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Cell Culture

All biotechnological processes are performed within bioreactors (may be a culture vessel, an open tank or sophisticated fermenters) containing correct medium provided with optimum growth conditions, like pH, temperature, aeration, light (for photosynthetic organisms), etc, The growth of the organisms is the increase of cell material that can be measured in terms of many parameters, like mass (dry weight or fresh weight), total amount of proteins, photosynthetitic pigments, number of cells, etc., Doubling time is the period required for doubling the biomass (due to cell division as well as due to cell growth) which varies from organism to organism, e.g., for bacteria it is about 0.25-1.00 h, for yeast 1-2 h, for plant cells 20-70 h and for animal cells 15-48 h (generation time is the time period required for doubling of the cell number due to cell division).

Plant cells (similar to microorganisms ) are mainly grown in liquid or solidified nutrient medium for various purposes, like artificial micropropagation of certain plants, production of valuable compounds (e.g., perfumes), for genetic engineering purposes, etc. (Discussed in the chapter “Biotechnology in Agriculture and Forestry”).

Animal and human cell cultures are grown in liquid or solid media and these cells are used for the production of many important organic compounds (e.g., vaccines, interferons, hormones, monoclonal antibodies, etc.

1. Nutrient Medium

The basic nutritional requirement for different types of cells is different and includes the components, like a carbon source (e.g, glucose, lactose, starch, NaHCO3, CaCO3, etc.), a nitrogen source (e.g., NaNO3), a phosphorus source (e.g., NaHPO4) and other elements. Generally, the carbon and nitrogen sources are derived from cheap natural products or by-products. The liquid medium is known as broth, Solidification of the medium can be achieved by adding 1-2% agar.

Many biotechnological processes involve the growth of a single or group of inoculated or
indigenous microorganisms on solid substrates, e.g., agricultural and food processing industrial wastes used for biogas production, composting and mushroom cultivation, fermentation processes in the batter of idli, cheese, etc.

2. Bioreactor (Fermenter)

For the culturing of cells, correct environment for the growth is provided in different types of bioreactors, normally ranging between two important types:

Simple Bioreactors: These are manually stirred or non-stirred, non-aseptic open containers e.g., shallow tanks made in open sunlight are used for cultivation of Spirulina  and cyanobacterial biofertilizer sewage treatment plants., (Fig.14.1)

An Erlenmyer flask

Spirulina grown in artificial tanks.

Fig. 14.1 Some simple bioreactors.

(i)                 Complex Bioreactors: They may be computerized, sterilized systems with controlled parameters like pH, temperature, etc., where mechanical agitation and aeration is provided by pumping, e.g., used for the production of antibiotics, vitamins, etc., (Fig. 14.2), Here, the contamination by unwanted microorganisms and possibility of release of the culture organisms into the environment (particularly for genetically engineered organisms) is very small.

Cross section of a common bioreactor.

A photobiorector.

Fig. 14.2 Some complex bioreactors.

In the bioreactors, microorganism are either inoculated in aqueous nutrient medium or the indigenous microorganisms, present in the solid medium, perform the fermentation process (e.g., promotes growth rates by mixing uniformly the nutrients, gases and wastes, both in aerobic and anaerobic systems.

3. Culturing of Cells

For the growth of an organism, the conditions are not always ideal to permit unlimited growth. Thus, at sometime a few factors (e.g. a particular essential nutrient) become limiting for the growth of the cells (as the concentration of this nutrient decreases a certain level). Two main ways of growing microorganisms in the bioreactors are:

(i)                 Batch Culture: In this method of growth, the microorganisms are inoculated (added) into a fixed volume of artificial nutrient medium. Initially, the inoculated cells try to adjust in the new environment and do not show any growth, and this period is called lag phase. Thereafter, the growth occurs exponentially, proceeding with the maximum speed as the conditions are ideal for growth (optimum amount of nutrients with minimum toxic wastes), and the phase is known as exponential phase. As the growth proceeds, various nutrients get consumed and different metabolites (mostly toxic) are accumulated leading to continuous change of the nutrient medium, pH, etc. Gradually, the cell multiplication decreases and finally ceases due to the exhaustion of nutrients and accumulation of toxic wastes. At this time, the growth becomes stationary and the phase is known as stationary phase. Finally, the death phase starts because of decreasing metabolism and lysis of cells (Fig. 14.3).

In industries, this system of cell cultivation is most commonly employed and the harvesting phase depends upon the type of product. When the biomass is concerned as the product, harvesting is done near the end of continuous exponential phase when the yield is generally maximum, whereas if a particular metabolite is of interest its maximum yield at a particular phase depends upon the kind of metabolite (e.g., antibiotics are optimally formed during the stationary phase of growth cycle).

Fig. 14.3 A batch culture in Erlenmeyer flask (left) and growth curve in batch culture (right).

(i)                 Continuous Culture: In this method of culturing, after the lag phase, when the exponential phase starts, fresh sterile medium is allowed to enter through an inlet with a steady flow rate and at the same time the culture (cells along with nutrient medium and wastes) is withdrawn through an outlet with the same rate, so that the volume of medium in the bioreactor remains constant (Fig. 14.4). Thus, the cells grow in the ideal growth conditions of nutrients and remain in exponential phase indefinitely. In industries the continuously operated systems are of limited use e.g., single cell protein production, activated sludge process in oxidation ponds (secondary treatment) of sewage treatment plants, biogas plants, etc.

Fig. 14.4 Continuous culture devices.

4. Scale-up

Fermentation processes are generally developed in three stages

 i. Initial Stage: Screening process is performed in small containers, like culture tubes, Petridishes, Erlenmeyer flasks, etc.

ii. Middle Stage: Screening process is followed by a pilot plant investigation to know the optimal operating conditions in 5-200 litres volume fermenters.

 iii. Final Stage: It involves the transfer of technology thus developed to the actual production site.

 5. Downstream Processing

After the growth of the cells in bioreactor, the desired end product commercial use it extracted and purified from the biomass, and this process is known as downstream processing, e.g., human insulin isolation from recombinant E.coli cells. The cost of product mainly depends upon the technique used for its production.

i. Separation of cells from the liquid medium in bioreactor, that can be achieved by different ways depending upon the system. e.g.,

a)     Gravity sedimentation of larger heavier cells.

b)     Floatation of lighter cells.

c)     Cheap cloth filters, e.g., used for Spirulina.

d)      Centrifugation of smaller cells, e.g., for Chlorella cells, that is costly.

 ii.   Purification of desired product (e.g., antibiotics, enzymes, etc.) from the liquid medium can be achieved by various processes depending upon the size of the product:

a)      Sedimentation (10-1 –103µm)

b)      Screens (105 – 107 11 µm)

c)      Centrifugation (102 – 105 µm) where the liquid sample is rotated with a high speed in tubes using the instrument, centrifuge, so that the heavier product gets settled at the bottom of the tube and the liquid remains at the upper part (Fig 14.5).

Fig. 14.5 Centrifuge machine (left) and centrifuge tubes (right).

d.      Microfilt.crs (10-103 µm)

e.       Ultracentrifugation(10-103 µm)

f.        Gel electrophoresis (10-103 µm) where the molecules of various sizes are separated as bands by running the mixture over a solid material (e.g., agarose, polyacrylamide) under an electric field (Fig.14.6).

g.      Distillation (1-103 µm)

h.      Ultrafiltration (1-103 µm)

i.        Reverse osmosis (1-102 µm)

Osmosis is the flow of solvent along the concentration / pressure gradient (e.g., flow of water from a region of high solvent concentration to a region of low solvent concentration, that are separated by a membrane), but if the pressure is applied, the direction of flow gets reversed and it is called reverse osmosis (Fig. 14.7).

Fig. 14.6 Gel electrophoresis apparatus (left) and agarose gel with DNA bands (right).

(i)                 Drying of the final product can be achieved by many ways, e.g., freeze drying, where, the product is first frozen and then allowed to evaporate at very low pressure. Lowering of pressure increases the melting point, so that increasing of temperature leads to faster evaporation of water of solid phase without getting converted into the liquid form (sublimation).When biomass is the commercial product, it can be packed after drying (either in sun or in oven) with or without any processing, e.g., Spirulina and Chlorella can be used as food or feed after sun drying the biomass (Fig. 14.8).

Fig. 14.8 Sun drying of harvested Spirulina.

Many products are sold in liquid preparations without drying.

QUESTIONS

1. What do you understand by batch culture and continuous culture techniques?

2. Write short notes on:

(i)  Bioreactors

(ii) Downstream processing.