Microbial Growth

Standard bacterial growth curve

(i) Lag Phase (A)

(ii) Log (logarithmic or exponential) phase (B)
(iii) Stationary Phase (C)
(iv) Decline (death) phase (D)

D
Ln X
B

Time (H)

A standard bacterial growth curve

Log phase

This lag in division is associated with a physiological adaptation to the new

environment, by the cells, prior to their resumption of division that is, cells may
increase in size during this time, but simply do not undergo binary fission.

Log phase (logarithmic phase, exponential phase)

Lag phase is followed by log phase during which binary fission occurs. This phase of
growth is called logarithmic or exponential because the rate of increase in cell
number is a multiplicative function of cell number
This can be seen in a graph of cell number versus time where cell numbers increase
at ever increasing rates with time or generation; that is, the rate of increase is a
function of absolute cell number such that the more cells present, the faster the
population of cells increases in size (at least, during log phase) Generation time is
the period a bacterial takes population to double in size (number) during log-phase
growth.
Note that the time it takes for the population to double in size does not change with
cell number (so long as cells remain in log phase)
That is, with exponential growth, the absolute increase in cell number increases as
cell number increases while the relative increase remains invariant
Typically, generation times range from 20 minutes to 20 hours depending on the
bacterial species/strain and the conditions during which log-phase growth is
occurring.

Stationary phase

Stationary phase is a steady-state equilibrium where the rate of cell growth

(division) is exactly balanced by the rate of cell death (i.e., increase in cell number
due to cell division exactly balanced by a decrease in cell number due to death)
Note that the simplest conditions that will result in a stationary phase is when both
the rate of cell increase and the rate of cell death together equal zero.

Decline phase (death phase)

Stationary phase, in a standard bacterial growth curve, is followed by a die-off of

cells.
Cell death in bacteria cultures basically means that the cells are unable to resume
division following their transfer to new environments
Typically this die-off occurs exponentially. This death occurs because vegetative cells
cannot survive exposure to harsh conditions (few nutrients and too-many toxins) for
only so long
 FERMENTATION KINETICS

EXPONENTIAL GROWTH LAW:

In fermentation the conditions required for growth of microorganisms are given as
follows:
 Viable Inoculum
 Energy Source (substrate)
 Essential nutrients to produce material for biomass synthesis.
 Suitable physiochemical conditions. (PK, temp.)
 Absence of toxins, inhibitors etc.

If all the requirements for growth are satisfied then “the rate of
increase in biomass concentration during a small interval of time i.e. dx/dt is
directly proportional to the biomass present at the beginning of that interval
time”. This is called exponential growth law.

dx / dt ∞ X
dx/dt = µ . x eq.1
dx/dt is called population growth rate and µ = Specific growth rate

dx = µ dt . x eq.2
µ = dx . 1
dt x

µ represents the growth rate per unit biomass. Its dimensions are h -1 (reciprocal
time).

Microbial growth that follows the exponential growth law is called or logarithmic

growth. So during the exponential growth phase “µ”remains constant provided that
conditions and the constitution of the biomass remains uniform. When µ is constant,
equation can be integrated as.
x t
x
O
∫ dx = ∫ µdt
o

ln x/xo = µ t
ln x - lnxo = µ t
ln x = µ t + lnxo eq.3

Where
X0 = biomass at the beginning of interval.

Eq. 3 is a straight line eq. and plot of ln x against time “t” will be a straight line with
slope equal to µ and intercept equal to lnxo.

y
y/x = slope = µ
ln x x

lnx0

DOUBLING TIME

Time during which biomass is doubled is called doubling time.

t = td
x = 2x0

ln x = µt + ln x0

ln x – ln x0 = µt

ln x = µt
x0
ln 2x0 = µ td
x0
ln 2 = µ td
0.693 = µ td
td = 0.693
µ
DEGREE OF MULTIPLICITY

Degree of multiplicity may be defined as “No of generations of microorganisms

produced in a particular time interval.”

0.693 = 2n
µ

n = 3.32 log x
x0
Bacterial and yeast cultures are well known to have exponential growth phases. But
the law is universal for all prokaryotes and eukaryotes provided that two conditions
are not.
Growth of filamentous fungi (molds) often shows divergence from the exponential
law because of formation of pellets in which biomass is heterogeneously dispersed.
Moreover in rapidly growing fungal cultures oxygen supply is growth limiting factor.
Different scientists have demonstrated that protozoan and animal cells also obey the
exponential law.

Microbial growth and nutrient Consumption

Substrate is used for the following purposes by the microorganism during

fermentation.
1 – Metabolic function (energy production)
2 – To increase biomass.
3 – Product formation.

Increase in microbial biomass is a function of substrate utilization.

dx ∞ dS
where
dx = change in biomass concentration
ds = change in concentration of substrate
dx = -y dS
-ve sing is because X and S change in opposite sense
Y = - dx
ds
Here y is growth yield.
Growth Yield (Y)

Increase in biomass per unit decrease in substrate concentration

is called growth yield.

Y = -dx
ds
Growth yield is an important mean of expressing quantitative requirements of
microorganisms. When conditions of microbial growth and biomass constitution are
maintained constant, growth yield (y) becomes constant and reproducible. It is
characteristic of every microorganism under specified conditions.

Economic Coefficient

Amount of substrate consumed per unit increase in biomass is called economic

coefficient. It is reciprocal of growth yield and is equal to 1/y.

1/y = -ds
dx
Note:-
High growth yield and low economic coefficient are required when we are
interested in biomass and biomass related products, such as in backers yeast
production
Now at (to) the cell concentration is ‘x0’ but after time (t) it changes to ‘x’.
And ‘S0’ is substrate concentration at t =0 which changes to S at time t.

dx = x - x0
Ds = S – S0
Putting 2 and 3 in equation
Y = - (x – x0)
(S –S0)

(x – x0) = - y(S –S0)

(x – x0) = y(S0 –S)
There are more than one substrates in the medium and one of these may be a
limiting nutrient (substrate). Concentration of biomass will be dependent on the
limiting nutrient.
Now if S is limiting nutrient then biomass will achieve its maximum value (X m) on
complete consumption of the limiting nutrient (S=0), that is:
When
S = 0
then
x = xm

Hence

Xm - X0 = YS0
Xm = YS0 + X0

It is equation of straight line.

Xm = YS0 + X0
Xm is dependent variable and S0 is independent variable.

Xm

X0

S0

Intercept = X0
Slope = Y = growth yield.
Substrate Consumption

(Substrate consumption rate) ds - x

dt
ds
= qx q = metabolic quotient
dt specific metabolic rate

Substrate Consumption and metabolic quotients

Rate of substrate consumption during fermentation depends upon microbial activity.
q = ds
dt . x

q is also called as Specific metabolic rate.

In simple words it is grams of substrate used by one gram of microorganisms in unit
time.
If biomass constitution and growth conditions are constant, q must be constant. q
can be related to specific growth rate (µ) and growth yield (y).

dx
µ =
dt.x
µ = Specific growth rate
dx
Y =-
ds
Y = growth yield
Multiplying equation (1) by dx
Dx
ds dx
q= *
dt.x dx
dx ds
q= *
dt.x dx
dx ds 1
As = µ and = /
dt.x dx y

So
µ
q =
y
 1/ µ

µ(max)
1/[s]

Slope = Ks
Intersept = µ(max)
ETHANOLIC FERMENTATION

Ethanol is produced both by fermentation and chemical method. The Microbial

method has definite advantages over the chemical method and involves renewable
sources. It is the one of the best known industrial fermentations.

PRODUCER ORGANISMS

Industrial production of ethanol is carried out by certain species of yeasts. Most

commonly employed brewer’s yeast is Saccbaromyces cerevisiae. Other import
species of yeast used for brewing are:
S. fragilis, S. Pombe, S. anamensis, Torulopsis cremoris, I. sphaerica etc. certain
bacteria has also been reported to produce ethanol but yet there are not used on
industrial scale. In any case the selected strain must be vigorous high alcohol
tolerant and capable of producing high yields of alcohol.

RAW MATERIALS

The raw materials used for ethanolic fermentation are of three types that is:-
(i) Materials containing sugars or saccharine material such as molasses, fruits
(ii) Starchy materials such as wheat, sweet potatoes, corn, barley etc
(iii) Cellulosic materials such as sulphite liquor etc.
Materials containing sugars such as molasses need no preliminary treatment other
than dilution. Starchy and cellulosic materials, however, must be hydrolysed to
fermentable sugars before they can be utilized by the yeast.

PROCESS

The process for the production of ethanol through fermentation involves following
stages.
1. Preparation of Fermentation Medium:-
Different raw materials contain different concentrations of sugars. For example cane
molasses contain about 55% sugars. The level of sugars is brought to 6-15% by
dilution. Salts like (NH4)2SO4, (NH4)3 PO4 etc are added to the fermentation medium
to supply Nitrogen and phosphates.
pH of the fermentation medium is set at about 4-5. This low pH is necessary to
inhibit bacterial contamination.

2. Sterilization of the fermentation Medium & the fermenter:-

The fermentation medium is either sterilized in a separate sterilizer or in a fermenter

by passing super heated steam through costs. Complete sterilization of the
fermentation medium (which takes place at 121c) is not achieved in many cases
because heavy Inoculum and low pH prevent bacterial contamination.

3. PREPARATION OF INOCULUM

The producer strain of the yeast is maintained on potato-dextrose agar slants or

malt-agar slants. From the cultured slant yeast cells are aseptilly transferred to a
lube containing coml sterile fermentation medium. The lube is inoculated at ± 2˚c for
24 to 36 hours. The cells of this lube are then transferred to about 200 ml sterile fermentation
medium and inculcated. This process is repeated and finally the volume of the Inoculum is
raised to 200 litres.

4. FERMENTATION

On industrial scale ethanolic fermentation is carried out in 10,000 to100,000 litre fermenter
(ravi Rayon Ltd has 100,000 litre fermenter) The fermentation medium, after partial
sterilizations and cooling is inoculated with starter or seed culture (developed as discussed
above in NO3). Fermentation is allowed to take place for 48 to 54 hours. During fermentation
heat is produced. The system is cooled down to maintain the temperature at 28±2˚c. The
fermented liquor certain 8 to 12% ethanol.

5. DISTILLATION
The fermented liquor is distilled to produce concentrates called ‘high wines’ having 60-90%
ethanol. Fractional distillation using rectifying column produces 95-96% ethanol (called
rectified alcohol).
Rectified alcohol is redistilled with quick lime (CaO)to yield 99.5% ethanol called ‘absolute
alcohol’ treatment with Na-metal and reinstallation produces 99.99% ethanol.

Biochemical pathway for ethanol production from cane molasses:-

Sucrose + H2O Invertase Fructose + Glucose

Hexokinose Hexokinase

Phosphohexose
Isomerase
Glucose – 6 – phosphate
Fructose – 6 – phosphate

Phosphohexokinose

Fructose – 1,6 – Diphosphate

Aldolase

Dihydroxy acctone phosphate Glyceraldhyde – 3 phosphate

Isomerase

NADtH3PO4
Triosephosphate
NADH+Ht
dehydrogenase

Glycerate -1,3 - diphosphate

ADP
Phosphoglycerate
ATP Kinase

Glycerate -3 – phosphate

Mutase
 Glycerate -2 – phosphate

Enolase
Enol pyruvate phosphate

Pyruvate kinase

Spontaneous
Pyruvic Acid Enol pyruvic acid

Carboxylase

Acetaldehyde

NADH+Ht

NADt

Ethanol