1. Fermentation Technology
623311
Yalun Arifin
Chemical Engineering Dept.
University of Surabaya

2. Course content Introduction General aspects of fermentation processes
Quantification of microbial rates
Stoichiometry of microbial growth and product formation
Black box growth
Growth and product formation
Heat transfer in fermentation
Mass transfer in fermentation
Unit operations in fermentation (introduction to downstream processing)
Bioreactor

3. Chapter I
Introduction

4. What is fermentation?
Pasteur’s 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

5. What is fermentation techniques (1)?
Techniques for large-scale production of microbial products. It must both provide an optimum environment for the microbial synthesis of the desired product and be economically feasible on a large scale. They can be divided into surface (emersion) and submersion techniques. The latter may be run in batch, fed batch, continuous reactors
In the surface techniques, the microorganisms are cultivated on the surface of a liquid or solid substrate. These techniques are very complicated and rarely used in industry

6. What is fermentation techniques (2)?
In the submersion processes, the microorganisms grow in a liquid medium. Except in traditional beer and wine fermentation, the medium is held in fermenters and stirred to obtain a homogeneous distribution of cells and medium. Most processes are aerobic, and for these the medium must be vigorously aerated. All important industrial processes (production of biomass and protein, antibiotics, enzymes and sewage treatment) are carried out by submersion processes.

7. Some important fermentation products
Organism
Use
Ethanol
Saccharomyces cerevisiae
Industrial solvents, beverages
Glycerol
Production of explosives
Lactic acid
Lactobacillus bulgaricus
Food and pharmaceutical
Acetone and butanol
Clostridium acetobutylicum
Solvents
-amylase
Bacillus subtilis
Starch hydrolysis

8. Some important fermentation products

9. Some important fermentation products

10. Some important fermentation products

11. Winemaking fermenter

12. General Aspects of Fermentation Processes
Chapter II
General Aspects of Fermentation Processes

13. Fermenter The heart of the fermentation process is the fermenter.
In general:
Stirred vessel, H/D  3
Volume m3 (80 % filled)
Biomass up to 100 kg dry weight/m3
Product 10 mg/l –200 g/l

14. 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
The size is few liters (laboratory use) - >500 m3 (industrial applications)

15. Cross section of a fermenter for Penicillin production ( Copyright:  

16. Cross section of a fermenter for Penicillin production ( Copyright:  

17. Flow sheet of a multipurpose fermenter and its auxiliary equipment

18. 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.

19. 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

20. 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)

21. Required value generation in fermenters as a function of size and productivity

22. Quantification of Microbial Rates
Chapter III
Quantification of Microbial Rates

23. Microbial rates of consumption or production
H2O H+
C, N, P, S source
biomass
CO2 O2
product
heat

24. What are the value of rates
What are the value of rates? Rates of consumption or production are obtained from mass balance over reactors
Mass balance over reactors
Transport + conversion = accumulation
(in – out) + (production – consumption) = accumulation
Batch: transport in = transport out = 0
Chemostat: accumulation = 0, steady state
Fed batch: transport out = 0

25. How are rates defined?
Rate (ri) = amount i per hour / volume of reactor
Biomass specific rate (qi)
qi = amount per hour / amount of organism in reactor
Thus:
Substrate (-rS) = (-qS)CX
Biomass rX = CX
Product rP = qPCX
Oxygen (-rO2) = (-qO2)CX
ri = qi CX

26. Yield = ratio of rates Yij =
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]

27. Stoichiometry of Microbial Growth and Product Formation
Chapter IV
Stoichiometry of Microbial Growth and Product Formation

28. 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).

29. Composition of biomass
Molecules
Protein %
Carbohydrate 5-30 %
Lipid 5-10 %
DNA 1 %
RNA 5-15 %
Ash (P, K+, Mg2+, etc)
Elements
C %
H %
O %
N %
P %
Ash %
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?

30. Anabolism Amino acids  protein Sugars  carbohydrate
Fatty acids  lipids
Nucleotides  DNA, RNA
Sum of all reactions gives the anabolic reaction
(…)C-source + (…)N-source + (…) P-source + O-source
C1H1.8O0.5N (…)H2O + (…)CO2
Thermodynamically, energy is needed. Also for cells maintenance
energy

31. 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/HCO3-
acceptor couple: O2/H2O
Glucose  (…)HCO3- + (…)ethanol
acceptor couple: CO2/ethanol
The catabolism produces Gibbs energy (Gcat.reaction)

32. 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.

33. Several examples stoichiometry of growth Aerobic growth on oxalate
5.815 C2O NH O H H2O
C1H1.8O0.5N HCO3-
What is C-source? N-source? Electron donor? Electron acceptor?
YSX = 1 C-mol X / mol oxalate = 1 C-mol X / C-mol oxalate
Catabolic reaction for oxalate:
C2O O2 + H2O  2HCO3-
or H2C2O O2  H2O + 2CO2

34. Aerobic growth on oxalate
Catabolism
3.715 C2O O H2O  7.43 HCO3-
Anabolism (total-catabolism)
2.1 C2O NH H H2O
C1H1.8O0.5N HCO3-
Fraction of catabolism: 3.715/5.815 = 64 %
Fraction of anabolism: 2.1/5.815 = 36 %

35. 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:
N-source, H2O, HCO3-, H+ and biomass are always present
Only substrate and electron acceptor are case specific
YSX is mostly available, all other coefficients follow the element or charge conservation

36. Aerobic growth of Pseudomonas oxalaticus using NH4+ and oxalate (C2O42-)
Electron donor couple?
Electron acceptor couple?
C-source? N-source?
YSX is gram biomass/ gram oxalate and biomass has 5 % ash. Biomass molecular weight = 24.6 g/C-mol X
YSX = C-mol X/mol oxalate

37. 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
f = mol oxalate/C-mol X
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

38. Microbial growth stoichiometry
Degree of reduction (i)

39. 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):
HCO3-/CO2
H+/OH-
NH4+/NH3
SO42-
Fe3+
N-source for growth
atom i C +4 H +1 O -2 N -3 S +6 Fe +3
+ charge -1
- charge
NH4+ as N-source
N2 as N-source
NO3- as N-source +5

40.  for compounds -balance 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)

41. Example Catabolism of glucose to ethanol in anaerobic culture
-C6H12O6 + aC2H6O +bCO2 + cH2O +dH+
glucose = 24,  ethanol = 12,  balance = a = 0, a = 2
b, c, d follow from C,O, and charge conservation
Thus: -C6H12O6 + 2 C2H6O + 2 CO2
Try to solve:
Catabolism of ethanol to acetate (C2H3O2-) using O2/H2O
Catabolism of H2S to S- using NO3-/NO2-
Anabolic reaction, glucose as C-source and electron donor
Complete growth reaction, aerobic growth on oxalate (C2O42-)

42. 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:
TOC, Carbon balance
Kj-N, Kjeldahl-nitrogen for all reduced nitrogen (organic bound and NH4+), N-balance
ThOD, COD balance (similar to  balance)