Material Day 2 (MHD Taherzadeh)

27th July – 6th August
2021
www.taherzadeh.se Mohammad.Taherzadeh@hb.se
Tel/WhatsApp: +46-70-7171032
SSF for
Bulk Chemicals and Products
Borås
Mohammad J. Taherzadeh
Swedish Centre for Resource Recovery
University of Borås
Sweden
Mohammad.Taherzadeh@hb.se
Whatsapp: +46-707171032
Tel/WhatsApp: +46-70-7171032
List of content
• Introduction about Sweden, Univ. of Borås
• Our research activities incl. SSF
• Definition of SSF & SmF
• Factors to control SSF
• SSF in wastewater treatment: Trickling filter
• SSF for biogas: Dry digestion
• SSF for enzymes
• SSF for biopolymers
• SSF for biosurfactants
• SSF for organic acids
• SSF for pigments
Tel/WhatsApp: +46-70-7171032
Sweden
Tel/WhatsApp: +46-70-7171032
University of Borås
Tel/WhatsApp: +46-70-7171032
• Advanced area of research:
– Textile
– Library
– Resource Recovery
Tel/WhatsApp: +46-70-7171032
Wastes and by-
products
(Municipal, agricultural,
forest, industries)
Social Thermal Chemical Biological Material Logistic

aspects processes processes processes Recycling optimization
Biopolymers
Chemicals Feed
Biogas Ethanol
Heat Diesel Gasoline Biochemicals Food Metals
Chill Plastics
Electricity Papers
Glass
Sustainable
Society
Tel/WhatsApp: +46-70-7171032
crr.se
.s
www
Tel/WhatsApp: +46-70-7171032
Educations within
PhD in Resource Recovery
MSc in Resource Recovery (2 years)

- Sustainable Energy Processes
- Biotechnology and Bioeconomy
- Polymer Materials for the Circular Economy
BSc in Chemical Engineering–Applied Biotechnology

BSc in Energy Engineering
Tel/WhatsApp: +46-70-7171032
Unique PhD program in
Resource Recovery
A multidiciplinary PhD-program with
specialities in:
• Industrial Biotechnology
• Polymer technology
• Energy technology
• Resource Management
• Sustainable Built Environment
• Consumer behaviour
Tel/WhatsApp: +46-70-7171032
Biotechnology group (Apr. 2021)
• Seniors:
– Mohammad Taherzadeh • Current PhD students:
– Ilona Sarvari Horvath – Rebecca Gmoser
– Patrik Lennartsson – Gülru Bulkan
– Akram Zamani – Mohsen Parchami
– Päivi Ylitervo – Sofie Svensson
– Jorge Ferreira
– Hanie Moshtaghian
– Amir Mahboubi
– Neda Rousta
– Swarnima Agnihotri
– Sajjad Karimi
• Postdocs/Researchers:
– Clarisse Uwineza
– Lukitawesa
– Tugba Sapmaz
– Taner Şar
– Steven Wainaina – Ricky Wang
• + Visiting PhD students – Md Tahir Nazir
– Negar Basereh
– Normally 4-5 persons in Borås
– (from Indonesia, Poland, Brazil, Iran, – MT Asadollahzadeh
Malaysia, Germany Turkey, Nigeria, – Milad Parchami
China…)
Tel/WhatsApp: +46-70-7171032
Biotechnology group graduated PhDs since 2010
1. Mohammad Pourbafrani, 2010 16. Julius Akinbomi, 2015
2. Akram Zamani, 2010 17. Karthik Rajendran, 2015
3. Azam Jeihanipour, 2011 18. Jorge Ferreira, 2015
4. Patrik Lennartsson, 2012 19. Jhosane Pagés Díaz, 2015
5. Gergely Forgacs, 2012 20. Ramkumar B. Nair (2017)
6. Supansa Youngsukkasem, 2012 21. Regina J. Patinvoh (2017)
7. Anna Teghammar, 2013 22. Osagie Alex Osadolor (2018)
8. Isroi, 2013 23. Pedro Ferreira (2018)
9. Johan Westman, 2014 24. Veronika Bátori (2019)
10. Solmaz Aslanzadeh, 2014 25. Amir Mahboubi (2019)
11. Hamidreza Barghi, 2014 26. Konstantinos Chandolias (2019)
12. Päivi Ylitervo, 2014 27. Mostafa Jabbari (2020)
13. Mofoluwake Ishola, 2014 28. Steven Wainaina (2020)
14. Rachma Wikandari, 2014 29. Lukitawesa (2020)
15. Maryam Mohseni Kabir, 2015
Tel/WhatsApp: +46-70-7171032
Our vision:
Waste is a ”Resource”
but our knowledge is not enough to
utilize it!
Tel/WhatsApp: +46-70-7171032
Challenging wastes = Research subjects
Tel/WhatsApp: +46-70-7171032
Biotechnology group
Hydrogen
& VFA
Biogas &
Fertilizer
Residuals Biotech.
Ethanol
& Wastes group
Feed/Food
Biopolymers
Tel/WhatsApp: +46-70-7171032
Research platforms
Anaerobic digestion Fungi
• VFA • Waste as raw materials

• H2 • Ethanol & Feed/Food
• Membrane bioreactors • Biopolymers
• Syngas fermentation • Process development
• Rapid fermentation • Pellet formation
• Dry digestion • Pigment
• Textile reactors • Membrane bioreactors
• Pretreatment • Integration 1st & 2nd generations
• Process simulation ethanol
• Process simulation
Tel/WhatsApp: +46-70-7171032
Solid State Fermentation
vs
Submereged fermentation
Tel/WhatsApp: +46-70-7171032
Submerged fermentation (SmF)
https://popups.uliege.be/1780-4507/index.php?id=12469
Tel/WhatsApp: +46-70-7171032
Solid state fermentation (SSF)
Tel/WhatsApp: +46-70-7171032
Biofilm fermentation
Tel/WhatsApp: +46-70-7171032
SSF process categories
• Treatment and no product (e.g. wastewater):
– Trickling filters or filter bed using bacterial biofilms
• Production using bacteria:
– Dry digestion using bacteria to produce biogas
• Processes using fungi
– Enzymes
– Organic acids
– Food
– ….
Tel/WhatsApp: +46-70-7171032
SSF important factors to consider
• Batch or continuous process
– How to feed? (gas, liquid or solid)
– How to take out the products? (gas, liquid or solid)
Tel/WhatsApp: +46-70-7171032
• Water activity (aw)
– Honey (aw ≈ 0.6)
– Humid air (aw ≈ 0.7),
• the honey absorbs water from
the air.
– Salami (aw ≈ 0.87)
– Dry air (aw ≈ 0.5),
• the salami dries out
Tel/WhatsApp: +46-70-7171032
Tel/WhatsApp: +46-70-7171032
• Rule of thumb:
– Bacteria: More humid
– Fungi and molds: less humid
Tel/WhatsApp: +46-70-7171032
• Temperature:
– Microbial growth produce heat
– We will have large temperature gradients
– How to control the temperature?
Tel/WhatsApp: +46-70-7171032
How are these factors controlled
in a trickle filter?
Tel/WhatsApp: +46-70-7171032
Open bed biofilter to clean gas
with e.g. stinky odor
• We should keep it humid
(e.g. sparkling water)
• Gas to blow from bottom
• To consume
– NH3, VOCs, H2S
– Acetic acid, …
• Bacteria stick to solid
(biofilms)
Tel/WhatsApp: +46-70-7171032
• We should keep it humid
(e.g. sparkling water)
• Gas to blow from bottom
• To consume
– NH3, VOCs, H2S
– Acetic acid, …
• Bacteria stick to solid
(biofilms)
Tel/WhatsApp: +46-70-7171032
• Temp control by gas movement
• Beads to grow biofilms are e.g.
– Wood chips
– Bricks parts
– Metals etc.
Tel/WhatsApp: +46-70-7171032
Traditional Anaerobic digestion
Tel/WhatsApp: +46-70-7171032
Overview of biogas industry (biochemical processes)
Feeding Vehicle
in gas fuel
grid
Agricultural residues
(manure, slurry, etc.)
& biowastes
D
i Biogas upgrading
g
e
s Cooking
Biogas
Energy crops, grass t cleaning
Biogas
utilisation
Direct
burning
Lighting
Cooling
from Landscape Heating
maintenance (grass, i
maize, beets)
o
n Cogeneration
Electrica Thermal
Transpor Storage energy
t l energy
Spreadin Separation
g
liquid components back to the

Agriculture Gardening solid components
fermentation or spreading
become compost
Tel/WhatsApp: +46-70-7171032
Anaerobic digestion (AD) for methane production
Food wastes Sludge Lignocelluloses Manure…
Organic waste
Hydrolysis
Smaller compounds
(sugars, fatty acids and amino acids)
Acidogenesis Acetogenesis
H2 & Volatile fatty acids
(butyric, propionic, acetic acid etc.)
Methanogenesis
Biogas
(methane and CO2)
Tel/WhatsApp: +46-70-7171032
Dry or wet
digestion?
Tel/WhatsApp: +46-70-7171032
Wet digestion Total solid (TS)?
D
i
g
e
s
t
TS=10-12% i
o
r ? n
t e r ?
wa a te
of te w
n s
d i ti o w a
d TS=3-4% i th
A o w
o d
t t
h a
W
Tel/WhatsApp: +46-70-7171032
Dry digestion:
New technology with high TS!
Tel/WhatsApp: +46-70-7171032
Dry digestion Total solid (TS)
D
i
g
e
s
t
TS=30-50% i
o
d ed n
a d
e r r ry
a t lu
w o s
n o r n
o r TS=5-17% o
s s s
s l e
L e c h
M u
Tel/WhatsApp: +46-70-7171032
Dry digestion: Batch process
http://www.bioferm-energy.com/
Tel/WhatsApp: +46-70-7171032
Continuous dry digestion (horizontal)
Tel/WhatsApp: +46-70-7171032
Dry digestion (batch & continuous)
Novel textile bioreactor
Tel/WhatsApp: +46-70-7171032
Continuous reactors in lab
Tel/WhatsApp: +46-70-7171032
Example of anaerobic digestion results in
batch reactor
Novel textile bioreactor
v Feedstock – manure with straw

v No mixing
v Total volume – 90 L, working volume – 30 L
Tel/WhatsApp: +46-70-7171032
batch reactor
Total solid at different batch setup
Setup Feedstock Inoculum Reactor
(total solid) Mixture
TS (%) TS (%) TS (%)
Unacclimatised 22 7 10
inoculum
Acclimatised 30 9.6 17
(High TS)
Tel/WhatsApp: +46-70-7171032
batch reactor
22%; 10%
22%; 10% 30%; 17%
400
Theoretical methane yield
Cumulative methane yield (Nml/gVS)
82 %
350 theoretical
300 Acclimatised (High TS)
250
52 %
200 Unacclimatised Inoculum
150
100
50
0
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140
Batch duration (days)
(Paper IV and V)
Tel/WhatsApp: +46-70-7171032
Enzymes by SSF
Microorganisms produce external enzymes

mainly to degrade substrates
(macromolecules) and assimilate them
Tel/WhatsApp: +46-70-7171032
Share of enzyme production
Global enzymes market: USD 9.9 billion (in 2019)
Tel/WhatsApp: +46-70-7171032
Enzymes by SSF:
Cellulase family
• Cellulases, including exo- and endo-glucanase,
cellobiase, xylanase, beta-xylosidases…
https://doi.org/10.1016/j.biteb.2021.100652
Tel/WhatsApp: +46-70-7171032
Enzymes by SSF:
Cellulase family
Tel/WhatsApp: +46-70-7171032
Enzymes by SSF:
Cellulase family
Tel/WhatsApp: +46-70-7171032
Enzymes by bacteria (SSF & SmF):
Cellulase family
Ref: DOI: 10.9734/BMRJ/2013/2367
Tel/WhatsApp: +46-70-7171032
Enzymes by SSF:
Amylases act on starch
• Amylases, including:
– α-Amylase:
• breaks down long-chain saccharides
– β-Amylase:
• breaks starch into maltose (two glucose units)
– γ-Amylase
• cleave α(1–6) glycosidic linkages, and the last α-1,4 glycosidic bond at the
nonreducing end of amylose and amylopectin, yielding glucose
Tel/WhatsApp: +46-70-7171032
Enzymes by SSF:
Amylases act on starch
Bacteria Fermentation pH optimal/stability
Chromohalobacter sp. SSF 7.0 - 9.0
Bacillus sp. SSF 6.5
Bacillus subtilis SSF 7.0
Bacillus subtilis DM-03 SSF 6.0–10.0
Bacillus sp. AS-1 SSF 6.5
Fungi and yeast

Thermomyces lanuginosus SSF 6.0
Aspergillus niger SSF 5.5
Aspergillus sp. AS-2 SSF 6.0
Aspergillus oryzae 5.0 – 9.0
Aspergillus oryzae CBS570.64 SSF 7.0
Aspergillus oryzae NRRL 6270 SSF
Aspergillus oryzae CBS 125-59 SSF 6.0
Pycnoporus sanguineus SSF 5.0
https://doi.org/10.1590/S1517-83822010000400004
Tel/WhatsApp: +46-70-7171032
Microorganism
Other enzymes by SSF
Substrate Product
Bacillus subtilisNRC1aza Starch Levansucrase
Aspergillus heteromorphus MTCC 8818 Rosewood saw dust Tannase
Kluyveromyces marxianus var. marxianus Press mud Inulinase
Trichoderma harzianum Castor oil cake and sugarcane bagasse Lipase
Bacillus subtilis GXA-28 Soybean residue Fibrinolytic enzymes
Natural microbial fauna from raw sludge Soy fibers residue Alkaline protease
Aspergillus oryzaeMTCC 5341 Wheat bran Acid protease
Bacillus sp. UEB-S Millet Lichenase
Cladosporium sp. Wheat bran l-Asparaginase
Cladosporium sp. Wheat bran l-Glutaminase
Aspergillus niger Citrus peel Phytase
Aspergillus niger Apple pomace β-Mannanase
Pleurotus ostreatus Sugarcane bagasse Laccase
Colletotrichum lindemuthianum Shrimp shell chitin waste and wheat bran Chitin deacetylase
Aspergillus caespitosus Wheat bran Invertase
Trichoderma koningii Wheat bran and chitosan Chitosanase
Oerskovia xanthineolytica Chitinase
Aspergillus oryzae Red gram plant waste-wheat bran based medium α-Galactosidase, invertase
Thielavia terrestrisNRRL 8126 Chick pea seed α-Galactosidase
Streptomyces griseoloalbus Soybean flour α-Galactosidase
Streptomyces sp. NRC 13S Chicken feather Keratinase
Penicillium viridicatumRFC3 Orange bagasse and wheat bran Pectate lyase
Aspergillus fumigatusASH Agro-residues Homocysteine γ-lyase
https://doi.org/10.1016/j.bej.2013.10.013
Tel/WhatsApp: +46-70-7171032
Downstream processing: various options
https://doi.org/10.3390/su9020224
Tel/WhatsApp: +46-70-7171032
Biopolymers by SSF
• Exopolysaccharides (EPS):
– Applications e.g.antitumor, hypoglycemic, and
immunostimulant
– Produced by
• Fungi, especially mushrooms,
• bacteria, such as lactic acid bacteria (LAB)
Tel/WhatsApp: +46-70-7171032
Biopolymers by SSF
• Polyhydroxyalkanoates (PHA):
– Normally produced by SmF, but it is
expensive, so SSF can be the solution.
DOI: 10.1016/j.biortech.2009.03.088
Tel/WhatsApp: +46-70-7171032
Biosurfactants by SSF
• BioSurfactants are microbial compounds that lower
the surface tension (or interfacial tension) between two
liquids, between a gas and a liquid, or between a liquid
and a solid. BioSurfactants may act as
– detergents,
– wetting agents,
– emulsifiers,
– foaming agents,
– dispersants.
Tel/WhatsApp: +46-70-7171032
• Sophorolipids:
– glycolipids consist of a hydrophobic fatty acids (16-18 carbon)+
hydrophilicn head of Sophorose (a dimer of glucose)
• Surfactins:
– A powerful surfactant commonly used as an antibiotic
• Lipopeptides:
– A secondary metabolites with antimicrobial or cytotoxic activities.
• Rhamnolipids:
– With good sequestering, detergency, demulsifying, foaming,
emulsifying, thickening, metal, solubilizing, wetting, vesicle
forming and phase dispersion, are used in cosmetics, food,
pharmaceutical formulations etc.
Tel/WhatsApp: +46-70-7171032
Microorganism Substrate Biosurfactant
Starmerella bombicola Wheat bran, isabgol husk Sophorolipids
Bacillus amyloliquefaciens Rice straw and soybean flour Surfactins
Bacillus amyloliquefaciens Rice straw and soybean flour Lipopeptides
Pretreated molasses-based
Brevibacterium aureum Lipopeptides
medium
Okara and sugarcane bagasse

Bacillus pumilus Surfactins
based medium
Sugarcane bagasse and corn

Pseudomonas aeruginosa Rhamnolipids
bran based medium
Tel/WhatsApp: +46-70-7171032
Organic acids by SSF: Citric acid
Tel/WhatsApp: +46-70-7171032
Tel/WhatsApp: +46-70-7171032
• Major producer:
– Aspergillus niger
• Tray SSF:
– Example: Max citric acid (123.9 g/kg) was
achieved on peat moss with the aeration
rate of 0.84 vvm, bed depth of 22 cm and
fermentation temperature of 32 °C
• Rotary drum SSF:
– Max citric acid (220.6 g/kg dry solids) was
produced on apple pomace with
supplementation 3% methanol, intermittent
agitation of 1 h after every 12 h at 2 rpm, 1
vvm of aeration rate and 120 h incubation
time.
Tel/WhatsApp: +46-70-7171032
Horizontal drum bioreactor:

1, compressor; 2, air filter; 3, humidifier; 4, horizontal drum bioreactor;
5, axis; 6, motor; 7, speed controller; 8, air discharge; 9, silica gel column;
10, automatic injector; 11, gaseous chromatograph; 12, computer.
DOI: 10.1385/abab:118:1-3:293
Tel/WhatsApp: +46-70-7171032
Various steps in recovery of citric acid
https://biologyreader.com/production-of-citric-acid.html
Tel/WhatsApp: +46-70-7171032
Other organic acids by SSF
• Succinic acid
– Approved additive (E363), with a global production ca 25,000 tons per
year, used as an acidifier and taste enhancer in food like relishes,
beverages, and sausages.
– produced by Aspergillus awamori, A. oryze, A. succinogenes, ….
• Lactic acid
– Normally produced by SmF by Lactobacillus strains
– The bacteria on solid bed of e.g. tea wastes or pine needles + glucose
could grow and produce lactic acid, but still early phase
– Fungi e.g. Rhizopus oryzae produce lactic acid, but lower yield than
bacterial
• Several other organic acids e.g. oxalic acid or fumaric acids
are also produced by SSF
Tel/WhatsApp: +46-70-7171032
Natural pigments
• Many fungi produce pigments as secondary metabolites e.g.:
– Monascus purpureus
• Red pigments
– Neurospora intermedia
• Yellow to red pigments
Tel/WhatsApp: +46-70-7171032
Pigments are stress responds
RESULTS
• Pigments as secondary
metabolites: Light
– Additional protection against pH
environmental stresses, extreme C source
cultivation conditions. N source
• Many factors affect their C/N ratio
Temp.
production Metal ions
H2O2
Salts
Oxygen
https://doi.org/10.1007/s12010-019-02961-y
67
www.taherzadeh.se
Tel/WhatsApp: +46-70-7171032
Pigments are stress responds
EtOH
6.3 g/L thin stillage
Thin Stillage
Distillation
Separation Syrup Feed (DDGS)
Submerged
Fungal Biomass Fungal Product
(5 g/L) 33% proteins
fermentation
1.2 mg carotenoids/g total material
Surplus Bread
Solid-state fermentation
68
www.taherzadeh.se
Tel/WhatsApp: +46-70-7171032
Conclusion
• SSF gets more attention for production of various
metabolites (gas, liquid, solid)
• Environmental aspects and water use are main
driving forces
• SSF processes are developing to be more
economically feasible
• There are many products on the market by SSF
Tel/WhatsApp: +46-70-7171032
Thank you!
Questions?
Tel/WhatsApp: +46-70-7171032

Material Day 2 (MHD Taherzadeh)

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Material Day 2 (MHD Taherzadeh)

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Available Formats

27th July – 6th August

Social Thermal Chemical Biological Material Logistic

MSc in Resource Recovery (2 years)

BSc in Chemical Engineering–Applied Biotechnology

• VFA • Waste as raw materials

liquid components back to the

Food wastes Sludge Lignocelluloses Manure…

Novel textile bioreactor

v Feedstock – manure with straw

TS (%) TS (%) TS (%)

Microorganisms produce external enzymes

Global enzymes market: USD 9.9 billion (in 2019)

Ref: DOI: 10.9734/BMRJ/2013/2367

Fungi and yeast

Starmerella bombicola Wheat bran, isabgol husk Sophorolipids

Bacillus amyloliquefaciens Rice straw and soybean flour Surfactins

Bacillus amyloliquefaciens Rice straw and soybean flour Lipopeptides

Okara and sugarcane bagasse

Sugarcane bagasse and corn

Horizontal drum bioreactor:

Separation Syrup Feed (DDGS)

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