Advanced

Chemical Reaction Engineering

Topic 2c
Bioreactors and Biosynthesis-
Cell Growth and Kinetics

(Chapter 9 Fogler’s Essentials)

Slides courtesy of Prof M L Kraft, Chemical & Biomolecular Engr Dept, University of Illinois, Urbana-Champaign.
 L10-2

Topic Learning Outcomes

After completing this topic, students will be able to:
• Discuss the pseudo-steady-state-hypothesis and
explain how it can be used to solve reaction
engineering problems.
• Write reaction pathways for complex reactions.
• Explain what an enzyme is and how it acts as a
catalyst.
• Describe the Michaelis-Menten enzyme kinetics and
rate law along with its temperature dependence.
• Discuss how to distinguish the different types of
enzyme inhibition.
• Discuss the stages of cell growth, the rate laws used to
describe growth and determine kinetic parameters
• Write material balances on cells, substrates, and
products in bioreactors to size chemostats and plot
concentration-time trajectories in batch reactors.
Slides courtesy of Prof M L Kraft, Chemical & Biomolecular Engr Dept, University of Illinois, Urbana-Champaign.
 Microbial Kinetics and Bioreactors
• Introduction to Process Biotechnology
– Advantages of Bioprocesses
– New Developments
– Overview of a Bioprocess
• Microbial Kinetics and Bioreactors
– Phases of Growth
– Growth Kinetics
– Batch Bioreactor
– Continuous Bioreactor: Growth and Substrate Uptake
– Monod kinetics with Inhibition
– Product Formation kinetics
• Various Bioreactor Designs and Applications
3
 Introduction to
Process Biotechnology

4
 Biochemical Engineering History
• 5000 to 10,000 BC: yogurt, cheese and soy products, wine
and beer.
• 1683:.Leeuwenhoek first names Fermentation: from Latin ->
”fervere” -> to boil (describing the anaerobic process of
yeast producing CO2 on fruit extracts)
• 1856 Pasteur demonstrates that different organisms
produce different fermentation products. (His commercial
applications include the "pasteurization" of wine as well as
milk.)
• In early 20th century: pure bakers yeast were being
produced in tanks and sold.
• In world war I: fermentation was used to produce chemicals
needed for war.
• World War II: antibiotics production on the commercial scale.
• 1970s: recombinant DNA technology
 Commercially important
Bio-Processes

1. Microbial cells or biomass as the product: e.g. Bakers yeast

2. Microbial enzymes: e.g. Amylase, protease, glucose isomerase

3. Microbial metabolites:
A. Primary metabolites: e.g. Ethanol, citric acid, amino
acids, vitamins
B. Secondary metabolites: All antibiotic fermentations

4. Recombinant products: e.g. Insulin, interferon

5. Bio-transformations: e.g. Steroid transformation

6
 Growth of Process Biotechnology
• The number of chemicals, agricultural products, and food
products produced by biosynthesis has risen dramatically.
• In 2007, companies in this sector raised over $24 billion of
new financing.
• Both microorganisms and mammalian cells are being used to
produce a variety of products, such as insulin, most
antibiotics, and polymers.
• It is expected that in the future a number of organic chemicals
currently derived from petroleum will be produced by living
cells.

7
 Advantages of Bioprocesses
• The advantages of bioconversions are mild reaction
conditions; high yields (e.g., 100% conversion of glucose to
gluconic acid with Aspergillus niger); and
• the fact that organisms contain several enzymes that can
catalyze successive steps in a reaction and, most
importantly, act as stereospecific catalysts.
• A common example of specificity in bioconversion
production of a single desired isomer that when produced
chemically yields a mixture of isomers is
• the conversion of cis-proenylphosphonic acid to the
antibiotic (-) cis- I ,2-epoxypropyl-phosphonic acid.

8
 Industrial Biotechnology- Recent Developments
• Bacteria can also be modified and turned into living
chemical factories . For example, using recombinant DNA,
Biotechnic International engineered a bacteria to produce
fertilizer by turning nitrogen into nitrates.
• Sapphire energy, the world’s first integrated algae-oil
production facility, has pilot growth ponds in New Mexico.
• In this process, they grow the algae, flocculate and
concentrate, extract and then refine it and convert it to fuel
oil in a liquid phase flow reactor.
• In 2009, ExxonMobil invested over 600 million dollars to
develop algae growth and harvest in waste ponds. It is
estimated that one acre of algae can provide 2,000 gallons
of gasoline per year.

9
 Overview of a Bioprocess
• In biosynthesis, the cells, also referred to as the biomass,
consume nutrients to grow and produce more cells and
important products.
• Internally, a cell uses its nutrients to produce energy and
more cells.
• This transformation of nutrients to energy and bioproducts is
accomplished through a cell's use of a number of different
enzymes in a series of reactions to produce metabolic
products.
• These products can either remain in the cell (intracellular) or
be secreted from the cells (extracellular). In the former
case, the cell must be lysed (ruptured) and the product
filtered and purified from the whole broth (reaction mixture).

10
Overview of a Bioprocess
UPSTREAM Stock
culture
Raw material Seed Fermenter

Shake
Medium preparation Flask
Inoculum
Sterilization

Bioreactor

DOWNSTREAM
Separation

Biomass Supernatant

Product Purification
 Microbial Growth - Growth Media
1. Main Constituents of Growth Media
 Water
 Nitrogen sources: nitrogen-containing compounds such as proteins
 Carbon & energy sources: such as carbohydrates, lipids, and proteins
 Mineral salts: phosphates, sulfates, potassium, magnesium, manganese &
iron
 Growth factors: may include vitamins, amino acids & nucleotides
2. Examples of Media Categories

A. Nutrient Broth: suitable for growth of many bacterial species

B. Selective Media: favor the growth of certain microorganisms and
prevent others
C. Differential Media: distinguish different growing microorganisms
12
 Growth media used for Bioreactors
There are two types of growth media used for production
1. Defined/Synthetic media
2. Complex media
Defined media contain specific amounts of pure chemical
compounds with known chemical compositions.
• Have better control of the bioprocess. Results are more
reproducible.
• Recovery and purification is much more cheaper and easier.
Complex media contain natural compounds whose chemical
composition is not exactly known e.g. yeast extract, peptone,
molasses, or corn steep liquor or palm oil.
• Can provide necessary growth factors, vitamins, hormones, and
trace elements.
• Result higher cell yields compared to defined medium. Product
Recovery is complex
13
3. Natural Raw Materials for Biotechnology:
• Growth medium can be formulated based on natural raw materials

• These may be:

1. Wastes: unwanted or unusable materials
2. By-products: a secondary product deriving from a manufacturing process

• They are mainly of agricultural, industrial or domestic origins

• They could be economic nutrient sources for industrial fermentation

• Getting benefits from a waste turns it into a by-product (it became

utilizable)

• Their utilization eliminates a major source of environmental pollution

• May require chemical or enzymatic pretreatments to become utilizable

 • Many of these substrates were successfully converted to:
Fuels (e.g. ethanol, methane)
Animal Feeds (e.g. single cell protein)
Enzymes (e.g. proteases, amylases)
Fertilizers (compost)

• Examples of natural raw substrates:

Nutrient Compound Raw material Origin

Carbon Sucrose • Sugarcane Agriculture

• Molasses Industry
Lactose • Milk/cheese whey Industry
Fats • Vegetable oil Industry
Hydrocarbons • Petroleum Industry
fractions
Starch & Cellulose • Straw Agriculture
• Maize cobs
Nitrogen Protein • Soybean meal Industry
• Wheat bran
• Milk/cheese whey
Types of Cells and
their Chemistry

16
 Classification of Microbes and Other Cells
• Taxonomy: deals with the organization of knowledge, naming
& classication of microbes. Important in patent litigations.
• Naming cells: e.g. Escherichia coli K 12
genus species strains sub-strains

• Broad Classification of microbes is based on morphology

(physical form and structure of microbial cells): Procaryotes
and Eucaryotes
• Morphology means physical form and structure of microbial
cells. Morphology affects mass transfer of nutrients into the
cells and fluid properties and fluid mechanics of the cell
suspension.
• Procaryotes: Bacteria, Actinomycetes & Blue green algae
• Eucaryotes: Fungi (yeast and mold), algae & protozoa.
• Animal cells(10 µ) and Plant cells(20 µ) are also eucaryotic.
• Viruses are very small and are obligate parasites. Viruses are
important in Bioprocessing, as they can destroy a whole batch.17
 Procaryotes & Eucaryotes
• Procaryotes
1) Simple structure:
size: 0.5 – 3µ
2) No nuclear membrane–
single chromosome
3) no organelles – (i.e. no
membrane-enclosed
structures which
carry out specialized
functions).
4) Mostly single-celled except
actinomycetes.
5) Can utilize a variety of
nutrients such as
carbohydrates, hydrocarbons,
proteins and CO2 as carbon
source
• Eucaryotes
1) More complex structure
size: 2 – 200 µ
2) True nuclear membrane
>1 chromosome
3) Contain specialized
organelles– mitochondria,
endoplasmic reticulum,
etc.
4) Can be both single-celled
organisms e.g. Yeast,
algae, protozoa; and multi-
cell systems e.g. Molds
(Fungi), Plants and
animals (multi-cell).
18
 Structure of a Procaryotic cell

CELL MEMBRANE: separates the inside from the cell’s

outside; CYTOPLASM: a homogeneous, clear jelly-like
material that fills cells. RIBOSOMES: make Proteins
NUCLEAR REGION: holds DNA
GAS VACUOLES: for buoyancy
SPORES & ENDOSPORES: resistant to heat & desiccation
19
INCLUSION BODIES: storage of energy or building blocks
 Organelles of a Eucaryotic Cell
NUCLEUS: holds
chromosomes (DNA)
MITOCHONDRIA:
perform respiration for
energy generation
ENDOPLASMIC
RETICULUM: holds
ribosomes for protein
synthesis
GOLGI BODY: packages
& modifies proteins for
secretion.
Structural features
Storage bodies: for
storage of energy.
20
 Chemistry of Life
• Life's Hierarchy
– Living systems are broken up into the following generalized
hierarchical levels:
– Moleculesorganelles  cells  tissues  organs, 
organisms  populations  communities  ecosystems 
biosphere.
• Cell: Cells are the fundamental units of life, because a cell is
the simplest unit capable of independent existence. All living
things are made of cells.
• Cells are 90% water. Of the remaining molecules
present, the dry weight is approximately:
– 50% protein (polymers of amino acids)
– 15% carbohydrate (mono and polysachharides)
– 15% nucleic acid (nucleotides, DNA & RNA)
– 10% lipid (polymers of fatty acids)
21– 10% miscellaneous items
 Cell’s Elemental Composition
• C, H, O & N are the four elements that make up most of the
composition of all living organisms.
• The other notable elements are phosphorus and sulphur.
• Total approximate composition by element:
– 8% H
– 20% O
– 50% C
– 14% N
– 3% P
– 1% S
– Small amounts of K+, Na+, Ca2+, Mg2+, Cl-, and vitamins.

22
Basics of Growth

23
Growth: basic concepts

Precursors

Anabolism = biosynthesis
Catabolism = reactions to
recover energy (often ATP)
 Batch Bioreactor

25
 Bioreactors

inoculum

t=0 time 

26
Cells + Substrate  More Cells + Product
 Bacterial growth
Most bacterial cells reproduce asexually by binary fission.
This involves several stages:

(i) increasing cell size (growth)

(ii) DNA replication, and
(iii) division (septum formation)
 Generation Time:
Time required for a cell population to double
DNA

DNA Replication

Cell Elongation
12

9 3 Septum Formation
6

Cell Separation
 Geometric progression
24
22 23

21

4
8 16
Bacterial growth

• The time taken for a microbial population to double in

number is called the doubling time. The time taken
for a single cell to divide is called the generation time

2N
1N
N DT
time
• The mean generation time of a population is equal to
the doubling time.

• Doubling time is a measure of growth rate

a short doubling time implies a fast growth rate.
 Use of generation time to compare
growth of different bacteria

Microorganism Temp oC Generation Time

B. stearothermophilus 40 11 min
Escherichia coli 40 20 min
S. aureus 37 28 min
P. aeroginosa 37 36 min
Lactobacillus acidophilus 37 75 min
M. tuberculosis 37 720 min
 Temperature & pH
• Temperature has a large effect on growth and survival of
microbes. Each organism shows a characteristic
temperature optimum, a maximum, and a minimum.
– Thermophiles & Hyperthermophiles: Organisms that
grow best at high temperatures (>50oC)
• Bacillus stearothermophilus, Thermococcus celer,
Pyrodictium brockii
– Mesophiles: Organisms that grow best at moderate
temperatures (20 – 50oC), e.g. Escherichia coli
– Psychrophiles: Organisms that grow best at low
temperatures (< 20oC) e.g. Flavobacterium sp.

• pH: Each organism has a pH range within which growth is

possible, and usually has a well-defined pH optimum.
– Acidophiles: organisms that grow best at low pH (fungi)
– Alkaliphiles: organisms that grow best at high pH
(Bacillus). 32
Microbial Growth
Microbial growth: Increase in cell number, not cell size!

Physical Requirements for Growth: Temperature

 Minimum growth temperature
 Optimum growth temperature
 Maximum growth temperature
Five groups based on optimum growth temperature
1. Psychrophiles
2. Psychrotrophs
3. Mesophiles
4. Thermophiles
5. Hyperthermophiles

Fig. 6 .3
Microbial Growth
Kinetics
 Growth Kinetics
Introduction
- Autocatalytic reaction: The rate of growth is directly related
to cell concentration

substrates + cells → extracellular products + more cells

∑S + X → ∑P + nX

S: substrate concentration (g/L); X: cell mass concentration (g/L);

P: product concentration (g/L); n: increased number of biomass.

Net specific growth rate (1/time): 1 dX

net 
X dt
t: the time
 Enzyme Kinetics r  r  Vmax S
KM  S
P S

Microbial growth Kinetics

cells more cells + Products

Nutrient (S) +

CC C S
rg   max  CC  x
K S  CS

36
 Typical Growth Curve for a Bacterial
Population
100
Stationary Phase
Log No. of Viable cells

80 Death Phase

60

40 Exponential Phase Biomass (g/L)

20
Lag Phase
0
0 2 4 6 8 10 12 14
Time in (hrs)
 Batch Growth Kinetics
Lag phase
A period of adaptation for the cells to their new
environment
• New enzymes are synthesized.
• A slight increase in cell mass and volume, but no
increase in cell number
• Prolonged by low inoculum volume, poor inoculum
condition (high % of dead cells), age of inoculum,
nutrient-poor medium
• Multiple lag phases: (diauxic growth) if medium contains
more than one carbon source, there is a lag phase after
each carbon source is exhausted.
 Diauxic growth

Typical growth curve for a bacterial population

Typical growth curve for a bacterial population
 Batch Growth Kinetics
Exponential growth phase
In this phase, the cells have adjusted to
their new environment and multiply rapidly
(exponentially)
• Balanced growth –all components of a cell
grow at the same rate.
• Growth rate is independent of nutrient
concentration, as nutrients are in excess.
 Batch Growth Kinetics
Exponential growth phase: The balance of cell mass in a
batch reactor during exponential phase gives:

dX
  net X , X  X 0 at t  0
dt
Integration of the above equation yields :
X
ln  μnet t , or X  X 0 e  net t
X0
X and X 0 are cell concentrations at
time t and t  0,
X is the same as CC
 The slope net is constant.

Typical growth curve for a bacterial population

 Batch Growth Kinetics
Exponential growth phase
net  μm
μm is the maximum specific growth rate (1/time)

Doubling time of cell mass: the time required to double

the microbial mass:

ln X / X 0 ln 2 0.693
d   
 net  net  net
Typical growth curve for a bacterial population
 Batch Growth Kinetics
Deceleration growth phase

Very short phase, during which growth decelerates

due to either:
• Depletion of one or more essential nutrients
• The accumulation of toxic by-products of growth
(e.g. Ethanol in yeast fermentations)
• Period of unbalanced growth: Cells undergo
internal restructuring to increase their chances
of survival
 Batch Growth Kinetics
Stationary Phase:
With the exhaustion of nutrients (S≈0) and build-up
of waste and secondary metabolic products
- The growth rate equals the death rate.
- There is no net growth in the organism population.
- Cells may have active metabolism to produce secondary
metabolites.
Primary metabolites are growth-related: ethanol by S.
cerevisae.
Secondary metabolites are non-growth-related:
antibiotics, pigments.
 Batch Growth Kinetics
Stationary phase
- Cell lysis (death) may occur and viable (living) cell mass may
drop. A second growth phase may occur and cells may grow
on lysis products of lysed cells (cryptic growth)
- Endogenous metabolism occurs by catabolizing cellular
reserves for new building blocks and energy-producing
monomer (maintenance energy).
The rate describing the conversion of cell mass into
maintenance energy or the loss of cell mass due to cell lysis:
dX
 k d X
dt
kd is the rate constant for endogenous metabolism .
 Growth Kinetics
Net specific growth rate (1/time):

net   g  kd

g : Gross specific growth rate (1/time)

kd : The rate of loss of cell mass due to cell death

or endogenous metabolism

Endogenous metabolism: during the stationary phase,

the cell catabolizes cellular reserves for new building blocks and
for energy-producing monomers.
 Batch Growth Kinetics
Death Phase:
The living organism population decreases with time,
due to a lack of nutrients and toxic metabolic by-
products.
The rate of death usually follows:

dN
 k d N
'

dt
'
k d is the first - order death rate constant.
 Semilog Plot using natural logarithms

100
Natural log Cell biomass

10 Natural Log
Gradient =  (ln)

1
0 1 2 3 4 5 6 7 8 9 10
Time (Hrs)
 L10b-52

Quantifying Growth Kinetics

• Relationship of the specific growth rate to substrate concentration
exhibits the form of saturation kinetics
• Assume a single chemical species, S, is growth-rate limiting
• Apply Michaelis-Menten kinetics to cellular system→ called the
Monod equation
max CS
Monod equation: rg  CC
K s  CS
•max is the maximum specific growth rate when S>>Ks
•CS is the substrate concentration, CC is the cell concentration
•Ks is the saturation constant or half-velocity constant. Equals the rate-
limiting substrate concentration, S, when the specific growth rate is ½
the maximum
•Semi-empirical, experimental data fits to equation, assumes that a
single enzymatic reaction, and therefore substrate conversion by that
enzyme, limits the growth-rate

Slides courtesy of Prof M L Kraft, Chemical & Biomolecular Engr Dept, University of Illinois, Urbana-Champaign.
 L10b-53

Monod Model (1942) – Nobel Prize

First-order kinetics:
mCS r  C mCS Zero-order kinetics:
CS K S  rg  CC
CS  KS  rg  m
g C
Ks K s  CS

m
Exponential
phase

 decelerating
phase

KS CS
Slides courtesy of Prof M L Kraft, Chemical & Biomolecular Engr Dept, University of Illinois, Urbana-Champaign.
 ks
• Bacteria with a high affinity for substrate has
a low Ks and vice versa

• The higher the affinity the less growth is

affected until substrate levels are very low
Growth Kinetics- Dissolved oxygen
Effect of factors:
- Dissolved oxygen (DO)
- aerobic fermentation requires oxygen
- oxygen gas is sparingly soluble in water
- specific growth rate may be limited by DO if DO is
below a critical oxygen concentration.
Critical oxygen concentration: Above the critical DO, the
growth rate becomes independent of DO concentration.
bacteria and yeast: 5%-10% of the saturated DO
mold: 10%-50% of the saturated DO
The saturated DO in aqueous solution is 7 ppm at 25oC
and 1 atm.
 Growth Kinetics
Effect of factors:
- Dissolved oxygen (DO)

The rate of oxygen transfer (OTR) from the gas to liquid phase is
given by:
OTR = No2 = KLa (CL*-CL)

KL is the oxygen transfer coefficient (cm/h),

a is the gas-liquid interfacial area (cm2/cm3)
KLa is the volumetric oxygen transfer coefficient (h-1)
CL* is saturated DO concentration (mg/l);
CL is the actual DO concentration (mg/l);
No2 is the rate of oxygen transfer (OTR) (mgO2/l.h)
 Growth Kinetics
Effect of factors: Dissolved oxygen (DO)
Oxygen Uptake Rate (OUR) is oxygen consumption rate by
microbes. If the maintenance requirement of O2 is negligible
compared to growth, then
g X
OUR  qo2 X  (mg O2 /h)
YX / O2
qo2 is the specific rate of O 2 consumptio n (mg O 2 /g dw cells - h)
When oxygen transfer is the rate-limiting step, at steady state,
the rate of oxygen consumption is equal to the rate of oxygen
transfer.

g X
 K L a(C * C ) Sufficient oxygen supply:
YX / O2 OTR ≥ OUR
 Growth Kinetics
Effect of factors: Dissolved oxygen (DO)
• Question: Oxygen is to be supplied for yeast production. If
oxygen uptake rate (OUR) is 15g/L medium-h for a required
yeast growth, and the oxygen transfer rate (OTR) is 10 g/L
medium-h.
• Is such oxygen transfer rate sufficient to maintain the
required yeast growth? If the required growth has to be
maintained, how to improve the oxygen transfer rate?
• Answers: OUR=15g/L medium-h > OTR=10 g/L medium-h
insufficient oxygen supply rate.
Oxygen transfer rate is limiting.
Increase kLa so that g X
 k L a(C * C)
YX / O2
 Yield coefficients
Yield coefficients: defined based on the amount of
consumption of another material.
X
Growth yield : YCells / Substrate  YC / S  Y X / S  
S
P
Product yield : Y P / S  
S
Growth yield based on consumption of oxygen :
X
Y X / O2 
O2
S  S assimilation S assimilation S growth energyS maintenance
into biomass into an energy
extracellular
product
 Yield coefficients:
• Yield coefficients: defined based on the amount of
consumption of another material.
• For most bacteria and yeast:
Yx/s =0.4-0.6 g/g glucose
Yx/O2=0.9 – 1.4 g/g O2
At the end of the batch growth period, the measured yields are
apparent as of endogenous metabolism occurring, Kd > 0,
which changes the metabolic pathways of the substrate.
e.g.
YX / S  YX / S
M Ap p
 Bioreactors
3) Stoichiometry
A) Yield Coefficients
1
mass of new cells formed Y 'S C 
Y 'C S  Y ' X S  Y 'C S
mass of substrate to produce new cells

mass of product formed

Y 'P S 
mass of substrate consumed to form product
B) Maintenance: In addition to consuming substrate to produce new cells,
part of the substrate must be used just to maintain a cell’s daily activities.
The corresponding maintenance utilization term is
mass of substrate consumed for maint ainence
5 m
mass of cells time

 rS  rg / YX / S  rP / YP S  mC X 61
 Fed-Batch Bioreactor
Nutrients are continuously or semi-continuously fed,
while effluent is removed discontinuously.
- overcome substrate inhibition or catabolite repression by
intermittent feeding of substrate
for production of secondary metabolites e.g. antibiotics,
lactic acid, E. Coli making proteins from recombinant DNA
technology.
 Continuous
Bioreactor

07 Oct 2011 Prof. R. Shanthini 63

 Continuous Bioreactors
v0
Volume=V
CS0

V=V0 v
CC
CS

64
 Bioreactors
3) Stoichiometry
Rate of Substrate Consumption
 rS  rg / YX / S  rP / YP S  mC X
Can’t separate substrate consumption used for cell
growth from that used for product formations during
exponenial growth.

 rS  rgY 'S C  mCC

(grams of substrate consumed)

Y 'S C 
(grams of cells produced)

65
 Continuous Bioreactors

• Start as batch fermentations but exponential

growth can be extended by addition of fresh broth
• Reactor is continuously stirred and constant
volume is maintained
• Steady state conditions exist
• The rate of addition of fresh broth controls growth
 Chemostat / Turbidostat
• Chemostat Device for maintaining a bacterial
population in the exponential growth phase by
controlling nutrient input and cell removal.

• Turbidostat The concentration of cells is kept

constant by controlling the flow of medium such that
the turbidity of the culture is kept within certain limits
Cells Growth in Continuous Culture
Continuous culture: fresh nutrient medium is
continually supplied to a well-stirred culture and
products and cells are simultaneously withdrawn.
At steady state, concentrations of cells, products
and substrates are constant.
In batch culture: the culture environment changes
continually.
growth, product formation and substrate
utilization terminate after a certain time interval.
A continuous-culture laboratory setup

medium

Products,
cells
Ideal Chemostat

Same as perfectly mixed continuous-flow, stirred-tank

reactor (CFSTR).
- Control elements: pH, dissolved oxygen, temperature
- Fresh sterile medium is fed to the completely mixed
and aerated (if required) reactor.
- Suspension is removed at the same rate.
- Liquid volume in the reactor is kept constant.
 Cell Growth in Ideal Chemostat
A material balance on the cell & substrate concentration around the chemostat
yields,
[In] - [Out] + [Growth] - [Death] = Accumulation
dX
FX 0  FX  VR  g X  VR kd X  VR
dt

[In] - [Out] + Consumption for Growth - Consumption for Product = Accumulation

1 1 dS
FS0  FS  VR  g X M
 VR q p X  VR
Yp / s dt
Y
X /S

F is the volumetric flowrate of nutrient solution (l/h);

VR is the culture volume (l) (constant); X is the cell concentration (g/l);
P is the extracellular product (g/l);
µg and kd are growth rate and endogenous rate constant, respectively (h-1).
qp is the specific rate of extracellular product formation (g P/g cells-h)
Yp/s is the product yield coefficient (g P/g S).

Subscript 0 denotes the parameters at the feed medium.

 Cell Growth in Ideal Chemostat
without cell death and Product Formation
At steady state with sterile feed X0=0, assuming kd ≈ 0, qp=0 , and Monod
equation applied,

0 0 0
dX
FX 0  FX  VR  g X  VR kd X  VR
dt
0 0
1 1 dS
FS0  FS  VR  g X M
 VR q p X  VR
Yp / s dt
Y
X /S
m S
g 
KS  S
Cell Growth in Ideal Chemostat
without cell death and Product Formation
At steady state, X0=0, kd ≈ 0, qp=0 , Monod equation applied,

m S KS D
g  D  S
KS  S m  D
M M Ks D
X Y ( S0  S )  Y ( S0  )
X /S X /S m  D
Cell Productivity: DX

Ks
Dopt   m (1  )
d ( DX ) K s  S0
 0  Dopt
X opt  Y X / S ( S 0  K s ( K s  S 0 )  K s )
M
dD

D=F/VR, is dilution rate (1/time), the reciprocal of residence time.

 Cell Growth in Ideal Chemostat
Washed out: If D is set at a value greater than µm (D > µm),
the culture cannot reproduce quickly enough to maintain itself.

4 0.3
µm = 0.2 hr-1 DX
3.5
0.25
3
0.2

DX (g/L-hr)
2.5
S, X (g/L)

X S
2 0.15
1.5
0.1
1
0.05
0.5
0 0
0 0.05 0.1 0.15 0.2 0.25
D (1/hr)
 Maximum Product Flow Rate
DCC

 max CS0
DW 
K S  CS0
 K 
D max prod   max 1  S 

 K S  CS 0 

D
75
Dmaxpro DW
d
Continuous Stirred Tank Reactor (CSTR) at steady-state
Exercise

The growth rate of E. coli be expressed by Monod kinetics

with μm = 0.935 hr-1 and KS = 0.71 g/L.

Assume that YX/S is 0.6 g dry cells per g substrate.

The feed is sterile (CXi = 0) and CSi = 10 g/L.

show how CX and CS change with dilution rate.

Determine washout dilution rate.

76
Exercise 2 worked out using the calculator/spread sheet:

Plot the following using excel / MATLAB

From (60): CS = 0.71 D g/L

0.935 - D

(
From (64): CX = 0.6 10 - 0.71 D
0.935 - D ) g/L

From (65): DC = CSi μm / (KS + CSi)

= 10 x 0.935 / (0.71+10) = 0.873 per h

07 Oct 2011 Prof. R. Shanthini 77

Exercise 2 worked out using the calculator/spread sheet:

07 Oct 2011 Prof. R. Shanthini

DC = 0.873 78
Critical (Washout) dilution rate
• The dilution rate at which x = zero is termed
the critical dilution rate Dcrit

• Dcrit is affected by the constants max and Ks

and the variable Sr,
the larger Sr the closer Dcrit to max
 Microbial Kinetic
Parameter
Determination-
Material from Shuler’s
Bioprocess Engineering

80
 Determination of Monod Parameters in Ideal
Chemostat without cell death and Product Formation

In Chemostat: µg=D, varying D obtains D~S

m S
g  D 
KS  S
1 KS 1 1
  ( Lineweaver - Burk)
D m S m

Chemostat technique: reliable, constant environment,

operation may be difficult.
 Determination of Monod Parameters in a
Batch Bioreactor
Batch: X, S, t → lnX ~ t , get µm (slope) from data
in exponential phase.
X
ln  μnet t  μmt
X0
dX m S
 g  , Kd  0
Xdt KS  S
1 KS 1 1
  ( Lineweaver - Burk)
g m S m
S KS S
  (Hanes - Woolf)
g m m
 Determination of Monod Parameters in a
Batch Bioreactor
μm, Ks, Yx/s may be from nonlinear regression on S ~ t. In batch
culture, dX
  net X
dt
dX  S
When Kd, is small compared with μg,  m
X
dt K s  S

Since X  X 0  Yx / s (S0  S )
dS m S X 0
 [  ( S 0  S )]
dt Ks  S Y X / S
Regress the parameters by fitting the equation to the
experimental data S ~ t. Use numerical methods.
Monod model modified for substrate inhibition:
Monod model does not model substrate inhibition.
Substrate inhibition means increasing substrate concentration
beyond certain value reduces the cell growth rate.

1
0.8
μ (per h)

0.6
0.4
0.2
0
Prof. R. Shanthini 0 5 10
84
being modified Cs (g/L)
Monod model modified for cell growth with
noncompetitive substrate inhibition:
μm
μ=
(1 + KS/CS)(1 + CS/KI )

μm CS
=
KS + CS + CS2/KI + KS CS/KI

μm CS
If KI >> KS then μ=
KS + CS + CS2/KI

where KI is the substrate inhibition constant.

Prof. R. Shanthini 85
being modified
Monod model modified for cell growth with
competitive substrate inhibition:
μm CS
μ=
KS(1 + CS/KI) + CS

where KI is the substrate inhibition constant.

Prof. R. Shanthini 86
being modified
Monod model modified for cell growth with product
inhibition:
Monod model does not model product inhibition (where
increasing product concentration beyond certain value reduces
the cell growth rate)
For competitive product inhibition:
μm CS
μ=
KS(1 + Cp/Kp) + CS
For non-competitive product inhibition:
μm
μ=
(1 + KS/CS)(1 + Cp/Kp )
where Cp is the product concentration and Kp is a product
inhibition constant.
Prof. R. Shanthini 87
being modified
Monod model modified for cell growth with product
inhibition:
Ethanol fermentation from glucose by yeasts is an example of
non-competitive product inhibition. Ethanol is an inhibitor at
concentrations above nearly 5% (v/v). Rate expressions
specifically for ethanol inhibition are the following:

μm CS
μ= (1 - Cp/Cp*)n
(KS + CS)

μm CS
μ= exp(-Cp/Kp)
(KS + CS)
Where Cp* is the product concentration at which growth
stops. typical inhibition parameters are n 0.5 and 93 g/dm3
Prof. R. Shanthini 88
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Filamentous Organisms:
• Types of Organisms
– Moulds and fungi
– bacteria or yeast entrapped in a spherical gel particle
– formation of microbial pettlets in suspension

• Their growth does not necessarily increase the number of

cells, but increase them in length, and hence there will be
changes in physical properties like density of the cell mass
and viscosity of the broth
dR
• Model - no mass transfer limitations  k  const
dt

where R is the radius of the cell floc or pellet or mold colony

Prof. R. Shanthini 89
being modified
Product Formation Kinetics
Product Formation:
• The product formation may be growth associated, which
means rate of product formation is proportional to the cell
growth rate (i.e., product is formed as a result of the primary
metabolic function of the cell)
r P =  rX
It happens mostly during the exponential growth phase

Examples:
- production of alcohol by the anaerobic fermentation of
glucose by yeast
- production of gluconic acid from glucose by Gluconobactor

Prof. R. Shanthini 91
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Product Formation:

• The product formation may be non-growth associated,

which means rate of product formation is proportional to the
cell concentration rather than cell growth rate (i.e., product is
formed as a result of the secondary metabolism)
rP = β CX

It happens at the end of the exponential growth phase or only

after entering into the stationary phase

Examples:
- production of antibiotics in batch fermentations
- production of vitamins in batch fermentations

Prof. R. Shanthini 92
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Product Formation:

Luedeking-Piret model:

rP =  rX + β CX

Used for lactic acid formation by Lactobacillus debruickii

where production of lactic acid was found to occur semi-
independently of cell growth.

Prof. R. Shanthini 93
being modified