sited along well-established trade routes. The variety ostriniae
was first found in China; var. pakistani in Pakistan; and var. wuhanensis in China.

b) Bacillus moritai: is used in Japan for the same purpose as B.thuringiensis serotypes
H3 and H3A.
c) Bacillus popilliae: this is an obligate pathogen of the Japanese beetle Popilla japonica
against which it is used. This microbial control agent was the first successful and well-
documented bioinsecticide in the developed world. The beetle was accidentally imported
into the USA.
Larvae feed for 2 or 3 years on roots, particularly those of grasses. They ravage pastures,
scrubland and high quality grass, such as lawns. Adults feed on foliage and damage crops
as well. B.popilliae and related species were introduced into infested areas and spread
rapidly amongst the larvae. The effective spread was possible because:
(1) the bacteria kill larvae by slow infection, producing mainly large cadavers packed with
spores;
(2) spores survive many years in soil and at high concentrations where a larvae has died
in untilled soil;
(3) larval populations were usually high, allowing an extensive reservoir of spores to build
up;
(4) most grassland was economically unaffected by subsequent small surviving larval
populations, which maintained spore reservoirs;
(5) some surviving larvae became infectred adults, which flew long distances and spread
the bacteria widely beyond inoculated areas.

Unfortunately, the production of the bacteria was a limiting factor. Although the bacterium
was able to grow well in complex medium, spore formation on an economical scale could
only be carried out using live larvae and adults.
d) Bacillus thuringiensis var. israelensis. t has proven to be very effective in killing
mosquito larvae and the black fly (Simulium spp). Unlike the classical B.thuringiensis it
does not produce a beta-toxin. ts killing effect is therefore based principally on its
crystalline delta-toxin, which is resistant to both heat (80
o
C for 10 min) and UV light.

n the USA and elsewhere commercial products were produced in a remarkably short time
of 6 years from the date of the first discovery of this new variety. t is used as a larvicide
both of nuisance species and vectors of human disease. ts biggest single market and
immediate application was in the river-blindness eradication program against blackflies in
the Upper Volta region of Africa. This large program, implemented by WHO had already
been in progress for 7 years by 1982 when it was threatened with collapse by appearance
of serious resistance to the organophosphate insecticide, temephos, and other acceptable
chemicals in two important blackfly species over about 25% of the treated area. The var.
israelensis became commercially available just in time to prevent loss of control of the
blackflies. Because it was relatively expensive, it was used only in the dry season, when
water areas requiring treatment were at their lowest. n the wet season another
organophosphate, chlorphoxim was used, but resistance to this chemical also became
serious. Luckily, this blackfly resistance proved to be transient and was abated by the use
of B.thuringiensis in the following summer, so that subsequently the bacterium and the
chemical could be alternated seasonally. This demonstrates an important principle for
successful pest control, the regular alternation in time of biological agents and chemicals,
to combat appearance of resistance to chemical insecticides.
e)Bacillus sphericus has been shown to be as highly specific for mosquito larvae as
B.thuringiensis var. israelensis. However, whereas the lethality of B.t.i. resides in toxic
protein crystals formed during the cell wall formation of the cell, the toxin of B.sphericus
resides in the cell wall of the organism. The toxin of B.sphericus works slowly (8-40 hrs)
compared with that of B.t.i. (2-10 hours).
B.sphericus can be produced in bulk by fermentation and formulated like B.thuringiensis
var. israelensis. A proteinaceous toxin is located mainly in the spore coat and, in some
strains, in crystals as well, serving a similar function to that of the 27.t.i. However, the
bacterium multiplies freely in larval cadavers and recycles in larvae but probably
inefficiently to maintain long-term effective control. The spore persists in the environment
for a long time, but tends to accumulate in bottom sediments away from feeding zones of
mosquito larvae.
4.3.2 Viruses A large number of viruses have been isolated from insects. The advantage
of viruses as biological control agents is that they are specific. Seven groups of insect-
pathogenic viruses have been identified. The most useful of them for biological control
purposes are the bacuIoviruses, which are easily recognizable because the virus particle
are included within a proteinaceous inclusion body large enough to be seen under a light
microscope. The baculoviruses are the best candidates for insect control because they are

a) effective in controlling insect populations;
b) restricted to a host range of invertebrates;
c) relatively easy to produce in large quantities;
d) stable under specific conditions because of the inclusion bodies.

Several experimental preparations are available and at least two have been produced on
commercial scale. The preparations are ingested when the insects consume leaves and
other plant parts on which the virus particles have been sprayed. After ingestion the
polyhedral inclusion bodies dissolve within the mid-gut; the released virions pass through
the mid-gut epithelial cells into the haemocoel. Death of the larvae occurs four to nine days
after ingestion.
Baculoviruses protect their infective DNA in lipoprotein-sheathed particles, themselves
embedded in a proteinaceous matrix to form an inclusion body. n Eurpe and North
America, inclusion bodies of a baculovirus of the gypsy moth, Lymantria dispar, L., have
been produced to control this severe forest pest. Virus inclusion bodies are harvested by
blending thawed insects in water, straining through cheesecloth, centrifuging and drying
overnight under a hood fed with a laminar flow of sterile air. After grinding, quality control
consists of checking potency of the powder by bioassay, and ensuring absence of
dangerous bacteria by plating on selective bacteriological media and injection into mice.
Field application is by conventional ground spraying, or by aircraft sprayin g in carefully
monitored meteorological conditions. For example, two aerial applications of 2.5 x 10
11

inclusion bodies/ha with, for instance, molasses and a suitable sticker are recommended,
the first timed when leaves are partly expanded and larvae in development stages 1 aqnd
2 are present. The second is timed 5-10 days later, but before larvae grow to stage 4. This
treatment should reduce gypsy moth egg masses in the next generation by 75% or more
and give acceptable reduction of tree defoliation. 'Gypcheck' is used strictly as a viral
insecticide.
Various strains of baculovirus have been developed against various insects in various
countries. t is impossible to deal with all of them as research is still continuing.
Here in SEAsia and the Pacific, the best example is the accidental introduction of the
coconut rhinoceros beetle, Oryctes rhinoceros L. from ndia into Western Samoa and other
sland Nations. This beetle completely obliterated the coconut palm. A special baculovirus
saved the coconut industry in the 1970s.
4.3.3 Fungi All the four major groups of fungi, Phycomycetes, Ascomycetes, Fungi
Imperfecti, Basidiomycetes contain members pathogenic to insects. The great difficulty
with using fungi for biological control is that environmental conditions including
temperature and humidity must be adequate for spore germination and insect cuticle
penetration by the hyphae. Since these environmental conditions are not always assured
the result is that fungi are used for biological control only in few countries, especially the
former Soviet Union. Fungi which have been most widely used are Beauvaria bassiana,
Metarrhizium anisopliae, Hirsutella thompsonii. The latter is being developed commercially
as acaricide, for killing mites which attack plants. H.thompsonii has been found particularly
active against mites which attach citrus. t is applied as a conidial powder and maximum
effectiveness occurs at 27
o
C and under moist conditions or at relative humidities of 79-
100%. Coelomomyces sp. is very effective against mosquitos but its production is difficult
because of the need for a secondary host. Most effective and specific against mosquitos
are Culicinomyces sp. which was isolated in Australia and produced a mortality rate of 90-
100%.

4.3.4 Protozoa Protozoa are pathogens of insects. However, in contrast to the rapid action
of viruses and spore-forming bacteria, killing by protozoa is slow and may take weeks.
Furthermore they are difficult to produce, being accomplished only in vivo. Nevertheless
they have been produced and successfully used experimentally for store-product pests
(Matosia trogoderina), mosquitos (Nosema algerae) and grasshoppers (Nosema pyrasta).
So far, however, protozoa have not been produced on an industrial scale for biological
control.
4.4 Production of BioIogicaI Insecticides
Microbiological insecticides are produced in one of three ways: submerged fermentation,
surface or semi-solid fermentation; and in vitro production. The first two are for facultative
pathogens and the third is for obligate pathogens.

4.4.1 Submerged Fermentations This type of fermentation has been used for the
production of Bacillus spp (excluding B.popillae) and to a lesser extent, fungi.
Medium. n fermentation for Bacillus thuringiensis the active principle sought is the delta
toxin found in the crystals. Media for submerged fermentation have been compounded by
various workers in a number of patents. n one such preparation, the initial growth in a
shake flask occurred in nutrient broth; in the second shake flask, and in the seed fermenter
beet molasses (1%), corn steep liqour (0.85%) and CaCO
3
(0.1%) were used. A typical
medium for production would be beet molasses (1.86%), pharmamedia (1.4%) and CaCO
3

(0.1%).
Other production media contain corn starch (6.8%), sucrose (0.64%), casein (1.94%), corn
steep liquor (4.7%), yeast extract (0.6%) and phosphate buffer (0.6%).
A third medium contained soya bean meal (15%), dextrose (5%), corn starch (5%), MgSO
4

(0.3%), FeSO
4
(0.02%), ZnSO
4
(0.02%) and CaCO
3
(1.0%).
These media were used for agricultural strains of B.thuringiensis but could no doubt be
used also for B.thuringiensis var. israelensis.
Bacillus thuringiensis var israelensis and Bacillus sphericus do not require carbohydrates
for growth and can grow well and produce materials which will kill the larvae of mosquitoes
in a variety of proteinaceous materials such as commercial powders of soy products, dried
milk products, blood and even materials from primary sewage tanks.
At the end of fermentation, the active components of the broth are recovered by
centrifugation, vaccum filtration with filter aid or by precipitation. Precipitation has been
carried out with CaCl
2
, but acetone can also be used. The fermentation beer may readily
be diluted and used directly.
4.4.2 Surface CuIture Surface culture techniques are used for fungi and for spore-
formers. The organisms after shake flask growth are cultured in a seed tank from where
the broth is transferred to flat bins with perforated bottoms. The semi-solid medium is a
mixture of an agricultural by-product such as bran, an inert product such as kieselgur, soy
bean meal, desxtrose and mineral salts. The use of this medium increases the surface
area and hence aeration because of the thinness of its spread in the bins. Hot air is
passed through the perforations to dry the material. t is ground, assayed and
compounded to any required strength with inert material.
4.4.3 In vivo CuIture In vivo culture methods are used for producing caterpillar viruses,
mosquito protozoa and Bacillus popillae. The method is labour-intensive and could be
easily applied for suitable candidates in developing countries where expertise for
submerged culture production is usually lacking.
Once the organism has been obtained in a sufficient quantity to last for several years it is
lyophilised and stored at low temperature. The viruses are introduced into the food of the
larvae and the dead larvae are crushed, centrifuged to remove large particles and the rest
are dried. The amount of viruses in each larvae is variable but the virus content of between
one and one hundred caterpillars should be sufficient to treat one acre in the case of
cotton moths. Usually separate facilities are used for rearing the caterpillars. for infecting
them and for the extraction of the virus particles. The preparation is then bioasssayed and
mixed with a suitable carrier.
4.5 Bioassays
t is obvious that a reference standard must be set up against which various preparations
can be compared. The standard will differ with each particular bioinsecticide. Thus
standards exist for B.thuringiensis serotypes H3 and H3A used against caterpillars andn a
standard for B.thuringiensis var. israelensis against mosquito exists. Both standards are
prepared and deposited at the nstitut Pasteur in Paris. n the simplest terms a standard is
based on the LD50, the dose of the insecticide which will kill 50% of the population must
be clearly defined; the age and type of insect to be used; the food of the insect; the
temperature conditions and a host of other parameters.
4.6 FormuIation and use of bioinsecticides
The formulation of the bioinsecticides is extremely important. An insecticide shown to be
highly potent under laboratory experimental conditions may prove valueless in the field
unless the formulation has been correctly done. Since microorganisms cannot by
themselves be patented, industrial firms producing insecticides depend for their profits on
the efficiency of their formulation (i.e. the inert material which ensures adequate
presentation of the larvicide to the target insect). The inert material is referred to as a
carrier or an extender. Carriers or extenders are the solids or liquids in which the active
principle is diluted. When the carrier is a liquid and the active principle is suitable in it, the
application is a spray. There are thus two types of formulation: (a) powders and dusts; (b)
flowable liquid; which of the two is manufactured depends to a large extent on the method
of production and intended use of the insecticide.
Dusts
Semi-solid preparations based on waste plant products usually are compounded as dusts
or powders because making them into liquid causes the bran to absorb water and prevent
free flow thuis leading to the clogging of conventional liquid applicators. The advantage of
dusts is greater stability of the preparation. They are also useful when the insecticide is
intended to reach the underside of low lying crops such as cabbage. Heavy rains
unfortunately wash off dusts. They may also lead to inhalation of the bioinsecticides by the
persons applying them. Diluents which have been used in commercial dust of Bacillus
thuringiensis are celite, chalk, kaolin, bentonite, starch and lactose. When the active
principle is adsorbed on to the extender (or filler), the extender is referred to as a carrier.

Liquid formulation
Liquid formulations are normally made from water in which both the crystal and spores are
stable. Sometimes oils of water/oil emulsions may be used. When liquids other than water
are used it must be ascertained that they do not inactivate the active agent. Emulsifiers
may be added to stabilize emulsions when these are used. Dome emulsifiers which have
been used for B.thuringiensis and viruses are Tween 80, Triton B1956 and Span 20.
The nature of the surface on which the insecticide is applied and which may be oily,
smooth or waxy may prevent the liquid from wetting the sprayed surface. Spreaders or
wetting agents which are surface-tension reducers may be added.
To prevent run-off of liquids or wettable powders, stickers or adhesives are added to hold
the insecticide to the surface. Stickers which have been used include skim milk, dried
blood, corn syrup, casein, molasses, and polyvinyl chloride latexes.
Protectants are often added to insecticides which protect the active agent from the effects
of ultraviolet light, oxidation, desiccation, heat and other environmental factors which
reduce the effectiveness of the active agent. These are usually trade secrets and their
composition is not disclosed.
4.7 Safety Testing of bioinsecticides
Many individuals on first learning of the use of microorganisms to control insect pests and
vectors of disease express fear about the effect of these entomopathogens or their
effective components. Testing at the second level is usually carried out by industry. The
tests include feeding by mouth, inhalation, intraperitoneal, intradermal and intravenous
inoculations as well as cancerogenicity testing.
4.8 Future
The need for effective quality control of products is a major consideration in local
production. Paramount is the safety aspect to ensure that a dangerous organism is not
produced in error, or that a dangerous contaminant is not present. t is also important to
ensure that the product is efficaceous. Existing production of insect pathogens in
developing countries has largely ignored quality control. As these countries advance
towards standards required, quality control technology will be needed and the scale of
production will have to be increased to encompass the extra costs.
The scale of crop production in a developing country will influence the approach taken to
local production of microbial insecticides. Some crops grown on large plantations may
have a microbial pesticide requirement sufficient to warrant local production.
Research on-site is desirable at all levels. An important basic research activity is collection
of new isolates and species of m icroorganisms as potential control agents. Local trials of
microbial agents are essential, particularly for those most sensitive to environmental
conditions, with special emphasis on the possible roles of agents in relation to regional
crop husbandry. Locally-orientated practical work is likely to give the quickest return for
effort in the developing countries. Thus some teaching of insect pathology should be
incorporated in university curricula.
Recent highlights of fundamental work in developed countries offer prospects of far
reaching advances. These highlights include research on the mode of action of pathogens
and toxins, chemistry of toxins, genetics and genetic manipulation. Microbial control has
the great advantage of specificity to pests, but this also creates the problem of limiting the
market size for individual microbial insecticides. Genetic recombination has created strains
of B.thuringiensis with improved host ranges. The B.thuringiensis endotoxin gene has
been inserted and expressed in the tobacco plant to create a plant systemically protected
from caterpillar attack. The toxin might possibly be inserted into blue green algae to
engineer mosquito-larvicidal algae that grow freely in natural waters. To protect plant roots
from root-feeding caterpillars, the toxin has been transferred to soil bacteria that grow
around the rhizosphere. Some 30% of the protein in the B.thuringiensis cell becomes
crystal toxin and the plyhedrin protein in which virus particles are embedded in baculovirus
inclusion bodies far exceeds in quantity the actual virus particle. Thus, both types of insect
pathogen have very powerful producer sequences. These sequences can be used to
amplify expression of the products of introduced genes
5. BibIiography
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(A.H.Rose,ed.), Economic Microbiology 3,151-238, Academic Press
Singapore
DoeIIe,H.W.,D.A.MitcheII and C.L.RoIz(eds.) 1992 - Solid Substrate Cultivation. Elsevier
Applied Science, London
Ebner,H. 1982 - Vinegar. n Industrial Microbiology (G.Reed, ed.) The AV Publishing
Company, p. 802-834
Ebner,H., S.SeIImer, and H.FoIImann 1996 - Acetic Acid. n Biotechnology (H.J.Rehm,
G.Reed, eds), Vol 6,381-402. VCH Verlagsgesellschaft mbH, Weinheim, Germany
Esaki,N., S.Nakamori, T.Kurihara, S.Furuyoshi and K.Soda 1996 - Enzymology of
Amino Acid Production. n Biotechnology (H.J.Rehm, G.Reed, eds), Vol 6,503-560. VCH
Gutierrez,S., F.J.Casqueiro and J.F.Martin - 2003 - Production of Antibiotics. n
Biotechnology (H.W.Doelle, E.J.DaSilva, eds.), Encyclopedia for Life Support Systems,
EOLSS Publishers, Cambridge, UK
HostaIek,Z., M.BIumauerova snd Z.Vanek 1979 - Tetracycline Antibiotics. n Secondary
Products of Metabolism (A.H.Rose, ed.). Economic Microbiology 3,294-354, Academic
Press
Kapoor,K.K., K.Chaudhary and P.Tauro 1982 - Citric Acid. n Industrial Microbiology
(G.Reed,ed.), p. 709-748. The AV Publishing Comp.
Kasak,J.S., J.Kominek and M.Roehr 1996 - Lactic Acid. n Biotechnology (H.J.Rehm,
G.Reed, eds.), Vol. 6,293-305. VCH Verlagsgesellschaft, Weinheim, Germany
Kinoshita,S. and K.Nakayama 1978 - Amino Acids. n Primary Products of Metabolism
(A.H.Rose, ed.), Economic Microbiology 2,210-262. Academic Press
Kosaric,N. 1996 - Ethanol - Potential Source of Energy and Chemical Products. n
Biotechnology (H.J.Rehm, G.Reed, eds.), Vol. 6,121-204. VCH Verlagsgesellschaft gmbH,
Weinheim, Germany
Lawson,C.J. and I.W.SutherIand 1978 - Polysaccharides. n Primary Products of
Metabolism (A.H.Rose, ed.), Economic Microbiology 2,328-391. Academic Press
Leuchtenberger,W. 196 - Amino Acids - Technical Production and Use. n Biotechnology
(H.J.Rehm, G.Reed, eds.), Vol 6,465-502. VCH Verlagsgesellschaft gmbH, Weinheim,
Germany
Moazami,N. 2003 - Biopesticide Production. n Biotechnology (H.W.Doelle,E.J.DaSilva,
eds.), Encyclopedia for Life Support Systems, EOLSS Publishers, Cambridge, UK
Nakayama,K. 1982 - Amino Acids, n Industrial Microbiology (G.Reed, ed.), p. 748-800.
The AV Publishing Comp.
Okafor,N. 1978 - ndustrial Microbiology. Unoiversity of fe Press, fe-fe, Nigeria
Queener,S. and R.Swartz 1979 - Penicillins: Biosynthetic and Semisynthetic. n
Secondary Products of Metabolism (A.H.Rose, ed.), Economic Microbiology 3,55-122,
Academic Press
Rehm, H.J. 1996 - Microbial Production of Glycerol and other Polyols. n Biotechnology
(H.J.Rehm, G.Reed,eds.), Vol. 6,205-227. VCH Verlagsgesellschaft mbH, Weinheim,
Germany
Roehr,M., C.P.Kubicek and J.Kominek 1996 - Citric Acid. n Biotechnology
(H.J.Rehm,G.Reed, eds.), Vol. 6,307-346. VCH Verlagsgesellschaft gmbH, Weinheim,
Germany
Rose,A.H.(ed.) 1979 - Secondary Products of Metabolism. Economic Microbiology 3.
Academic Press
SteinbuecheI,A. 1996 - PHB and other polyhydroxyalkanoic acids. n Biotechnology
(H.J.Rehm, G.Reed, eds.), Vol. 6,403-464. VCH Verlagsgesellschaft mbH, Weinheim,
Germany
SutherIand,I.W. 1996 - Extracellular Polysaccharides. n Biotechnology (H.J.Rehm,
G.Reed, eds.), Vol. 6, 613-658. VCH Verlagsgesellschaft mbH, Weinheim, Germany

Deputy-Director, MIRCEN-Brisbane and Pacific Regional Network
Chapter 17
MicrobiaI Production of Food
Content
1. Introduction
2. Fermented Foods and CuIture
3.1 Ang-Kak
3.2 Bagoong
3.3 Doza & IdIi
3.4 Fish Sauce
3.5 Miso
3.6 Natto
3.7 Oncom
3.8 Puto
3.9 Soy sauce
3.10 Tempeh
4. African Region
4.1 Gari
4.2 Ogi
4.3 OIive fermentation
5. European Region
5.1 Bread
5.2 Cheese
5.3 Yoghurt
5.4 ButtermiIk
6. BibIiography

1. Introduction
Fermented foods, whether from plants or animal origin, are an intricate part of the diet of
people in all parts of the world. t is the diversity of raw materials used as substrates,
methods of preparation and sensory qualities of the finished products that are so
astounding as one begins to learn more about the eating habits of various cultures. The
preparation of many indigenous or traditional fermented foods and beverages remains
today as a household art. The preparation of others, e.g. soy sauce, has evolved to a
biotechnological state and is carried out on a large commercial scale. This chapter will
bring a selection of these traditional fermented foods from different regions of the world.

2. Fermented Food and CuIture
Since his appearance, man has always lived in an uncertain, sometimes precarious,
symbiosis with nature, obtaining his nourishment needed from plants and animals
personally accessible to him. n accordance with the climatic and environmental
conditions, the search for food (life as Nomades) soon developed into actively growing,
storing and preparing the food (life as Settlers and Farmers). Water, sun availability and
soil conditions determined the type of original food for the particular society.
t was the farmer, who was responsible for the growth and harvesting of the crops,
whereas the families used their own recipes for the conversion of the agricultural products
into edible and palatable products for themselves, their animals and later the market place.
This practice is still followed today in many societies in SEAsia, Africa and Latin America.
The culture of biotechnology originated therefore in the rural areas, where people
experimented with the regeneration of soil fertility, breeding of new crop varieties and
fermentation for a palatable and well digestible food. Whereas cold and temperate zones
used mainly grain for their food and fermentation, tropical zones of SEAsia and Africa
produced numerous foods from rice, soybean, cassava [manihot] and other plants.
A second type of fermentation technology was accidentally introduced very soon in form of
beverages such as wine, met [honey beer], and beer and in different types of food such as
bread, milk products such as cheese, butter and yoghurt. Whereas the societies of cooler
climates preferred beer and wine from barley and grapes, respectively, it was pulque from
the sweet juice of the Mexican Agave in Aztec countries of Latin America, the saki from
rice in SEAsia and the palm wine from palms in some societies of Africa. Other societies in
Africa did not encourage this second type of fermentation out of personal and/or religious
beliefs.

Each region of the globe developed its own fermentation technology producing
characteristic food and drink for the local population [Table 1].

Since food preparation and fermentation was carried out according to 'local society
tradition', handed down from generation to generation, these complex preparations were
much more an art than a science. Neverthless, the traditionally fermented protein-rich
foods are highly acceptable to millions of people until today, because they are easily made
and are generally more attractive to the consumers tyhan the cooked original substrates.
The organoleptic characteristics of the substrate are improved by the fermentation
process. These fermentations also increase the nutritional value of the substrate, since the
amount of vitamins are significantly increased as well as the digestibility. f properly
fermented, these foods are not hazardous to health since the microorganisms responsible
for these processes are not toxin producers (Wang & Hesseltine 1982).
t was not before A.van Leeuwenhoek in the 18
th
century was able to construct the first
microscope and Louis Pasteur in the 19
th
century found the reasons for the fermentations
to occur (Doelle & DaSilva 2003), was the complexity of the traditional food fermentation
realised.
Biotechnology processes, which include these fermentation technologies are now
recognised as the translations of basic scientific research achievements in the field of
biology into practical and/or commercial applications using low, medium or high capital
technology. They unquestionable generate many benefits, but can also be seen to bring
certain dangers or potential threats with them (Doelle 1998) depending on which strategies
are adopted by the individual community.

TabIe 1: Ancient Fermented Foods in different Societies of the world. [adapted from
Spicher and Bruemmer 1995; Beuchat 1995;Wang and Hesseltine 1982]

3.1 ANG-KAK
Ang-kak or red rice has been used in the fermentation industry for the preparation of red
rice wine and foods such as sufu, fish paste and red soybean curd. Pigments produced by
Monascus purpureus and Monascus anka on a rice substrate have been used as
household and industrial food colourants.

Figure 1: Production of Ang-kak [Hesseltine 1965]

Rice is first washed, soaked in water for about 1 day and drained. The moist rice is then
transferred to a glass beaker or suitable container to allow plenty of air space above it.
This is autoclaved for 30 min at 121
0
C. Unpon cooling the rice is inoculated with a sterile
water suspension of ascospores removed from a 25-day old culture of M.purpureus grown
on Sabourand agar. At the time of inoculation, the rice should appear rather dry. The
inoculated rice is thoroughly mixed and then incubated at 25-32
0
C for about 3 days. The
rice will show a red colour. t should then be stirred and shaken to redistribute moisture
and kernels. Within 3 weeks, the rice should take on a deep purplish red colour and
kernels should not stick together. After drying at 40
0
C, the kernels are easily crumbled by
slight force and may be reduced to a powder. Monascus produces large quantities of
hydrolytic enzymes such as alpha-amylase, beta-amylase, glucoamylase, protease and
lipase to break down the rice constituents.n addition to its value as a colourant, angkak
may also possess therapeutical properties and also has considerable antibacterial activity.
3.2 Bagoong
Bagoong is a fish paste prepared in the Philippines from sea fish, achovies, ambassids, or
shrimp. Salt is mixed with three parts fish, placed in clay vats and left undisturbed for 3
months. The resulting past-like product is either eaten raw or cooked.
3.3 Puto
Puto is a fermented rice cake in the Philippines and is usually prepared from one-year-old
rice that is ground with sufficient water to allow fermentation before steaming (see Figure
2). The product is similar to tofu prepared from rice in Thailand.

Figure 2: The production of Puto [Beuchat 1995]
The quality of puto is dependent upon the microflora present in the milled rice as well as
the variety of rice used as a starting material. The preparation of puto takes approx. 42
hrs. This time can be shortened to 21 h using a starter culture containing Leuconostoc
mesenteroids, Streptococcus faecalis and Saccharomyces cerevisiae. A sensory
evaluation showed no significant difference in general acceptability, texture, flavour and
volume expansion.
3.4 Doza and IdIi

dli is a steamed fermented dough made from various proportions of rice and black gram
flours. t is typically eaten for breakfast and is especially popular in South ndia. The
proportions of rice to black gram cotyledons used depends upon taste preference and
availability between 1:4 and 4:1. Acidification and leavening are the most important
processes which occur during the fermentation (Figure 3).

Figure 3: The production of Dosa and dli (Reddy et al. 1986)
While several reports have been made on the microbiology of idli fermentation, no
comprehensive studies are available. The identified bacteria are Leuconostoc
mesenteroides, Lactobacillus delbrueckii, L. lactis, Streptococcus faecalis and
Pediococcus cerevisiae together with the yeasts Geotrichum candidum, Torulopsis
candida and Trichosporon pullulans (Beuchat 1995). The lactic acid bacteria are obviously
responsible for the sour taste. Soured buttermilk or yeast are sometimes added to the
dough to reduce the fermentation time which, of course, influences the microbial profile of
the total fermentation process.
3.5 Fish Sauce
Fermented fish sauce and paste are very popular condiments prepared and consumed in
the SEAsia region. They make the bland rice and vegetable diet more palatable. Fish
sauce and fish paste made in various countries are known under different names, such as
nuoc-mam and mams in Cambodia and Vietnam, patis and bagoong in the Philippines,
and nampla and kapi in Thailand (Wang & Hesseltine 1995). Although the basic principles
of making these products are the same, a large number of variations developed.
The production of nuoc-mam is a very active industry in Vietnam and thus best known. t is
produced commercially in large plants as well as in so-called cottage industries. n the
most primitive way, small fish are kneaded and pressed by hand, salted and tightly packed
into earthenware pots which are sealed and buried in the ground for several months.
When the pots are opened, the supernatant liquid formed is decanted and represents the
fish sauce.
n the commercial production (Van Veen 1953) cylindrical vats made with local wood and
encircled with twisted bamboo are used. The vats are equipped with taps at the bottom.
The fresh uncleaned fish are mixed with a small amount of salt and packed in the vat in
alternative layers with more salt and a final layer of salt is placed on the top. After 3 days,
the collected liquid is drained off and reserved for later use. Meanwhile the fish have
settled below the top of the vat and the salt has almostg disappeared. The fish are now
packed down thoroughly, and the surface is smoothed. The contents are covered with a
layer of coconut leaves and then with bamboo trays on top of which weights are placed.
The drained liquid from above is poured back into the vat so that a layer of liquid covers
the fish. The fish are now left to ferment.
Fermentation time varies with the kind of fish used ranging from a few mponths to a year.
After maturing, the liquid is run off through the tap at a rate of 300-400 liters per day. The
residue is in many countries used as fish paste.
The hydrolysis of the fish protein appears mainly due to the enzymes in the fish.n addition
it was found that proteolytic enzymes from Aspergillus oryzae greatly reduced the
fermentation time and increases the yield. Furthermore it is thought that the presence of
anaerobic spore-forming bacteria belonging to the Clostridium group may be responsible
for the typical flavour. About 70% of the microflora found after the fermentation were
halophiles belonging to the bacillus-type of bacteria.

3.6 Miso

Miso is the name given to paste-like products made by fermenting cereal, soybeans and
salt with molds, yeasts and bacteria (Wang & Hesseltine 1995). As was the case with fish
sauce, each nation has its own name for the product. Literally they all mean 'bean paste' .
t has the consistency of peanut butter and its colour varies from light yellow to reddish-
brown. According to Ebine (1971), miso is categorised into 3 types based on the raw
material used, eg rice miso made from rice, soybean and salt, barley miso made from
barley, soybeans and salt and soybean miso made from soybeans and salt. The most
used type is undoubtedly the rice miso.
As is shown in Figure 4, the production of rice miso consists of cooking soybeans,
preparing what is called koji from rice and mixing both with salt, fermenting and ripening in
a tank.

Figure 4: Production of miso (Beuchat 1995)
Soybean quality is of utmost importance in miso fermentation. The soybean variety was
found to significantly affect the quality and organoleptic scores of the final product.
3.7 Natto
n SEAsia soybean fermentations, molds usually dominate, but natto fermentation is an
exception in which bacteria dominate. The bacterium Bacillus natto, identified as B.subtilis
is the organism responsible for this fermentation. Natto therefore possesses the
characteristic odour and persistent musty flavour of this organism and is also covered with
viscous, sticky polymers that this organism produces.
n Japan, natto is seasoned with soy sauce, salt or sometimes mustard and served with
rice. Making natto is a very simple operation and can easily be done at home. The
soybeans are soaked, boiled, drained, cooled, wrapped in rice straw and kept in a warm
place for 1-2 days. The quality is then ascertained by the stickiness of the beans and their
flavour.
3.8 Oncom
Oncom or ontjom is a popular ndonesian food and closely related to tempeh. Ontjom is a
solid like product and is commonly served deep fat-fried or cooked in other local dishes.
Peanut press cake is generally used as substrate in oncom fermentation, although coconut
press cake, cassava press cake and residues from tofu are sometimes used.
The fermentation is carried out by strains of Neurospora or Rhizopus. Neurospora
fermentation results in a pink or orange cake, whereas with Rhizopus the cake has an ash-
grey colour.
As can be seen in Figure 5, the peanut press cake is broken into pieces and soaked in
water for 24 hrs or until it is soft. They are washed, drained and gently pressed to remove
excess water. This cake is now mixed thoroughly with cassava press cake and steamed
for 1-2 hours and transferred into a mould forming a flat cake. After cooling, the cakes are
inoculated with powdered ontjom from an earlier preparation and placed in bamboo trays
which are kept for 1-2 days at 25 30
0
C.
Typical oncom cultures are Neurospora sitophila or N.intermedia and Rhizopus
oligosporus.
Fresh oncom has a moisture content of 57%, 13% protein, 6% fat and 22% carbohydrates.

3.9 SOY SAUCE
Soy sauce appears as shoyu in Japan, chiang-yu in China, kecap in ndonesia, kanjang in
Korea, toyo here in the Philippines, and see-ieu in Thailand. The written records of the
Chinese show that they have been using soy sauce for over 3,000 years. Two distinct
processes can be used to prepare soy sauce. The first involves fermentation with
microorganisms and the second is a chemical method, which involves the use of acids to
promote hydrolysis of ingredient constituents. We are, of course, looking only at the first
process, as the chemical method also is regarded to be of inferior quality (Figures 6 and
7).

Figure 5: The production of Oncom [Ontjom]

Figure 6: Production of shoyu [soy sauce]

Figure 7: Soy sauce production (Beuchat 1995)

Whole soy beans or meal are soaked for 12-15 h at ambient temperature or, preferably at
30
0
C until a 2.1-times increase in weight occurs. Soaking is done either by running water
over the beans or by changing water every 2-3 hrs. f the water is not changed, spore-
forming Bacillus may proliferate to levels eventually deleterious to endproduct quality. The
swollen beans or meal are then drained,, covered with water again and steamed to
achieve further softening and pasteurization, until soft enough to easily press flat between
the thumb and finger. The procedure used for cooking is very critical to fermentation
patterns and endproduct quality. Rapid cooling on an industrial scale is done by spreading
the beans in about a 30 cm layer on tray-like platforms and forcing air through them (see
also 'solid substrate cultivation' in chapter 7). t is important to reduce the temperature to
less than 40
0
C within a few hours.
Concurrent with the preparation of soybeans is the roasting and crushing of wheat. The
roasting of wheat contributes to aroma and flavour of soy sauce. Characteristic breakdown
and conversion products produced by cooking wheat include the guaiacyl series of
compounds, such as vanillin, vanillic acid, ferulic acid etc.
The word koji, meaning 'bloom of mould', refers to the enzyme preparation produced on
cereals that is used as a seed or starter for larger batches of plant seed substrates when
making several kinds of traditional fermented foods. n the case of soy sauce, seed koji is
produced by culturing a number of mixed strains of Aspergillus oryzae or Aspergillus sojae
on either steamed, polished rice or a mixture of wheat bran and soybean flour.
The strains of mold used as a starter culture must have high proteolytic and amylolytic
activities and should contribute to the characteristic aroma and flavour of soy sauce.
Lipase and cellulase may also be produced. Several acid, neutral and alkaline proteases
as well as peptidases are known to be produced by both strains of Aspergillus. On an
industrial scale, the koji substrate, a 1:1 soybean:wheat mixture, is spread in 5 cm layers
in trays, inoculated with the seed koji; a temperature of 30
0
C for 2-3 days is preferable. A
moisture content of 27-37% is necessary to maintain good enzyme activity. Mature koji is
clear yellow to yellowish-green in colour.
When the koji is mature, it is ready for brining. The koji is mixed with an equal amount or
more (up to 120% by volume) of saline to form the mash or moromi. The NaCl content of
the mash should range from 17-19%. Concentrations less than 16% salt may enable
growth of undesirable putrefactive bacteria during subsequent fermentation and aging. On
the other hand, concentrations in excess of 23% may retard the growth of desirable
osmophilic yeasts and halophilic bacteria. The mycelium of koji mold is killed during the
very early stages of mash preparation.
f the fermentation is allowed to proceed naturally without temperature control, a period of
12-14 months is necessary for both the fermentation and aging process [home production].
f the temperature is maintained at 35-40
0
C, this period can be reduced to 2-4 months.
During the early stages of fermentation, koji enzymes hydrolyse proteins to yield peptides
and free amino acids. Starch is converted to glucose, which in turn is fermented to lactic
acid, glutamic and other acids as well as alcohols and carbon dioxide. As a consequence,
the pH drops from around pH 7 to 4.5. Since elevated carbon dioxide levels enhance
growth of certain unwanted anaerobic bacteria, which form undesirable flavour to the
product, some stirring is required to foster carbon dioxide escape.
The microbiology of the fermentation is not as yet well understood. t is known that various
groups of bacteria and yeasts predominate in sequence. t is perhaps Pediococcus
halophilus and Lactobacillus species which produce lactic and other organic acids
contributing to the flavour. However, it is strongly believed that the yeasts probably
contribute to most of the flavour. The optimal aging period can be determined in part by
analyzing for free glutamic acid content.
The raw soy sauce is pasteurized at 70-80
0
C thus killing vegetative cells of
microorganisms. Alum or kaolin may be added to enhance clarification. The sauce is the
filtered, bottled and marketed. Advances in fermentation technology have enabled
manufacturers to produce soy sauce with consistent quality.
3.10 TEMPEH
Tempeh is made by fermenting dehulled soybeans with various Rhizopus species (Figure
8). Soybeans are soaked in water at ambient temperature overnight or until hulls can be
easily removed by hand. Lactic acid, Enterobacteriaceae and yeasts are predominant in
water in which the soybenas have been soaked. Lactobacillus casei, Streptococcus
faecium, Stapylococcus epidermidis and Streptococcus dysgalactiae dominate
fermentation, but significant contribuitons come from Pichia burtonii, Candida diddensiae
and Rhodotorula rubra.

Figure 8: The production of tempeh
After the hulls are removed from the soaked soybeans, cotyledons may be pressed slightly
to remove more water and then mixed with small pieces of tempeh from a previous batch
or ragi tempeh, a commercial starter. The inoculated beans are then spread onto bamboo
frames, wrapped in banana leaves and allowed to ferment at ambient temperature for 1-2
days. At this point, the soybeans are covered with white mycelium and bound together as
a cake.
As with other indigenous fermented foods, the temperature and moisture content of the
fermenting substrate are critical if a good quality tempeh is to be obtained. The most
desirable temperature is between 30 and 38
0
C at which fermentation is complete within 1-
2 days.
Although other genera of molds are occasionally found in tempeh, none of them in pure
culture, except species of Rhizopus, can produce tempeh.
4. Africa Region
4.1 Gari
A stable food prepared by fermenting the root of the cassava (manioc, tapioca) plant
(Beuchat 1995) is known as gari in West Africa. t has been estimated that approx. 70% of
the cassava grown in Nigeria is used for gari manufacturing (Figure 9).

Figure 9: The production of gari
The traditional preparation of gari (Olayide et al. 1972) consists of the following stages:

(1) the corky outer peel and the thick cortex are removed and the inner portion of the root
is grated by hand on homemade raspers.
(2) The grated pulp is then packed into jute bags and weights are applied to express some
of the juice
(3) Fermentation takes place over a 3-4 day period at which time the cassava is sieved to
remove coarse lumps and heated while constantly turning over a hot steel pan or an oven.
The moisture content of gari is reduced to about 10% to yield a final product known as
gari.

While it has been recognised that fresh cassava roots contain cyanogenic glucosides, it is
also known that these glucosides decompose during traditional procedures for preparing
gari with the liberation of gaseous hydrocyanic acid (Collard & Levi 1959). Studies of the
microbiology of gari showed the participation of a bacterium Corynebacterium manihot and
a fungus Geotrichum candidum during the process and thus the latter is often referred to
as a two-stage process with C.manihot being responsible for the starch hydrolyses.
Lactobacillus plantarum and other lactic acid bacteria contribute significantly to decreasing
the pH, which causes spontaneous hydrolysis of cyanogenic glucosides with the liberation
of gaseous hydrocyanic acid. The acid condition in turn favours the growth of Geotrichum
candidum which produces aldehydes and esters which gives gari its characteristic aroma
and flavour.
For gari production it is very important to use cassava varieties with a relative low
cyanogenic glucoside content and to ferment for 4 days or longer in order to make sure
that the final product is free of cyanide or any cyanide-yielding glycosides. Short cuts and
the use of varieties of high cyanogenic glycosides have let to many deaths in Nigeria
through cyanide poisoning.
4.2 Ogi
Maize or corn is eaten in West Africa principally in the form of a porridge known as ogi in
Nigeria, and kenkey in Ghana . The Bantu equivalent to ogi in South Africa is called
mahewu.
n order to prepare ogi, maize kernels are soaked in warm water for a few days, after
which they are wet-milled and sieved through a screen to remove fiber, hulls and much of
the germ (Akinrele 1970).The filtrate is fermented to yield a sour, white, starchy sediment
known as ogi, which is marketed as a wet cake wrapped in leaves.
The fermentation proceeds naturally without the addition of inoculants or enzymes.
Microorganisms responsible were found to be Lactobacillus plantarum, Cporynebacterium
and Aerobacter, the yeasts Candida mycoderma, Saccharomyces cerevisiae and
Rhodotorula and the moulds Cephalosporium, Fusarium, Aspergillus and Penicillium
species.
Ogi is an important traditional food for weaning babies and a major breakfast cereal for
adults.
4.3 OIive fermentation
The preservation of foods by fermentation is thought to have originated in Asia well before
recorded history (Fleming 1982). Salting (brining) is a requisite for preserving vegetables
and certain fruits, such as olives, by fermentation because it helps to direct the course of
the fermentation and prevent softening and other degradative changes in plant tissues.
The type and extent of microbial action in salted vegetables is highly dependent on the
concentration of salt.
Brining and fermentation were primary methods for preserving vegetables throughout the
world prior to the advent of canning and freezing. Although secondary to modern
preservation methods, brining and fermentation remain important methods for preserving
certain vegetables even in highly developed countries because it imparts certain desired
organoleptic qualities and provides a means for extending the processing season for fruits
and vegetables.
Comprehensive reviews are available on the fermentation of sauerkraut and cucumbers as
well as many other vegetables (Fleming 1982).
4.4 PaIm Wine
Palm wine is the general name for alcoholic beverages produced from the saps of palm
trees (Okafor 1987). t differs from the grape wines in their opaque colour. Palm wine is
drunk all over the tropical world in Africa, Asia and South America.
The sap of the poalm is obtained from a variety of positions: the stem of the standing tree,
the tip or trunk of the felled tree and the base of the immature male inflorescence. Which
method is used depends on the particular country, but most studies have centred on
influorescence wine. The sap produced by this method contains about 12% sucrose and
about 1% of the other sugars frustose, glucose and raffinose.
Palm wine is fermented without the addition of any microorganisms. t is believed that a
succession of microorganisms are involved, such as gram-negative bacteria, lactic acid
bacteria and yeasts and finally also some acetic acid bacteria. These organisms find their
way into the wine from natural sources, such as air, tapping utensils etc. The wine
contains 3% alcohol and since the bacteria and yeasts are consumed , it is a source of
protein as well as vitamins.
The great problem with palm wine is its short shelf-life. t has to be consumed within 48
hours and not later than 5 days after tapping. Fully fermented palm wine has 5% to 8%
alcohol and can be distilled for kai-kai, a gin with a distinct fruity flavour.
5. European Region
5.1 Bread
Baked foods (see also chapter 13) are produced and consumed in most countries of the
world. Considerable variation exists in the type of baked foods that are made from country
to country, and often among regions within a given country.
Means of producing these baked foods also vary considerably. Large, highly mechanised
bakeries account for much of the developed countries, whereas they are very small in
developed countries. A common denominator in many of these countries is that baked
foods, in particular bread, has traditionally been an important factor in human nutrition
(Ponte & Reed 1982; Spicher & Brmmer 1995).
Modern baked goods are produced from the milled products of wheat, rye or other grains,
potable water, salt, leavening, and some optional ingredients. Rye breads also require the
addition of acids. n spite of the great variability, one can basically distinguish between
wheat doughs and rye doughs. Wheat doughs are leavened with yeast, whereas rye
doughs require besides yeast some acidification either by use of a sour dough starter or by
addition of an acid. Mixed grain breads which may contain between 20-80% rye flour and
80-20% wheat flour must also be acidified according to their content of rye flour.
The production of bread and all other baked goods consists therefore basically of 5 steps:

1. Preparation of raw materials
2. Dough formation (kneading, maturing)
3. Dough processing, consisting of fermentation and leavening, dividing and shaping;
4. Baking
5. Final preparation, which consist of steps for retention of quality, slicing, packaging,
sterilisation or pasteurization etc.

Various mechanical, physical, chemical, biochemical and microbiological processes occur
during the production of baked goods, which act either at the same time or in succession.
For the production of bread and rolls, leavening is carried out by microbial fermentation.
The main yeast strains used belong to the top fermenting species of Saccharomyces
cerevisiae, which has an optimum temperature for growth and fermentation between 28
and 32
0
C with an optimum pH between 4 and 5. Leavening of doughs requires the
addition of 1-6% yeast based on the weight of the flour. Yeasts are available as 'yeast
cake', 'bulk yeast', 'yeast cream', and 'active dry baker's yeast'.
Sour dough starters are also commercially available under various designations. The 'sour
dough bacteria' are not an independent group of microorganisms occurring only in sour
dough. They are strains specially adapted to the sour dough as their medium, and belong
to the lactobacilli which also occur on other products such as vegetable fermentation. They
belong to the genus Lactobacillus and are relatively well characterised. They are Gram-
positive rods, non-motile, and do not form spores. Furthermore they are anaerobes or
microaerophiles, acid tolerant and capable of intensive carbohydrate fermentation [see
chapter 10]. Some sour dough starters contain a wide spectrum of homo- and
heterofermentative species. Lactobacillus brevis var lindneri can be considered as the
representative microorganism for production of sour doughs in Central Europe.
5.2 Cheese
Despite the enormous heterogeneity of cheese varieties, there are common ingredients
and process that apply to all cheeses [Figure 10;Olson 1995].
Treatments of milk before cheese manufacturing vary with the type of cheese, but some of
the most common treatments are
a) heating, including pasteurisation, for the reduction of bacterial populations and heat-
labile enzymes;
b) adjustment of the milk composition by removing milk fat by centrifugation and by adding
non-fat solids or cream

The first step in cheese manufacturing concerns the addition of lactic acid bacteria. Acid-
producing activity and metabolism of lactic starter cultures are the most important factors
to control, since they are responsible for cheese manufacturing efficiency, quality and
safety of the final cheese.
Uniformity of clotting and strength of the milk gel is critical for maximum retention of milk
protein and milk fat. Milk-clotting enzymes are handled to avoid exposure to high
temperatures and to oxidising agents.
Whey is rapidly expelled from the curd formed after cutting. Most of the lactic acid bacteria
are trapped in the curd and ferment lactose to lactic acid, which diffuses from the curd.
The relationship between the rate of moisture [and thus lactose] removal versus rate of
lactic acid production to lower the curd pH has profound effects on the characteristics of
the final cheese.
When the appropriate moisture and pH levels have been attained in a particular type of
cheese curd, curd particles and whey are separated. Sodium chloride may be applied in
the crystalline form to curd after whey drainage or as brine to cheese after manufacturing.

Figure 10: Cheese manufacturing process (Olson 1995)
5.3 Yoghurt
Yoghurt is a semi-solid fermented product made from a heat-treated standardised milk mix
by the activity of a symbiotic blend of Streptococcus salivarius subsp. Thermophilus and
Lactobacillus delbrueckii subsp. Bulgaricus (Chandan & Shahani 1995; Chandan 1982).
Yoghurt is produced from milk of various animals such as cow, water-buffalo, goat, sheep
etc in various parts of the world. Cow's milk is the predominant starting material. As can be
seen from Figure 11, plain yoghurt is an integral component for the manufacture of frozen
yoghurt. Plain yoghurt normally does not contain any added sugar or flavours in order to
offer the consumer natural yoghurt flavour for consumption.

Figure 11: Manufacture of low fat plain yoghurt (Chandan 1982)
Several variations of yoghurt do exist with the addition of various flavour and/or fruit
components to give the yoghurt a fruity taste.

5.4 CuItured ButtermiIk
Cultured buttermilk is obtained from pasteurised skim milk or part skim milk cultured with
lactic and aroma producing microorganisms such as Streptococcus lactis, Streptococcus
cremoris, Streptococcus lactis subsp. diacetylactis and Leuconostoc cremoris. Cultured
buttermilk is a viscous, cultured, fluid milk, containing a characteristic pleasing aroma and
flavour.

Figure 12: Flow sheet for the manufacture of cultured buttermilk [adapted from Chandan
1982]
The processes used in the manufacture of buttermilk (Figure 12) include pasteurisation,
homogenisation, and culturing systems. The ingredients are skim milk, low-fat milk, cream,
condensed skim milk, nonfat dry milk, culture and salt. The addition of 0.2 0.25% sodium
citrate to milk provides a precursor to enhance flavour production by the culture. A proper
buttermilk flavour is produced by maintaining high standards of sanitation, an active starter
and a uniform temperature of 22
0
C. t is possible to improve the buttermilk flavour by the
addition of citric acid, sodium citrate, sodium chloride and/or cream.
Buttermilk possesses a characteristic fluidity. The viscosity is directly related to acidity.
Under refrigeration, the keeping quality of cultured buttermilk is extended to 3-4 weeks.

6. BibIiography
AkinreIe, I.A. 1970 Fermentation studies on maize during the preparation of a traditional
African starch-cake food. J.Sci.Food Agric. 21,619-625
Beuchat, L.R. 1995 ndigenous Fermented Foods. n Biotechnology, 2
nd
ed. (H.J.Rehm,
G.Reed, eds.), Vol. 9,505-559, VCH Verlagsgesellschaft mbH, Weinheim, Germany
Chandan,R.C. 1982 Other Fermented Dairy Products. n Industrial Microbiology, 4rh ed.
[G.Reed, ed.], p. 113, The AV Publishing Company, Westport, USA
Chandan,R.C. and Shahani,K.M. 1995 Other Fermented Dairy Products. n
Biotechnology, 2
nd
ed. [H.J.Rehm, G.Reed, eds.] Vol. 9, 385-419, VCH
CoIIard,P. and Levi,S. 1959 A two-stage fermentation of cassava. Nature 183,620-621
DoeIIe,H.W. and E.J.DaSiIva 2003 Biotechnology. n Encyclopedia for Life Support
Systems, EOLSS Publishers, Cambridge UK
Ebine,H. 1971 Miso. n Conversion and Manufacture of Foodstuffs by Microorganisms
(Kawabata,T., Fujimaki,M., Mitsuda,H., eds.), p. 127 pp Tokyo: Saikon Publishing Co.
FIeming,H.P. 1982 Fermented Vegetables . n Fermented Foods. Economic
Microbiology 7,227-258
HesseItine,C.W. 1965 A millennium of fungi, food and fermentation. Mycologia 57,149-
197
Okafor, N. 1987 ndustrial Microbiology. University of fe Press Ltd, fe-fe, Nigeria
OIayide,S.O., OIatunbosun,D., Idusogie,E., Abiagom,J.D. 1972 A quantitative
analysis of food requirements, supplies and demands in Nigeria 1968-1985, Lagos,
Nigeria: Federal Department of Agriculture
OIson,H.S. 1995 Use of enzymes in food processing. n Biotechnology, 2
nd
ed.
(H.J.Rehm, G.Reed, eds), Vol 9,663-735, VCH Verlagsgesellschaft mbH, Weinheim,
Germany
Ponte jr,J.G. and G.Reed 1982 Bakery Foods. n Industrial Microbiology, 4
th
ed.
[G.Reed, ed.], p 246, The AV Publishing Company, Westport, USA
Spicher,G. and J.M.Brmmer 1995 Baked Goods. n Biotechnology, 2
nd
ed. (HJRehm,
G.Reed, eds), Vol. 9,241-320, VCH Verlagsgesellschaft mbH, Weinheim, Germany
Steinkraus, K.H. 1983 Handbook of ndigenous Fermented Foods. Marcel Dekker, New
York
Van Veen,A.G. 1953 Fish preservation in Southeast Asia. Adv. Food Res. 4,209-231
Wang, Hwa L. and C.W.HesseItine 1982 Oriental Fermented Foods. n 'Industrial
Microbiology ' (G.Reed, ed.) , The AV Publishing Co., Westport, USA, Chapter 12

Horst W.DoeIIe, DSc, DSc[hc]
Deputy-Director, MIRCEN-Biotechnology Brisbane and Pacific Regoinal Network;
Chapter 18
Socio-economic strategies for ruraI farming and agro-
industriaI processing industries
[This chapter is an assortment of lectures and publications on the topic]
Content:

1. Introduction
2. Impact of fermentation technoIogy, and the industriaI revoIution on cuIture and
society in the now deveIoped countries
3. Present situation in deveIoping countries
3.1 TechnoIogy transfer
3.2 New trends in microbiaI biotechnoIogy
4. Impact of integrated ruraI fermentation technoIogy on cuIture and society in
deveIoping countries
4.1 TropicaI wet zones
4.2 TropicaI arid zones
5. Joint venture capitaI investment for cIean technoIogies
5.1 CIean TechnoIogies and Eco-efficiency
5.2 Infrastructure
5.3 Existing joint venture probIems
6. Socio-ecoIogiaI strategies for future sustainabiIity
6.1 Information TechnoIogy coordinators for education and discussions on
sustainabIe biotechnoIogy
6.2 HistoricaI DeveIopment of Internet Conferences on Bio-integrated
Systems
6.3 Scope and purpose of internet conferencing
6.4 IndustriaI Ecosystems
6.6 ConcIusions
7. BibIiography

1. Introduction
Any technological development is aimed at improving the quality of life of a community of
people. t may lead to longer life expextancy and higher survival rates through better
health conditions (DaSilva et al. 1992; Doelle et al. 1987).
Since his appearance, man has always lived in an uncertain, and sometimes precarious,
symbiosis with nature (King & Cleveland 1980), obtaining his nourishment and the small
amount of energy needed from plants and animals personally accessible to him.
Fermentation technology was accidentally introduced very soon in the form of wine,
followed by the brewing of met (honey beer), beer, the making of bread, the development
of milk products such as yoghurt and cheese, the development of a variety of meat
products, and the fermentation of the sweet juice (aguamiel) extracted from the heart of a
Mexican Agave to produce pulque, an alcoholic beverage, in Aztec countries of Latin
America. n SEAsia and Africa, fermentation technology produced numerous foods from
rice, soybean and other plants, the best known products being the Soy sauce, gari, miso
and the alcoholic rice beverage sake.
Biotechnology processes, which include the fermentation technologies, are recognised as
the translation of basic scientific research achievements in the field of biology into practical
and/or commercial applications using low, medium or high capital technology. They
unquestionably generate many benefits, but can also be seen to bring certain dangers or
potential threats with them (Doelle 1998) depending on which strategies are adopted by
the community.
Within developing countries, technological independence is increasing which gives them
the ability to design appropriate strategies which take advantage of biotechnological
processes tailored to their needs. Whilst avoiding imitating the mono-product strategy of
many industrialised countries, the search for appropriate solutions will then lead the
developing countries to participate in the general advancement of scientific knowledge
needed for progress in biotechnology (DaSilva & Sasson, 1989; Doelle, 1982, 1991).
During the past decades, production systems have been based on the assumption that
wastes are an unavoidable part of our daily lives but that ecological and environmental
destruction could be avoided because of the planet's vast natural resources. However,
ecosystems and renewable resources are being destroyed at an increasingly rapid rate
and the problems of pollution and waste disposal are growing. t is thus becoming
increasingly evident that new production methods must be devised to fulfil society's basic
needs. n order to maintain the vitality of the rural sector and preserve the environment, a
system must be devised in which energy, food, animal feed, fuel and fertiliser
requirements can all be met from renewable resources used at a sustainable level (Doelle
1982,1989; Raymond & Larvor 1986; Szmant 1986; White & Plaskett 1981; Moo-Young &
Gregory 1986; Sasson 1990).
The new new socio-economic concept is based on the requirement for full exploitation of a
harvested renewable resource and the replacement of monoculture/monoproduct farming
with a multiproduct system. Because it produces a variety of products, this system will
hopefully enjoy a constant and reliable market demand and will be able to secure income
for the rural sector as well as for joint venture industries.
2. Impact of fermentation technoIogy, and the industriaI revoIution on
cuIture and society in the now deveIoped countries
The impetus of the industrial revolution during the 18th and 19th centuries transformed the
very nature of society in many parts of the world, which are now referred to as the
developed countries. Society was now not only using renewable resources, but also
consuming vast amounts of non-renewable resources. The industrial society developed by
the accumulation of scientific knowledge, the spread of technological innovations, and the
exploitation of enormous natural resources. Traditional vegetable and animal fibres were
increasingly replaced, or extended, by synthetics manufactured by an ever increasing
chemical industry from coal and petrochemicals. This development in the 20th century
fundamentally altered the pattern of consumption, land use and international trade, and the
distribution of wealth (King & Cleveland 1980). Longer life expectancy and higher survival
rates followed through better housing and sanitation, and the production of antibiotics and
vaccines. The quality of life was improved by the introduction of petrol (gasoline) and the
motor industry among others. The impact on society was dramatic and on culture
devastating as a large proportion of the traditional way of life was lost through this
development owing to an ever increasing urbanisation.
The reasons for such an impact on culture and society are manifested in the principles of
the 'industrial systems' organisation' (Fernandez & Ocampo 1980), which dominates
todays' society in the developed countries. This organisation is based on short-term profit,
with a production to sell attitude, with preference given to the production of luxury
consumer goods over goods required for basic needs, particularly at the level of the large
energy systems, such as coal, hydrocarbons, and nuclear energy. Such a production is an
obstacle to the total realisation of individuals and society.
The results of these chemical and manufacturing industries are accompanied by ever
increasing amounts of effluents of both heat and toxic substances, many of which are non-
biodegradable. Modern agriculture is now strongly based on the application of chemical
fertilisers and ever-increasing amounts of organic pesticides, mainly as a consequence of
an enormous and rapid expansion in world population demanding an ever greater quantity
with increasing quality of food and goods of all kinds. This, in turn, encourages the use of
still further quantities of non-renewable resources and energy. This development led in the
1970s to a turning point in the perception of man's relation to his natural environment, the
biosphere, as well as a shift in man's relationship to the man-made environment, the
technosphere (King & Cleveland 1980). The question was raised whether the earth and its
atmosphere can provide an infinite sink and absorb the waste products of industry,
agriculture and urban living as they become more and more prevalent. The processes of
physical planning are now challenged and well established procedures are under severe
scrutiny. Whereas a successful community has been judged by the amount of resources it
would usefully consume, in the future it will succeed only if it manages to conserve
resources without loss in life quality (Meier 1980).
3. Present situation in the deveIoping countries
The less developed or developing countries have, in general, been bypassed by the
industrial revolution and chemical industry development. Starvation on some continents, or
at best malnutrition, together with a rising population and rising prices for non-renewable
or traditional energy sources, coupled with a more or less complete dependency on the
importation of goods have led to neglect of agriculture, but a built-up of a large urban
population has brought disaster to the economy and society of many developing countries
(see also Chapter 4). The majority of these developing countries lie in two climatic zones,
the tropical wet zone and the tropical arid zone. Can scientific knowledge and technology
improve their quality of life, life expectancy, and increase survival rates without repeating
the disastrous effect of the industrial revolution on culture and society ?
n order to be able to develop joint ventures in developing countries, one has to know the
situation in those countries. Despite their social and political diversity, the less developed
countries of Asia share a number of common characteristics:

a) they are densely populated: while these countries represent only approximately 13% of
the land area, their combined population in 1980 accounted for 50% of the world
population;
b) they are characterized by low-income economies;
c) they have predominantly agrarian economies in which 50-75% of the population
depends on agriculture;
d) agriculture generates 33-50% of the domestic products;
e) there level of literacy as well as technical capability is low;
f) with some exceptions, the economies of these countries are growing at a much slower
rate compared to the world economy (slam & Kaya 1985)
These common characteristics, many of which are also common to those in the African,
Latin American, South American and Pacific Region, stress the importance of applied
microbiology in these developing countries particularly in the fields as diverse as
agriculture, public health, water supply and sanitation, environmental conservation and
resource management, as well as the production of food, fodder, and energy (DaSilva
1986). n many of the developing countries, indigenous fermented foods are very
important, but they are socio-culturally bound (Doelle et al. 1987), especially in rural
household and village community traditions.
Many of the developing countries base their diets on low-protein staple foodstuffs such as
cassava, plantains, yams and taro. These roots, tubers and fruits are rich in starch and
suitable for microbial protein enrichment. Cassava particularly is an abundant root crop in
tropical areas with possibilities of being transformed into high protein feed (Carrizales &
Jaffe 1986; Sukara & Doelle 1989).
The disposal of sewage wastes is one of the priority needs of less developed countries. As
mentioned in an earlier chapter (see chapter 4), only about one-third of the population of
the developing countries has adequate sanitation services. Due to a constant rural to
urban migration, large low-cost urban areas have developed where less than 50% of the
urban poor have suitable waste disposal facilities (Biswas et al. 1985).
3.1 TechnoIogy transfer
Technology transfer could form a basis for joint ventures and has been and still is, a rather
popular slogan in both developed and developing countries. Under this term one
understands, that a biotechnological process developed in one country is transferred as a
whole to another country, which then will modify the process according to its own
conditions (Doelle 1982). t is not surprising that in most cases governments of developing
countries have had frustrating experience of failure in assimilating such imported
technology (slam & Kaya 1985), because:
a) the developed countries have a biotechnology-oriented programme which is more
toward industrial, business and economical advances and contain little or no socio-
economical or socio-ecological orientation (Doelle 1989a; Bunders et al. 1989);
b) most of the technologies are high-capital technologies in contrast to low-capital
technology requirement (DaSilva 1981);
c) the lack of sufficient national scientific and technological infrastructure to assimilate any
imported technology;
d) the lack of units capable of advanced research and development in biotechnology
whether at universities, national laboratories or industries (Zilinska 1988; Chakravarty
1988);
e) legal trends preventing the developing countries' researchers from access to important
information sources (Barton 1989);
f) the relatively high expenditure required for the transfer with the existence of a lack of
hard currency (Lamptey & Moo-Young 1987; Stolp & Bunders 1989).
n some developing countries a new technology has been diffused to farmers on a large
scale without adequate investigations of its effect on the socio-cultural, economic and
physical-biological environment. A large-scale adoption may adversely affect the social
and cultural system or have unwanted economic effects such as input shortages or surplus
products (Wilson et al. 1986).
The vast majority of the populations in developing countries live in villages and the
importance of rural technology development in those countries cannot be overstated
(DeBruin & DeBoer 1986). n the context of technological development it is generally
considered imperative to see that such developments are of benefit to their lives as well. t
certainly is recognised that rural life is affected by many factors, such as political and
social institutions, rural economic structures, communication, education and technology.
An initial step is therefore that building up the rural technology capacity is one of the tools
for development. Another step is the recognition that the technology employed or
developed should suit locally available resources and skills and be in harmony with local
culture (DeBruin & DeBoer 1986; Doelle et al. 1987). DeveIoped countries shouId act
more in an advisory capacity rather than seIIing their technoIogy at aII cost.
A joint microbial biotechnological venture in developing countries has therefore to come
from within the country. The motivation for any joint venture development effort must be
based on either satisfying basic needs, such as food, shelter and human life conditions or
improving standards by providing more material or intellectual goods, by improving
working conditions or by increasing public participation in decision making and discussions
of long range social planning (Doelle 1982; Ul Haq 1988). Future development depends on
proper planning and optimal utilisation of local talents and resources.Judicious selection is
required to determine those biotechnological processes that will provide net-positive socio-
economic returns from the investment (Lamptey & Moo-Young 1987).
3.2 New trends in microbiaI biotechnoIogy
The fast development in fermentation and thus microbial biotechnology has emerged from
a social evaluation of technology. Simmonds (1980) explained this new trend by saying
that efficiency or generation of wealth, in its technical and economic sense, will clearly
continue to be desirable, but that it will be placed in balance with equity or distribution of
wealth, and survival or continuation of wealth. 'We have lived through a period
characterized by one major goal, economic growth, fuelled by one source of energy,
petroleum hydrocarbon, with materialism as king and consumers as his loyal subjects.
The bioresources development will be different. The availability of renewable substrates is
much more restricted (vory & Siregar 1984) and should therefore be exploited much more
carefully. Such a careful exploitation requires a market rethinking of the scientist and
technologist, as the newly developing fermentation technology must use the vast potential
of the microbe to provide fuel, food, fertiliser, and feed supplements from the renewable
resource (DaSilva 1979; DaSilva & Doelle 1980). The social evaluation thus turns into a
socio-economic bioresources development with multiple goals in place of one goal, the
utilisation of several sources of energy rather than one, acceptance of heterogeneity as
normal rather than homogeneity, a goal of greater overall well-being rather than just more
money or possession (Simmonds 1980). Bioresource development can therefore only
succeed if it is actively integrated into the culture of the developing countries. This is one
of the major reasons why new technological advances in developed countries should not
be transferred without adjustments to the local conditions.
n order to develop an appropriate biotechnology, resources available together with the
social structure of the population are of vital importance. t is often the need and not the
economics of the process, which is of importance (Sorensen 1979; Rolz 1980) Here
seems to lie one of the cardinal differences between the thoughts of appropriate
technology in developed, from those in developing countries. n this context, a new
concept has been developed specifically for rural communities: the so-called integrated
rural biotechnology systems. The development of these systems depends primarily on
the climatic conditions of the regions.
4. Impact of integrated ruraI fermentation technoIogy on cuIture and
society in deveIoping countries
ntegrated rural fermentation technology aims at rural progress, conservation of the rural
environment and rural self-reliance in relative primitive economies (DaSilva 1981).
Biological waste conversion into food products, fuel and fertilizer could be the basis of a
long-term strategy to alleviate not only the 'food crisis', but also the 'energy crisis'. Long-
term strategies require not only a proper technological and economical assessment, but
also a deep evaluation of the social and environmental impact produced by the strategy
(Olguin 1982). Conventional and non-conventional food and feed production, together with
a considerable production of energy and fertiliser would have a positive economic and
environmental impact (Olguin 1978; Olguin & Vigueras 1981). Caution should, however,
be incorporated when choosing the best and most adequate technological alternatives for
waste processing to ensure the social relevance of the final product.
The development of an integrated rural fermentation technology with a certain flexibility in
product formation according to social demand and relevance depends primarily on the
climatic conditions of the regions.
4.1 TropicaI wet zones
Pilot plant schemes for the introduction of integrated systems have been developed in
Mexico and the Philippines. The first integrated system approach is the connection of
animal waste with aquaculture and, more specifically, with algae and fish production, and
not just provision for biogas and fertiliser. There is no doubt that algae are among the most
efficient converters of radiant energy (DaSilva 1980; Olguin 1982; 1984), with a solar
energy conversion efficiency of approximately 7 percent. The cultivation of algae has also
the advantage of utilising semi-arid land. Such land utilisation s the most efficient by using
algae to produce protein in comparison to any conventional source. The production of one
tonne of Spirulina (Ciferri 1983) requires only 0.03 ha/year compared to 452.5 ha/year to
produce one tonne of beef or 1.55 ha/year to produce one tonne of soybeans (Leesley et
al. 1980). The integrated system consists of anaerobic digesters, similar to those used in
biogas production, from which the aqueous effluent is connected to algae cultivation
ponds. Whereas biogas and fertiliser are constantly produced, the type of algae used
depends on the demand of the social community. Spirulina platensis, for example, can be
used directly as a protein supplement for cattle, pigs, or poultry. Spirulina has also found
entry into health food shops in the developed countries owing to its high vitamin content
and represents the main staple food for the people of Chad in Africa. Spirulina cultivation
with a multi-purpose approach has been reported by Tel-Or and co-workers (1980),
whereby besides protein, chlorophyll-a, xanthophylls, and beta carotene can be produced.
This versatility of the algae Spirulina means that the village or society now has the
advantage of not only satisfying their energy and food requirements, but also obtaining
income from sales of the various products.
A change from Spirulina to other Microalgae leads to an excellent feed for fish production.
n the Philippines, an integrated livestock-meat processing and canning operation system
has been established at the Maya Farm in the Antipolo Hills outside Manila (Judan 1980;
Maramba 1978). n January 1983, this system had over 4000 sow units, 1000 duck or hen
units, and 25 cow units. From the animal waste, some 3,510 m3 biogas are produced
daily, which supplies all the energy requirements of the livestock farm and after increasing
its biogas output has become completely self-efficient with regard to its energy
requirements. n addition to its large integrated livestock-meat processing and canning
operation system, Maya Farms have developed a crop-livestock-fish-farming system,
which brings about intensive use of land, full utilisation of farm wastes, and near energy-
sufficiency of the farm.
4.2 TropicaI arid zones
n the arid zones of Mexico the situation is quite different from the tropical wet zones of this
country. Following the concept of maximum use of resources, saline water in these regions
could become an essential element for biomass production and integrated rural biosystem
of a different kind (Olguin 1986). This system takes advantage of those resources which
are most available to arid lands: solar energy, saline water, harvested rain water, organic
wastes, non-conventional crops, halophyte plants or microorganisms and energy crops.
The result of such an integrated system can lead to a wide diversity of products: food (fish,
cattle, pigs, chicken), feed (Spirulina, halophyte crops), fuel (biogas, ethanol), and
chemicals. The algae Dunaliella is able to accumulate large amounts of glycerol in
response to the externally imposed osmotic pressure caused by high salt concentrations.
The proper application of biotechnology in arid zones can therefore provide communities
or societies for the first time with energy, food, feed and fertiliser from either animal waste
or sea/saline water. The final pollutant free effluent can be used for irrigation of
halotolerant plants which in turn provide feed for animals.
The greatest challenge for integrated biosystems, however, still lies ahead of us.
Starvation in its greatest dimensions occurs in the arid and semi-arid areas which have a
high annual solar energy input, eg Ethiopia and surrounding countries in Africa. Dry scrub,
dry desert, and chaparral lands constitute 19% of the continental area, but only 2-3 % of
the primary photosynthetic productivity is found here (Bassham 1977). Without irrigation,
the photosynthetic productivity is naturally extremely low, and the availability of water
becomes the limiting factor.
Priorities in the field of biotechnology/fermentation technology are different in developed
and developing countries. Whereas in developed countries, the high value added
products, especially those for use in medical fields, may dominate the aspirations of
biotechnologists (Bull et al. 1982) in order to maintain the present living standards, it is the
basic needs of the society , without interference in the traditional culture, which will
dominate in the developing countries. Bioresource utilisation programmes (Hermosillo &
Gonzales 1981) are the best systems to combat malnutrition and starvation, and oif
coupled with waste treatments could lead to high public health standards. Multiple goal or
integrated system development can also lead to employment in the villages and higher
living standards, avoiding or reducing the trend of increasing urbanisation.
5. Joint venture CapitaI Investment for CIean TechnoIogies
5.1 CIean technoIogies and eco-efficiency
Two of the premises of sustainable development are that economic growth has to be in
harmony with the environment and that a rational and sustainable use of natural resources
has to be implemented (Olguin 2000). n congruence with such premises, industrial
development has to change from the degradative to the sustainable style, which requires
the adoption of cleaner production systems.
The United Nations Environment Program (UNEP) defines the cleaner production concept
as 'the continuous application of an integrated preventive environmental strategy to
processes, products and services to increase eco-efficiency and reduce risks to humans
and the environment' (UNEP 1996). One of its distinctive features is that reduction of the
quantity and toxicity of all emissions and wastes is made before they leave the process
stream. n the case of services, environmental concerns should be incorporated into
design and delivery.
Eco-efficiency, on the other hand, involves 'the delivery of competitively priced goods and
services that satisfy human needs and bring quality of life while progressively reducing
ecological impacts and resource intensity, throughout the life cycle, to a level at least in
line with the Earth's estimated capacity' (UNEP 1994).
t is becoming very clear that adoption of clean production systems by industries calls for
fundamental changes, not only at the technological level but also at the legislative level
(Olguin 2000). Cleaner bioprocesses are under intensive research and development
following the general guidelines for cleaner production.
5.2 Infrastructure
There is no doubt that any joint venture with promises of social advances and economic
benefits will have to be rural-based in most of the developing countries. Small farmers
(2.5-5 acres or 1-2 ha) account for 19% of the cultivated holdings and 12% of the
cultivated area in SEAsia with larger farmers making up the rest (Stolp & Bunders 1989),
thus 31% are under cultivation (Doelle & Gumbira-Said 1992). t is therefore necessary
that frequent communication occurs among the farming community, the government
representatives and the biotechnologist (Bunders et al. 1989) to discuss and establish joint
ventures between all three and convince industry and entrepreneurs to join in the venture
toward social advance and national economic benefits. t has to be the aim of these
bodies, in particular the researcher, to convince society that biotechnology is not a threat
to family or to molarity (Fleising 1989), but can bring enormous social and economic
advances for the individual as well as the country. The local or national cooperative joint
venture group should then seek cooperation or joint venture groups from the developed
countries to help achieve their goals.
A suggested scheme for joint microbial biotechnological ventures for developing countries
is given in Figure 1 (Doelle 1996; Doelle & Gumbira-Sa'id 1992).

Figure 1: Scheme for joint socio-economic strategy development (Doelle & Gumbira-Sa'id
1992)

n order to instigate rural technology in a particular developing country, national authorities
have to set their priorities in regard to social, economical and ecological development.
Such an authority should consist of farmers, farmer cooperatives, researchers, the
appropriate government agencies and financiers, such as bankers, industry, entrepreneurs
and others. n co-opting consultants in biotechnology, this Board of Development should
establish a national programme. This communication link between farmers, government
and researchers is vital for any success in the establishment of rural industries. Major
consideration should and must be given to the raw materials available, the local market
demand for feed, food, fertiliser, fuel and energy , to increase living standards.
Furthermore, consideration should also be given towards the savings which could be
achieved through less imports, which could be significant to the national or communal
economies, and the replacement of wood for energy which could stop further de-
forestation and save further deterioration of the environment.
All of these considerations have to be taken into account by the Board of Development,
which would give its final proposals to the Center for Development. This Center should be
a Biotechnology Research Centre devoted entirely to the development of rural
biotechnology with the largest component centred on microbial technology. Basic and
fundamental research should be contracted to university or other research institutes. Since
every joint microbial biotechnological capital venture investment system depends on
the availability of raw materials, including not only agricultural products but also
agricultural, human and animal wastes, This Centre may well be confronted additionally
with tasks of improving farm management and practices, soil denitrification, rhizobacterial
plant growth promotion, plant disease resistant breeding, and future planning and
development of arable agriculture. Such new developments must always take care that it
does not affect local traditional culture, although it should and can improve the conditions
of the society within its traditional culture.
Each developing nation should have one such Centre, which not only has a close link to
the nearby MRCEN-Network Centres, but is also responsible for the development of an
appropriate biotechnological system using, if possible, established technologies as a
whole or in part for adaptation to local conditions. Such a Centre should attract overseas
finance through aid programmes in addition to the input from the national government,
local governments and financiers. One of the major goals should be an Exchange
Programme, whereby local researchers are sent to specific laboratories and vice versa to
learn techniques vital for the rural process development. t is of great importance to ensure
that the social and economic capacity of rural communities is increased through the
application of science and technology, which is best served through the Centre for
Development.
Any system developed in the proposed Centre for Development, whether it is a simple or
integrated rural technology system, must go through pilot-scale and field trials. t is the trial
outside protective laboratory conditions that has to withstand rigorous social, economical
and ecological evaluation. No process should be acceptable that produces a product but
simultaneously causes severe pollution of air, soil or waterways. These field trials must be
able to exhibit to the farmer and the national and local governments the social, economical
and ecological benefits and promises for the farmer, the region and the nation as a whole.
The successful field trials make the process available to the farming community.
Depending upon the quantity of raw material available and the local market demand, the
Biotechnology Process Unit has to be tailored to these requirements. t would therefore be
of great benefit if each Biotechnology Process Ubit has an Industry Service Centre
(DeBruin & DeBoer 1986) attached for direct and indirect support activities. Whereas it is
the goal of the Process Unit to deliver the products from the available raw materials, the
Service Center could have a number of aims:
a) to help in the construction of the Process Unit;
b) to maintain continuously the equipment etc required in the Process Unit;
c) to train the farmers in the area;
d) to carry out extension work, which may lead to new methods of production, equipment
and cost-saving repairs.
The ndustry Service Centre should have a close communication link with the Centre for
Development. The establishment of both the Process Unit and the ndustry Service Centre
brings to the local society and local government further employment and education, social
advances and promises otherwise not available. This type of decentralisation of services
may stem the tide of migration from the rural to the urbanised cities.
5.3 Existing joint venture probIems
Joint ventures in developing countries are at present few and relatively restricted to certain
aspects of development. Most of these joint ventures suffer under so-called economic or
management problems. Many of these projects never leave the research establishments
and thus do not find their way into the applied field (Zilinskas 1988). Other projects mainly
financed by nternational Agencies to address possible food shortages (Swaminathan
1982) led to improved crop production through the creation of higher yielding strains, eg
rice, but these crops required a significantly higher quantity of fertiliser that the low-income
farmer could not afford.
The problems faced in each of the above joint venture cases are created by a lack of
socio-economic thinking together with a project analysis orientated more towards a single
purpose rather than multi-purpose projects with several outputs, the economist has to
attempt to establish the best mix of resource disposal, resource use and resource
recycling involving the translation of technical constraints into economic values (BOSTD
1981).
t is time that nternational and National Agencies start to realise that joint ventures in
biotechnologies in developing countries can only be successful and socio-economically
useful if multi-purpose projects in the form of integrated systems are considered (Little &
Muir 1987; Doelle 1989a) and analysed by the economist. The multipurpose projects start
from the natural resource crop improvement to its use and conversion to multiple products.
Such projects should be carried through to supply the rural and urban population with food,
feed, fertiliser and fuel.
6. Socio- ecoIogicaI strategies for future sustainabiIity
The significant availability of arable land for agricultural production, but nevertheless the
still increasing protein deficiencies of many of the world's population urges the need for
agricultural biotechnology development (Doelle 1989b; Raymond & Larvor 1986; El
Nawawy 1987; Senez 1987). Such a development faces two significant cost factors:
feedstock and transportation. n order to avoid an escalation of these costs, feedstock and
product must be made available in the producer and consumer region, which requires a
microbial or biotechnological process industry flexible in both, scale and product formation.
The first step towards a socio-ecological development was taken by plant geneticists
directing their work toward higher disease resistance and higher yields in crops, which
should eventually eliminate, or at least reduce, the use of pesticides in the agricultural area
(Cocking 1988; Rogers 1989).
The second step of development concerns the use of biological biodegradable pesticides.
Products from genetically improved Bacillus thuringiensis have been successfully applied
in the former USSR (Afrikian 1973) and is further developed in the Unesco sponsored
MRCEN of Teheran in the slamic Republic of ran.
ncreased monoculture with a single outlet however, will continue to cause problems to the
farmer. Price fluctuations and product quality, owing to changes in global weather patterns,
coupled with greater self-sufficiencies of less developed countries and the increased
usage of available land, will cause severe economic problems to the farming community in
developed countries and signs are already visible in the developing countries for a similar
development.
6.1 Information TechnoIogy - coordinators for education and discussion on
sustainabIe biotechnoIogy
Ancient civilisations have managed to sustain large human populations by mimicking
Nature's capacity in bioproduction and by integrating biological systems so that all
available resources and wastes are used. Such bio-systems have been practised
particularly in China for centuries and also in Latin America, Africa and Asia. They are
currently used by resource-poor communities in low-input-high-output agriculture, by
farmers to produce organically grown agricultural products and to reduce operational costs
by using less chemicals and turning to biological pest control, fertilisation and using
renewable resources. Examples of such multidisciplinary approaches using ecological
engineering, systems ecology and ecotechnology are provided in this paper to illustrate
how they are used to develop socio-economic strategies for future sustainability in both
industrial and rural eco-systems.
ndustry is a major polluter and consumer of raw materials mainly because of its linear
approach in production where extraction or processing only uses a small portion of the raw
materials and the residues or by-products are considered as wastes for disposal. These
industrial and especially biological waste materials are finding value added to them via the
application of integrated bio-systems because they can be used to generate bio-energy,
be converted into food and feeds, or their nutrients recycled via a chain of biological sub-
systems. This approach has allowed industries to aim for zero wastes or zero emissions
which find a use for all wastes.
6.2 Historic DeveIopment of internet conference on bio-integrated systems
The tremendous loss of biodiversity after the introduction of the Green Revolution required
some international action. During conferences in 1970 and 1972, the United Nations called
for the establishment of centers in developing regions of the world to conduct an integrated
program for the preservation and use of microbial resources. n cooperation with UNEP
and CRO, Unesco established between 1975 and 1995 a global network of 31 Microbial
Resources Centres [MRCENs] in 25 countries, with UNDP helping in the establishments
of the MRCENs in Lubljana, Budapest and Teheran (ASM 1997). n order to foster
communication between developed and developing regions and exchange of ideas, ten
(10) conferences on 'Global mpact of Applied Microbiology and Biotechnology' [GAM]
were held between 1963 and 1995 and the MRCEN-Journal of Applied Microbiology and
Biotechnology, which is now the World Journal of Microbiology and Biotechnology, was
established in 1994 and printed by Oxford University Press in association with Unesco.
nternational conferences have unfortunately become unaffordable to most of the
researchers from developing countries owing to the ever increasing costs and reduced
availability of funds. The 10th and last conference of GAM in Elsinor (Denmark) in 1995
highlighted also the widening gap in the research areas and interests between the
developed and the developing world. The main papers delivered at this conference can be
found in Volume 12 of the World Journal of Microbiology and Biotechnology of 1996.
MRCEN-Bioinformatic in Stockholm in 1983 and later under the direction of Mr.Eng-Leong
Foo, launched a pioneering programme from 1984, to organise a series of
electronic/computer conferences that were aimed to enable any scientists with access to
email to participate. The target was on junior researchers and scientists in developing
countries and several approaches were tried. The first approach was the 1983 ' Computer
Conference on the Bioconversion of Lignocellulose for Fuel, Fodder and Food needed for
Rural Development in Poor Countries' (D.A.Balson) , which was jointly organised by the
MRCENs in Stockholm and Guelph. Three conferencing systems (QZCOM, EES, CoSy)
were used. Connection was either via packet switched networks or direct telephone dial-up
using 300-1200 bps modems. Messages had to be manually copied into each of the three
systems. The conference was for 10 months and enabled about 107 people to participate.
The conference ended with a physical gathering of participants for a week in locations
Ottawa, Guelph, Manchester, Stockholm, Frankfurt, Moscow, Bangkok, Manila, and Tokyo
in order to conduct a week of intensive exchanges. Since 1984, MRCEN-Stockholm
continued the effort to reach scientists in developing countries by developing a programme
to

(a) extend face-to-face conferences electronically by making available the abstracts via
email and enabled participants to interact with the authors who are gathered at the
physical conference venues;
(b) organise electronic seminars;
c) electronic workshops; and
d) electronic forum.

The type of activities were
(1) anaerobic digestion in 1984-1987;
(2) biological nitrogen fixation in 1991-1994;
(3) lactic acid bacteria from 1993 onwards;
(4) ecotechnology from 1994 onward and
(5) integrated biosystems starting in 1995.

About 11 electronic extensions of physical conferences, numerous electronic seminars, 3
workshops and 6 conferences have been organised in the last 15 years [
http://home2.swipnet.se/~w-25860/jacky/conf.htm].
n cooperation with the Biofocus Foundation under the directorship of Prof. Karl-Goran
Heden [also one of the founders of the nternational Organisation of Biotechnology and
Bioengineering] the 1st EIectronic Conference on EcotechnoIogy for sustainabIe
deveIopment in 1994 was organised. n 1995, the Biofocus Foundation and MRCEN-
Bioinformatic Stockholm were invited by the United Nations University (Tokyo) to establish
an electronic networking and conferencing program for its ' Zero Emissions Research
nitiative [ZER] project. This led to the establishment of the integrated bio-systems
Network [BSnet], which also strengthened efforts of other members of the MRCEN
network in advocating for a more integrated system approach in environmental, applied
and biotechnological research (DaSilva & Doelle 1980, Doelle 1982, 1989). The BSnet
activities included the ' EIectronic Conference on Zero Emissions by Eco-Breweries
(1996)', ' EIectronic Workshop on Carbon Sinks (1996)', and culminated in the 'Internet
Conference on Integrated Biosystems' in 1998. A follow-up activity in 2000 will be the
'Internet Conference on MateriaI FIow AnaIyses of Integrated Bio-systems'.
6.3 Scope and purpose of internet conferencing
The purpose of the conference was to permit access via the internet to a comprehensive
documentation of past and current work on integrated bio-systems, to enable authors to
share their experiences and to interact with participants, and to foster the development
and cooperation in projects that may result from this conference (Doelle & Foo 2000).
The conference also intended to draw the attention of funding agencies to their potential
roles in funding projects on integrated biosystems because of their increasing importance
as a solution to ensure livelihood and food security of farmers and the poor as well as in
providing an ecologically sound environment and to contribute to sustainable eco-
development.
The internet conference did permit free access to information to the general public,
academic institutions and any organisation that is connected wit improving the
environment, sustainable development, food security and production, alleviation of
poverty, natural resource conservation, coast management, aquaculture, natural resource
management and others.
The scope of the conference covered the science, technology and practice of integrated
bio-systems in agriculture, aquaculture, horticulture, forestry, industry, built structure and
natural ecosystems. The conference emphasised the use of integrated bio-systems in
human activities and their roles in creating a better environment and to sustain
development. The main sessions encompassed

(1) integrated biosystems for agriculture, aquaculture, horticulture and forestry;
(2) integrated biosystems for treatment and utilisation of industrial/municipal solid wastes
and wastewaters
(3) integrated biosystems for management of natural resources
(4) integration of biosystems into built structures.

n nature, seasonal changes may affect the availability of a resource and a change in the
population of a species that use it as a food source may occur. This may further lead to a
series of changes if it is in the food chain for other species in a system. Human beings
have constantly been causing changes in the environment and the increased activities
from an overpopulation will have many consequences, one of them in the accumulation of
by-products, solid and liquid wastes. They have caused great environmental concerns
about the future of sustainable human development on Earth. Another important issue is
the need of increasing local food production in many developing countries by 100-300% in
the next 15 years. ntegrated biosystems will play an important role as they can use by-
products and wastes and convert them into feed.
6.4 IndustriaI Ecosystems
n starting with the developed world approach of sustainable systems for waste
management (Diaz et al. 1998; Riggle 1998), most of the discussion papers centred
around the use of aquaculture principles in greenhouse mesocosm (Guterstam & Forsberg
1998; Todd & Josephson 1998), and new integrated sanitation and waste management
(Otterpohl et al. 1998; Birley & Lock 1998; Riggle 1998).
From a cumulative experience of over 25 years of designing and testing integrated living
technologies based upon an ecosystem approach (Todd & Todd 1980; 1984;1994), twelve
(12) key factors were discussed for the design of task-oriented mesocosms (Todd &
Josephson 1998): mineral diversity, nutrient reservoirs, step gradients, high exchange
rates, periodic and random pulses, cellular design and mesocosm structure,
subecosystems, microbial communities, photosynthetic basis, animal diversity, biological
exchanges beyond the mesocosm and the mesocosm/macrocosm relationship. A 'living
machine' system (Todd & Todd 1995) for the treatment of sewage and the production of
fish and horticultural products operational since 1989 was described. t produces high
quality of water irrespective of season. The data presented depict a robust, self-organising
technology capable of handling the mixed waste stream of a New England industrial city in
the USA.
A very similar approach could be found for the Stensund Folk College system in Sweden
(Guterstam & Forsberg 1998) and in other parts of Europe (Otterpohl et al. 1998; Riggle
1998). Similar to the Kalundborg ndustrial Ecosystem (Ehrenfeld & Gettler 1997), the
Stensund Folk College near the Baltic Sea was used as a model community for studies of
flows of energy and materials as well as on the development of recycling technologies for
waste and wastewater. The wastewater aquaculture project was initiated in 1989, using
greenhouses for treating wastewater representing an intensive indoor-technology as
opposed to extensive outdoor technology (Etnier & Guterstam 1991). The results indicate
that such an indoor-technology is feasible as the constructed mesocosm functions as a
treatment plant for 6 m
3
/day of household wastewater from 40 persons. t produces new
plant and fish biomass and exports heat energy during the summer.
The importance of differentiating the management of water and waste in urban areas was
stressed by Otterpohl and co-workers (1998). New integrated sanitation and waste
management systems will mostly have to expect different qualities of matter from human
settlement, eg. blackwater with biowaste [= sewage or manure], grey water [= kitchen,
bath], stormwater run-off and non-biodegradable waste. First priority is given to sanitation,
sending all blackwater through anaerobic digestion for biogas production (= energy) or
composting (= fertiliser for garden). n comparing an advanced traditional with the new
sanitation system, the cumulative savings of emission to the seas and of energy-material
usage for an average lifetime of 70 years for a single person were calculated as about 700
m3 freshwater, 200 kg COD, 4.2 kg of Phosphorous, 37 kg of Nitrogen, 91 kg of K
[potassium], 15,000 KWh of energy and about 160 tons of material usage. These saved
emissions can certainly replace fertiliser production from fossil resources which would
represent a further energy saving of 7,000 KWh (Boison, 1996).
f, however, the wastewater comes from industry, it will contain significant amounts of
organic matter. n these cases both, municipal solid waste (MSW) and wastewater have to
be submitted to anaerobic digestion (Riggle 1998). Anaerobic digestion of MSW in most of
these cases use different types of digesters depending on the COD content and the
quantities to be treated. This paper submission to the conference not only described the
basic microbiology involved in anaerobic digestion, but more importantly drew the attention
to the different types of technologies available, eg. CSTR or contact process, UASB,
fluidised bed, dry continuous and batch digestion, leak-bed, wet continuous and multistage
wet digestion.
These process designs and management were all concerned with urban areas. A very
important aspect of peri-urban health and natural resource production drew the attention
to the plight of people living on the outskirts of the cities in a semi-rural environment (Birley
& Lock 1998). These peri-urban areas may produce a significant amount of products for
the cities, but are at the same time also sinks for the city's waste. The health issues of the
rural-to-urban transition include communicable disease (eg. malaria), non-communicable
disease (eg. poisoning), injury, malnutrition and psychosocial disorders. Attention is drawn
to the urgent need of research between natural resource and health specialists.
There is now emerging evidence that regenerative and resource-conserving technologies
and practices can bring both environmental and economic benefits for farmers,
communities and nations (Pretty 1998). The best evidence comes from countries of Africa,
Asia and Latinamerica, where the concern is, apart from practically non-existing sanitation,
to increase food production in areas where farming has been largely untouched by modern
packages of externally supplied technologies. Farming communities were able to
substantially improve agricultural yields by adapting regenerative technologies. t is
therefore not surprising that the majority of papers came from rural and agro-industrial
areas in developed and developing countries, emphasising a large number of different
designs for integration of biosystems to eliminate health hazards in favour of food, feed,
fuel, fertiliser and energy production.
The practice of integrated biosystems in China can be traced back almost 3,000 years
(Wang et al. 1998; Li Kangmin 1998). Chinese philosophers elaborated the harmonious
relationship of Tian (heaven or universe), Di (earth or resource) and Ren (people or
society) into a systematic set of principles for managing the relationships between man
and its environment. Thus rice-fish culture is an age-old practice in China, and represents
one of the integrated biosystems in China. Rice-fish culture means raising aquatic animals
in irrigated rice fields to obtain aquatic products in addition to rice production. t has been
suggested that about 136,000 ha of irrigated rice fields in SEAsia are used for culturing
fish (Baharin et al. 1997), representing 0.65% of the total irrigated fields. China is striving
to develop its fishing industry to help feed its growing population (Li Kangmin 1998). n
1997, the area of rice-fish culture reached 1.67 million ha with a total fish production of
700,000 tons from rice fields of the total aquatic production of 29 million tons. Outside
China in SEAsia, aquaculture in rice fields is declining due to the introduction of high-
yielding and short-stem rice varieties calling for a thin water sheet and use of agricultural
chemicals which are toxic to water animals (Vincke & Micha 1985). The question whether
this introduction by developed countries is of benefit to the farming community.
Apart from fish, livestock is another large protein supplier for human populations.
Dramatical increases in livestock in developing countries, often to replace the diminishing
fish protein resource, leads to enormous surplus of animal manure. n the case of pigs, the
surplus manure can either be separated into the liquid and solid fraction before treatment
(Bonazzi & Piccinini 1998) with composting or anaerobical digestion with biogas
production (Piccinini et al. 1998).
The reason for separating pig manure slurry into a liquid and solid fraction appeared to be
justified in areas where the amount is so large that transportation to other areas have to be
considered. After the separation, the solid fraction is composted, whereas the liquid
fraction is treated aerobically and added to a sewage system or landspreading. However,
the creation of centralised treatment plants is not possible because of the opposition of the
residential population and the high environmental impact due to the residual load of
treated effluent.
At the end of the eighties, a new generation of biogas system for animal (mainly pig) slurry
were developed, which are simple and low cost, involving the use of a plastic cover over a
slurry tank. For about 10 years, more than 100 farm biogas plants and about 25 large
agro-industrial plants were built (Piccinini et al. 1998). A large farm located in the province
of Parma in taly is described , where the biogas was produced from a total useful volume
of 600 m3 and used for a co-generator that can supply about 50 KW of electric power and
120 KW of thermal power. The pay back time of such a system producing an electric
energy of about 191,000 KWh/year is 4.5 years. Since energy saving is not a very big
issue in taly, such operational plants will decline rather than increase.
The situation in SEAsia is very different from the one described in Europe. With a present
energy and pollution problem, conversion of livestock wastes as a source of energy and
fertiliser offers a great advantage for the livestock industry. A total of 99 biodigesters were
installed in the Philippines (Moog et al. 1998). The Philippines has 8.33 millions pigs and
63 million chickens with an estimated amount of 28,960 tons of manure produced daily or
10.1 million tons/year. Of all the livestock, 85% of it can be found on smallholder farms.
The cost effectiveness of biogas digesters was significantly increased through the use of
tubular polyethylene digesters [see chapters 15 and 20], thus using a low-cost biogas
technology (Bui Xan An et al. 1997). The promotion of this technology through seminars
and study tours has been very effective. The socio-economic aspects of using these TPE
digesters was assessed through questionnaires which established that the savings on fuel
per family was around 160 Pesos (= US$ 6.00) per month and the expenses for the biogas
plant were paid back in 11 months. t was also established that 6 pigs would be sufficient
to supply the daily requirement for cooking for a 6-member family.
Our attention was drawn to the fact that the tropics present great opportunities for
sustainable development thanks to the enormous cultural and biological riches (Rodriguez
et al. 1998; Bin Xuan An et al. 1998). A rationaI expIoitation of IocaI feeds and IocaI
breeds of Iivestock wiII support much more sustainabIe production systems in the
medium and Iong term. These have received insufficient attention in the past and
have not been considered seriousIy because of the introduction of ' exotic' systems
based on high inputs, high technoIogy and ' breeds of high genetic merit' . As a
resuIt, IocaI breeds in many tropicaI countries have disappeared or their popuIation
is decreasing drasticaIIy. With the worId popuIation rising it is a fundamentaI issue
that any intervention invoIving Iivestock must be predicted on their synergistic roIe
in benefit of the whoIe farming system rather than as producers of meat, miIk or
eggs using foods which are in competition with human needs. The compIexity of
this reaIity shouId make scientists think more carefuIIy about the appropriate
strategy that wiII get peopIe out of poverty.
As living standard rise, so does the consumption of livestock products. But the feeding
systems to produce these products, especially in the industrial countries, use the same
feed resources as are eaten by humans. Such a competition can cause severe problems
(FAO 1999). t is estimated that nearly 50% of the world grain supply is consumed by
livestock (Sansoucy 1995) and it has also been argued (Preston 1995) that if all the
world's grain production was reserved for human consumption, there would be enough to
feed 9-10 billion inhabitants.

Figure 2: Diagrammatic ntegrated Biosystem [Rural Ecosystem]

The sustainable use of renewable resources will be facilitated when the feed is grown, the
animals are fed and the excreta is recycled on the farm in ways that minimise the use of
imported inputs including energy (Preston & Murgueitio 1994; Doelle 1989, 1994).
Strategies are outlined with an emphasis on local conditions. t is a realisation that in
respect to livestocks, cattle are mainly in the hands of the wealthy people, while poor
people start with ducks (Bui Xuan An et al. 1998; Chen 1998) and chickens followed by a
few goats, milk cows and a bullock or two to plough the fields (Rodriguez et al. 1998). This
concept means to start with small livestock and women and then the household will step
by step get out of poverty. The present households keep only poultry and these
households were those most dependent on common property resources for their living.
One of the best examples of such a strategy was reported from Bangladesh, where small
scale rural poultry production has lifted millions of women out of absolute poverty (Jensen
1998) as the poultry enterprise gave a 35% increase in household income and family food
intake was increased (Alam 1997).
There is no doubt that the beneficiaries of such strategies with the local resource use
(cassava, duckweed, sugarpalm etc.) in integrated farming systems are the farm families
and society as a whole. Firewood, the collection and use of which is done by women, can
be totally replaced by biogas.
A very similar concept of integrated biosystems (Figure 2) is beginning to generate interest
in the Pacific sland Region (Ajuyah 1998). Based on commercial pig and poultry
population from six sland Nations (Fiji, Samoa, Tonga, Solomon slands, Vanuatu and
Cook slands), the estimated manure output for 1991 was 83 tons/day for chicken and 299
tons/day for pigs (Ajuyah 1996).

Figure 3: Overview of an ntegrated Biosystem [Ajuyah 1998]

The use of agricultural wastes in integrated biosystems has been reported by Okafor
(1998) from Africa. The use of cassava waste generated through the production of a
fermented food gari [see chapter 17] which is a very popular food in West Africa, would
not only eliminate ecological problems, but at the same time benefit the producer in form of
bioenergy and animal feed production.
Biogas is, however, not the only source of energy from waste. A number of papers were
looking at biofuel production from agricultural raw materials and wastes. n combining
methane, ethanol and biodiesel production (Bekers & Viesturs 1998) using agricultural raw
materials could provide the region not only with energy, but also reduces at the same time
air pollution through the use of biofuels. Biofuels provide an alternative to fossil fuel
dependency. Sweden plans to replace 15% of the fossil fuel consumption in the transport
sector with alternate fuels by 2010 (Mansson & Foo 1998). Sweden as well as Latvia
(Telysheva et al. 1998) have a huge amount of lignocellulosic material, which requires a
different technology to the biogas production system.
6.6 ConcIusions
If one combines aII these efforts reported into an socio-economic strategy (DoeIIe
1998, DoeIIe et al. 1998; 2000), farms and/or farm cooperatives as weII as agro-
industries are abIe to combine naturaI renewabIe resource production with
bioenergy, food, feed, biofueI and fertiIiser production. Such a system must be and
can be fIexibIe and shouId be adaptabIe to IocaI conditions. The new term Bio-
Refinery has been given to these systems and a number of exampIes of such bio-
refineries wiII be given Iater. The exampIes which wiII be presented have their core
in the biogas production to eIiminate heaIth hazards and secure sufficient sanitation
in the ruraI areas with aquacuIture and/or greenhouse mesocosm systems. It aIso
uses the wastes from our renewabIe human food production to secure animaI feed
and soiI fertiIisers. In aII our consideration one shouId never forget the naturaI
cycIes of matter and the microbiaI soiI popuIation, both so important for the
production of our renewabIe resources, maintenance, environment, and
improvement of Iiving standards.
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Chapter 19
Concept of a Bio-Refinery
I. Processing of LignoceIIuIosic Biomass, Human and AnimaI Waste with ControI of
Pathogens
Content:
1. Introduction
2. Community invoIvement and joint venture capitaI
4.1 Energy [eIectricity, heat, peIIets, charcoaI]
4.1.1 Direct combustion
4.1.2 Cofiring
4.1.3 Cogeneration
4.1.4 Gasification
4.2 Mushrooms
4.3 FertiIiser [Composting]
4.4 AnimaI Feed [SiIage]
5. Products from Human, AnimaI and AgricuIturaI Waste Biomass
5.1 Energy [biogas] and FertiIiser
5.2 Food [fish and aIgae through aquacuIture]
6. References

1. Introduction
The processing of biomass and control of pathogens in waste management depends
entirely on the particular socio-economy adopted for rural and urban sustainability. n order
to sustain life and improve the standard of living, the general advancement of scientific
knowledge should be used for the application of clean technology strategies, which
support the natural cycles of matter [Doelle et al. 2002]. These strategies should drive
towards a pollution free environment, resulting in the prevention of diseases and an
improvement of our natural cycles of matter to increase our renewable resource
production. At the same time they need to be flexible to guarantee the rural farmer and the
urban workforce a sustainable per capita income.
Sustainability refers therefore to the ability of a society, ecosystem or any such ongoing
system to continue functioning into the indefinite future without being forced into decline
owing to exhaustion of key resources, weather conditions or world market price forces
[Chantalakhana 1998; Doelle et al. 1994]. A sustainable community effort consists of a
long-term integrated system approach to developing and achieving a healthy community
by jointly addressing economic, environmental, and social issues. Fostering a strong
sense of community and building partnerships and consensus amongst key stakeholders
are important elements of such efforts. The focus and scale of sustainability efforts depend
therefore on local conditions, including resources, politics, individual actions, and the
unique features of the community.
Socio-economic strategies have therefore to be designed in a way that individual choices
are shaped by values, emotions, social bands and judgements rather than a precise
calculation of self-interest [Doelle 1982; 1989; Doelle and Foo 2000].
Such a strategy requires a sound knowledge of the natural cycles of matter as well as
consumer demand and gives domestic markets a much higher priority over foreign or
export markets. Furthermore, it is highest time that we make everybody, including many
scientists and the majority of expert advisers, aware of the fact that microorganisms are
the most powerful creatures in existence as they play an integral part in determining life
and death on this planet. As we know, some can kill merciless (=pathogens), but the vast
majority can be harnessed to sustain life. We have also to realise and to admit that over
the past decades we have managed to foster the killer-type pathogens through population
growth and density, overuse of antibiotics, use of raw waste onto farms, and reduce and/or
eliminate the beneficial type (eg soil microflora) through overuse of chemical fertilisers,
pesticides and farm management resulting in ever increasing areas of soil infertility. The
salt problems experienced with large areas of agricultural land together with the increasing
effects of chemical fertiliser leaching effects on the Great Barrier Reef in Australia are
strong reminders. n addition, our present waste management destroys immense natural
resources for energy and value-added product formation. Here have only to draw your
attention to the flares over our sewerage plants, use of anaerobic ponds, reduction of BOD
through aeration etc etc.
n order to reverse this trend, it should be a first priority of any region, eg sugarcane area,
grain area, vegetable area, urban area etc to establish so-called Bio-Refineries, where
agriculturists [responsible for maximal biomass production], microbiologists [responsible
for beneficial and control of pathogenic microorganisms] and chemical engineers
[responsible for plant design and construction] together with other advisers work together
to fully exploit our natural renewable resources using clean technologies with no useable
waste accumulation. Such a bio-refinery is therefore totally based on biomass, human and
animal wastes as raw materials.
2. Community invoIvement and joint venture capitaI [see aIso chapter 18]
Success and failure of any socio-economic strategy towards sustainability, that is food,
feed, energy, biofuel and fertiliser production, depends on the initiatives and involvement
of local communities and government agencies [Doelle 1996; Doelle and Gumbira-Sa'id
1992]. t is therefore very important that frequent communication occurs among the rural
and urban communities, government representatives and biotechnologists to discuss and
establish joint ventures with all four and convince industry and entrepreneurs to join in the
venture towards social advance and national economic benefits. t has to be the aim of
these bodies, in particular the scientists/biotechnologist, to convince society that the new
technologies are not a threat to families or societies, but can bring enormous social and
economic advances for the individual as well as the country. This is a very important, if not
the most important aspect, considering the presently existing controversies surrounding
the use of genetically modified/engineered seeds, plants and microorganisms for food and
feed production.

Figure 1: Scheme for joint socio-economic strategy development

n order to investigate rural technologies, national authorities have to set their priorities in
regard to social, economic and ecological development. Such an authority [eg Board of
Development; Figure 1] should consist of farmer and urban dweller representatives,
researchers, appropriate government agencies and financiers. Such a communication link
is vital for any success in the establishment of social-economic strategies in rural and
urban areas. Major consideration should be given to health, raw material available and the
local market demand. n order to become self-efficient, which means that health, supply
and demand for domestic consumption are guaranteed, each government or local
authority must strive and direct all its efforts towards increasing production, maintaining or
reducing its demand by staple food diversification together with a microbial waste
management .
The Board of Development would give its final proposals to the Center of Development.
This center should be a Biotechnology Research Center devoted entirely to the
development of rural biotechnology. Since every joint microbial biotechnological capital
venture investment system depends on the availability of raw materials, including
agricultural, human and animal wastes, this Center may well be confronted additionally
with tasks of improving farm practices, soil denitrification, rhizobacterial plant growth
promotion, plant disease resistant breeding, and future planning and development of
arable agriculture. Such developments must always take care that it does not affect local
traditional culture, although it should and can improve the conditions of the society within
its traditional culture [Doelle et al. 1987; 1994].
Any system developed in the proposed Centre of Development, whether it is a simple or
integrated rural technology system, must go through pilot scale and field trials. t is the trial
outside protective laboratory conditions that has to withstand rigorous social, economical
and ecological evaluation by the local community and their leaders. No process should be
acceptable that produces a product or products, but simultaneously causes severe
pollution of air, soil or waterways. The trial must exhibit to the farmer and the national
and/or local governments the social, economical and ecological benefits to the farmer, the
region and the nation as a whole.
Any fermentation technology developed from bioresources, which fully exploits the
substrates with the result of multiproducts and no pollution effluent on the ground, in the
water or air falls into the category of bio-integrated system technology [Doelle and Foo
2000]. Bioresource utilisation programmes are the best systems to combat malnutrition
and starvation as well as infectious diseases and, if coupled with waste utilisation and
water purification could lead to high public health standards.
The biggest problem for a joint venture community involvement and venture capital
investment is not so much the lack of entrepreneurs or Government initiatives, but rather
the lack of trained scientists capable of converting scientific knowledge into a clean socio-
economic technology.
Biomass in the form of plants and trees capture solar energy through photosynthesis and
stores it as chemical energy in the bonds between the carbon, hydrogen, nitrogen and
oxygen atoms that form lignocellulosic plant material together with starchy, sugary, fatty as
well as proteinaceous crops. Biomass is solar energy stored in a chemical form, which is
available for bioenergy, biofuel, food, feed, fertiliser and many other products formation
[NREL Biomass 2000].
t should be the aim of each bio-refinery management to assure self-efficiency in food,
feed, fuel, fertiliser and energy production with marketing products depending on the
surplus encountered after the first priority, which must also include improved health
standards, has been satisfied.

Figure 2: Processing of lignocellulosic , simple polymeric and waste products with control
of pathogens in a Bio-refinery

n order to establish such bio-refineries, a sound knowledge is required in
1. land availability
2. biomass availability
3. biodiversity in crop production
4. maintenance of high soil fertility
5. maintenance of high crop yields
6. population growth and demand
7. type of animal production (sheep, chicken, pigs, beef etc)
8. type and amount of any waste accumulation from the production unit, human and
animal population

Figure 2 exhibits what regard as a general outline for the functioning of a bio-refinery.
Each region consists of biomass, people and animals. Let us concentrate first on the pIant
biomass itself, which consists of about 25% lignin and 75% carbohydrates or sugars. The
lignin holds the carbohydrates cellulose and hemicellulose together in the form of
lignocellulose . Other carbohydrate molecules such as starch, sucrose and free cellulose
can be found in the fruit or crops of the plant.
The lignocellulosic material can be used very efficiently for energy, food, feed and fertiliser
production. Energy production, mushroom production, composting and silage are very
important industries.
4.1 Energy [eIectricity, heat, peIIets, charcoaI]
Lignocellulosic biomass such as trees, grasses, agricultural by-products of food, fibre and
forest production as well as household wastes [eg paper] are the most economical
biomass fuels for generating electricity [FAO-RWEDP 1999]. There are four primary
classes of BioPower systems: direct combustion, cofiring, cogeneration and gasification
[see also chapter 15].
4.1.1 Direct combustion. Direct combustion deals mainly with primary fuels in the form in
which it is available in nature or after some form of processing [briquetting, pelleting, heat,
charcoal]. Briquetting and pelleting are densification processes of loose organic materials
such as rice husks, saw dust, coffee husks, municipal wastes etc, aiming to improve
handling and combustion characteristics for stoves, fireplaces, kiln etc. [FAO-RWEDP
1999]. The biomass fuel is burned in a boiler to produce high-pressure steam. This steam
is introduced into a steam turbine, where it flows over a series of aerodynamic turbine
blades, causing the turbine to rotate. The turbine is connected to an electric generator and
electricity is produced. Biomass-fired power plants have been installed in a number of
countries in Asia and Europe. These plants have the option to deliver electricity to the grid,
so-called dendropower, utilise the electricity to satisfy the power demand of a stand-alone
production process or a combination of both.
4.1.2 Cofiring. Cofiring involves substituting biomass for a portion of coal in an existing
power plant furnace. Compared to the coal it replaces, biomass reduces sulfur dioxide,
nitrogen oxides and other air emissions. After turning the boiler for peak performance,
there is little or no loss in efficiency from adding biomass. Extensive demonstration trials
have shown that the effective substitution can be made in the range of 10-15% of the total
energy input.
4.1.3 Cogeneration. Cogeneration is the simultaneous production of electricity, heating
and cooling in a single process and with an overall efficiency exceeding 70%
[Cogeneration at http://www.localpower.org ]. Such combined heat and power plants [CHP
plants] , often integrated with a pellet-manufacturing process have been installed in
Scandinavia [CADDET 95; 96] and are becoming increasingly popular also in Asia
[Coovattanachai 1998].
At a cost of SEK 216 million [approx. A$ 41 million], a Swedish company uses
unprocessed biomass residues producing 120 GWh electricity and 210 GWh heat or in an
integrated operation 170 GWh electricity, 230 GWh heat with 130,000 tonnes of pellets.
The electricity consumption in the pellet plant is around 100 kWh/tonne. Whereas the heat
is connected to a district housing system for heating houses and schools, the surplus
electricity goes into the grid and the pellets are transported to the market place [CADDET
136].
The world's first straw-fired CHP plant was constructed in 1989 in Denmark [CADDET 96].
The plant uses about 26,000 tonnes of straw annually and has a nominal production
capacity of 5 MWe and 13 MJ/s heat. The annual electricity production is around 17 GWh,
corresponding to the consumption of around 3,000 households. Heating output in 1998
was around 228 TJ. Around DKK 102 million [approx A$ 22 million] has been invested in
the plant itself and around DKK 12 million in transmission pipes.
Co-generation of both heat and power is increasingly applied in various wood and
agroprocessing industries such as sugar, palm oil and rice mills in Asia, in particular in the
Philippines [FAO Field Document No. 57].
t is encouraging to learn that the Australian Government is at last introducing programmes
which will require the electricity industry to achieve a significant increase in the contribution
of renewable energy generators. Currently the dominant fuel in the Australian biomass
industry is sugarcane bagasse. About 60 years ago, mills started to generate electricity
mainly for their own needs. Bagasse currently fires 14% of Australians cogeneration
capacity with 302.8 MWe installed. Estimates made by the Sugar Research nstitute
suggests that there is enough waste bagasse currently produced in Australia to provide
fuel for an additional 3,000 MW [The Australian Renewable Energy Site
http://renewable.greenhouse.gov.au ].

Figure 3: Generalised Gasification scheme

4.1.4 Gasification. Gasification (see Figure 3) is a form of pyrolysis and is the complete
breakdown of biomass into a combustible gas, volatiles and ash in an enclosed reactor or
gasifier [Turare 1997; EREN 2000]. The gas produced can then be used either for heat or
for power generation. A wide range of biomass material [wood, charcoal, coconut shells,
rice husks] can be used to fuel gasifiers. Typically 1 kg of air dried biomass gives 3-3.6
kWh heat or 0.7-0.9 kWh electricity plus 1.4 kWh heat. Gasifier units are becoming very
popular in ndia, Thailand and ndonesia [FAO-RWEDP 1999].
4.2 Mushrooms
Cereal straws and other plant by-products are either burnt in the field or utilised as feed for
low producing ruminants. More than 3.5 billion tons per annum of agricultural by-
products are produced in the world. A more efficient way of utilising lignocellulosics is in
the cultivation of edible fungi. By suitable treatment, lignocellulosics can be converted into
substrates for the cultivation of higher fungi. Fruiting bodies serve as delicious food, spent
substrate can be used as feed or as humus fertiliser.

Figure 4: The cultivation of mushrooms

Mushroom production is a multimillion dollar industry because of its nutritive value,
particularly in our region of SEAsia and Asia itself [Chang 1980; 2002; Chang and Miles
1991]. Mushrooms are rich in vitamins and contain, on a dry basis, 20-35% protein. They
are palatable and can be eaten directly in their natural form. Especially valuable is their
ability to produce a wide range of extracellular enzymes which degrade complex organic
substances into soluble substances which the mushroom can absorb as nutrition. Thus
mushrooms can convert lignocellulosic materials and other organic wastes, which have
little or no market value and are inedible for humans into higates. The production of edible
mushrooms could make important contributions to the nutrition and economic welfare of
the population, while simultaneously reducing pollution [Chang and Miles 1989]. t is an
evergrowing industry, because of the protein and medicinal value of these fungi. A new
development in the mushroom industry is the production of nutriceuticals [Chang and
Buswell 1996]. The residuals from a mushroom cultivation are excellent fertilisers, or can
be additives for composting and/or anaerobic digestion [Chang 1980].
The conversion of plant by-products by edible fungi has many remarkable advantages:

1. ndustrial plant residues and by-products (e.g. bagasse, sawdust, etc.) can efficiently be
extracted and reintegrated into the ecosystem through natural processes;
2. Solid and liquid waste (e.g. cereal straw, sawdust, sulfite liquor and other residues from
the paper industry) can be directly converted into fungal substrate;
3. Carbon sources of lignocellulosics, which have an unsuitable low digestibility can be
transformed into consumable biomass (i.e. fruit bodies);
4. Harvesting of flesh fruit bodies from the surface of the substrate (pure microbial
biomass) can be obtained without expensive separation;
5. Edible fungi represent a well characterised microbial biomass, which will be generally
accepted by the consumer. The presently cultivated species are:

TabIe 2: Edible Fungi [mushrooms]
Edible fungi are cultivated worldwide under various climatic conditions. n Europe, North
America, Asia and Australia, Agaricus bisporus is traditionally grown. n the last decades,
the People's Republic of China and Taiwan became the second or third largest producers
of mushrooms in the world.
The most important step in the whole process of mushroom cultivation is the production of
the spawn. This is obtained under axenic conditions in specially equipped facilities of the
mushroom spawn industry.
The inoculation of the sterilised spawn with the starter culture takes place in special rooms
with laminar flow hoods or on clean benches under axenic conditions. A high efficiency
of spawn production is achieved onIy if the transfer of the starter cuIture is carried
out under strictIy aseptic conditions [Figure 5]

Figure 5: Scheme for Spawn Production [acc. to Zadrazil et.al.1992]
noculation is followed by incubation, which is executed in rooms under controlled humidity
and temperature. Mechanical shaking of the container encourages homogenous growth of
mycelium throughout the substrate.
Due to the high sensitivity of the spawn during the production process (inocuIation
and incubation) to competitive microorganisms, steriIity and the quaIity of the
spawn have to be ensured.
Finished containers of Agaricus bisporus, Pleurotus spp., Flammulina velutipes, and
Lentinus edodes spawn are stored in the refrigerator at a constant temperature of 2-5
o
C.
During this time, the mycelium remains viable, but in a latent state. Spawn should be
transported in air-conditioned vehicles, because every fluctuation of the temperature
diminishes the quality.
4.3 FertiIiser [Composting]
Composting is the controlled microbial bio-oxidation process involving biodegradable
organic matter, conducted under controlled environmental conditions [Manderson 1997;
Starbuck 2001; Trautmann and Olyncin 1999]. The oxidation produces a transient
thermophilic stage which is followed by a period of cooling of the now degrading organic
matter. The material is held at ambient temperatures for maturation purposes, which
results in a stable, volume-reduced, hygienic, humus-like material, that has retained the
mineral elements beneficial to soil and plants (see also chapter 14). Emphasising a
'controlled' process distinguishes composting from uncontrolled rotting or putrefaction of
organic matter. This oxidative metabolism of beneficial microorganisms is exothermic and
the heat produced is sufficient to increase the temperature of organic matter to between 60
and 75
o
C, thus offering a self-sanitising mechanism by which pathogens, seeds and heat-
labile microbial and plant toxins will be destroyed. The final humus-like material, the
compost, is a dark, crumbly, earthy material usually containing less than 2% (w/w) each of
nitrogen, potassium and phosphorous. Apart from their availability to plants, the compost
offers improved soil structuring characteristics.
The related process of vermicomposting, ie. composting which involves the use of
earthworms in conjunction with aerobic microorganisms to bring about the bio-oxidation
and subsequent stabilisation of biodegradable organic matter, requires the addition of
anaerobic digester sludge and increases the valuable humic acid content of the compost.
Vermicomposting is becoming increasingly popular in SEAsia and the USA on small to
large scale. Earthworms can also be used as a supplement to animal feed [Rodriguez
1997a,b].
A relative new development in composting, particularly of putrescent wastes, is the use of
the black soldier fly [BSF, Hermetia illucens, a tropical fly found throughout the world
(Olivier 2004). This process is housed within a container that resembles a small plastic
garbage bin. The unit has no moving parts and requires no maintenance. Since it must be
emptied once a year, it eliminates altogether the collection, transport and landfilling of food
wastes. This bioconversion process reduces the weight and volume of food waste by over
95% within a matter of a few hours. t requires no energy, no electricity, no chemicals, not
even water and is totally self-contained. t produces no methane or other greenhouse
gases. t can be situated out-of-doors in a shaded area, and any number of units can be
coupled together to handle unlimited quantities of waste. One unit sells for US $80 and
can handle the daily food waste of more than 25 people.

4.4 AnimaI Feed [SiIage]
Silage is forage, crop residues or agricultural and industrial by-products preserved by
acids, either added or produced by natural fermentation (see chapter 14 for details). Fresh
forage is harvested, or crop residues and by-products are collected, the material may be
chopped or conditioned, additives may be added, and it is then stored in the absence of air
so that facultative anaerobic bacteria, present in the forage, or added as inoculants, can
rapidly convert soluble carbohydrates into acids. The resulting pH of a well-ensiled product
becomes so low that all life processes come to a halt and the material will be preserved so
long as it remains in airtight storage. Silage making is practised widely in intensive animal
production systems in temperate regions, mainly to bridge periods of the year when there
is no high quality feed available in the fields and to supplement feed to improve milk
production in the dairy industry [Suttie 1999; t'Mannetje 1999; Elferink et al. 1999].
Bioenergy production, composting, silaging and mushroom production ensure that no
lignocellulosic biomass is wasted, but fully exploited.
5. Products from human, animaI and agricuIturaI waste biomass
Turning to the right hand side of figure 2, people and animal waste management for such a
bio-refinery are outlined. Household wastes can either be used for electricity generation
with the lignocellulosic material and/or mixed with the human and animal manure waste
transferred into the anaerobic digestion reactor for biogas production [Gustavsson 2000;
TC 1998; SAT 1999]. t is important to stress, however, that anaerobic digestion, similar
to composting, is a self-sanitising system and very important for the health standard of
both people and animals. Under no circumstances should any of the raw waste be used
directly for whatever purpose. We have to realise that the days of our grandparents are
gone and we cannot use the techniques used in those days. Our grandparents and
parents had no antibiotic resistant strains of pathogens and none of the virulent mutants.
The incredible death toll in developing countries amongst children and the re-occurrence
of rare infectious diseases in developed countries has always been traced back to
reluctant self-sanitation systems.
5.1 Energy [Biogas] and fertiIiser production
Anaerobic digestion (see also chapter 10 and 15) is an integrated part of, what we call
today, environmental biotechnology, as it concerns itself predominantly with the use of
mixed microbial populations for waste and effluent treatment. t is another cycle of matter
existing in mangroves and wherever organic matter is decomposed in the absence of
oxygen. This natural cycle has been improved and used very successfully over many
centuries for the benefit of mankind.
n rural communities recycling of human, animal and vegetable wastes has been practised
for centuries by man and nature itself, providing in many cases valuable fertilisers and/or
cooking gas [fuel]. Growth in urban communities has generally been matched by a
concomitant formation of a wider range of waste products, many of which cause serious
environmental pollution if they are allowed to accumulate in our ecosystem
Mainly by empirical means a variety of biological treatment systems have been developed,
ranging from cesspits, septic tanks and sewage farms to gravel beds, percolating filters
and activated sludge processes coupled with anaerobic digestion. The primary aim of all of
these systems is to alleviate health hazards and to reduce the amount of oxidisable
compounds and thus produce a final effluent or outflow which can be discharged into the
natural environment without producing any adverse effects.
f we look at nature, it has provided us already with a mixed microbial population capable
of anaerobic digestion, that is the conversion of organic matter to methane by
fermentation (Figure 6). This natural process occurs in marshes, organic sediments in
aquatic systems and in the rumen of cattle and sheep. n these anaerobic environments, a
large variety of microorganisms develop, degradation of macromolecules occur through
the action of heterotrophic extracellular enzyme producing bacteria, fermentation of
sugars, amino acids, fatty acids and alcohols follows and finally a special consortium of
bacteria collaborate to stabilise these volatile fatty acids to give methane and carbon
dioxide. The success of an anaerobic digestion resides in maintaining a balance of acid
producers and methane formers. The microbiology of methane formation is therefore as
complex as each cycle of matter and can be visualised to occur in four stages, based on
the metabolic characteristics of the microorganisms involved:
1. Those microorganisms which are capable of producing extracellular enzymes
[proteases, lipases, amylases, etc] for the breakdown of polysaccharides and other
polymers into monomeric structures, such as sugars, amino and fatty acids; this
process is referred to as liquefaction;
2. those microorganisms which are capable of fermenting the monomeric structures
produced in (1) to volatile fatty acids, eg fermentative bacteria [Escherichia coli,
Bacillus sp., Enterobacter spp.etc]; a process referred to as fermentation;
3. those microorganisms which are capable of converting all the volatile fatty acids
other than acetic acid into acetic acid and carbon dioxide, eg acetogenic bacteria
[Syntrophobacter wolinii, S.wolfei, Syntrophonomas wolfei]; a process referred to as
acetogenesis;
4. those microorganisms which are capable of converting acetic acid, carbon dioxide
and hydrogen into methane plus carbon dioxide, eg methanogenic bacteria; the
process is referred to as methanogenesis.
Polymeric substances are usually in solid or semi-solid form and, in order to become
available to bacterial degradation, must be broken up into their monomers by extracellular
enzymes produced by bacteria. The monomers will then be dissolved into the water which
surrounds them. This hydrolysis of polysaccharides, fats or proteins occurs outside the
cell. Thus, in a digester, the rate of hydrolysis of these polymers can influence the rate of
growth and metabolism of a large part of the digester flora.
During the second stage of the anaerobic digestion, all the monomeric substances are
degraded in the normal basic anaerobic fermentation fashion to a variety of acids,
hydrogen and alcohols.

Figure 6:Methanogenesis in Nature.
Although most of the carbohydrates finish up in acetic acid and carbon dioxide, fat and
protein metabolism in general form higher fatty acids such as propionic, butyric, valeric
and other volatile fatty acids. n methanogenic environments it is therefore not unusual for
the methanogens, which can only utilise acetate and carbon dioxide, to co-exist in close
association with another metabolically specific bacterium. This close association is called
syntrophism and is based upon closely integrated biochemical features of the two
bacteria in this anaerobic consortium. One member of this pair is called 'methanogen',
because it generates methane, whereas the other members is called an acetogenic
hydrogen plus acetic acid producing bacterium or short 'acetogen'. The most important
feature in this syntrophism is the interspecies hydrogen transfer, without which no
methane can be formed.
t is first the role of the acetogens during stage 3 to degrade alcohols and fatty acids to
hydrogen and acetic acid:

Consequently, acetogens are capable of converting the products of other microbial
degradation reactions to substrates easily metabolisable by the methanogens.
The role of the methanogens in the syntrophic relationship is to utilise the hydrogen and
carbon dioxide to form methane and to also convert the acetic acid to methane:

Methanotrix soehngii uses the acetoclastic (acetate to methane) reaction almost
exclusively, Methanosarcina barkeri is most versatile and uses all substrates, whereas
Methanobacterium thermoautotrophicum uses only the autotrophic reaction.
Biogas is a mixture of roughly 70 per cent methane and 30 per cent CO
2
, is colourless,
odourless and inflammable. n its crude form, it is largely used for cooking, lightning and to
power stationary engines for the generation of electricity. One m
3
biogas is equivalent to
0.7 l fuel oil or 0.82 l petrol or 2.7 kg dry firewood or 0.3 m
3
propane gas and can be
harnessed to generate 1.5 KWh electricity. The residual sludge is virtual free of pathogens.
n order to have a good biogas formation, the C/N ratio of the raw material should be 20
and not exceed 30. Waste materials with lower C/N ratios should be used for composting
or enriched with a nitrogen-containing source, whereas materials with high C/N ratios
should be diluted with water.
The loading of the digester [or anaerobic fermenter] is determined by the solids of the
influents, the retention time, digester size and temperature. During the digestion it is
important to control the process, which normally is done by pH and total volatile fatty acid
(VFA) determination.
The most impressive installations of methane producing fermenters can be found in the
Peoples Republic of China. Digesters up to 400 m
3
in series of 5-6 are a common side on
the outskirts of Shanghai or in rural areas. t is in the rural areas where there exist some 4
million family size plants in China today.

Figure 7: Diagrammatical Biogas Production from animal and human wastes
The Maya Farm on the outskirts of Manila in the Philippines [Maramba 1978] and biogas
plants in Sweden [CADDET 112] are typical examples of the utilisation of methane
produced from animal waste for electricity, power, and heat generation.
The following two examples should demonstrate the usefulness of biogas
1. Each person in the UK needs an average of about 5500 BTUs or 5830 kJ day
-1
for
cooking purposes, which corresponds to about 225 litres of biogas. This amount of biogas
can be produced from two pigs, allowing one-third of the gas to be returned for heating the
digester.
2. A farm of 1000 pigs therefore would generate enough energy to service about 500
people with cooking gas. Biogas can also be used to heat water for central heating. Gas-
fired boilers have a similar heat conversion efficiency of 75 percent. Since the normal
range of central heating systems require an energy input of 42,000 - 131250 kJ h
-1
or
1876-5825 l h
-1
, such a system could be serviced from the waste of 250-780 pigs.

The recent development of polyethylene biodigesters has reduced the cost of biogas
production significantly [Bui Xuan An et al. 1997]. These digesters have become very
popular in poor countries such as Vietnam, Bangladesh and Cambodia and can be used
up to 20 m
3
for a construction price under US $100.

Figure 8: Polyethylene anaerobic digester tube

Figure 9: Polyethylene anaerobic digester

The solids and liquid effluents from the anaerobic digester can safely be used directly as
fertiliser, but would be much more effective in the soil after vermicomposting. t is of
interest to note that comparative analyses of energy production from municipal wastes
lead to the conclusion that biogas production is the most economic and effective way of
producing energy (Murphy and McKeogh 2004)
5.2 Food [Fish and aIgae through AquacuIture]

The liquid effluent from the anaerobic digesters has a great potential for food production
and should not be wasted onto the fields. The liquid effluent is an excellent source for
aquaculture, in particular algae, which are an excellent feed source for aquaculture [fish
production] and is also rich on protein, vitamin and carotene, which has an excellent
market in health food stores. Algae are phosphate and nitrogen scavengers, cleaning up
our water resources. n particular Spirulina can have a protein content of up to 72%, a
remarkable resource for feed supplementation and vegetarians. Some countries, eg Chad,
use algae as their main protein source, other countries love fish or beef, pigs and other
meat.
Algae have long been recognised as practical sources for the production of traditional
foods, polysaccharides, and single cell protein from minerals, CO
2
, and light. Algal
properties such as high growth rates, adaptability to sewage, resistance to contamination
and environmental fluctuations, relative non-toxicity, and ease of harvesting and
processing, have resulted in their mass-cultivation in open, low-energy, labour-intensive
systems suitable for developing countries, as well as in enclosed systems (photoreactors)
in developed countries. nterest in algae has recently expanded to include algal lipids and
pharmaceuticals.
Algae have also been used for centuries as natural organic nitrogen fertiliser in rice
paddies in Southern China [Anabaena, Nostoc]. The algae Spirulina is very popular in its
dry form [Ciferri 1983] in health stores for human consumption. Algae are excellent
scavengers of P and N, in particular the mentioned genus Spirulina, which can reach a
protein content of up to 72 percent [Olguin et al. 1994]. The use of this genus could solve
many constraints in some anaerobic digester effluents and at the same time serve as an
excellent food and feed supplement.
n China, bio-integrated systems of waste management are using algae as biofertiliser, fish
feed or even in polyculture for fish production [Li Kangmin 1997].
Here again it is very important to realise that a proper selection of microorganisms is vital,
since some algae are also producing toxins, as we have experienced all over the world
with the algae bloom occurring due to the waters being rich on phosphates from the
leaching of phosphate fertilised soils. t is important to realise and pay respect to the
microorganisms to be used in any process, whether natural or biotechnological, as they
are a part of the waste stream and have to be re-used or destroyed.
The implementation of clean and healthy sustainable technologies means therefore

a) that the selected microbial catalyst has to be a non-pathogenic, non-toxic producing
natural, not genetical engineered strain, if it is being recycled as a protein supplement to
the animal [MBP = microbial biomass protein]
b) that the use of genetically engineered microorganisms in processes requires special
precautions, as they cannot be used as protein supplements or fertiliser and must be
incinerated.

t is very distressing to realise that Culture Collections, who have the expertise in
microbiology, are not receiving the support required because of a lack of understanding of
the important part microorganisms play in our environment and the application of clean
technologies. Furthermore it should be the task of international organisations such as
Unesco and FAO to foster and re-introduce these proven old but improved
biotechnological systems, before any improved plant or animal is being introduced, in the
overall concept of integrated biotechnology and the biorefinery concept.
6. References
Bui Xuan An, T.R.Preston and DoIberg,F. 1997 - The introduction of low-cost
polyethylene tube biodigesters on small scale farms in Vietnam. Livestock Res. For Rural
Development 9:2 http://www.cipav.org.co/lrrd/lrrd9/2/an92

CADDET 95 - Straw-fired CHP plant in Rudkobing - Providing environmentally-friendly
energy. Technical Brochure 95; http://www.caddet-re.org

CADDET 96 - The World's first straw-fired CHP plant offers environmental benefits.
Technical Brochure 96 ; http://www.caddet-re.org
CADDET 112 - Biogas combined heat and power in Sweden. Technical Brochure 112;
http://www.caddet-re.org

CADDET 136 - A multipurpose Bioenergy Plant Producing Electricity, Heat and Biopellets.
Technical Brochure 136; http://www.caddet-er.org

Chang,S.T. 1980 - Mushroom Production in SEAsia. Mushroom Newsletter for the Tropics
1, 18022

Chang,S.T. & MiIes,P.G. 1991 - Recent Trends in World Production of Cultivated Edible
Mushrooms. J.Mushroom 504,15-18

Chang, S.T. 2002 - Mushroom Production. n 'Biotechnology', in Encyclopedia of Life
Support Systems, Eolss Publishers, Oxford, UK

Chang,S.T. & MiIes,P.G. 1989 - Edible Mushrooms and their cultivation. CRC Press nc.
Florida

Chang,S.T. & BusweII,J.A. 1996 - Mushroom Nutriceuticals. World J. Microbiol.
Biotechnol. 12 ,473-476

ChantaIakhana,C. 1998 - Sustainable agriculture as an approach to economic self-
sufficiency. Songklanakarin J.Sci.Technol. 30,31-40

Ciferri,O. 1983 - Spirulina, the edible microorganism. Microbiol. Revs. 47,551-578

Cogeneration at http://www.localpower.org

Coovattanachai,N. 1998 - Electricity from biomass: The expanding role of gasification.
Songklanakarin J.Sci.Technol. 30 ,65-86

DoeIIe,H.W. 1982 - Appropriate Biotechnology in Less Developed Countries. Conservation
and Recycling 5 ,75-77

DoeIIe,H.W. 1989 - Socio-ecological biotechnology concepts for developing countries.
MIRCEN J. Appl. Microbiol. Biotechnol. 5 , 391-410

DoeIIe,H.W. 1996 - Joint venture capital investment for clean technologies and their
problems in developing countries. World J.Microbiol.Biotechnol. 12,445-450

DoeIIe,H.W. and Foo,E.-L 2000 - Socio-Ecological strategies for future sustainability. A
review of an internet conference. Acta Biotechnol. 20, 203-218

DoeIIe,H.W. & Gumbira-Said,E. 1992 - Joint Microbial Biotechnological Ventures in
Developing Countries: Social Promises and Economic Consideration. n 'Biotechnology:
Economic and Social Aspects [E.J.DaSilva, C.Ratledge, A.Sasson, eds], pp 235-265.
Cambridge Univ. Press

DoeIIe,H.W. & Prasertsan,P. 2003 - Environmental Biotechnology - Socio-economic
Strategies for Sustainability. n 'Biotechnology' [H.W.Doelle, ed.], in Encyclopedia of Life
Support Systems, Eolss Publishers, Oxford, UK

DoeIIe,H.W., OIguin,E.J. & Prasertsan,P. 1987 - Fermentation Technology and ts
mpact on Culture and Society. n 'Microbial Technology in the Developing World'
[E.J.DaSilva, Y.R.Dommergues, E.J.Nyns, C.Ratledge, eds], pp 209-225, Oxford Science
Publications

DoeIIe,H.W., OIguin,E.J. & DoeIIe,M.B. 1994 - A Model for Future Microbial Process
Systems as a Clean Environmental Technology for Sustainable Development. n
'Environmental Biotechnology: Principles and Applications' [M.Moo-Young, W.A.Anderson
& A.M.Chakrabarty, eds.],pp. 712-722, Dordrecht Luwer Academic Publishers

EIferink,S.J.W.H.O., Driehuis,F., GottschaI,J.C. & SpoeIstra,S.F. 1999- Silage
fermentation processes and their manipulation. Electronic Conf. On Tropical Silage at FAO
Rome 1999:
http://www.fao.org/waicent/faoinfo/agricult/agp/agpc/gp/silage/contents.htm

EREN 2000 - Gasification Technology for clean, cost-effective biomass electricity
generation. Energy Efficiency and Renewable Energy Network, US Dept. of Energy at
http://www.eren.doe.gov/biopower/bplib/library/li_gasification.htm

FAO-RWEDP 1999 - Biomass Energy Technology.
http://www.rwedp.org/d_technology.html

FAO FieId Document No. 57 - options for Dendropower in Asia. http://www.rwedp.org

Gustavsson,M. 2000 - Biogas Technology - Solution in search of is problem. A study of
small-scale rural technology ntroduction and ntegration. http://www.he.gu.se

ISAT [nformation and Advisory Service on Appropriate Technology] - http://gate.gtz.de/isat

ITC 1998 - Anaerobic Digestion. http://www.dmu.ac.uk/ln/itc/itc.htm

Li Kangmin 1997 - Fish polyculture in China. n UNDP/UNU-ZERI Indo-Pacific Workshop,
May, Fiji

Manderson,G.J. 1997 - Biosolids Processing with particular reference to Composting.
ASM/Unesco/MRCEN Workshop seminar on 'General Aspects of available
Biotechnological Systems for a Sustainable Development of the Pacific Island Nations,
February-March 1997, Suva, Fiji; Apia, Western Samoa and Nuku'Alofa, Tonga

Maramba,F.D. 1978 - Biogas and Waste Recycling. The Philippine Experience. Regal
Printing Comp.

Murphy,J.D. and E.McKeogh 2004 - Technical, economic and environmental analysis of
energy production from municipal wastes. Renewable Energy 29,1043-1057

NREL Biomass - Biomass, Nature's Renewable Storehouse of Solar Energy and
Chemical Resources. Natural Renewable Energy Laboratory, US Department of Energy
at:
http://www.nrel.gov/research/industrial_tech/biomass.html

OIivier,P.A. 2003 - The Bio-conversion of putrescent wastes.
http://www.esrla.com/brazil/frame.html or http://webber.biotech.kth.se/iobb/news/e-sem-03.html

Rodriguez,J.L. 1997a- Use of Earthworms: A case study. UNDP/UNU ZERI Indo Pacific
Workshop, May 1997, Fiji

Rodriguez,J.L. 1997b - Recycling in ntegrated Farming Systems. UNDP/UNU ZERI Indo
Pacific Workshop, May 1997, Fiji

Starbuck,C.J. 2001 - Making and using compost.
http://muextension.missouri.edu/xplorpdf/agguides/hort/G06956.pdf

Suttie, J.M. 1999 - Hay and Straw Conservation for small scale farming and pastoral
conditions. FAO Rome 1999, Electronic Conference On Tropical Silage at:

The AustraIian RenewabIe Energy Site
http://renewable.greenhouse.gov.au/

t'Mannetje,L. 1999 - ntroduction to the conference on silage making in the tropics.
Electronic Conf. On Tropical Silage, FAO Rome 1999:

Trautmann,N. & OIyncin,E. 1999 - Compost Microorganisms. Cornell Composting
Science & Engineering at
http://www.cfe.cornell.edu/compost/microog.html

Turare,Ch. 1997 - Biomass gasification: technology and utilisation.
http://members.tripod.de/cturare/bio.htm
Chapter 20
Concept of Bio-Refineries
II. Processing of SimpIe PoIymer Biomass [starch, sugar, oiI, protein] from various
agricuIturaI crops and residuaI wastes
Content:

1. Introduction
2. Enzyme Production
3. Starch containing crops
3.1 Introduction
3.2 Grain
3.4 SagopaIm
4. Sugar containing crops: Sugarcane
5. Fatty acid and oiI containing crop: OiI PaIm
6. Fish processing Industries [protein]
7. BibIiography
1. Introduction
Depending on the crop cultivated in the region, it will consist of either the polymer starch,
sucrose, protein or oil. Although all of these polymers are useable as food for the people,
any excess can be transformed enzymatically into monomers, which are the preferable
raw materials for microbial conversion into hundreds of different products [Doelle
2002].Nature has also provided us with starchy crops which are not very popular for food
consumption in certain communities, eg sagopalm, which could be exploited for product
formation as they would not compete with natural food. The higher the crop yields, the
more products can be produced.
2. Enzyme production
n order to obtain these monomers economically, each bio-refinery should have its own
enzyme manufacturing facility to produce the starchy enzymes alpha- and glucoamylase,
proteinases as well as esterases. The microorganisms to be used for these enzyme
production units should be selected not only for their production rate, but also for non-
pathogenicity and non-toxin producing capability which makes them available for feed
supplementation after the production process. t is often forgotten that microbial biomass
can also be a serious environmental pollution waste. As an example for such a selection,
may mention amylase production, which at present uses the strain Aspergillus niger, a
fungus with strong capabilities for the production of the toxin 'aflatoxin' . A simple change
to Aspergillus oryzae or Rhizopus oligosporus [Sukara & Doelle 1989b] would solve the
problem.
The monomers can now be converted into products of demand, ranging from antibiotics,
biopolymers, surfactants to enzymes, alcohols, amino acids, organic acids and new
products depending upon the choice of microorganism and the demand by people,
animals and market place. All these technologies outlined in Figures 1 and 2 are readily
available and are proven technologies. t is, however, important to realise that bio-
refineries must work on a multi-product system in order to be sustainable. Past agricultural
practises have clearly shown that monocultures are far too vulnerable to pest control,
weather conditions and soil conditions.
3. Starch containing crops
3.1 Introduction

Starch, because of its wide distribution in nature, has been widely iused since early times
not only as food, but also as a useful product in various practical and industrial
applications.
The potential use of single-cell protein (SCP) or microbial biomass protein (MBP) as an
alternative supplement in animal and human diets has promoted research on the microbial
fermentation of various starchy substrates (Reade & Gregory 1975; MacLennan 1976;
Azoulay et al. 1980; Opoku & Adoga 1980; Yousri 1982; Touzi et al. 1982; Neumann et al.
1984; Tan et al. 1984; Tuse 1984; Ringpfeil & Heinritz 1986).
Starch saccharification by acid or enzyme hydrolysis is commonly used in the industrial
ethanol and fructose sweetener production in the USA (Torrey 1983) and
amyloglucosidase production uses mainly Aspergillus niger (Alazard & Raimbault 1981;
Fogarty & Benson 1983), although other fungi, in particular Aspergillus oryzae and
Rhizopus oligosporus have also been investigated (Kassim 1983; Saha & Ueda 1983;
Grigorov et al. 1983,1986; Doelle & Sukara 1989).
A very simplified fermentation diagram is given in Figure 1:

Figure 1: A simplified diagram of multiproduct formation from starch using fermentation
technologies
The most common starch-based renewable resources are corn (maize), wheat, barley,
sorghum, sago palm, potato and cassava (also called tapioca or manihot). Whereas the
former are grains, potato and cassava are root crops and the sagopalm is a tree.

3.2 Grain

Grains such as corn [maize], wheat, barley and sorghum can be dry milled or wet milled
[Figure 2]. The wet milling process separates the starch from the fibre first and allows the
production of fIour (for baking), oiI and gIuten ( a type of protein). The residue consists of
lignocellulosic fibre [see Section 4]. The dry milling process does not separate any
component out, but the milled grain is transferred into a cooker, which not only heats the
milled corn, but also liquefies the corn mash with the enzyme -amylase at elevated
temperatures of 90-950
0
C for 1-1.5 hrs. After cooling the mash to approximately 50-600
0

C, the second enzyme amyloglucosidase is added to convert the starch to glucose. This
latter process can also be performed using the pure flour [starch] from the wet milling
process.
Only the glucose obtained from the wet milling process is pure enough for the production
of gIucose syrup or the enzymatic conversion to fructose or fructose syrup using the
enzyme glucose isomerase. The fructose is very important for people with diabetes.
As was mentioned, the conversion of starch into marketing products requires the
conversion of the polymer starch into gIucose, which can only be done economically and
fast using the two mentioned enzymes -amylase to loosen the structure of the molecule
and thus lowering the viscosity and amyloglucosidase for the final formation of glucose.
Both enzymes can easily be produced from the starch using the non-pathogenic fungus
Rhizopus oligosporus, the same fungus producing the excellent SEAsian food tempeh.
The additional microbial biomass formed contains about 42 percent protein and can safely
be used as an animaI feed additive [Sukara & Doelle 1989a]. The liquid effluent from this
process can either be recycled into the fermenter or added to the anaerobic digester.
GIucose is one of the most common and best substrate for any microorganism. n
choosing the proper bacterium, yeast or fungus, almost any product can be produced
including the biofueI ethanoI [Mathewson 1980; Doelle et al. 1989; Millichip & Doelle
1989] given as an example in Figure 2.
A dry-milling process carries the grain through the fermentation process and extracts the
unfermented grain residue at the end of the fermentation process, drying it to produce
either a dried cattle feed, referred to as DDG [Distillers Dried Grain] [Ains et al. 1986] or
DDGS, if syrup (S) from the separation of the solubles is added.
Many reviews have appeared as to the economics of the ethanol fermentation process,
which only consider the starch or sugar to ethanol conversion ignoring any further product
cross-subsidisation or socio-economic benefits [AFDC 1999; Kosaric et al. 1981; Maiorella
et al. 1981]. These reports catalysed research work into the improvement of the
fermentation process [Viikari 1988; Atthasampunna et al. 1987; Doelle et al. 1993].
The development of a new bacterial ethanol fermentation technology using Zymomonas
mobilis was sparked by the realisation that the kinetics of glucose batch fermentations
allows significantly higher rates of ethanol production (eg 5.6 g/g biomass/h) compared to
yeast (eg 0.67 g/g biomass/h), produces less biomass, possesses high ethanol tolerance
and has a higher protein content with a much superior amino acid profile [Doelle 1986;
Lawford 1986; Rogers et al. 1984]. During trials it was realised that the DDG, to be used
for animal feed, contains 3% higher protein values [Doelle 1989] and no glycerol and the
ethanol contains no fusel oils (higher alcohols) and is thus close to pharmaceutical grade.
These findings increased the economic values of both products.

Figure 2: Grain Bio-Refinery
Any ethanol production has, of course, waste products such as carbon dioxide and thin
stillage, which have to be removed in a socio-ecological system. Whereas carbon dioxide
can be sold as dry ice or channelled into a lagoon for algal production, thin stillage can be
recycled to at least 30% into the fermenters saving considerable water usage and energy
costs. Both waste products can also be used as a carbon and nutrient source for algal
biomass production. A great number of algae are autotrophic and thus use carbon dioxide
as their carbon source and photosynthetic using light as their only energy source (Shelev
& Soeder 1980) . The algae themselves are a good vitamin source for human consumption
and also an excellent protein source for aquaculture, such as fish production. Algae can
therefore be used very effectively as a human protein food, as a protein supplement in
aquaculture and as a nitrogen fertiliser because of their nitrogen fixing ability.
The production of 1 tonne of the alga Spirulina [Ciferri 1983] requires only 0.03 ha/year
compared to 452.5 ha/year to produce 1 t of beef or 1.55 ha/year to produce 1 t of
soybeans [Leesley et al. 1980].


n Southeast Asia as well as Central and South America and the Pacific, rice, cassava and
sago are the main staple crops compared with potatoes in cooler climates. Of these,
cassava, potatoes and sagopalm are inexpensive and not nearly as agriculturally intensive
than grain or rice, making them obvious candidates for further diversification and
exploitation as starch sources.
The root tubers of cassava and potato are more restrictive in their versatility because as a
tuber they do not carry out photosynthesis and thus contain only a small amount of
lignocellulosic material. However, an efficient multiproduct conversion process can still be
developed using either root crop and following figure 1. The fermentation of cassava or a
similar starch-based source using the fungus Rhizopus oligosporus can easily be made ro
yielod different products through the addition of minerals to the fermentation mixture
(Sukara & Doelle 1989). For example, the addition of zinc or zinc plus iron to a
combination of calcium plus magnesium switches the fermentation from glucose
production to microbial protein production (Sukara & Doelle 1989a) resulting in 24 g
biomass containing 30% true protein/100 g cassava starch, which is equal to 7.45 g
MBP/100 g substrate in 24 hours. n the absence of zinc or zinc and iron, almost 80% of
the starch accumulates as glucose owing to repression of growth and high amylase
production (Garg & Doelle 1989). A third fermentation condition was established with the
same fungus, whereby the production of microbial cell protein was combined with a
commercial amyloglucosidase production (Sukara & Doelle 1989b). The growth of the
fungus is a strictly aerobic process with a fermentation time of 12-15 hrs at 37-42 C. The
mycelium can easily be separated through a cheesecloth and the enzyme purified by ultra-
membrane filtration. From pilot plant experiments it was calculated that 1 ha bearing 65 t
of cassava tuber can produce 3,500 kg of microbial protein and a significantly quantity of
highly active amyloglucosideas capable of converting 39,000 tons of cassava (the harvest
of approx. 1200 ha) into glucose. This quantity of glucose could then be converted to 15.6
million litres of ethanol using Zymomonas mobilis.
n the case of potato, one kg of potato mash contains approximately 142 g starch, which is
equivalent to about 155 g of glucose availability. This potato mash, however, is so thick
even after liquification, that sufficient stirring is impossible and a 1:1 dilution is required,
which in turn reduces the glucose availability. The problem is the dilution, which allows
only a fermentable mash from 478 g/l potato mash, which gives maximal 4.6% (v/v)
ethanol (Richard & Doelle 1989). n order to obtain a sufficiently high ethanol
concentration, the mash has to be enriched with starch from grain to give values of at least
7-10% (v/v) to be economical. For example, when the potato mash was enriched with
starch from maltrin to give a final concentration of 186.8 g starch/litre, a 96.6% conversion
efficiency was obtainable with Zymomonas mobilis resulting in an ethanol yield of 12.7%
(v/v)

3.4 SagopaIm
3.4.1 Starch extraction
n Southeast Asia and the Pacific, rice, cassava, and sago are the main staple food crops.
Of these, sagopalm and cassava are inexpensive and not nearly as agriculturally intensive
as rice, making them obvious candidates for further diversification and exploitation as
starch sources. The sago palm [Figure 3] grows well in swampy areas [Figure 4], which
can only be developed for other crops at high cost. t is perennial, very suitable for humid

Fig. 3: Sagopalm [courtesy of Dr.Kopli Bujang, UNMAS]

Fig. 4: Sagopalm plantation in Sarawak (East Malaysia) [courtesy of Dr.Kopli
Bujang,UNMAS]

tropical low lands and is already available in areas which are in urgent need of economic
development. There exist at present an estimated two million hectares of natural or wild
stands of sago palm compared to only 200,000 ha of cultivated sago palm.

The production capacity of the sago palm varies between 2-5 tons of dry starch/ha in the
wild to 10-25 t/ha in cultivated crops (Flach 1983). Clump densities of 590 palms/acre or
1480 palms/ha would allow a yearly harvest of 125-140 palms/year (Tan 1983). A well
attended farm can produce 175 kg starch/palm, giving a total yield of 25 tons of starch/ha.
At present only 3,460 ha of sagopalm are being cultivated, but a total of 61,980 ha are
estimated to be available for production (Maamun & Sarasutha, 1987).
After the removal of cortex, rachis and leaflets from the pith, which is probably the most
labour intensive part of the sago palm processing, starch has to be extracted from the pith.
Whereas the non-pith parts of the sago paIm trunk form

(1) excellent building materials for local and urban houses, sheds or other
buildings;
(2) resources for composting [biofertiliser];
(3) resources for gasification and energy production;
(4) resource for animal feed (EI-Nawawy 1992; ZadraziI 1992);

the trunks have to be cut into 1 - 1.5 m length for transportation into the regional
processing plant.

Figure 5: Transportation of sagopalm trunks down the river.
After debarking the trunk [Figure 6], the pith [Figure 7] consists mainly of starch, which
has to be separated from the cellulosic cell walls of the trunk. The residue from this starch
extraction is a very strong pollutant because of its cellulosic fibrous material. n ndonesia,
such material coming from the cassava (= manihot or tapioca), is being used as an animal
feed additive. We suggest, however, that it should form the basis for a mushroom
industry. Almost purely cellulosic in nature, mushroom would thrive on this waste. The
cultivation of edible mushrooms from lignocellulosic and cellulosic residues is well-known
(Chang 1980; Chang & Buswell 1996; Chang & Miles 1989; Zadrazil et al. 1992) and
represents the only current large-scale controlled application of microbial technology for
the profitable conversion of agroindustry-waste. A third application would be the use as
additional carbon in an anaerobic digester for the production of biogas

Fig. 6: Debarking of the sagopalm trunks [courtesy of Dr.Kopli Bujang, UNMAS]

Figure 7: The pith of the debarked Sagopalm trunk [courtesy of Dr.Kopli Bujamg,
UNMAS]
The fIexibiIity, simpIicity and Iow cost aIternate usage of the residue not onIy
removes a severe heaIth hazard to the community but, more importantIy, increases
the seIf-efficiency of the processing pIant and increases the farmer's income
through mushroom production.
The starch obtained from the sago palm processing unit can easily be transported to
regional centres for further processing. The starch flour or meal can either be used and/or
sold for breadmaking or as staple food with the surplus being channelled into further
bioprocessing.
3.4.2 MicrobiaI Bioprocessing of starch
The conversion of starch into marketing products [Figure 8] requires the conversion of the
polymer starch into glucose, which can only be done economically on larger scale using
two enzymes, alpha-amylase to loosen the structure of the molecule and thus lowering the
viscosity and amyloglucosidase for the final formation of glucose.

Figure 8: Sagopalm Bio-Refinery [adapted from Doelle 1998]

Process 1: Enzyme production
The fungus Rhizopus oligosporus, producer of the delicious tempeh food, is a prolific
amylase enzyme producer and is known to be free of mycotoxin production, such as
aflatoxin. From pilot plant experiments with cassava tuber containing 65% starch it was
calculated that 1 ha bearing 65 t cassava tuber can produce 3,500 kg of microbial protein
with highly productive amylase enzymes to convert approximately 39,000 t of grain or
cassava tuber into glucose (Sukara & Doelle 1989a,b), which is equivalent of a 1200 ha
harvest and a glucose yield required for the production of 15.6 million litres ethanol.
Microbial biomass protein (MBP) as well as amylase enzymes could become income-
producing products in the local and export markets.
At present ndonesia alone spends millions of US dollars for the importation of these
enzymes.
The aqueous effluent can be used for ponding, as will be outlined below, as it contains
only nitrogen and phosphorous with traces of carbon.
Process 2: Ethanol production
Ethanol is gaining an ever increasing importance as fuel additive or even conventional
non-renewable fuel replacement. Ethanol is able to reduce significantly the oil import into
developing countries or can replace the present fuel allowing the government to save large
import costs or increasing the export market of their own oil, both of which will contribute
significantly towards a strengthening of foreign currency exchange (Doelle 1994).
There are two technologies available at present, the old traditional yeast [Saccharomyces
cerevisiae or others] fermentation and a newly developed bacterial ethanol fermentation
technology using Zymomonas mobilis (Doelle et al. 1993) isolated from tropical fruits.
Whereas the bacterium allows significantly higher ethanol production rates, produces less
biomass, has a higher ethanol tolerance and has a high protein content with a much higher
amino acid profile and no glycerol as by-products, the presently operating plants are using
the old traditional yeast technology.
The yeast technology converts approx. 90% of all glucose carbons into ethanol with the
bacterium increasing this to up to 98%.
Whichever technology is used, by-products [some call it wastes] are formed, mainly CO
2
,
microbial protein and aqueous effluent [or stillage]. Microbial Biomass Protein [MBP]
can be used as animal feed additive as the solid residue contains between 30-34%
protein, CO
2
can either be compressed to dry ice or transferred into a pond system (see
below) for algal cultivation. The stillage can be recycled partly, with yeast only about 30-
40% and for the bacterial fermentation up to 80%. Otherwise the stillage contains enough
nitrogen and phosphorous etc to be transferable as biofertiliser or into ponds.
EthanoI is onIy one of the many products [see chapter 16] which can be produced
depending on the IocaI market demand
Process 3: Biogas production
The basic core unit of any socio-economic integrated biosystem should be anaerobic
digestion, because the biggest and most health hazardous waste is the animal and
human waste. Anaerobic digestion (see also chapter 15 and 19) can now be carried out
mesophilic (35-40
o
C) and thermophilic (around 50
o
C). Here it is suggested to implement
the simplest and most proven technology of mesophilic anaerobic digestion. Depending on
the available waste, fermenter sizes in use at present range from a small family 6 m
3
to
commercial 1500-2000 m
o
. A very well managed anaerobic digester should produce 1 m
3

gas/m
3
volume and the biogas mixture should be 70% methane plus 30% CO
2
(Hobson &
Wheatley 1993).
Biogas is an excellent energy source and can be used to run generators for electricity
production as well as cooking in the households. Biogas behaves similar to natural gas,
but has a slightly higher calorific value. [see chapter 19]
Anaerobic digestion aIso heIps in prevention of infectious diseases caused by
pathogens occurring in human and animaIs wastes. The strict anaerobic conditions
required for a successfuI methane production kiIIs most pathogens responsibIe for
infectious diseases to deveIop.
Like all processes, anaerobic digestion also has unwanted products as it reduces the COD
in general only by 60%. There are solids as well as liquid effluent. Whereas the solids can
be used directly as biofertiliser, it would be preferred to be used as an enrichment of
composting first before utilising it as a biofertiliser. Composting (Miller 1991; Stentiford &
Dodds 1992) adds to the removal of pathogens, making the biofertiliser even safer.
The liquid effluent with its nitrogen and phosphorous content and high alkalinity is an
excellent source for algal production, which not only oxygenates the shallow pond but in
turn can also be used as an animal and/or fish feed (Thirumurthi 1991; Vonshak 1992;
Olguin et al. 1994).
Anaerobic digestion not onIy removes heaIth hazardous waste, but serves as an
exceIIent source of bioenergy, biofertiliser, compost, algae and fish production.
An algal waste treatment process can therefore be converted into a waste utilisation for
the production of high-quality protein and in the case of blue-green algae can be made into
a biofertiliser production unit to provide nitrogen replacing our chemical fertilisers.

In summary, the sagopaIm can provide the community with sago fIour for food, a
mushroom industry, bioenergy, enzyme industry, microbiaI biomass protein for
animaI feed additive, compost and effIuent for biofertiIiser and ethanoI as biofueI
amongst many other products [Doelle et al. 1993].It shouId be mentioned here, that
gIucose is an ideaI substrate for aII microorganisms and thus can be used to a
variety of product formations, incIuding biopoIymers such as dextran, antibiotics,
acetone, butanoI etc., some of which may require a too expensive downstream
processing, as weII as microbiaI biomass protein (EI-Nawawy 1992)..
4. Sugar containing crop - Sugarcane
Sugarcane is probably the most efficient plant in regards to photosynthesis in the plant
kingdom. t is also the most efficient plant for sugar and biofuel ethanol production.
The sugarcane is harvested with the tops and trash removed from the stem. The
sugarcane has to reach the sugarmill within 12 hrs to avoid the polymer dextran formation.
The stem is crushed and the juice is separated from the fibrous, lignocellulosic material
[see Figure 6] referred to as bagasse. Tops, trash and bagasse are predominantly
lignocellulosic by nature and can be used to produce energy through gasification [see
Section 4.1]. f enough energy through gasification is provided to make the mill self-
efficient, the excess material forms a good substrate for compost [see Section 4.3] or
mushroom production [see Section 4.2]. The sugarcane juice can either be used straight

Figure 9: Sugarcane Biorefinery [adapted from Doelle et al. 2000]
for biofueI ethanoI production as is practised in Brazil, or is being clarified first before
sugar production can commence. The resulting filter cake can be added to the top, trash
and bagasse, to enrich mushroom production or compost formation. The clarified
sugarcane juice, usually containing 10-14 per cent sucrose, is now concentrated by
evaporation to a sugary syrup containing approximately 82 percent fermentable sugars.
For the production of top quality sugar, the sugar is crystallised out and bleached, resulting
in a residue or A-molasses containing about 70-72 percent fermentable sugars. Depending
on the country and efficiency of sugar mills, lower grade sugar can be extracted resulting
in B- or C-molasses, the latter of which is also often referred to as blackstrap molasses.
Each step requires, of course, further concentration of the juice, which also increases salt
concentration and furfural formation. The residual molasses can be used as animal feed
addition or for human consumption.
Sugarcane juice as well as the different types of molasses can be fermented to biofuel
ethanol using either the old traditional yeast or new Zymomonas technology. A mutant of
the bacterium Zymomonas is also able to produce fructose and ethanol simultaneously
[Doelle et al. 1993; Doelle & Doelle 1989; Johns et al. 1991]. Using a combination of 350
g/l C-grade molasses with 20.8 g/l sugarcane syrup resulted in a 94.2 g/l fructose
accumulation giving a recovery yield of 95.7% (Doelle and Doelle 1991). At 300 g/l C-
grade molasses, the bacterial strain succeeded in a 99% fructose recovery yield with a
95% glucose to ethanol conversion efficiency.
Stillage, also known as mosto, vinasse or rum slopes, is the most problematic effluent in
the sugar industry, due to the high volume produced (12-13 l/ l ethanol), its high
biochemical oxygen demand (BOD) in the range of 30-40 g/l, and chemical oxygen
demand (COD) in the range of 60-100 g/l. (Olguin et al. 1995)
There are at least five different alternative technologies to recycle this highly pollutant
effluent. These alternatives range from low capital cost options to high capital cost
technologies. The simplest approach is the use of stillage as fertilizer which returns most
of the minerals back into the soil for the next crop. This option is being used by some
factories in Mexico and other coutries such as Zimbabwe (Boot and Lightfoot 1988).
However, the fertilizer option can develop problems by poor management of the irrigation
system, which has to be strictly controlled. Poor management can result in groundwater
contamination with nitrate and in soil column disruptions. Repeated applications of
sulphate rich stillages, for example, may result in a drop of the soil pH (Sastry and
Mohanrao 1964), whereas the application of anaerobically digested molasses stillages
may significantly increase the soil pH (Sweeney and Graetz 1988). n order to avoid the
problems derived from high mineral contents, a significant detoxification using nitrifying
bacteria such as Nitrosococcus oceanus) has been suggested (Aroral et al.1992).
Whenever capital investment is feasible, there are other technological options, which allow
a greater degree of resource recovery from stillage.. The choice of any of them may
depend on the available market for animal feeds, and the local requirement for energy,
and their economical feasibility.
t appears that recycling of stillage as a source of energy offers more advantages than
disadvantages. Here again, there is a low capital option which consists of evaporating the
residual stream to a solids content ranging from 50-60% prior to being injected into the
stillage fired boiler. Apart from generating energy, a high percentage of potassium salt may
be recuperated in the ash and sold as fertiliser.
Methane generation through anaerobic digestion has become one of the more convenient
options. Large-scale commercial reactors, such as the 3.5 million gallons anaerobic filter
set up by Bacardi Cooperation in Puerto Rico, have shown the viability of such systems
(Szendry 1984). The residual stillage from the anaerobic digestion can then be channelled
into high rate oxidation ponds as a polishing step with the recuperation of protein rich
Microalgae such as Spirulina (Olguin et al. 1994).
Sugarcane farms and mills can therefore be totally independent in regard to energy
requirements and biofertilisation and are also able not only to produce sugar, but also
mushrooms, compost and bioenergy.

5. Fatty Acid containing crop - OiI PaIm

Oil palm plantations and palm oil mill industries in SEAsia can predominantly be found in
Malaysia, Thailand and ndonesia. The important oil yield represents only 20% of the
original fresh fruit bunches [FFB], which includes the kernel oil [Kirkaldy and Sutanto
1976]. n Thailand alone, the oil palm plantations have a production yield of 1.5 x 106 t of
fresh fruit bunches for a total production of 304,000 t crude palm oil [Prasertsan and
Prasertsan 1996]. Each ton of FFB produces 280 kg empty fruit bunches [EFB), 120 kg
pericarp fibre, 80 kg shells as solid waste and 1 t of palm oil mill effluent [POME] as liquid
waste. This 1 t of liquid effluent has a BOD load of 27 kg, a COD load of 52 kg, 23 kg
suspended solids and 8 kg of oil and grease [Hanpongkittikun et al. 1996].

Figure 10: nput and Output of a Palm Oil Mill [Kirkaldy & Sutanto 1976]. Figures in
parenthesis indicate weight portions of original Fresh Fruit Bunches.
n order to overcome the massive solid waste problem, the technology of utilising EFB,
fibre and shells in boilers and incinerators was adopted (Sulaiman and Shafii 1987). To
produce 1 million t of palm oil, 5 million tons Fresh Fruit Bunches (FFB) are required,
producing 500,000 tons Empty Fruit Bunches (EFB), 500,000 tons pericarp fibre and
800,000 t palm shells, which produce altogether 211 MW electricity (Doelle et al. 2000).
The ash (5%) contains a substantial amount of potash fertiliser and is usually recycled to
the palm oil plantations (about 200,000 tons).
The first priority must be the cessation of incinerators, which burn the surplus solid
material not required for energy production in the mill and thus eliminate the worst air
polluter. n order to do so, a good look at the material indicates that the solid material could
be divided into two groups on their chemical composition. Whereas EFB and the fibre are
mainly lignocellulosic in nature, the main compponents of the shell and cake are volatile
materials and carbons.
The solid material (Figure 10), consisting mainly of lignocellulose, are very good resources
for mushroom production [apart from energy production as outlined in chapter 19), which is
a well established in industry in many countries around Thailand and is a growing industry
due to an increasing demand [see chapter 19]. The residue from the mushroom industry
can now be used for compost production [see chapter 19]. The humus obtained from
composting can further be improved through the use of earthworms [Rodriguez 1997a,b].
Eisenia foetida transforms the swallowed material into humic acid, an excellent soil
conditioner.
Whereas the solid material can be used for energy, mushroom and fertiliser production, a
solution has to be found for the liquid waste [POME]. The energy supply of the industry
can be more than satisfied using the alternative bioenergy methane or biogas produced
from liquid effluent using an anaerobic digester. Assuming 1 m
3
biogas is equivalent to 0.7
l fuel oil or 0.8 l petrol or 2.7 kg dry firewood or 0.3 m
3
propane gas [Petitpierre 1982], and
that the processing of 1 t FFB produces 1 t or 1 m
3
liquid waste [POME] with a COD of 52
kg, the production of biogas would be 52 x 0.3 = 15.6 m
3
/ t FFB. Since an average palm
oil mill has a capacity of processing 20 t FFB/h or 50,000 t FFB/year, 780,000 m
3
biogas
equivalent to 546,000 l fuel can be generated, if all this effluent is treated by closed
anaerobic digestion [Doelle et al.1998]. f the 780,000 m
3
biogas are run through an
electricity generator, it would operate a 200 kW (250 kva) generator for 9,828 h or in
excess of 24 h/d (fuel consumption being 0.275 l/kW) This energy consumption is far in
excess of the electricity required [Doelle et al. 2000; Doelle et al. 1998].
The sludge from the biodigester can now safely be used as biofertiliser, and the liquid
effluent containing approx. 40% of the original BOD can now be transferred into shallow
ponds for the production of algae. Algae such as Dunaliella, Spirulina, Azolla, Anabaena
etc are excellent nitrogen and phosphorous scavengers [Olguin et al. 1988; 1995, 1994;
srael 1997a,b]. They are an excellent source for protein, vitamins and carotene, and can
be used as health food, or as an animal feed supplement (eg. shrimps). Algae can also be
used as feed for the production of fish [Thirumurthi 1991; Vonshak 1992] where the
effluent from the shallow pond is transferred into a deeper pond [Chan 1993; Li Kangmin
1997; Wang et al. 1998; Moi 1987]. The design and cost of such systems can be relatively
low [Khan 1996] and of significant benefit to the industry [Chan 1997].

Figure 11: The use of clean microbial processes in the Palm Oil ndustry for a sustainable
and pollution-free industry with significant economic benefits (Doelle et al. 2000)

The kernel oil and any surplus from the palm oil produced can be used for biodiesel
production (see also chapter 15). Biodiesel can be made from new or used vegetable oils
[Biodiesel; Biofuels] and animal fats. Currently, biodiesel is being produced by a process
called transesterification. The vegetable oil is first filtered, then preprocessed with alkali to
remove free fatty acids. t is then mixed with an alcohol (methanol or ethanol) and a
catalyst (usually sodium or potassium hydroxide). The oil's triglycerides react to form
esters and gIyceroI, which are then separated from each other and purified.. Biodiesel is
given to these esters when they are intended for use as fuel. Glycerol, which is extensively
used in pharmaceuticals and cosmetics, is produced as a co-product. Biodiesel is
biodegradable, requires minimal engine modification when used either as blend or as is,
and is cleaner burning than the diesel it replaces.
The use of biodiesel [AFDC 2000; NREL 2000a,b] has grown dramatically during the last
few years particularly in the US and in Europe, where rape seed oil is the most common
raw material [Louwrier 2003].

6. Fish processing industry

Solid wastes in a seafood processing plant range from whole fish, off-cuts of fish, bone,
skin, and intestinal organs to heads, shells and pieces of shrimp waste. The non-shrimp
solid wastes are mainly sold to fishmeal factories, while shrimp wastes are used for the
production of chitin and chitosan, as well as chicken feed.

Figure 12: A simplified Bio-refinery scheme for a sustainable fish processing industry with
increasing economic benefits [adapted from Doelle et al. 2000]
These solid wastes are very rich in protein, one of the reasons why fish meal and fish
sauce has been known for a long time in SEAsia. Because of the relatively high hydrolytic
activities in these solid wastes, fish silage, fish sauce and protein hydrolysate production,
chitin and chitosan isolation and production of many enzymes, microbial biomass protein
(MBP), and oil flavour compounds could form a new waste re-use industry [Doelle et al.
2000; Figure 12).
The most useful microbial process for the treatment of the liquid waste, which is enormous
in the seafood industry, is the anaerobic digester system. Experiments have already
shown [Prasertsan et al. 1994] that a higher than 75% COD reduction could be obtained
up to an organic loading rate of 1 kg m
-3
day
-1
with an HRT of 11 days. ncreasing the
organic loading to 1.3 kg m
-3
day
-1
corresponds to an HRT of 6.6 days with a maximal
biogas production of 1.5 m
3
m
-3
day or a 1.3 m
3
biogas kg
-1
COD with a 65 % COD
reduction.
Tuna condensate could also be used first for the production of MBP such as the
photosynthetic bacterium Rhodocyclus gelatinosus [Prasertsan et al. 1993a,b], Bacillus
subtilis, Candida tropicalis [Sangsri et al. 1996] or other yeasts [Sujarit et al. 1996].
The effluent from the biodigester can then be transferred into a shallow pond for the
production of alga such as Chlorella [Ratanapradit et al. 1996] and photosynthetic
bacteria. Both microorganisms have a high content of chlorophyll, protein, vitamin and fatty
acid and would make an excellent feed for fish production in a second deeper pond or
could be sold to shrimp farmers.
Fish processing waste is therefore one of the richest resource materials for further
enzymatic and microbial process development [Dermlin et al. 1999]. These additional
product formation would undoubtedly raise the economic benefits, viability and
sustainability of the industry in a clean ecological environment.

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MICROBIAL METABOLISM AND BIOTECHNOLOGY

Horst W.DoeIIe, DSc, DSc [hc]

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