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Course content
I. II. III. IV. Introduction General aspects of fermentation processes Quantification of microbial rates 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
What is fermentation?
Pasteurs definition: life without air, anaerobe red ox reactions in organisms New definition: a form of metabolism in which the end products could be further oxidized For example: a yeast cell obtains 2 molecules of ATP per molecule of glucose when it ferments it to ethanol
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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
Simple fermenters (batch and continuous) Fed batch fermenter Air-lift or bubble fermenter Cyclone column fermenter Tower fermenter Other more advanced systems, etc
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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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Chapter III
Quantification of Microbial Rates
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biomass CO2
O2 heat
product
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reactor
Biomass specific rate (qi) qi = amount per hour / amount of organism in reactor Thus: ri = qi CX
kg.i / hour kg. X
qj qi
YSX = rate of biomass production / rate of substrate consumption [g biomass/g substrate] YOX = rate of biomass production / rate of oxygen consumption [g biomass/g oxygen]
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Chapter IV
Stoichiometry of Microbial Growth and Product Formation
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Introduction
Cell growth and product formation are complex processes reflecting the overall kinetics and stoichiometry of the thousands of intracellular reactions that can be observed within a cell. Thermodynamic limit is important for process optimization. The complexity of the reactions can be represented by a simple pseudochemical equation. Several definitions have to be well understood before studying this chapter, for example: YSXmax, YATP X, YOX, maintenance coefficient based on substrate (ms).
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Composition of biomass
Molecules Protein 30-60 % Carbohydrate 5-30 % Lipid 5-10 % DNA 1 % RNA 5-15 % Ash (P, K+, Mg2+, etc) Elements C 40-50 % H 7-10 % O 20-30 % N 5-10 % P 1-3 % Ash 3-10%
Typical composition biomass formula: C1H1.8O0.5N0.2 Suppose 1 kg dry biomass contains 5 % ash, what is the amount of organic matter in C-mol biomass?
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Anabolism
Amino acids protein Sugars carbohydrate Fatty acids lipids Nucleotides DNA, RNA
()C-source + ()N-source + () P-source + O-source C1H1.8O0.5N0.2 + ()H2O + ()CO2 Thermodynamically, energy is needed. Also for cells maintenance
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Catabolism
Catabolism generates the energy needed for anabolism and maintenance. It consist of electron donor couple and electron donor acceptor couple For example: Glucose + ()O2 ()HCO3- + H2O
donor couple: glucose/HCO3acceptor couple: O2/H2O Glucose ()HCO3- + ()ethanol donor couple: glucose/HCO3acceptor couple: CO2/ethanol The catabolism produces Gibbs energy ((Gcat.reaction)
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Coupled anabolism/catabolism
C-source (anabolism) and electron-donor (catabolism) are often the same (e.g. organic substrate) Only a fraction of the substrate ends in biomass as C-source, while the rest is catabolized as electron-donor to provide energy for anabolism and maintenance YSX is the result of anabolic/catabolic coupling.
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H2O + 2CO2
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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 ! 0 .172 C-mol X/mol oxalate 24 . 6
YSX =
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Set up the general stoichiometric equation f C2O42- + a NH4+ + b H+ + c O2 + d H2O HCO3 Use YSX to calculate f f=
1 YSX 1 ! ! 5.815 mol oxalate/C-mol X 0.172
C1H1.8O0.5N0.2 + e
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
RQ ! qCO2 qO2
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K for compounds
For example: glucose (C6H12O6) K glucose = 6(4) + 12(1) + 6(-2) = 24 = 4/C-glucose Biomass? O2? Fe2+? Citric acid? Ethanol? Lactic acid?
K-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)
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Example
Catabolism of glucose to ethanol in anaerobic culture -C6H12O6 + aC2H6O +bCO2 + cH2O +dH+ K glucose = 24, K ethanol = 12, K 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-/NO2c. Anabolic reaction, glucose as C-source and electron donor d. Complete growth reaction, aerobic growth on oxalate (C2O42-)
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Further reading
Stoichiometry calculations in undefined chemical systems for fermentation with complex medium, biological waste water treatment, and soluble and non-soluble compounds Measurements of lumped quantities: 1. TOC, Carbon balance 2. Kj-N, Kjeldahl-nitrogen for all reduced nitrogen (organic bound and NH4+), N-balance 3. ThOD, COD balance (similar to K balance)
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