What are the Basic Techniques of Microbial Culture?

The technique of microbial culture is a multistep process and requires media formulation, sterilization, environmental control and operation of bioreactor, etc. These steps are discussed in this section:

1. Growth Media :

Microorganisms require several nutrients (e.g. carbon, nitrogen, phosphorus, minerals) and oxygen for growth and yield. The nutrient formulations which support optimum microbial growth and yield are called growth media. On the basis of purity of chemical compounds used, media are grouped into the following three types:

(i) Synthetic Media:

Microbes are cultured on a small scale in laboratory on artificially devised nutrient media by using pure chemicals. Such media are called synthetic media such as Czapek Dox agar medium for isolation of fungi.

(ii) Semi-synthetic Media:

The media which contain pure form of chemicals as well as complex compounds are called semi-synthetic media; for example nutrient broth, brain heart infusion broth, etc. In these media the complex compounds are beef extract, yeast extract, peptone, potato or casein digest. Now these media are commercially available.

(iii) Natural Media:

The media prepared by using the natural complex compounds are called natural media e.g. soybean seed extract broth, V8 juice broth, soil extract broth, etc.

These media are suitable for growth of microorganisms in laboratory only. However, these are not used on a large scale because these are not economical.

Such media are formulated for use on a large scale that are economical and of consistent quality, and available -throughout the year. Besides, the raw materials are also pre-treated before use if desired.

The following carbon and nitrogen sources used in media formulation are cheaper and economical and available throughout the year.

2. Sources of Nutrition :

There are different sources of nutrients required by different types of microorganisms. These are given below:

(i) Carbon Sources:

The carbon sources used for large scale microbial culture in fermentor are sugarcane molasses, beet molasses, vegetable oil, starch, cereal grains, whey, glucose, sucrose, lactose, malt, hydrocarbons, etc.

(ii) Nitrogen Sources:

The nitrogen sources are corn steep liquor, slaughterhouse wastes, urea, ammonium salts, nitrate, peanut granules, soyabean meal, soya meal, yeast extract, distilled solubles, etc.

(iii) Growth Factors:

There are certain microorganisms which are not capable of synthesising vitamins or amino acids. Therefore, to achieve optimum growth , media are supplemented with the growth factors.

(iv) Trace Elements:

Microorganisms also require certain trace elements (e.g. Zn, Mn, Mo, Fe, Cu, Co, etc.) in trace amount. Because these are associated with stimulation of metabolism or enzymes (metallo-enzymes) and proteins (leg-haemoglobin).

(v) Inducers, Repressors and Precursors:

The catabolic enzymes are induced only in the presence of inducers. For example, yeast extract induces streptomycin fermentation. Production of catabolic enzymes and secondary metabolites is repressed by the presence of certain compounds in the culture medium.

For example, rapidly utilised carbon sources repress the formation of amylases, griseofulvin, penicillin, etc. Besides, production of certain products is increased if precursor metabolite is supplemented in medium. For example, addition of phenyl acetic acid enhances penicillin fermentation.

(vi) Antifoams:

Protein sources in culture media (i.e. products of medium produced by microorganisms) cause foaming. Mostly foaming creates problem in microbial process. Therefore, to check the foaming problem, some antifoams i.e. fatty acids (such as sunflower oil or olive oil) are added in the culture medium.

(vii) Water:

Water is the most important component of the living cells. Because all metabolic activities occur in cytosol. Water-soluble ionic forms of nutrients are absorbed by the cell. In laboratory single or double distilled water is used for preparation of culture media.

But for large stage industrial production clean water of consistent composition is required. Dissolved chemicals and pH of water are measured. Water is also needed for ancillary activities for example, cleaning, washing, rinsing, cooling, heating, etc.

3. Procedures of Microbial Culture

Following are the procedures of microbial cultures:

(a) Sterilisation:

Sterilisation is a process of complete eradication of microorganisms from a given place or source. For small scale culture of microorganisms in laboratory in culture tubes or flasks (in 100-1000 ml), the growth media are sterilised by autoclaving at 15 psi (i.e. pound per square inch) for 15-20 minutes. At this pressure, temperature reaches to about 120°C.

According to requirement a pressure cooker or an autoclave is used. However, for large fermentation thousand to millions of liters of culture medium is used. Large sized fermentor and huge amount of medium are sterilized by using steam.

Besides, if medium is sterilised in a separate vessel, the fermentor must be sterilised by steam before passing the sterilised medium into it. Steam is spared into the fermentor from its all entries. Steam pressure of 15 psi is maintained for 20-30 minutes.

Then the steam is allowed to come out from air outlet. A sparger is attached at the bottom of fermentor. It is a tube containing several holes for coming out the steam. For detail description see preceding section and Fig. 6.2.

(b) Control of Environmental Conditions for Microbial Growth:

The success of fermentation to produce biomass and products depends upon the defined environmental conditions that exist inside the fermentor.

Therefore, temperature, pH, agitation, 0₂ concentration, etc. should be maintained during the process through careful monitoring of the fermentation. Microbial growth is significantly influenced by pH of the medium and temperature. Bacteria prefer neutral pll, while acidic pH favours the growth of yeast and fungi.

Therefore, pR of the growth medium should be maintained before autoclaving as required by the microbe to be used in fermentor. During the process, suitable temperature of the medium should also be maintained. For example, temperature regime of different groups of microorganisms varies as given on next page.

i. Psychrophiles 5-15°C

ii. Mesophiles 15-35°C

iii. Thermophiles 45-65°C

(c) Aeration and Mixing:

Aeration and mixing are done in laboratory by keeping the flasks on shakers. Hence, it is called shake culture. On the other hand, in large sized fermentors the oxygen should be well mixed and dispersed in the medium so that it may be available to microbial cells.

Therefore, the fermentors are equipped with stirrers for oxygen mixing and baffles for increasing the turbulence. Thus adequate mixing is done by the stirrers and baffles. Sufficient mixing and oxygen requirement are also met through forced aeration. The oxygen molecules come in the contact of cells and diffused inside the cell wall.

(d) Vessels for Microbial Cultures:

Microorganisms are grown on different types of vessels according to requirement. For example, in laboratory a single microbial culture is grown and maintained on slant of culture tubes. They are also grown in simple Erlenmeyer flasks of different volumes (100-1000 ml). Growth in laboratory grown cultures can be improved simply by designing the flask using shakers at controlled temperature as given below:

(i) Baffle Flasks:

When the sides of the flask are indented or V-shaped notch is produced, such flasks are called baffle flasks. The V-shaped notch or indentation increases turbulence of the agitated culture medium. This increases the efficiency of oxygen transfer and improves growth of the microorganisms in the culture.

(ii) Shakers:

There are different types of shakers used in laboratory. Shakers continuously agitate (100-120 throws/minute) the culture medium and efficiently transfer oxygen. This results in an improvement in microbial growth.

(iii) Fermentors (bioreactors):

Fermentors are the closed vessels which are used for production of products (cell mass and metabolites) on a large scale. Fermentors are of different capacities. Small scale fermentors (10-100 litres) are used by scientists in research laboratories for optimisation of different parameters of microbial growth and production of products.

Besides, large scale fermentors (thousand to million litres capacity) are used in industries for production of commercial products.

It shows a basic design of a fermentor. It is facilitated to meet several requirements such as pH and temperature control, aeration and agitation, drain or overflow and sampling facility. Interestingly, fermentor can be operated aseptically for many days under the controlled conditions.

Regular monitoring for physical, chemical and biological parameters is done through control systems of the fermentor. Because these parameters influence the growth of microbial cells. Therefore, a maximum microbial yield can be achieved through monitoring these parameters.

As per requirement and use of types of microorganisms, there are different types of fermentors. But the most common among these is the stirred tank fermentor where the impellers are fitted to stir the medium. Stirring of medium is done forcibly.

Otherwise the increasing concentration of microbial cells will deplete the dissolved 0₂ concentration resulting in creation of anaerobic condition. Aerobic microbe will never grow under anaerobic condition.

Therefore, microbial growth will decrease with simultaneous decline in product production. Hence forced aeration favours rapid growth of microorganisms. Secondly, pH of the culture medium also declines sharply with rapidly growing microbial cells. Therefore, a pH probe fitted with the fermentor regularly monitors the pW and maintains at optimum by adding acid or alkali.