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623311
Yalun Arifin
Chemical Engineering Dept.
University of Surabaya
1
Course content
I. Introduction
II. General aspects of fermentation processes
III. Quantification of microbial rates
IV. Stoichiometry of microbial growth and product
formation
V. Black box growth
VI. Growth and product formation
VII. Heat transfer in fermentation
VIII. Mass transfer in fermentation
IX. Unit operations in fermentation (introduction to
downstream processing)
X. Bioreactor
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Chapter I
Introduction
3
What is fermentation?
4
What is fermentation techniques (1)?
5
What is fermentation techniques (2)?
6
Some important fermentation products
8
Some important fermentation products
9
Some important fermentation products
10
Winemaking fermenter
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Chapter II
General Aspects of Fermentation
Processes
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Fermenter
The heart of the fermentation process is the fermenter.
In general:
• Stirred vessel, H/D 3
• Volume 1-1000 m3 (80 % filled)
• Biomass up to 100 kg dry weight/m3
• Product 10 mg/l –200 g/l
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Types of fermenter
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Cross section of a fermenter for Penicillin production ( Copyright:
http://web.ukonline.co.uk/webwise/spinneret/microbes/penici.htm)
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Flow sheet of a multipurpose fermenter and its
auxiliary equipment
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Fermentation medium
• Define medium nutritional, hormonal, and
substratum requirement of cells
• In most cases, the medium is independent of the
bioreactor design and process parameters
• The type: complex and synthetic medium (mineral
medium)
• Even small modifications in the medium could
change cell line stability, product quality, yield,
operational parameters, and downstream processing.
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Medium composition
Fermentation medium consists of:
• Macronutrients (C, H, N, S, P, Mg sources water,
sugars, lipid, amino acids, salt minerals)
• Micronutrients (trace elements/ metals, vitamins)
• Additional factors: growth factors, attachment
proteins, transport proteins, etc)
For aerobic culture, oxygen is sparged
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Inoculums
Incoculum is the substance/ cell culture that is
introduced to the medium. The cell then grow in the
medium, conducting metabolisms.
Inoculum is prepared for the inoculation before the
fermentation starts.
It needs to be optimized for better performance:
• Adaptation in the medium
• Mutation (DNA recombinant, radiation, chemical
addition)
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Required value generation in fermenters as a
function of size and productivity
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Chapter III
Quantification of Microbial Rates
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Microbial rates of consumption or production
H2O
H+
C, N, P, S source
biomass
CO2
O2 product
heat
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What are the value of rates?
Rates of consumption or production are obtained from
mass balance over reactors
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How are rates defined?
kg.i / hour
Rate (ri) = amount i per hour / volume of reactor m3 reactor
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Chapter IV
Stoichiometry of Microbial Growth and
Product Formation
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Introduction
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Composition of biomass
Molecules • Elements
• Protein 30-60 % • C 40-50 %
• Carbohydrate 5-30 % • H 7-10 %
• Lipid 5-10 % • O 20-30 %
• DNA 1 % • N 5-10 %
• RNA 5-15 % • P 1-3 %
• Ash (P, K+, Mg2+, etc) • Ash 3-10%
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Several examples stoichiometry of growth
Aerobic growth on oxalate
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Aerobic growth on oxalate
Catabolism
3.715 C2O42- + 1.8575 O2 + 3.715 H2O 7.43 HCO3-
Anabolism (total-catabolism)
2.1 C2O42- + 0.2 NH4+ + 0.8 H+ + 1.700 H2O
C1H1.8O0.5N0.2 + 3.2 HCO3-
Fraction of catabolism: 3.715/5.815 = 64 %
Fraction of anabolism: 2.1/5.815 = 36 %
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Microbial growth stoichiometry using
conservation principles
The general equation for growth stoichiometry
-1/YSX substrate + (…)N-source + (…)electron acceptor +
(…)H2O + (…)HCO3- + (…)H+ + C1H1.8O0.5N0.2 +
(…)oxidized substrate + (…)reduced acceptor
(…) > 0 for product, (…) < 0 for reactant
Note:
1. N-source, H2O, HCO3-, H+ and biomass are always present
2. Only substrate and electron acceptor are case specific
3. YSX is mostly available, all other coefficients follow the
element or charge conservation
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Aerobic growth of Pseudomonas oxalaticus
using NH4+ and oxalate (C2O42-)
Electron donor couple?
Electron acceptor couple?
C-source? N-source?
YSX is 0.0506 gram biomass/ gram oxalate and biomass has 5
% ash. Biomass molecular weight = 24.6 g/C-mol X
0.0506 * 88 * 0.95
YSX = 0.172 C-mol X/mol oxalate
24.6
36
• Set up the general stoichiometric equation
f C2O42- + a NH4+ + b H+ + c O2 + d H2O C1H1.8O0.5N0.2 +
e HCO3-
• Use YSX to calculate f
1 1
f= 5.815 mol oxalate/C-mol X
YSX 0.172
• There are 5 unknowns (a, b, c, d, e) and 5 conservation
balance (C, H, O, N, charge). For example:
C : 2f = 1 + e
H? O? N? charge?
• Solve for a, b, c, d, and e!
• What is the value of respiratory quotient (RQ)? Remember
qCO2
RQ
qO2
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Microbial growth stoichiometry
38
What is degree of reduction (i)?
• It is about proton-electron balance in bioreactions
• Stoichiometric quantity of compound I
• Electron content of compound i relative to reference
The references (i = 0): atom i
HCO3-/CO2 C +4
+ - H +1
H /OH
O -2
NH4+/NH3 N -3
SO42- S +6
Fe3+ Fe +3
+ charge -1
N-source for growth
- charge +1
NH4+ as N-source -3
N2 as N-source 0
NO3- as N-source +5
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for compounds
For example: glucose (C6H12O6)
glucose = 6(4) + 12(1) + 6(-2) = 24 = 4/C-glucose
Biomass? O2? Fe2+? Citric acid? Ethanol? Lactic acid?
-balance
It is used to calculate stoichiometry
It follows from conservation relations (C, H, O, N, charge, etc)
by eliminating the unknown stoichiometric coefficient for
reference compounds
It relates biomass, substrate/donor, acceptor, product
(H2O, H+, HCO3-, N-source are always absent) 40
Example
Catabolism of glucose to ethanol in anaerobic culture
-C6H12O6 + aC2H6O +bCO2 + cH2O +dH+
glucose = 24, ethanol = 12, balance = -24+12a = 0, a = 2
b, c, d follow from C,O, and charge conservation
Thus: -C6H12O6 + 2 C2H6O + 2 CO2
Try to solve:
a. Catabolism of ethanol to acetate (C2H3O2-) using O2/H2O
b. Catabolism of H2S to S- using NO3-/NO2-
c. Anabolic reaction, glucose as C-source and electron donor
d. Complete growth reaction, aerobic growth on oxalate
(C2O42-)
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Further reading
42