The deliberate culture of living cells is perhaps the oldest type of biotechnology. Brewing, baking, cheese making and the production of other ‘fermented’ foods such as youghurt and bean curd, all involve the cultivation of micro-organisms in specific media. Now natural media have been replaced by synthetic media, either aqueous or semisolid agar.

Industrial fermentation for non-food products began in the mid 1940s with the production of glycerol and acetone for the second World War. After the discoveries of penicillin and streptomycin, the production of a vast number of antibiotics has revolutionized modern medicine. The techniques of genetic engineering and cell fusion would not be possible without cell culture on a laboratory scale. Without the ability to grow the genetically manipulated organisms in large quantities using bioreactors, these methods would remain purely a matter of academic interest.

In any of these cultivation processes, the cells being cultured may be isolated from natural sources or they may be from strains improved by selection, mutation or genetic engineering. Whatever their source, in biotechnology the cells are grown for one of three purposes:

a) to produce specific useful metabolites such as interferon or penicillin;

b) to convert a substrate into a more useful product such as alcohol from glucose or fructose from sucrose; and

c) to convert the substrate medium into more cells (‘biomass’) thereby either rendering it more useful (food proteins from glucose or methanol) or less toxic (water treatment and attenuated pathogens for vaccines).

The term ‘single-cell protein’ (SCP) comes from the cultivation of unicellular organisms, like Chlorella, as an edible protein source for humans or animals. Biomass can also be used as a source of fuel. Cell cultures could also produce a wide range of organic chemicals which are now manufactured from petroleum feed stocks.

4 IMMOBILISATION

When cultured cells are being used to effect a chemical transformation, it may be convenient and economic to bind them to a specific medium to immobilise them. The means used for immobilisation are gelifying material like agar, alginates, carrageenan, polyacrylamide, etc. Nylon or polymer memberanes may also be used. Fluidised beds use microbeads for immobilisation. Immobilisation makes the separation and recovery of biochemical products from the synthesising units, easier and efficient, particularly when the products are secreted into the medium. Else, product recovery is through cell destruction. Immobilization allows the cells to be reused. Living cells may be immobilised but most processes use killed cells or purified enzymes. Many immobilised enzyme preparations do not use pure enzymes but rather relatively impure forms from dead cells. Immobilised enzyme processes have been particularly successful in the production of sugar syrups such as high-fructose corn syrup, using glucose isomerase.

4 SUSPENSION CULTURES AND BIOREACTORS

Cells or cell free systems that are cultured, in liquid media as suspensions, are called suspension cultures. A bioreactor is a device of industrial application that employs organisms, cells, enzymes, etc., for the large scale production of biochemicals. Bioreactors are now at the heart of many biotechnical procsesses and devices, which are used in industrial, agricultural and medical applications. They rely on the bioconversion functions of agents in the form of living cells or cell-free systems which transform raw materials into useful products and/or less hazardous by-products (as in waste management). A wide variety of compounds like the antibiotics, organic acids, alcohols, proteins, enzymes, monoclonal antibodies and several therapeutically active compounds are sought to be mass produced through bioreactor processes.

Bioreactors differ from fermenters in that they are used for the mass culture of plant or animal cells, instead of micro-organisms. The chemical compounds synthesised by these cultured cells, such as therapeutic agents, can be extracted easily from the cell biomass. Use of bioreactors affords the following advantages:

a) this process eliminates the necessity of cultivating the concerned plants in the conventional way, thus reducing the demand on land use;

b) bioreactors provide for clearly defined and closely controlled parameters that would promote the performance of the cells in their metabolic activity and biochemical transformations which would enhance the purity of the desired compounds;

c) The production time is drastically reduced, in some cases, by years;

d) bioreactors have an edge over the other methods of cell culture like shake-flask culture as they afford better conditions for multiplication of suspension cultures under parameters defined and set for the production of specific compounds; and

e) a very large scale culture of tissues like meristems is also possible in bioreactors.

All aspects related to bioreactors cannot be discussed in detail here. The fundamental issues on bioreactors were well covered by Moo-Young (1988), but there is extensive literature on bioreactors both before and after this publication. Many bioreactor systems employ ‘immobilised cells and/or enzymes’, which means that they are biocatalytic systems in which the bio-reagents are segregated and/or attached to solid support carriers in contrast to the more conventional systems in which the bio-agents are in free suspension (cells: animal, plant, bacteria, yeasts, fungi) or are dissolved in a bulk aqueous medium (enzymes). The capacities, configurations, types of operations of bioreactors are varied and many.

The synthetic process in a bioreactor may be terminated at fixed periods for the recovery of the product and a fresh cycle is started because the culture was grown in a fixed volume of the medium which restricts cell proliferation and product synthesis. This is called ‘batch processing’. The alternative method, called ‘continuous processing’, involves a continuous influx of the fresh medium and a withdrawal of the spent medium. In one version, the ‘closed continuous processing’, the cells/products are harvested at one time while in the ‘open continuous processing’, the harvest is periodical.

Attractive and promising, bioreactor processes may seem, but they are very involved technically and financially. The success achieved with antibiotics is rarely matched elsewhere. A considerable lot of preliminary and preparatory work needs to be done for each species separately. A number of technical problems may crop up. For example, certain compounds like terpenoids do not accumulate unless the cells are differentiated but differentiated cells are normally unsuitable for suspension cultures, because a considerable amount of the nutrient supply is spent on differentiation rather than on biosynthesis of the desired product. Certain compounds like essential oils accumulate faster under stressful environments. Charlwood and Charlwood (1991) discussed the conditions required for suspension culturing of over a dozen species with particular reference to terpenoids. The biochemical protocols and/or design engineering of the bioreactors change very rapidly. The problems that now hinder a more common use of bioreactor processes will certainly be overcome in course of time.

4 ELICITORS

It is possible to induce production or enhance production of a compound in cultures by using elicitors, which may be micro-organisms. For example, Saccharomyces cervisiae was an efficient elicitor in the production of glyceollin (Glycine max) and berberine (Thalictrum rugosum). Rhizopus arrhizus trebled diosgenin production by Dioscorea deltoidea. The production of morphine and codeine by Papaver somniferum was increased 18 times by Verticillium dahliae.

4 BIOTRANSFORMATIONS

Immobilised plant cells may be used for biotransformations (or bioconversions). Using alginate as the immobilising polymer, digitoxin from Digitalis lanata was converted into digoxin, which is a therapeutic agent in great demand. Similarly codeinone was converted into codeine and tyrosine from Mucuna pruriens was converted into DOPA.

4 TISSUE CULTURE

That any single cell of a plant contains the whole genome of the plant, and that this genome can totally express to develop into a plant with fully differentiated organs, a capability called ‘totipotency’, is the basis of tissue culture.

In tissue culture, a mass of undifferentiated cells (callus) is formed from the explant (the starting material from the donor plant), which is a bit of a tissue or organ of the plant desired to be cultured. The callus initially produces small heart-shaped structures, the somatic (vegetative) embryos called ‘embryoids’, which can be made to differentiate into numerous plant-lets, each of which can be separately grown in the soil to become fully independent plants, that would complete their life cycle. This process is ‘micropropagation’ which helps in producing very large numbers of plants from an explant. The products of micropropagation are genetically identical to the explant donor. When the meristem is used as the explant, the products are also disease free.

Embryoids can be individually encapsulated in gels. These are the ‘synthetic seeds’, which can be stored conveniently for longer periods and can be developed into plants when required.

When pollen are used in tissue culture, the resultant plants are haploid. This is the easiest and surest way of obtaining haploids, though the pollen of all species do not respond with the same ease. If the haploid plants resulting from pollen are diploidised using chemicals like colchicine, the resulting diploid is totally homozygous, for all the alleles. Such plants are very useful, where uniform genotypes are advantageous.