Professional Documents
Culture Documents
Biotechnology Ebook
Biotechnology Ebook
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.
MICROBIAL METABOLISM AND BIOTECHNOLOGY
Horst W.DoeIIe, DSc, DSc [hc]
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
2.2.1 Nitrogen fixation
2.2.2 Symbiotic Nitrogen fixation
2.2.3 Ammonification
2.2.4 Nitrification
2.2.5 Denitrification
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
3.2.2 DetrimentaI 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.
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)
2.2.1 Nitrogen fixation
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
2.2.3 Ammonification
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.
2.2.5 Denitrification
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.
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
2. Present DeveIopment
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.
2.1 FundamentaIs in BiotechnoIogy
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).
2.2 AgricuIturaI BiotechnoIogy
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.
2.3 MedicaI BiotechnoIogy
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.
2.4 IndustriaI BiotechnoIogy
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.
2.5 EnvironmentaI BiotechnoIogy
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).
3. Trends for Future DeveIopment in BiotechnoIogy
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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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.
5. RecycIing CuItivation System
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).