Microbial Cultures

Microbial populations dominate the biosphere in terms of metabolic impact

and numbers. Among the various types of microbes, prokaryotes are the

most pervasive lifeform on the planet, often tolerating extremes in pH,

temperature, salt concentration, etc. Metabolic diversity is greater among

prokaryotes than all eukaryotes combined. Men have long been utilizing the

bacterial and yeast and fungal population for the manufacturing of various

chemicals, biochemicals, antibiotics, beverages, etc.

Production of antibiotics, alcohols, vinegar, amino acids, vitamins, therapeutic antibodies, acetone and other solvents, and recombinant proteins is accomplished by the large-scale cultivation of microbial cells such as bacteria, algae, yeast, and fungus on industrial scale.

In all these industrial applications the metabolic activities or the biochemical pathways

are used for the production of specific chemicals with the consumption of the

substrates or a carbon source such as sucrose. Here, the microbial culture acts as a

factory, where the substrate is the raw material. It is converted into the product

and secreted into the media. The product can be recovered from the media with a

process called downstream processing. There is a limitation for a single cell to

convert the raw material into products in a given period of time. It is possible to

calculate the rate of product formation by a single cell under a specific metabolic

condition, if we know the quantity of product formed over a period of time and

the number of cells in the culture. If we want to produce a specific quantity of the

product over a period of time it is possible to calculate the number of bacterial or

microbial cells required to operate the bioprocess on an industrial scale.

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?Types of Microbial Cultures

?The culturing of the microbial system can be achieved in different ways. The type

of culture method sometimes depends on the type of the microbial system or on

the type of the product that we expect. For example, one can get two entirely

different products from the same organism by changing the nutritional and other

parameters or even culturing vessels.

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1. Batch culture- This is a small-scale laboratory experiment in which a microbial

culture is growing in a small volume flask. It consists of a limited volume of

broth culture in a flask inoculated with the bacterial or microbial inoculum

and follows a normal growth phase. It is a closed-culture system because the

medium contains a limited amount of nutrients and will be consumed by the

growing microorganisms for their growth and multiplication with the excretion

of certain metabolites as products. In batch cultures, the nutrients are not

renewed and the exponential growth of cells is limited to a few generations.

The growth phase of the culture consists of an initial lag phase, a log phase or

the exponential growth phase, and a stationary phase. During the log phase

the consumption of the nutrients will be the maximum resulting in the

maximum biomass output with the excretion of the product. At the stationary

phase the rate of growth decreases and becomes zero. This is because at the stationary phase the cells are exposed to a changed environment where there

is only a small amount of nutrients and more cells along with the accumulation

of metabolites, which may have a negative effect on the growth of the cells.

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2. Fed-batch culture. The batch culture can be made into a semi-continuous

culture or fed-batch culture by feeding it with fresh media sequentially at the

end of the log phase or in the beginning of the stationary phase without

removing cells. Because of this the volume of the culture will go on increasing

as fresh media is added. This method is specially suited for cultures in which

a high concentration of substrate is inhibitory to cell multiplication and biomass

formation. In such situations the substrate can be fed at low concentrations to

achieve cell growth. This method can easily produce a high cell density in the

culture medium, which may not be possible in a batch fermentor or shake

flask culture. This is especially important when the product formation is

intracellular to achieve maximum product output per biomass.

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3. Continuous culture- ?Bacterial cultures can be maintained in a state of

exponential growth over long periods of time using a system of continuous

culture, designed to relieve the conditions that stop exponential growth in

batch cultures. Continuous culture, in a device called a chemostat, can be used

to maintain a bacterial population at a constant density, a situation that is, in

many ways, more similar to bacterial growth in natural environments.

This is a very convenient method to get continuous cell growth and product

formation over a long period of time. In continuous culture, the nutrient

medium including the raw material is supplied at a rate that is equal to the

volume of media with cells and product displaced or removed from the culture.

The volume removed and the volume added is the same. In effect there is no

change in the net volume as well as the chemical environment of the culture.

?????? In a chemostat, the growth chamber is connected to a reservoir of sterile

medium. Once the growth is initiated, fresh medium is continuously supplied

from the reservoir. The volume of fluid in the growth chamber is

maintained at a constant level by some sort of overflow drain. Fresh medium

is allowed to enter into the growth chamber at a rate that limits the growth of

the bacteria. The bacterial cells grow (cells are formed) at the same rate at

which bacterial cells (and spent medium) are removed by the overflow. The

rate of addition of the fresh medium determines the rate of growth because

the fresh medium always contains a limiting amount of an essential nutrient.

Thus, the chemostat relieves the insufficiency of nutrients, the accumulation

of toxic substances, and the accumulation of excess cells in the culture, which

are the parameters that initiate the stationary phase of the growth cycle. The

bacterial culture can be grown and maintained at relatively constant conditions,

depending on the flow rate of the nutrients.

If the chemical environment is constant in a chemostat continuous culture, the

cell density is constant in a turbidostat culture, which is also a continuous culture.

Since the culture is fed with the fresh medium at specific rate, a steady state of

growth and metabolism is achieved. At a steady state, the cell multiplication and

substrate consumption for growth and product formation occur at a fixed rate.

The growth rate is maintained constantly. The formation of new biomass is balanced

with the removal of cells from the outlet. Continuous culture is very suitable for

the production of cell biomass and products, if it is excreted into the medium. It is

widely used for the production of single-cell protein from liquid effluents as a

byproduct of the waste treatment. The organic waste present in the effluent is

converted into microbial biomass, which is known as single-cell proteins.

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?Applications Of Microbial Culture Technology

Microbial cultures have a large number of applications. Microbial cells can be used for the production of various substances, depending on the metabolic activities of the cells. ?

The following are the major applications of microbial cultures for basic research and industrial applications:

1. Production of whole microbial cells such as food and vaccines.

2. Production of primary metabolites such as acids, alcohol, enzymes, and

microbial polysaccharides.

3. Production of secondary metabolites such as antibiotics and biodegradable plastics.

4. Microbial leaching of metals, effluent and waste treatment.

5. Microbial cells such as agents of biotransformation of organic compounds.

6. As host cells for the production of recombinant proteins, gene cloning, and

other molecular biology research.

The oldest use of microbial cultures is to produce fermented foods such as

cheese and wine. Here, the whole cells are used. Whole cells are also used for the

production of single-cell proteins and certain vaccines such as tetanus vaccines,

typhoid vaccines, and tuberculosis vaccines. For the production of single-cell

proteins selected microorganisms such as yeast and spirulina are allowed to grow

in the culture (may be starch factory effluents) and the whole microbial cells are

dried and used as food material both for human consumption and cattle feed.

Production of organic acids, alcohol, various types of enzymes, and polysaccharides

are examples for primary metabolites. Antibiotics and other types of organic

molecules such as hydrocarbons are examples of secondary metabolites. Microbial

extraction of metals such as copper and iron and treatment of liquid waste or

effluents using microbial systems are examples where microbial metabolism is used

to convert the waste into useful products and thereby the disposal of the waste

without creating any environmental pollution. In modern biology, particularly in

molecular biology and recombinant techniques, certain microbial cells such as e.coli

are used as host cells for the cloning and expression of certain genes and the

production of recombinant proteins. Production of human insulin in e.coli and the

hepatitis B vaccine in yeast are good examples.

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Some microbial products and their organisms

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