Presentation on theme: "Fermentation Technology"— Presentation transcript:
1 Fermentation Technology 623311Yalun ArifinChemical Engineering Dept.University of Surabaya
2 Course content Introduction General aspects of fermentation processes Quantification of microbial ratesStoichiometry of microbial growth and product formationBlack box growthGrowth and product formationHeat transfer in fermentationMass transfer in fermentationUnit operations in fermentation (introduction to downstream processing)Bioreactor
4 What is fermentation?Pasteur’s definition: “life without air”, anaerobe red ox reactions in organismsNew definition: a form of metabolism in which the end products could be further oxidizedFor 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 reactorsIn 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 OrganismUseEthanolSaccharomyces cerevisiaeIndustrial solvents, beveragesGlycerolProduction of explosivesLactic acidLactobacillus bulgaricusFood and pharmaceuticalAcetone and butanolClostridium acetobutylicumSolvents-amylaseBacillus subtilisStarch hydrolysis
12 General Aspects of Fermentation Processes Chapter IIGeneral Aspects of Fermentation Processes
13 Fermenter The heart of the fermentation process is the fermenter. In general:Stirred vessel, H/D 3Volume m3 (80 % filled)Biomass up to 100 kg dry weight/m3Product 10 mg/l –200 g/l
14 Types of fermenter Simple fermenters (batch and continuous) Fed batch fermenterAir-lift or bubble fermenterCyclone column fermenterTower fermenterOther more advanced systems, etcThe 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 mediumDefine medium nutritional, hormonal, and substratum requirement of cellsIn most cases, the medium is independent of the bioreactor design and process parametersThe 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 InoculumsIncoculum 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 mediumMutation (DNA recombinant, radiation, chemical addition)
21 Required value generation in fermenters as a function of size and productivity
22 Quantification of Microbial Rates Chapter IIIQuantification of Microbial Rates
23 Microbial rates of consumption or production H2OH+C, N, P, S sourcebiomassCO2O2productheat
24 What are the value of rates What are the value of rates? Rates of consumption or production are obtained from mass balance over reactorsMass balance over reactorsTransport + conversion = accumulation(in – out) + (production – consumption) = accumulationBatch: transport in = transport out = 0Chemostat: accumulation = 0, steady stateFed batch: transport out = 0
25 How are rates defined?Rate (ri) = amount i per hour / volume of reactorBiomass specific rate (qi)qi = amount per hour / amount of organism in reactorThus:Substrate (-rS) = (-qS)CXBiomass rX = CXProduct rP = qPCXOxygen (-rO2) = (-qO2)CXri = 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 IVStoichiometry of Microbial Growth and Product Formation
28 IntroductionCell 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 MoleculesProtein %Carbohydrate 5-30 %Lipid 5-10 %DNA 1 %RNA 5-15 %Ash (P, K+, Mg2+, etc)ElementsC %H %O %N %P %Ash %Typical composition biomass formula: C1H1.8O0.5N0.2Suppose 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 lipidsNucleotides DNA, RNASum of all reactions gives the anabolic reaction(…)C-source + (…)N-source + (…) P-source + O-sourceC1H1.8O0.5N (…)H2O + (…)CO2Thermodynamically, energy is needed. Also for cells maintenanceenergy
31 CatabolismCatabolism generates the energy needed for anabolism and maintenance. It consist of electron donor couple and electron donor acceptor coupleFor example:Glucose + (…)O2 (…)HCO3- + H2Odonor couple: glucose/HCO3-acceptor couple: O2/H2OGlucose (…)HCO3- + (…)ethanolacceptor couple: CO2/ethanolThe 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 maintenanceYSX is the result of anabolic/catabolic coupling.
33 Several examples stoichiometry of growth Aerobic growth on oxalate 5.815 C2O NH O H H2OC1H1.8O0.5N HCO3-What is C-source? N-source? Electron donor? Electron acceptor?YSX = 1 C-mol X / mol oxalate = 1 C-mol X / C-mol oxalateCatabolic reaction for oxalate:C2O O2 + H2O 2HCO3-or H2C2O O2 H2O + 2CO2
34 Aerobic growth on oxalate Catabolism3.715 C2O O H2O 7.43 HCO3-Anabolism (total-catabolism)2.1 C2O NH H H2OC1H1.8O0.5N 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 reactantNote:N-source, H2O, HCO3-, H+ and biomass are always presentOnly substrate and electron acceptor are case specificYSX 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 XYSX = 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 ff = mol oxalate/C-mol XThere are 5 unknowns (a, b, c, d, e) and 5 conservation balance (C, H, O, N, charge). For example:C : 2f = 1 + eH? 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 bioreactionsStoichiometric quantity of compound IElectron content of compound i relative to referenceThe references (i = 0):HCO3-/CO2H+/OH-NH4+/NH3SO42-Fe3+N-source for growthatomiC+4H+1O-2N-3S+6Fe+3+ charge-1- chargeNH4+ as N-sourceN2 as N-sourceNO3- as N-source+5
40 for compounds -balance For example: glucose (C6H12O6) glucose = 6(4) + 12(1) + 6(-2) = 24 = 4/C-glucoseBiomass? O2? Fe2+? Citric acid? Ethanol? Lactic acid?-balanceIt is used to calculate stoichiometryIt follows from conservation relations (C, H, O, N, charge, etc) by eliminating the unknown stoichiometric coefficient for reference compoundsIt 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 = 2b, c, d follow from C,O, and charge conservationThus: -C6H12O6 + 2 C2H6O + 2 CO2Try to solve:Catabolism of ethanol to acetate (C2H3O2-) using O2/H2OCatabolism of H2S to S- using NO3-/NO2-Anabolic reaction, glucose as C-source and electron donorComplete growth reaction, aerobic growth on oxalate (C2O42-)
42 Further readingStoichiometry calculations in undefined chemical systems for fermentation with complex medium, biological waste water treatment, and soluble and non-soluble compoundsMeasurements of lumped quantities:TOC, Carbon balanceKj-N, Kjeldahl-nitrogen for all reduced nitrogen (organic bound and NH4+), N-balanceThOD, COD balance (similar to balance)