Fermentation Technology 623311 Yalun Arifin Chemical Engineering Dept

Fermentation Technology 623311 Yalun Arifin Chemical Engineering Dept. University of Surabaya 1 12/4/2020

Course content I. 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 2

Chapter I Introduction 3 12/4/2020

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 4

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 5

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. 6

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

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 12/4/2020

Fermenter The heart of the fermentation process is the fermenter. In general: • Stirred vessel, H/D 3 • Volume 1 -1000 m 3 (80 % filled) • Biomass up to 100 kg dry weight/m 3 • 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, etc The size is few liters (laboratory use) - >500 m 3 (industrial applications) 14

Cross section of a fermenter for Penicillin production ( Copyright: http: //web. ukonline. co. uk/webwise/spinneret/microbes/penici. htm) 15

Cross section of a fermenter for Penicillin production ( Copyright: http: //web. ukonline. co. uk/webwise/spinneret/microbes/penici. htm) 16

Flow sheet of a multipurpose fermenter and its auxiliary equipment 17

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. 18

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 19

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) 20

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

Chapter III Quantification of Microbial Rates 22 12/4/2020

Microbial rates of consumption or production H 2 O H+ C, N, P, S source biomass CO 2 product heat 23

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 24

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: ri = qi CX Substrate (-r. S) = (-q. S)CX Biomass r. X = CX Product r. P = q. PCX Oxygen (-r. O 2) = (-q. O 2)CX 25

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] 26

Chapter IV Stoichiometry of Microbial Growth and Product Formation 27 12/4/2020

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). 28

Composition of biomass Molecules • Protein 30 -60 % • Carbohydrate 5 -30 % • Lipid 5 -10 % • DNA 1 % • RNA 5 -15 % • Ash (P, K+, Mg 2+, etc) • • Elements C 40 -50 % H 7 -10 % O 20 -30 % N 5 -10 % P 1 -3 % Ash 3 -10% Typical composition biomass formula: C 1 H 1. 8 O 0. 5 N 0. 2 Suppose 1 kg dry biomass contains 5 % ash, what is the amount of organic matter in C-mol biomass? 29

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 energy C 1 H 1. 8 O 0. 5 N 0. 2 + (…)H 2 O + (…)CO 2 Thermodynamically, energy is needed. Also for cells maintenance 30

Catabolism generates the energy needed for anabolism and maintenance. It consist of electron donor couple and electron donor acceptor couple For example: • Glucose + (…)O 2 (…)HCO 3 - + H 2 O donor couple: glucose/HCO 3 - acceptor couple: O 2/H 2 O • Glucose (…)HCO 3 - + (…)ethanol donor couple: glucose/HCO 3 - acceptor couple: CO 2/ethanol The catabolism produces Gibbs energy ( Gcat. reaction) 31 12/4/2020

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. 32

Several examples stoichiometry of growth Aerobic growth on oxalate 5. 815 C 2 O 42 - + 0. 2 NH 4+ + 1. 8575 O 2 + 0. 8 H+ + 5. 415 H 2 O C 1 H 1. 8 O 0. 5 N 0. 2 + 10. 63 HCO 3 What is C-source? N-source? Electron donor? Electron acceptor? YSX = 1 C-mol X / 5. 815 mol oxalate = 1 C-mol X / 11. 63 Cmol oxalate Catabolic reaction for oxalate: C 2 O 42 - + 0. 5 O 2 + H 2 O 2 HCO 3 - or H 2 C 2 O 4 + 0. 5 O 2 H 2 O + 2 CO 2 33

Aerobic growth on oxalate Catabolism 3. 715 C 2 O 42 - + 1. 8575 O 2 + 3. 715 H 2 O 7. 43 HCO 3 Anabolism (total-catabolism) 2. 1 C 2 O 42 - + 0. 2 NH 4+ + 0. 8 H+ + 1. 700 H 2 O C 1 H 1. 8 O 0. 5 N 0. 2 + 3. 2 HCO 3 Fraction of catabolism: 3. 715/5. 815 = 64 % Fraction of anabolism: 2. 1/5. 815 = 36 % 34

Microbial growth stoichiometry using conservation principles The general equation for growth stoichiometry -1/YSX substrate + (…)N-source + (…)electron acceptor + (…)H 2 O + (…)HCO 3 - + (…)H+ + C 1 H 1. 8 O 0. 5 N 0. 2 + (…)oxidized substrate + (…)reduced acceptor (…) > 0 for product, (…) < 0 for reactant Note: 1. N-source, H 2 O, HCO 3 -, 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 35

Aerobic growth of Pseudomonas oxalaticus using NH 4+ and oxalate (C 2 O 42 -) 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 YSX = C-mol X/mol oxalate 36

• Set up the general stoichiometric equation f C 2 O 42 - + a NH 4+ + b H+ + c O 2 + d H 2 O C 1 H 1. 8 O 0. 5 N 0. 2 + e HCO 3 - • 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 : 2 f = 1 + e H? O? N? charge? • Solve for a, b, c, d, and e! • What is the value of respiratory quotient (RQ)? Remember 37

Microbial growth stoichiometry Degree of reduction ( i) 38 12/4/2020

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 HCO 3 -/CO 2 H +1 + H /OH O -2 NH 4+/NH 3 N -3 SO 42 S +6 Fe +3 Fe 3+ + charge -1 N-source for growth - charge +1 NH 4+ as N-source -3 N 2 as N-source 0 NO 3 - as N-source +5 39

for compounds For example: glucose (C 6 H 12 O 6) glucose = 6(4) + 12(1) + 6(-2) = 24 = 4/C-glucose Biomass? O 2? Fe 2+? 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 (H 2 O, H+, HCO 3 -, N-source are always absent) 40

Example Catabolism of glucose to ethanol in anaerobic culture -C 6 H 12 O 6 + a. C 2 H 6 O +b. CO 2 + c. H 2 O +d. H+ glucose = 24, ethanol = 12, balance = -24+12 a = 0, a = 2 b, c, d follow from C, O, and charge conservation Thus: -C 6 H 12 O 6 + 2 C 2 H 6 O + 2 CO 2 Try to solve: a. Catabolism of ethanol to acetate (C 2 H 3 O 2 -) using O 2/H 2 O b. Catabolism of H 2 S to S- using NO 3 -/NO 2 c. Anabolic reaction, glucose as C-source and electron donor d. Complete growth reaction, aerobic growth on oxalate (C 2 O 42 -) 41

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 NH 4+), N-balance 3. Th. OD, COD balance (similar to balance) 42