UNIT – I

PHYSIOLOGY OF MICROBIAL GROWTH

1. Batch culture
A technique used to grow microorganisms or cells. A limited supply of nutrients for
grow this provided when these are used up some other factor becomes limiting, the
culture declines. Cells or products that the organisms have made, can then be
harvested from the culture.
In batch cultivation, the bacteria are inoculated into the bio reactor(always stirred
tank bioreactor).Then, under certain conditions (temperature, pH ,aeration, etc.) the
bacteria go through all the growth phases(lag, exponential, stationary).At last, the
fermentation is stopped and the product is collected.Then, after cleaning and
sterilization of the fermenter is ready for another batch.

Physiological conditions:
Four typical phases of growth
 Lag phase
 Log phase
 Stationary phase
 Death phase
 Batch Culture Process
 A batch culture begins with sterilization, and the sterile culture is then
inoculated with microbes (about 2-5 % of the total volume).
 The percentages of nutrients, vitamins, and microbial cells in the reaction
mixture and the temperature difference during the reaction cycle.
 The proper mixing keeps them at acceptable concentrations and temperatures.
 The process is carried out in anaerobic conditions by bubbled oxygen in or out,
while acidic or alkaline solutions are added to control the PH.
 Antifoaming agents are added when indicated by a foam sensor.
 The microorganism growth is allowed to take place for days, weeks, or months.
 In the lag phase, little or no growth is observed at the beginning of
fermentation, depending on a physiochemical equilibrium between the
microorganism and the environment.
 Once the cells have adapted to the new conditions of growth, they enter the
exponential phase.
 Primary metabolites are produced during the log or exponential phase with
their formation decreasing when growth ceases. For
example, Saccharomyces cerevisiae produces ethanol as a primary metabolite.
 When the cells enter the stationary phase, secondary metabolites are produced.
Most antibiotics are metabolized as secondary metabolites. For
example, Penicillium chrysogenum produces Penicillin as a secondary
metabolite.
Advantages of batch culture:
1. Reduced risk of contamination .
2. cell mutation as the growth period is short.
3. Lower capital investment when compared to continuous processes for the same
bioreactor volume.
4. More flexibility with varying product/biological systems.
5. Higher raw material conversion levels, resulting from a controlled growth
period.
Batch Culture Applications
1. It is beneficial for the construction of biomass (Baker’s yeasts) and primary
metabolites (lactic acid, citric acid, acetic acid, or ethanol production).
2.In food industries, organic acids are used as preservatives or acidifiers(lactic
acids, citric acids, and acetic acids), alcoholic beverages (wine, beer, and distilled
spirits i.e. brandy, whisky, and rum), and sweeteners (e.g., aspartate) or amino
acids used as flavoring agents (e.g., monosodium glutamate)are the various
product manufactured by batch cultivation.
 Batch Culture Limitations
1. Due to nutrient consumption and waste build-up, microbes are exposed to
constantly changing environmental conditions in batch culture.
2. After reaching an endpoint, batch cultures must be restarted. In large
bioreactors, it takes a long time to empty, clean, and refill the reactor.
3. In batch culture, the low productivity is a consequence of the high downtime
(nonproduction time spent cleaning, sterilizing, and starting up another batch
cultivation) during two consecutive batches.

2.CONTINEOUS CULTURE
In continuous cultivation, the fresh medium flows into the fermentor continuously,
and part of the medium in the reactor is withdrawn from the fermenter at the same
flow rate of the inlet flow. The table below shows the advantages and disadvantages
of different modes of operation of the stirred tank reactor.

Process of Continuous Culture

 In continuous culture, an open system is set up in which one or more feed
streams containing the necessary nutrients are fed continuously, while the
effluent stream containing the cells, products, and residuals is continuously
removed.
 A steady-state is established by maintaining an equal volumetric flow rate for
the feed and effluent streams.
 The culture volume is kept constant, and all nutrient concentrations remain at
constant steady-state values.
 During this process, the exponential growth phase is prolonged and the
formation of by products is avoided.
 Continuous fermentation is monitored either by microbial growth activity or by-
product formation.

TYPES OF CONTINUOUS CULTURE

a. Chemostat

A chemostat(from chemical environment is static)is a bioreactor to which

fresh medium is continuously added, while culture liquid containing left over
nutrients, metabolic end products and microorganisms are continuously removed
at the same rate to keep the culture volume constant.
B. Turbidostat method
 Cell growth is controlled and remains constant in this system, but the flow
rate of fresh media varies.
 Cell density is controlled based on the set value for turbidity, which is
created by the cell population while fresh media is continuously supplied.
 Most turbidostats use as pectrophotometer/turbidometer to measure
the optical density for control purposes, there exist other methods, such
as dielectric permittivity.
 Medium and cells are continuously changing
 The cell density is constant
 Steady-state growth
 Open system

Applications of Continuous Culture

1. Continuous culture fermentation has been used for the production of single-
cell protein, organic solvents, starter cultures, etc.
2. It has been used in the production of beer, fodder yeast, vinegar, baker’s
yeast, etc.
3. In the industrial production of secondary metabolites (such as antibiotics from
a Penicillium or a Streptomyces sp.)
4. Continuous culture has been tested for L-lysine-
producing C. glutamicum mutant B-6
5. It has been used in municipal waste treatment processes.

Limitations of Continuous Culture

1. In the long-term cultivation process, sterility maintenance can be tricky, and
downstream processing can prove challenging.
2. Controlling the production of some non-growth-related products is not easy
Because of this, continuous culture often requires fed-batch culturing as well
as continuous nutrient supply.

3.Synchronous culture
A synchronous or synchronized culture is a microbiological culture or
a cell culture which contains the cells that are all in the same growth stage.

 The Synchronous culture also known as the synchronous growth.

 The synchronous growth of a bacterial culture means all the cells of the culture
remain at the same stage of growth and they grow all together from one phase to
another.
 A population can be synchronized by manipulating their physical environments or
physical composition of the medium.
 If we keep an organism under un favourable conditions there they will metabolize very
slowly, but will not divide. If we keep the organisms under favourable conditions, then
all cells undergo division and stay at the same phase.
 The cells of the synchronously growing culture divide at a time, their growth curve
forms a Zig Zag pattern.
 The easiest way to synchronize bacterial growth is to add some cytostatic agents so
that cells don’t divide and they all maintain the same state of metabolism and cell
cycle.
 When the cytostatic agent is removed, all cells start to divide at the same time.
 Synchronous culture/Synchronous growth of bacteria consists of two phases such as
stationary phase and exponential phase.



Method of Synchronous culture
 Physical selection method

 Blological selection method

1. PHYSICAL SELECTION METHOD -

 These methods involve separation of the cells on the basis of their size. Selection

of cells with uniform size and then their synchronous culture

 This is based on the fact that cells with same size occur in same state of cell

division and hence their use provides synchronized growth.

Cells with identical size are selected by various use provides methods:

o By use of membrane filter.

o By use of density gradient centrifugation.

A. SELECTION OF CELLS BY MEMBRANE FILTER -

Use of membrane filter with specific pore size will help in separating cells with uniform

diameter. Such cells are then used for obtaining synchronous growth.

Another method for obtaining cells with uniform size by use of membrane filter is

Helmstetter Cummings technique.

Helmstetter Cummings method for obtaining synchronous growth.

When heterogeneous culture is fltered through cellulose nitrate membrane filter, some

cells get adhered to the filter.

The filter is then inverted and fresh broth is allowed to low through. Initial flow removes

loosely adhered bacteria from the filter.

Then, the later flow will contain only these cells which have just separated after division

from adhered cells.

Therefore these cells will be in the same state of division. Hence, culture arising from

these cells will be growing as synchronized culture.

B. USE OF DENSITY GRADIENT CENTRIFUGATION

Another method of separation of cells having identical size is the use of centrifugation

technique. Thus, centrifugation of a culture allows one to separate cells with identical

size. They can yield synchronous growth upon inoculation to a fresh medium.

2. BIOLOGICAL SELECTION METHOD

Characteristic physiological properties of organisms can also be useful in obtaining

synchronised growth. Two methods based on these parameters are widely used.

a). Cyclic temperature shift method

b). Use of limiting growth substance.

A). USE OF CYCLLE TEMPERATURE SHIFT METHOD -

o The method is commonly used for obtaining synchronized growth of mesophilic

bacteria, where they are incubated for some time at 37°C and 20°C alternately.

o The fundamental property of the bacteria is that they can initiate cycle of new

chromosome replication and cell division only at a speciflc temperature ( at 37°C).

and not at lower temperature, i.e. at 20°C.

o But., once the cycle has initiated, it can continue till it is completed. Therefore, cells

which have been transferred to 20°C and kept for some time, all cells will continue

to complete their division cycle, but will not start new cycle of cell division.

o Thus, all cells are brought to same state of cell division. These organisms will

therefore give synchronized growth on further incubation.

B) USE OF LIMITING GROWTH SUBSTANCES -

o Supply of limniting growth substances in the medium controls the growth rate of

fastidious organisms.

o The organisms can be made to grow synchronously by supplying them with fresh

supply of this limiting growth substance in the nutrient medium after they have

entered stationary phase of growth. b.

o In stationary growth phase, bacteria stop growth because of non availability of

limiting growth substance. Hence, all cells may occur in same state of physiology

in this growth phase. The transfer of these cells to a fresh medium containing

limiting growth substance will allow them to divide at a time and hence will give

synchronous growth.

o In stationary growth phase, bacteria stop growth because of non availability of

limiting growth substance. Hence, all cells may occur in same state of physiology
in this growth phase.
o The transfer of these cells to a fresh medium containing limiting growth substance
will allow them to divide at a time and hence will give synchronous growth.

Applications of Synchronous culture

Synchronous culture helps in the separation of the smallest cells from an exponentially

growing culture.

Growth cycle or Growth Curve

 Bacterial growth is regulated by nutritional environment.

 When suitable environment is there that time bacterium is incubated its growth

leads to increase in number of cells which allow definite course.

 The growth curve has got four phases:

 Lag phase
 Log phase(logarithmic)or exponential phase
 Stationary phase
 Decline phase
 Lag Phase(1-4Hrs)
 Bacteria adapt themselves to growth conditions.
 It is the period where the individual bacteria are maturing and not yet able to divide.
 During the lag phase of the bacterial growth cycle,synthesis of RNA,enzymes and other
molecules occurs.
 Length of this phase depend on type of bacterial sepsis, culture medium, and
environmental factors.
Log Phase (8Hrs)
 Sometimes called the log phase or the logarithmic phase
 It is a period characterized by cell doubling.The number of new bacteria appearing per
unit time is proportional to the present population.
 If growth is not limited, doubling will continue at a constant rate so both the number of
cells and the rate of population increase doubles with each consecutive time period.
 For this type of exponential growth, plotting the natural logarithm of cell number against
time produces a straight line.
 The slope of this line is the specific growth rate of the organism, which is a measure of
The number of divisions per cell per unit time.
 The actual rate of this depends upon the growth conditions, which affect the frequency
of cell division event sand the probability of both daughter cells surviving.
 Under controlled conditions, cyanobacteria can double their population four times aday.
 Exponential growth can not continue in definitely, however, because the medium is soon
depleted of nutrient sand enriched with wastes.

Stationary phase
 Stationary phase is due to a growth-limiting factor; this is mostly depletion of a

nutrient, and/or the formation of inhibitory products such as organic acids.

 Unfortunately wide spread explanation is that the stationary phase results from a

situation in which growth rate and death rate have the same values (newly formed

cells per time=dying cells per time);but this is not logical, and it is better to forget this.

 Suchanexplanationwouldnotbeinaccordancewiththeobservedsubstratedepletion and

also could never explain the rather “smooth, ”horizontal line as part of the curve

during the stationary phase.

 Death of cells as a function of time is rather unpredictable and very difficult to explain.

 Another not realy logical explanation of the stationary phase is that there isn’t any

more enough space for the cells.

Decline Phase
 Bacteria run out of nutrients and die although number of cells remain constant.

 The decline phase is brought by exhaustion of nutrients, accumulation of toxic products

and autolytic enzymes

 Some times a small numbers of survivors may persist for month even after death of

majority of cells these few surviving cells probably grow at expence of nutrients release

MEASUREMENTS OF MICROBIAL GROWTH

 There are various ways to measures microbial growth for the determination of

growth rates and generation times.

  For the measurement of growth either mass or population number is followed

because growth leads to increase in both.

 Growth can be measured by one of the following types of measurements:

1. Cell count this method involves the measurement of growth either by

microscopy or by using an electronic particle counter or indirectly by a colony

count.

2. Cell mass in this growth can be measured directly by weighing or by a

measurement of nitrogen concentration in cells or indirectly by the determination

of turbidity using spectrophotometer.

3. Cell activity in this growth can be measured indirectly by analysis of the degree

of biochemical activity to the size of population.

METHODS TO MEASURE MICROBIAL GROWTH

1.Direct microscopic count
2.Electronic enumeration of cell numbers
3.The plate count method
4.Turbidity estimation of bacterial numbers
5.Determination of nitrogen content
6.Determination of dry weight of cells
7.Filtration method
8.Most Probable Number (MPN) Method

1.DIRECT MICROSCOPIC COUNT

 The most obvious way to count microbial numbers is through direct counting.

 Petroff-hausser counting is one of the easiest and accurate way to count bacteria.

 Side view of the chamber showing the cover glass and the space beneath it that

holds a bacterial suspension.

 A top view of the chamber. The grid is located in the center of the slide.

 An enlarged view of the grid. The bacteria in several of the central squares are
counted, usually at X400 to X500 magnification.
  Concentration of the cells can be calculated by using the average no. of bacteria
the avg. number of bacteria in these squares.
 There are 25 squares covering a part of area of 1 mm2, then the entire number of
bacteria in 1 mm2 of the chamber is (number/square) (25 squares). The chamber is
0.02 mm deep and thus, bacteria/mm3 = (bacteria/square) (25 squares) (50).
 The amount of bacteria per cm3 is 103 times this value. For example, imagine the
average count per square is 28 bacteria: bacteria/cm3 = (28 bacteria) (25 squares)
(50) (103) = 3.5X 107.

2.ELECTRONIC ENUMERATION OF CELL NUMBERS

 In this method of microbial growth measurement, bacterial suspension is kept inside

an electronic particle counter, within which the bacteria are passed through tiny

orifice 10 to 30 μm in diameter.

 This orifice is then connected to the two compartments of the counter which

contains an electrically conductive solution.

  The electrical resistance between two compartments will increases momentarily,

when bacterium passes through the orifice. This generates an electrical signal

which is automatically counted.

 The main disadvantage of this method is that there is no way to determine whether

the cell counted is viable or not.

THE PLATE COUNT METHOD

There are types of plate count method.
(a) The pour plate method.
(b) The spread plate method.
 This method allows the determination of the number of cells that will multiply under
certain defined conditions.
 Plate count method can be done in two ways either by spread plate method or by
pour plate method.
 This method of bacterial counting is most commonly used with satisfactory results
for the estimation of bacterial populations in milk, water, foods and many other
materials.
 This technique has some drawbacks because some relatively heat-sensitive
microorganisms may be damaged by the melted agar and will therefore be unable
to form colonies

TURBIDITY ESTIMATION OF BACTERIAL NUMBERS

  For some experimental work, turbidity is a only practical way of monitoring bacterial
growth.
 Actual way of monitoring bacterial growth. As bacteria multiply in a liquid medium,
the medium becomes turbid, or cloudy with cells.
 Turbidity is the Cloudiness or haziness of a media or fluid caused by large no. of
individual particles.
 The instrument used to measure turbidity is a spectrophotometer (or colorimeter).
 Microbial mass can be determined by determination of absorption of light.
 In the spectrophotometer, a beam of light is transmitted through a bacterial
suspension to a light-sensitive detector, as the bacterial numbers increase, less
light will reach the detector.
 As the population increases, absorbance of the light increases by the cells, so the
turbidity also increases. Turbidity can be measured by using an instrument
spectrophotometer.
 The absorbance is used to plot bacterial growth.

DETERMINATION OF NITROGEN CONTENT

 The major constituents of cell material are protein, and since nitrogen is
characteristics part of proteins.
 Bacterial population or cell crop can measure in terms of bacterial nitrogen.
 In this growth can be measured by first harvesting the cells and wash them free of
medium and then perform a quantitative chemical analysis of nitrogen.

DETERMINATION OF DRY WEIGHT OF CELLS

 For filamentous bacteria and molds, the usual measuring methods are less
satisfactory. A plate count would not measure this
increase in filamentous mass.
 In plate counts of actinomycetes and molds, it is mostly the number of asexual
spores that is counted instead.
 This is not a good measure of growth. One of the better ways to measure the
growth of filamentous organisms is by dry weight.
  In this procedure, the fungus is removed from the growth medium,
filtered to remove extraneous material, and dried in a desiccator, it is then weighed.
 Growth measurement by measuring cell mass is one of the easiest ways, a known
volume of culture sample from the ferment or withdrawn and centrifuged.
 It is the most direct approach for quantitative measurement of a mass of cells.

COUNTING BACTERIA BY FILTRATION METHOD

 When the number of bacteria is extremely few, as in lakes or relatively pure

streams, bacteria are often counted by filtration methods.

 During this technique, a minimum of 100 ml of water are passed through a thin
membrane filter whose pores are too tiny to permit bacteria to pass.

 After filtration bacteria are filtered out and present on the surface of the filter. Then
filter is transferred to a Petri plate containing a in liquid nutrient medium, where
colonies grow from the bacteria on the filter’s surface.

 This method is applied frequently to detection and enumeration of coliform bacteria,

which are indicators of fecal contamination of food or water.
MOST PROBABLE NUMBER (MPN) METHOD

 Another method for determining the number of bacteria in a sample is the most
probable number (MPN) method.
 This statistical estimating technique is based on the fact that the greater the number
of bacteria in a sample, the more dilution is needed to reduce the density to the
point at which no bacteria are left to grow in the tubes in a dilution series.
 The MPN method is most useful when the microbes being counted will not grow on
solid media (such as the chemoautotrophic nitrifying bacteria).
 It is also useful when the growth of bacteria in a liquid differential medium is used to
identify the microbes (such as coliform bacteria, which selectively ferment lactose to
acid, in water testing).
The MPN is only a statement that there is a 95% chance that the bacterial
population falls within a certain range and that the MPN is statistically the most probable
number.
Control OF microbial growth

These are the following methods through which microbial growth can be controlled.

Physical methods
 Heat (dry heat and moist heat)
 Radiation
 Filtration

Chemical methods
 Phenol
 Halogens
 Alcohols
 Heavy Metals
 Oxidizing agents

Physical methods
Sterilization by heat: Heat is the most widely and effective method to control microbial

growth. In this process, articles are exposed to dry heat, hot air oven, or moist heat for

sterilization.

Sterilization by dry heat: This method helps to kill the bacteria by oxidation effects. An

oven is used for this process and the items which need to sterilize are kept in the oven for

about 2 to 3 hours, from 160 degrees to 170 degrees celsius. The effectiveness of dry

heat is less than moist heat. Dry heat can either be done by the hot air oven or sterilizer

and by incineration.

Hot air oven or sterilizer: It is based on thermoregulation. There are several materials and

instruments which need a hot air oven or sterilizer for sterilization like Petri dishes,

forceps, scalpels. These materials are exposed to a hot air oven for about 1.5 hours to 3
hours at a temperature of 160 degrees Celsius to 180-degree celsius.
Incineration: This process is based on the burning of microorganisms by introducing the

needles into the flame of the Bunsen burner. This process is suitable for the destruction of

carcasses, infected laboratory animals.

Sterilization by moist heat

Microorganisms and their spores can be destroyed at lower temperatures with the help of

moist heat sterilization. Moist heat can be attained by pasteurization, autoclaving, and

tyndallization.

Autoclave

An autoclave is a useful device or an instrument and its structure is somewhat like a

pressure cooker. And it also follows the same principle to operate by filling with saturated

steam and desired temperature should be maintained to operate this device. This device is

effective to kill bacteria, viruses, fungi, and spores. It kills the microorganisms by providing

heat that has a moisture content at a temperature greater than 100 degrees celsius. The

nature of the materials that need to be sterilized, the type of vessel used, and the volume

alter the time of its operation.

Pasteurization

Many substances like milk, cream, and certain alcoholic beverages are exposed with

controlled heating at a temperature below boiling, which is known as pasteurization which

generally destroys microorganisms of certain types but does not act on all organisms.

Tyndallization

Tyndallization mainly involves heating the object at 100 degrees celsius for three days in a

row with an incubation period in between. The resistant spores can easily germinate

during an incubation period. The duration of the objects to be steamed for about 30-40

minutes on those three successive days

On the first occasion, vegetative bacterial cells are to be killed, resistant spores which

germinate during incubation period produce vegetative form and that is eliminated in

second or third steaming.

Radiation

Radiation is the transmission of energy through space in a variety of forms. UV radiation

(Non-Ionization) and Ionization radiations are most significant to sterilize or disinfect

objects.

UV radiation (Non- Ionization)

UV radiation around 2,650 angstroms is quite deathly but it is unable to penetrate dirt

films, glass, water, and other substances deeply. It damages DNA by inducing thymine

dimers in DNA and the interference with DNA replication of an organism.

Ionization radiation

It is a great sterilizing agent and can penetrate deep down into objects. It can easily

destroy bacterial endospores and vegetative cells, both prokaryotic and eukaryotic. It is

used in the sterilization of several antibiotics, hormones, and plastic non-reusable supplies

such as syringes and is also used to sterilize and pasteurize meat and other foods.

Filtration

It is a process to reduce the microbial population in a solution by the passage of a liquid or

air through a screen-like material with small pores. It is used to sterilize heat-sensitive

materials like vaccines, enzymes. These filters are used to sterilize pharmaceuticals,

ophthalmic solutions, culture media.

Chemical methods
Phenol: Its function is to destroy the plasma membrane and denatures protein. But this

chemical is not safe to use as it has a strong odor and can create skin irritation.
Halogens

 The tincture of iodine helps to control microbial growth by denaturing the proteins.

 Chlorine is mainly used to remove toxins and infections from drinking water, water

bodies, and sewage as it has bleaching properties.

Alcohols: Alcohols help to kill bacteria but are not effective in the case of endospores.

They denatured the proteins and disrupted the cell membranes. It is generally used to

wipe off microbes from the skin before injection or blood draw.

Heavy metals

Heavy metals and their compound are microbicidal, which can control microbial growth.

 Silver nitrate is used to protect infants against a harmful disease, gonorrheal eye

infections.

 Selenium is best to kill fungi and their spores, and it is used in the preparation of

dandruff shampoo.

 Zinc chloride is used as an antifungal agent in paints and also used in

mouthwashes to kill germs.

Oxidizing agents

Ozone: It helps to disinfect water, as it has more effective killing power than chlorine.

When ozone is dissolved in water, it produces a broad spectrum biocide that destroys all
viruses, bacteria, and cysts.

Hydrogen Peroxide: It is used as a disinfectant. It is powerful enough to kill viruses like the

H1N1 virus, Severe acute respiratory syndrome (SARS), and coronavirus. It can also kill

bacteria like staph.

Benzoyl peroxide: It is one of the important components of acne medicine. It kills the
bacteria underneath the skin. It also enables the skin to shed excess oil and dead skin.
Importance of control of microbial growth

These are the following importance of controlling microbial growth

 It has an immense role in controlling infectious and dangerous diseases.

 Prevents complications during surgery.

 Foods can be prevented from spoilage.