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Fermentation Technology

623311

Yalun Arifin, M.Sc

Chemical Engineering Dept.

University of Surabaya

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Course content

I. IntroductionII. General aspects of fermentation processesIII. Quantification of microbial ratesIV. Stoichiometry of microbial growth and product

formationV. Black box growthVI. Growth and product formationVII. Heat transfer in fermentationVIII. Mass transfer in fermentationIX. Unit operations in fermentation (introduction to

downstream processing)X. Bioreactor

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Chapter I

Introduction

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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

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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

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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.

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Some important fermentation products

Product Organism Use

Ethanol Saccharomyces cerevisiae

Industrial solvents, beverages

Glycerol Saccharomyces cerevisiae

Production of explosives

Lactic acid Lactobacillus bulgaricus

Food and pharmaceutical

Acetone and butanol

Clostridium acetobutylicum

Solvents

-amylase Bacillus subtilis Starch hydrolysis

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Some important fermentation products

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Some important fermentation products

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Some important fermentation products

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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

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

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

 

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

 

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Flow sheet of a multipurpose fermenter and its auxiliary equipment

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

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Chapter III

Quantification of Microbial Rates

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Microbial rates of consumption or production

C, N, P, S source

H2O

H+

O2

heat

product

CO2

biomass

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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

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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

reactorm

hourikg

3

/.

Xkg

hourikg

.

/.

ri = qi CX

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Yield = ratio of rates

Yij =

i

j

Xi

Xj

i

j

q

q

Cq

Cq

r

r

irate

jrate

.

.

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: YSX

max, 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

Sum of all reactions gives the anabolic reaction

(…)C-source + (…)N-source + (…) P-source + O-source

C1H1.8O0.5N0.2 + (…)H2O + (…)CO2

Thermodynamically, energy is needed. Also for cells maintenance

energy

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CatabolismCatabolism 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

donor couple: glucose/HCO3-

acceptor 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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Several examples stoichiometry of growthAerobic growth on oxalate

5.815 C2O42- + 0.2 NH4

+ + 1.8575 O2 + 0.8 H+ + 5.415 H2O

C1H1.8O0.5N0.2 + 10.63 HCO3-

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 C-mol oxalate

Catabolic reaction for oxalate:

C2O42- + 0.5 O2 + H2O 2HCO3

-

or H2C2O4 + 0.5 O2 H2O + 2CO2

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Aerobic growth on oxalate

Catabolism

3.715 C2O42- + 1.8575 O2 + 3.715 H2O 7.43 HCO3

-

Anabolism (total-catabolism)

2.1 C2O42- + 0.2 NH4

+ + 0.8 H+ + 1.700 H2O

C1H1.8O0.5N0.2 + 3.2 HCO3-

Fraction of catabolism: 3.715/5.815 = 64 %

Fraction of anabolism: 2.1/5.815 = 36 %

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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:

1. N-source, H2O, HCO3-, 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

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Aerobic growth of Pseudomonas oxalaticus using NH4

+ and oxalate (C2O42-)

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 oxalate172.06.24

95.0*88*0506.0

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• 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

815.5172.0

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SXY

2

2

O

CO

q

qRQ

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Microbial growth stoichiometry

Degree of reduction (i)

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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 +1

NH4+ as N-source -3

N2 as N-source 0

NO3- as N-source +5

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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, 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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ExampleCatabolism of glucose to ethanol in anaerobic culture

-C6H12O6 + aC2H6O +bCO2 + cH2O +dH+

glucose = 24, ethanol = 12, 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-/NO2

-

c. Anabolic reaction, glucose as C-source and electron donor

d. Complete growth reaction, aerobic growth on oxalate (C2O4

2-)

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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 balance)