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623311 Yalun Arifin Chemical Engineering Dept. University of Surabaya
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
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
It must both provide an optimum environment for the microbial synthesis of the desired product and be economically feasible on a large scale. fed batch. continuous reactors In the surface techniques.What is fermentation techniques (1)? Techniques for large-scale production of microbial products. The latter may be run in batch. the microorganisms are cultivated on the surface of a liquid or solid substrate. They can be divided into surface (emersion) and submersion techniques. These techniques are very complicated and rarely used in industry 5 .
the microorganisms grow in a liquid medium. Most processes are aerobic. All important industrial processes (production of biomass and protein. antibiotics. 6 . and for these the medium must be vigorously aerated.What is fermentation techniques (2)? In the submersion processes. Except in traditional beer and wine fermentation. enzymes and sewage treatment) are carried out by submersion processes. the medium is held in fermenters and stirred to obtain a homogeneous distribution of cells and medium.
beverages Glycerol Lactic acid Acetone and butanol -amylase Saccharomyces cerevisiae Lactobacillus bulgaricus Clostridium acetobutylicum Bacillus subtilis Production of explosives Food and pharmaceutical Solvents Starch hydrolysis 7 .Some important fermentation products Product Ethanol Organism Saccharomyces cerevisiae Use Industrial solvents.
Some important fermentation products 8 .
Some important fermentation products 9 .
Some important fermentation products 10 .
Winemaking fermenter 11 .
Chapter II General Aspects of Fermentation Processes 12 .
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 13 .
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.>500 m3 (industrial applications) 14 . etc The size is few liters (laboratory use) .
co.htm) 15 .uk/webwise/spinneret/microbes/penici.ukonline.Cross section of a fermenter for Penicillin production ( Copyright: http://web.
ukonline.htm) 16 .uk/webwise/spinneret/microbes/penici.Cross section of a fermenter for Penicillin production ( Copyright: http://web.co.
Flow sheet of a multipurpose fermenter and its auxiliary equipment 17 .
and downstream processing. yield. hormonal.Fermentation medium • Define medium nutritional. operational parameters. product quality. 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. and substratum requirement of cells • In most cases. 18 .
N. Mg sources water. H. P. transport proteins. vitamins) • Additional factors: growth factors. lipid. S. sugars. oxygen is sparged 19 .Medium composition Fermentation medium consists of: • Macronutrients (C. amino acids. attachment proteins. etc) For aerobic culture. salt minerals) • Micronutrients (trace elements/ metals.
Inoculum is prepared for the inoculation before the fermentation starts. chemical addition) 20 .Inoculums Incoculum is the substance/ cell culture that is introduced to the medium. The cell then grow in the medium. conducting metabolisms. radiation. It needs to be optimized for better performance: • Adaptation in the medium • Mutation (DNA recombinant.
Required value generation in fermenters as a function of size and productivity 21 .
Chapter III Quantification of Microbial Rates 22 .
P.Microbial rates of consumption or production H2O C. N. S source H+ biomass CO2 O2 heat product 23 .
steady state Fed batch: transport out = 0 24 .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.
X Thus: ri = qi CX Substrate (-rS) = (-qS)CX Biomass Product rX = CX rP = qPCX 25 Oxygen (-rO2) = (-qO2)CX .How are rates defined? Rate (ri) = amount i per hour / volume of reactor kg .i / hour kg .i / hour m3 reactor Biomass specific rate (qi) qi = amount per hour / amount of organism in reactor kg .
i ri qiC X 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] 26 .Yield = ratio of rates Yij = rj q jC X qj rate. j rate.
Chapter IV Stoichiometry of Microbial Growth and Product Formation 27 .
Several definitions have to be well understood before studying this chapter. maintenance coefficient based on substrate (ms).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. 28 . for example: YSXmax. The complexity of the reactions can be represented by a simple pseudochemical equation. Thermodynamic limit is important for process optimization. YOX. YATP X.
what is the amount of organic matter in C-mol biomass? 29 . Mg2+.2 Suppose 1 kg dry biomass contains 5 % ash. 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.Composition of biomass Molecules • Protein 30-60 % • Carbohydrate 5-30 % • Lipid 5-10 % • DNA 1 % • RNA 5-15 % • Ash (P.8O0.5N0. K+.
5N0. RNA Sum of all reactions gives the anabolic reaction (…)C-source + (…)N-source + (…) P-source + O-source energy C1H1.2 + (…)H2O + (…)CO2 Thermodynamically.Anabolism Amino acids protein Sugars carbohydrate Fatty acids lipids Nucleotides DNA. Also for cells maintenance 30 . energy is needed.8O0.
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.Catabolism Catabolism generates the energy needed for anabolism and maintenance.reaction) 31 .
organic substrate) Only a fraction of the substrate ends in biomass as C-source.Coupled anabolism/catabolism C-source (anabolism) and electron-donor (catabolism) are often the same (e. 32 .g. while the rest is catabolized as electron-donor to provide energy for anabolism and maintenance YSX is the result of anabolic/catabolic coupling.
63 HCO3What is C-source? N-source? Electron donor? Electron acceptor? YSX = 1 C-mol X / 5.5N0.5 O2 + H2O 2HCO3or H2C2O4 + 0.815 C2O42.8O0.815 mol oxalate = 1 C-mol X / 11.8575 O2 + 0.415 H2O C1H1.+ 0.2 NH4+ + 1.5 O2 H2O + 2CO2 33 .2 + 10.63 Cmol oxalate Catabolic reaction for oxalate: C2O42.+ 0.8 H+ + 5.Several examples stoichiometry of growth Aerobic growth on oxalate 5.
815 = 64 % Fraction of anabolism: 2.715 C2O42.2 + 3.715/5.+ 0.2 NH4+ + 0.700 H2O C1H1.+ 1.43 HCO3Anabolism (total-catabolism) 2.715 H2O 7.8O0.Aerobic growth on oxalate Catabolism 3.1/5.8575 O2 + 3.2 HCO3Fraction of catabolism: 3.1 C2O42.5N0.815 = 36 % 34 .8 H+ + 1.
HCO3-. (…) < 0 for reactant Note: 1.+ (…)H+ + C1H1. H2O.5N0. all other coefficients follow the element or charge conservation 35 .2 + (…)oxidized substrate + (…)reduced acceptor (…) > 0 for product. Only substrate and electron acceptor are case specific 3.8O0.Microbial growth stoichiometry using conservation principles The general equation for growth stoichiometry -1/YSX substrate + (…)N-source + (…)electron acceptor + (…)H2O + (…)HCO3. YSX is mostly available. N-source. H+ and biomass are always present 2.
6 YSX = 36 .0506 * 88 * 0. Biomass molecular weight = 24.0506 gram biomass/ gram oxalate and biomass has 5 % ash.6 g/C-mol X 0.95 0.172 C-mol X/mol oxalate 24.Aerobic growth of Pseudomonas oxalaticus using NH4+ and oxalate (C2O42-) Electron donor couple? Electron acceptor couple? C-source? N-source? YSX is 0.
charge).2 + e HCO3• Use YSX to calculate f f= 1 YSX 1 5.815 mol oxalate/C-mol X 0.• Set up the general stoichiometric equation f C2O42. c. N.172 • There are 5 unknowns (a. b. e) and 5 conservation balance (C. For example: C : 2f = 1 + e H? O? N? charge? • Solve for a. d.5N0.+ a NH4+ + b H+ + c O2 + d H2O C1H1. and e! • What is the value of respiratory quotient (RQ)? Remember RQ qCO2 qO2 37 .8O0. c. O. d. H. b.
Microbial growth stoichiometry Degree of reduction (i) 38 .
charge +1 -3 0 +5 39 NH4+ as N-source N2 as N-source NO3.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 atom i The references (i = 0): C +4 HCO3-/CO2 H +1 +/OHH O -2 NH4+/NH3 N -3 SO42S +6 Fe +3 Fe3+ + charge -1 N-source for growth .as N-source .
N-source are always absent) 40 . HCO3-. N. H+. substrate/donor. O. product (H2O. charge. H. 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. etc) by eliminating the unknown stoichiometric coefficient for reference compounds It relates biomass. acceptor.
balance = -24+12a = 0. Catabolism of H2S to S. c. a = 2 b. Complete growth reaction. Anabolic reaction.O. and charge conservation Thus: -C6H12O6 + 2 C2H6O + 2 CO2 Try to solve: a. ethanol = 12. glucose as C-source and electron donor d. d follow from C.Example Catabolism of glucose to ethanol in anaerobic culture -C6H12O6 + aC2H6O +bCO2 + cH2O +dH+ glucose = 24.using NO3-/NO2c. Catabolism of ethanol to acetate (C2H3O2-) using O2/H2O b. aerobic growth on oxalate (C2O42-) 41 .
COD balance (similar to balance) 42 . Kj-N. Carbon balance 2. TOC. N-balance 3.Further reading Stoichiometry calculations in undefined chemical systems for fermentation with complex medium. and soluble and non-soluble compounds Measurements of lumped quantities: 1. biological waste water treatment. Kjeldahl-nitrogen for all reduced nitrogen (organic bound and NH4+). ThOD.
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