Microbial cells

Prokaryotic cell
Found in two microbial groups: bacteria and blue-green
algae.
The cell is small and simple, not compartmentalized by
unit membrane systems.
The cell has only two structurally distinguishable
internal regions: cytoplasm and nuclear region
The cytoplasm has grainy dark spots as a result of its
content of ribosomes, which are composed of protein
and ribonucleic acid (RNA).
The ribosome is the site of important biochemical
reactions for protein synthesis.
The nuclear region is of irregular shape, sharply
segregated even though it is not bounded by
membrane.
Prokaryotic cell structure
 Prokaryotic cell
 The nuclear region contains deoxyribonucleic acid
(DNA), which contains genetic information

 The prokaryotic cell is surrounded with a cell wall and

a cell membrane.

 The cell wall, considerably thicker than the cell

membrane, protects the cell from external influences.

 The cell membrane (or cytoplasmic membrane) is a

selective barrier between the interior of the cell and
the external environment.
 Prokaryotic cell
 The largest molecules known to cross this membrane
are DNA fragments and low-molecular-weight
proteins.
 The cell membrane can be folded and extended into
the cytoplasm or internal membranes.
 The cell membrane serves as the surface onto which
other cell substances attach and upon which many
important cell functions take place.
 Eukaryotic cell
 The unit structure in plants, animals, protozoa, fungi,
and algae.
 The eukaryotic cell has internal unit membrane
systems that segregate many of the functional
components of the cell
 They are more complex and 1,000 to 10,000 times
larger than prokaryotic cells.
 The nucleus is surrounded by a double membrane
with pores 40 to 70 μm wide, containing cytologically
distinguishable chromosomes.
 The nucleus controls hereditary properties and all
vital activities of the cell.
Eukaryotic cell
 Eukaryotic cell
 The chromosomes are long and threadlike
bodies and are found in the nuclei of cells,
which contain the genes arranged in linear
sequence.

 The cytoplasm contains large numbers of

granules called ribosome, which are involved
in continuous reactions to synthesize cell
materials.
 Eukaryotic cell
The ribosome is especially concentrated along the
rough surface of the endoplasmic reticulum

The mitochondria contain the electron transport

enzymes that utilize oxygen in the process of energy
generation.

Vacuole and lysosome are organelles that serve to

isolate various chemical reactions in a cell.
 Microbial nomenclature
Taxonomy - science of classification of organisms
Nomenclature refers to the actual naming of organisms

 For microorganisms a binary name is used.

 Genus name and species name
Genus: a group of related species
Species: organisms that are substantially alike

Escherichia coli. Escherichia is the genus and coli the

species.
Bacillus subtilis, B. albus, and B. coagulans.
 Microbial nomenclature
Proper names of organisms are always italicized.
The genus is capitalized.
 Although organisms that belong to the same species
all share the same major characteristics, there are
subtle and often technologically important variations
within species.
 An E. coli used in one laboratory may differ from that
used in another. Thus, various strains and substrains
are designated by the
 Microbial nomenclature
Several genera are identified:
 Bacillus: a small rod
 Lactobacillus: a small milk rod
 Micrococcus: a small grain
 Clostridium: a small spindle
 Pasteurella: after Louis Pasteur, Latinized
 Salmonella: after Daniel E. Salmon, Latinized
 Saccharomyces: sugar fungus
 Bacteria
Shapes: Bacteria are unicellular microscopic
organisms.
The typical diameter of the cell ranges from
0.5 to 1 µm.
cocci: spherical or ovoid
bacilli: cylindrical or rod shaped
spirilla: helically coiled
 Reproduction
Bacteria reproduce predominantly by a process known
as binary fission
 Classification
Sources of energy
A. Phototrophs
organisms which are capable of employing radiant
energy.
B. Chemotrophs
organisms which obtain the energy for their activities and
self-synthesis from chemical reactions that can occur in
the dark.
 Classification
Sources of carbon
A. autotrophs:
organisms which can thrive on an entirely inorganic diet, using CO2
or carbonates as a sole source of carbon.

B. heterotrophs:
organisms which cannot use CO2 as a sole source of carbon but
require, in addition to minerals, one or more organic substances,
such as glucose or amino acids, as sources of carbon.

Sources of nitrogen
atmospheric nitrogen, inorganic nitrogen compounds, or other
derived nitrogen.
 Classification
Sources of sulfur and phosphorus
elementary sulfur, inorganic sulfur, or organic sulfur.

Sources of metallic elements

sodium, potassium, calcium, magnesium,
manganese, iron, zinc, copper, and cobalt.

Sources of vitamins
 Physical Conditions
Temperature
psychrophiles, mesophiles, or thermophiles.
 Response to oxygen
 Aerobic bacteria grow in the presence of free oxygen.
Anaerobic bacteria grow in the absence of free
oxygen.
Facultatively anaerobic bacteria grow in either the
absence or the presence of free oxygen.
Microaerophilic bacteria grow in the presence of
minute quantities of free oxygen.
 pH
For most bacteria the optimum pH for growth lies
between 6.5 and 7.5.
For most species the minimum and maximum limits
fall somewhere between pH 4 and pH 9
pH for different organisms

Bacteria 3-8
Yeast 3-6
Molds 3-7
Plant cells 5-6
Animal cells 6.5-7.5
 Effect of pH
 Only a few species can grow at pH values of less
than 2 or greater than 10
Acidophiles: organisms that grow at low pH
Alkaliphiles: optimal pH at 10-11

 Fungus tend to be more tolerant to acids than

bacteria.

 The internal pH of the cell must remain close to

neutral to prevent the destruction of the cell.
 Changes during growth
 The pH of the medium changes during most
fermentations
 Production of organic acids,CO2 and utilization of
amino acids affects the pH of the medium
 The utilization of nitrogen source also results in
either the release or removal of H ions.
 pH is mostly controlled by the use of an effective
buffer system
 Spore formation
Some bacteria form spores when growth ceases due to
starvation or other causes.
Spores are more resistant than normal cells to heat,
drying, radiation, and chemicals.
Spores can remain alive for many years; however, they
can convert back to normal cells at proper conditions.
Spore-forming bacteria are found most commonly in the
soil.
 Gram Reaction
Addition of dyes (staining) to distinguish
between bacteria based on presence and
composition of outer cell

gram-positive and gram negative

 Fungi
Fungi are plants devoid of chlorophyll and are
therefore unable to synthesize their own food.
 They range in size and shape from single-celled
yeasts to multicellular mushrooms.
Yeasts and molds are used industrially for many
fermentations
 Culture types
The growth of microbial population in artificial
environments is called cultivation.
A culture that contains only one kind of microorganism
is a pure culture.
A mixed culture is one that contains more than one
kind of microorganism.
 Steps for cultivation
 Preparing a culture medium in which a
microorganism can grow best.
 Sterilizing in order to eliminate all living organisms in
the vessel.
 Inoculating the microorganism in the prepared
medium.
 Culture media are usually prepared in a test tube, a
flask, a Petri dish, or a fermenter.
 Media types
 natural (or empirical, or complex)
 synthetic (or chemically defined)

 Compositions vary depending on the species of

organism to be cultivated and the purpose of the
cultivation.
 Natural media
 Used on the basis of experience and not on of
exact knowledge of their composition and action.
 Usually contain peptones, beef extract, or yeast
extract.
 When a solid medium is desired, a solidifying agent
such as gelatin or agar may be incorporated into
the medium.
 Examples are nutrient broth and nutrient agar.
Culture media
 Synthetic media
 Consist of dilute, reproducible solutions of chemically
pure, known inorganic and/or organic compounds.
 They are often required for research purposes.
 The medium may be as simple as inorganic
ammonium salt plus minerals and a sugar, or as
complex as purified casein with added vitamins,
minerals, and a sugar.
 These media have the added advantage that they
can be produced with a constant composition year
after year.
 Sterilization
 After a suitable culture medium is selected for the
cultivation of a specific microorganism, it is poured
into a culture vessel.
 The vessel is covered with a suitable closure to allow
for the exchange of gases with the atmosphere and
also keep foreign organisms out of the media.
 Various types of closures are used in the modern
laboratory including cotton plugs, plastic foam, screw
caps, metal caps, and aluminum foil.
 The medium is then sterilized to eliminate all living
organisms in the vessel.
 Sterilization
The most common method of sterilization is by moist
heat (steam under pressure) in an autoclave.
Generally, the autoclave is operated at approximately
15 psi at 121°C.
The time of sterilization depends on the nature of the
material, the type of container, and the volume.
For example, test tubes of liquid media can be
sterilized in 15 to 20 minutes at 121°C.
 Inoculation
Inoculation is the seeding of a culture vessel with the
microbial material (inoculum).
The inoculum is introduced with a metal wire or loop
which is rapidly sterilized just before its use by heating
it in a flame.
Transfers of liquid culture are often made by using a
sterilized pipette.
The inoculation is usually done in a laminar flow hood
to minimize the risk of contamination.
 Cell Growth
S+X Products + more cells (nX)

 Organisms extract nutrients from a suitable

medium and convert them into useful biological
components.
 energy production,
 biosynthesis
 product formation.
 Cell growth is a result of both replication and
change in cell size.
 Net specific growth rate
1 dX
 net 
X dt 1 dN
R 
X  cell mass conc (g/l) N dt
t  time (h), N  number of cells /L
 net  specific growth rate (h -1 )
 Batch Growth

Culturing cells in a vessel with an initial change of

medium that is not altered by further nutrient addition
or removal

 Lab and industrial.

Growth Patterns and Kinetics
Growth Patterns and Kinetics
 Lag phase
 Lag phase is a period of adaptation of cell to a new environment.
 Micro organisms re-organize their molecular constituents when
transferred to a new medium.

 Depending on the composition of the nutrients, new enzymes

may be synthesized, other enzymes may be repressed.
 Cell mass may increase a little but not the number density.
 Nutrients low in certain growth factors may lead to long lag
phases.
 The lag phase is also dependent on the age of the inoculum. Lag
phase increases with age of inoculum.
 Exponential phase cells are best to minimize lag phase.
 Also the use of large inoculum size decreases the duration of lag
phase.
 Lag phase
 Multiple lag phases may be observed when the
medium contains more than one carbon source –
diauxic growth.

 This is caused by a shift in metabolic pathways in the

middle of cell growth.

 The 1st carbon source is more readily utilizable than

the second and the presence of the readily available
carbon source represses the synthesis of the
enzymes required for the metabolism of the second
substrate.
 Exponential growth phase
 Cell mass and density increase exponentially
because the cell is now fully developed to its
environment.

 Balanced growth – all components of the cell grow at

the same rate.

 Composition of a single cell remains approximately

constant during exponential growth phase.

 In the presence of large nutrient concentration the

growth rate is independent of nutrient concentration.
 Exponential phase
𝑑𝑋
= 𝜇𝑛𝑒𝑡 𝑋, X = X0 at t = 0
𝑑𝑡
𝑑𝑋
න = න 𝜇𝑛𝑒𝑡 𝑑𝑡
𝑋
𝑋
𝑙𝑛 = 𝜇𝑛𝑒𝑡 𝑡 or 𝑋 = 𝑋0 𝑒 𝜇𝑛𝑒𝑡 𝑡
𝑋0

 To double the microbial cell mass,

𝑋
= 2 = 𝑒 𝜇𝑛𝑒𝑡 𝑡
𝑋0
𝑙𝑛2 0.693
⟹ 𝐷𝑜𝑢𝑏𝑙𝑖𝑛𝑔 𝑡𝑖𝑚𝑒, 𝑡𝐷 = =
𝜇𝑛𝑒𝑡 𝜇𝑛𝑒𝑡
 Example
The fermentation of corn steep liquor by a native
yeast strain gave the following results in the table:
Time (h) X (g/L) S (g/L)
0 0.200 9.23
2 0.211 9.21
4 0.305 9.07
8 0.980 8.03
12 3.200 4.60
14 5.600 0.92
16 6.150 0
Calculate:
a. The maximum growth rate (µmax)
b. The yield of biomass on substrate (YX/S)
c. The doubling time for the yeast (td)
d. Saturation constant Ks
e. The specific growth rate (µg) at t = 10 h
f. What maximum cell concentration could be
expected if 14 g/L glucose were used with the
same size of inoculum?
 Deceleration growth phase
 Growth decelerates due to the depletion of one or more
essential nutrients or the accumulation of toxic by-
products.

 Occur over a very short period and is not usually

observed
 Stationary phase
 Net growth rate is zero. Growth rate = Death rate

 Product of secondary metabolites

 Antibiotics
 Hormones
 Total cell mass concentration may stay constant but the
number of viable cells may decrease.

 Cell lysis may occur and viable cell mass may drop.

 A second growth phase may occur and cells may grow

on lysis product of lysed cells (cryptic growth)

 Cells may not be growing but may have active

metabolism to produce secondary metabolites.
 Growth Termination
 Exhaustion of an essential nutrient or accumulation of
toxic products.
 Ethanol production by yeast.

 During stationary phase, the cell catabolizes cellular

reserves for new building blocks and for energy
producing monomers. (Endogenous metabolism).

 Energy is spent to maintain the cell membrane,

transport of the nutrients, motility, repair of damaged
cellular structures
 Maintenance of energy.

𝑑𝑋
= −𝑘𝑑 𝑋, 𝑋 = 𝑋𝑆𝑂 𝑒 −𝑘𝑑 𝑡
𝑑𝑡
Xso = cell mass concentration at beginning of stationary phase
 Death phase
dN
 kd N
'

dt
 k d' .t
N  N se

Ns = concentration of cells at end of stationary phase

k’d is death rate constant
 Yield coefficients
 Yield coefficients are defined based on the actual consumption of
material.
∆𝑋
𝑌𝑋/𝑆 =−
∆𝑆
ΔX = change in cell mass
ΔS = change in substrate

 In batch growth, the yield coefficient is described as apparent

 The utilization of the substrate is to supply the following:

S  S assimilation  S assimilation into  S growth  S maintenance

into biomass an extracellular energy energy
product

 Culture conditions can alter the pattern of utilization

Yield coefficients
X
YX / O2 
O2
P
YP / S 
S

Y X / S  0 .4  0 .6 g / g
for most yeast and bacteria
YX / O2  0.9  1.4 g / g
Yield coefficients
 Example
The fermentation of corn steep liquor by a native
yeast strain gave the following results in the table:
Time (h) X (g/L) S (g/L)
0 0.200 9.23
2 0.211 9.21
4 0.305 9.07
8 0.980 8.03
12 3.200 4.60
14 5.600 0.92
16 6.150 0
Calculate:
a. The maximum growth rate (µmax)
b. The yield of biomass on substrate (YX/S)
c. The doubling time for the yeast (td)
d. Saturation constant Ks
e. The specific growth rate (µg) at t = 10 h
f. What maximum cell concentration could be
expected if 14 g/L glucose were used with the
same size of inoculum?
 Maintenance coefficient

m
ds 
dt m
X
 uptake of substrate for cellular maintenance

During stationary phase, endogenous metabolism of biomass is used for

maintenance energy.

Maintenance  repair damaged cellular components

 transfer some nutrients and products in and out of cells
for mobility
 adjust the osmolarity of the cell’s interior volume
 Products
Growth associated products  produced simultaneously
with microbial growth
1 𝑑𝑃
𝑞𝑝 = = 𝑌𝑃/𝑋 (𝜇𝑔 )
𝑋 𝑑𝑡
𝜇𝑔 = specific growth rate
For example production of constitutive enzyme

Non-growth associated product formation  During stationary phase

q p    constant
 Specific rate of product formation is constant.
 Mostly secondary metabolites, antibiotics
 Products
Mixed growth associated product formation 
During the slow growth and stationary phases.

q p   g  
 Examples: lactic acid fermentation, xanthan gum

Mixed-growth associated
Non-growth associated
Growth-associated product formation
 Effect of Temperature
Psychophiles (Topt <200C)
Mesophiles (Topt 200C-500C)
Thermophiles (Topt>500C)

For each organism, as the temperature is increased,

towards the optimum, growth rate approximately double for
every 10°C rise in temperature.
Above the optimal temperature, the growth rate decreases
 Net Replication Rate

 At high temperature, the thermal death rate exceeds the growth rate.

 The two(2) rate constant kd’ and µR’ vary with temperature
by the Arrhenius equation. ie:

 The activation energy  The activation energy is

is typically 10 to 20 typically 60 to 80
Kcal/mol Kcal/mol
Other effects of Temperature
 Product formation
 Yield coefficient
 Maintenance requirement of cell increases at high
temperature
 May affect the rate limiting step in fermentation process.
the rate of reaction might be higher than rate of
diffusion and diffusion becomes the rate limiting step
( eg: immobilized cell systems)
 Rate of oxygen Transfer

OTR is oxygen Transfer Rate

KL is oxygen transfer coefficient ( cm/h)
a is gas liquid interfacial area (𝑐𝑚2 /𝑐𝑚3 )
kLa – volumetric oxygen transfer coefficient
C*---- Saturated DO concentration (mg/L)
CL--- actual DO concentration in the broth
NO2– the rate of Oxygen transfer (mg O2/L.h)
OUR--- Oxygen Uptake rate

qO2 ----- Specific rate of oxygen consumption ( mg O2/g dw cells.h)

 Rate of oxygen Transfer
 When oxygen transfer is the rate limiting step, the rate of
oxygen consumption is equal to the rate of oxygen transfer

 If the maintenance requirement of O2 is negligible compared to

growth then;

OR
Heat Generation by Microbial Growth

Substrate + O2 Total combustion CO2 + H2O -------------- A

Microbial growth

Substrate metabolic heat Cells -------------- B

Cells +O2 Combustion of cells CO2 + H2O ----------- C

eqn A= eqn B+ eqn C

 Heat of combustion of the substrate is equal to the sum of the

metabolic heat and the heat of combustion of the cellular material.
 Heat Yield Coefficient
YH = (g cells/kJ)

---- heat of combustion of substrate (kJ/g substrate)

Yx/s --- substrate yield coefficient ( g cell/substrate)
• ----- heat of combustion of cells (kJ/ g cell)
1/YH ----- metabolic heat evolved per gram of cell mass produced (kJ/g cell)
 Heat Yield coefficient
 In a batch fermentation, the total rate of heat
evolution is:

 Metabolic heat removal

 Example
Pseudomonas putida with μm =0.5 h-1 is cultivated in a continuous
culture under aerobic conditions where D = 0.28h-1. The carbon and
energy source in the feed is lactose with a concentration of So =
2g/L. The effluent lactose concentration is desired to be S = 0.1 g/L.
If the growth rate is limited by oxygen transfer, use the information
below to answer the following questions:
a. Determine the steady state biomass concentration X and the
specific rate of oxygen consumption (qO2)
b. What should be the oxygen transfer coefficient (kLa) in order to
overcome oxygen transfer limitation (ie CL = 2 mg/L)

𝒀𝑴
𝒙/𝒔 = 𝟎. 𝟒𝟓𝒈𝑿/𝒈𝑺 𝒀𝑴
𝒙/𝑶𝟐 = 𝟎. 𝟐𝟓𝒈𝑿/𝒈𝑶 𝟐 𝑪 ∗ = 𝟖𝒎𝒈/𝑳
 Stoichiometry of
microbial growth/product
formation
 Stoichiometry

 Cells of different organisms vary in their elemental composition.

 A typical cellular composition is written as CHαOβNδ (eg CH18O0.5N0.2)

 Usually specified for 1 gram of atom in 1 mole.

 Consider the biological conversion

CHmOn + aO2 + bNH3 ------> cCHαOβNδ + dH2O + eCO2

 Performing elemental balances on C, H, O, N

C: 1 = c + e
H : m + 3b = cα + 2d
O : n + 2a = cβ + d + 2e
N : b = cδ
 Stoichiometry
 Five equations for the five (5) unknowns means, the equations can
be solved.

Respiratory quotient (RQ)

 RQ = e/a
 Moles of CO2 produced per mole of oxygen consumed
Microbial cell compositions
 Example
 Assume that experimental measurements for a certain organism
have shown that cells can convert two-thirds (wt/wt) of the
substrate carbon (alkane or glucose) to biomass.

(a) Calculate the stoichiometric coefficients for the following

biological reactions

(b) Calculate the yield coefficients YX/S (g dw cell/g substrate, YX/O2

(g dw cell/g O2) for both reactions. Comment on the differences
 Theoretical Prediction of Yield
coefficient
 In aerobic fermentation, the growth yield per available electron in
oxygen molecule is approximately 3.14 gdw cells/electron when
ammonia is used as the nitrogen source

 For O2, the number of available electrons is 4.

 Growth yield coefficient Yx/s can be calculated when the number of

oxygen molecules per mole of substrate considered is known.

C6H12O6 + 6O2 --------> 6CO2 + 6H2O

 Total number of available electrons in 1 mole glucose = 24

Yx/s = 24 (3.14) = 76 gdw cells/mol

Yx/s = 76/180 = 0.4 gdw cells/g glucose
Yield coefficients
Quantifying Growth Kinetics

Monod equation

 Equation assumes a single chemical species.

 [S] is growth limiting
 Monod equation
 μm--- maximum specific growth rate

 Ks---- saturation constant or half velocity constant

 Ks= S when μg = ½ μm

Generally μg=μm for S>> Ks and

μg = (μm/Ks)S for S<< Ks

 The equation describes substrate limited growth only

when growth is slow and population density is low
 Exponential growth
𝑑𝑋
= 𝜇𝑛𝑒𝑡 𝑋 = 𝑟𝑋
𝑑𝑡
𝑋 𝑑𝑋 𝑋 𝑑𝑋 𝑡
‫׬ = 𝜇 𝑋׬ = 𝑟 𝑋׬‬0 𝑑𝑡 (applicable when cells begin to grow)
0 𝑋 0 𝑋

A plot of 1/µX versus X yields batch time as the area

 Exponential growth
From the Monod kinetics
𝜇𝑚𝑎𝑥 𝑆
𝜇=
𝐾𝑠 + 𝑆
𝑋 𝑋 𝑡
𝑑𝑋 𝐾𝑠 + 𝑆 𝑑𝑋
න = න = න 𝑑𝑡
𝜇𝑋 𝜇𝑚𝑎𝑥 𝑆𝑋
𝑋0 𝑋0 0

Need relationship between S and X

∆𝑿 𝑿−𝑿𝟎
Define 𝒀𝑿Τ𝑺 = = = yield coefficient
−∆𝑺 − 𝑺−𝑺𝟎

𝑲𝒔 𝒀𝑿𝑺 𝑿 𝑲𝒔 𝒀𝑿𝑺 𝑺𝟎
𝒕 − 𝒕𝟎 𝝁𝒎𝒂𝒙 = +𝟏 𝒍𝒏 + 𝒍𝒏
𝑿𝟎 +𝑺𝟎 𝒀𝑿𝑺 𝑺𝟎 𝑿𝟎 +𝑺𝟎 𝒀𝑿𝑺 𝑺
 The Logistic Equation
1 𝑑𝑋
𝜇𝑔 =
𝑋 𝑑𝑡
𝜇𝑚 𝑆
𝜇𝑔 =
𝐾𝑠 + 𝑆

𝜇𝑚 𝑆 𝑑𝑋
⟹ 𝑋=
𝐾𝑠 + 𝑆 𝑑𝑡

𝑋 − 𝑋𝑜
𝑌𝑋/𝑆 =
𝑆𝑜 − 𝑆

𝑆𝑜 𝑌𝑋/𝑆 + 𝑋𝑜 − 𝑋
⇒𝑆=
𝑌𝑥/𝑠
 The Logistic Equation

Integrating
Other Forms
 Cell Growth in Continuous Culture
In batch culture, the medium is continuously altered by the
actions of the growing organism until it is no longer suitable to
support the growth.

A continuous culture is used when it is desired to keep the

culture in a constant environment for long times.

A continuous culture is essentially a flow system of constant

volume to which medium is added continuously and from which
a continuous removal of overflow occurs.

Cell numbers and nutrient status remains constant at steady

state.
Continuous
Process
 Chemostat
Equivalent to CSTR

Material balance
dx
Fxo  Fx  vR  g x  vR k d x  vR
dt
 g  growth rate (h -1 )
k d  death rate (h -1 )
 Chemostat
dx
 DX o  (  g  k d  D) X
dt
where D  F  dilution rate
VR
 reciprocal of residence time
 For a sterile feed medium Xo = 0.
 Neglecting death rate in comparison with growth rate, kd << µg

At steady state;
0  0  (  g  0  D) X
g  D
 In a chemostat, cells are removed at a rate equal to their
growth rate and the growth rate is equal to the dilution rate
 Chemostat
Growth rate can be manipulated as an independent
parameter.
Growth of the organism is also limited by at least one
substrate.
m S
g  D 
Ks  S
D greater than µm means the culture cannot reproduce
quickly enough to maintain itself and is washed out.

The effluent substrate concentration is given by;

Ks D
S
m  D
 Substrate Material Balance
1 1 dS
FS o  FS  VR  g x g X  VR q g X .  VR
YXM/ S YP / S dt
q p  specific rate of extra cellular product formation (gP/  g cells h)
YP / S  Yield coefficient (gP/gS)
dS
At steady state;  0.
dt
No extra cellular product formation.
 kd  0
 gX
D( So  S ) 
YXM/ S
 Material Balance

At steady state  g  D
 X Y M
X /S (So  S )
At D   m
Ks D
X Y M
(So  )
m  D
X /S

 YX/S is assumed constant when endogenous metabolism is

neglected
 Material Balance
Consider
D   net   g  k d
  g  D  kd
1
 D( So  S )  M
( D  kd ) X  0
Y X /S

So  S 1
 D( )  M ( D  kd )  0
X YX / S
1 D kd
or D( AP
)  M 0
Y X /S YXM/ S YX / S
1 kd 1
  
YXM/ S YXM/ S .D YXAP/ S
1 1 ms
 AP
 
Y X /S YXM/ S D
 Material Balance
kd
ms  M  maintenance coefficient based on substrate.
YX / S

 YAPX/S varies with growth conditions while YMX/S is constant.

 A plot of 1/ YAPX/S against D gives a slope of ms and the

intercept is 1/ YMX/S.
 In the presence of endogenous metabolism,

ks ( D  kd )
S and
m  D  kd

X Y M
So  S . D
D  kd
X /S
 Material Balance on Product
Model
Extracellular substrate converted instantaneously to product.

DP  qP X
1 dP
qP    g YP / X
X dt
substitute in substrate balance
1 1
D( So  S )  M
( D  kd ) X  qP X
Y X /S YP / S
 
 
 D 
X  YXM/ S ( S o  S ) M 
 D  k  YX / S q 
 d
YP / S
P

 Material Balance on Product
For biomass production, the optimum D is found by differentiating
DX with respect to D and solving at zero.

Ks
Dopt   m (1  )
K s  So
 Dissolved Oxygen( DO)
 Required for aerobic fermentations
 Oxygen is not very soluble in water
 At high cell concentration, the rate of oxygen
consumption may exceed the supply rate and the
growth may be limited by oxygen
 Oxygen is usually supplied by vigorously shaking
the flask on a shaker or by bubbling sterilized air into
the medium through a fine glass tube.
 Dissolved oxygen
 To overcome DO limitations:
 Use pure oxygen or oxygen enriched air
 Operate at high pressure (2-3 atm)

Other Factors:
Redox potential, DCO , Ionic strength
2
Rate of oxygen Transfer

OTR is oxygen Transfer Rate

KL is oxygen transfer coefficient ( cm/h)
a is gas liquid interfacial area (𝑐𝑚2 /𝑐𝑚3 )
kLa – volumetric oxygen transfer coefficient
C*---- Saturated DO concentration (mg/L)
CL--- actual DO concentration in the broth
NO2– the rate of Oxygen transfer (mg O2/L.h)
OUR--- Oxygen Uptake rate

qO2----- Specific rate of oxygen

consumption ( mg O2/g dw
cells.h)
 Rate of oxygen Transfer
 When oxygen transfer is the rate limiting step, the
rate of oxygen consumption is equal to the rate of
oxygen transfer

 If the maintenance requirement of O2 is negligible

compared to growth then;

OR
Critical Oxygen concentration
 Cell growth measurement
In any biological system, growth can be defined as the
orderly increase of all chemical components.
Increase of mass might not really reflect growth because the
cells could be simply increasing their content of storage
products such as glycogen or poly-β-hydroxybutyrite.
Balanced growth is defined as growth during which a
doubling of the biomass is accompanied by a doubling of all
other measurable properties of the population such as
protein, DNA, RNA, and intracellular water.
In other words, cultures undergoing balanced growth to
maintain a constant chemical composition.
 Cell growth measurement
In an adequate medium to which they have
become adapted, bacteria are in a state of
balanced growth.
To follow the course of growth, it is necessary
to make quantitative measurements.
Cell growth can be determined by measuring
cell number, cell mass, or cell activity.
 Measurement of Cell Number
 The number of cells in a population can be
measured
 under a microscope by counting cells placed
in special counting chambers.
 Measurement of Cell Mass
Cell dry weight can be measured directly by taking an
aliquot of cell suspension and centrifuging it.
After the supernatant is discarded, the cells are
thoroughly washed with distilled water to eliminate all
soluble matter.
The suspension is washed, re-centrifuged and the
settled cells are dried in an oven and weighed.
 Turbidity
The cell mass can be measured optically by
determining the amount of light scattered by a
suspension of cells.
The technique is based on the fact that small particles
scatter light proportionally, within certain limits, to their
concentration.
When a beam of light is passed through a suspension
of organisms, the reduction in the amount of light
transmitted as a consequence of scattering is thus a
measure of the cell density.
 Turbidity
 Such measurements are usually made in a
spectrophotometer, which reads in absorbance (A)
units.

 The absorbance is defined as the logarithm of the

ratio of the intensity of light striking the suspension to
that transmitted by the suspension

 A calibration curve can be obtained by measuring the

absorbance of the samples with known cell
concentration.

 The measurements are usually made at a

wavelength of 600 ~ 700 nm.
 Indirect Methods
 The indirect methods for measuring cell mass are
based on the overall stoichiometry for growth and
product formation,
 The change of the cell mass can be monitored
indirectly by measuring nutrient consumption, product
formation, cell components, heat evolution, or other
physical properties of broth.
 Functions of a Fermenter
 The main function of a fermenter is to provide a
controlled environment for the growth of
microorganisms or animal cells, to obtain a desired
product.

1. The vessel should be capable of being operated

aseptically for a number of days

2. Adequate aeration and agitation should be provided to

meet the metabolic requirements of the icroorganism.
However, mixing should not cause damage to the
organism nor cause excessive foam generation.

3. Power consumption should be as low as possible

 Bioreactor design
4. A temperature control system, both during
sterilization and fermentation, should be provided
5. A system of pH monitoring and control should be
provided
6. Sampling facilities should be provided
7. Evaporation losses from the fermenter should not be
excessive
8. The vessel should be designed to require the minimal
use of labor in operation, harvesting, cleaning, and
maintenance.
 Bioreactor design
9. Ideally the vessel should be suitable for a range of
processes, but this may be restricted because of
containment regulations.
10. The vessel should be constructed to ensure smooth
internal surfaces, using welds instead of flange joints
whenever possible.
11. The vessel should be of similar geometry to both
smaller and larger vessels in the pilot plant or plant
to facilitate scale-up
12. The cheapest materials, which enable satisfactory
results to be achieved should be used.
13. There should be adequate service provisions for
individual plants
 Bioreactor Design
9. Ideally the vessel should be suitable for a range of
processes, but this may be restricted because of
containment regulations.
10. The vessel should be constructed to ensure smooth
internal surfaces, using welds instead of flange joints
whenever possible
11. The vessel should be of similar geometry to both
smaller and larger vessels in the pilot plant or plant to
facilitate scale-up.
12. The cheapest materials, which enable satisfactory
results to be achieved should be used.
13. There should be adequate service provisions for
individual plants