Some of the most important types of fermentation are as follows:
1. Solid State Fermentation
2. Submerged Fermentation
3. Anaerobic Fermentation
4. Aerobic Fermentation
5. Immobilized Cell Bioreactors
6. Immobilized Enzyme Bioreactors
Solid State Fermentation
In solid state fermentations, microbial growth and product formation occur at the surface of solid substrates. Examples of such fermentations are mushroom cultivation, mold-ripened cheeses, starter cultures, etc. This approach is being used for the production of extracellular enzymes, certain valuable chemicals, fungal toxins and fungal spores. Traditional substrates are several agricultural products, e.g., rice, wheat, maize, soybean, etc. The substrate provides a rich and complex source of nutrients, which may or may not need to be supplemented. Such substrates selectively support mycelial organisms, which can grow at high nutrient concentrations and produce a variety of extracellular enzymes, e.g., a large number of filamentous fungi and a few bacteria (actinomycetes and one strain of Bacillus).
According to the physical state, solid state fermentations are divided into the following two groups:
(i) low moisture solids fermented without or with occasional/continuous agitation
(ii) suspended solids fermented in packed columns through which liquid is circulated.
The fungi used for solid state fermentations are usually obligate aerobes. Solid state fermentations on large scale use stationary or rotary trays. Temperature and humidity controlled air is circulated through the stacked solids. Less frequently, rotory drum type fermenters have been used. Solid state fermentations offer certain unique advantages but suffer from some important disadvantages. However, commercial application of this process for biochemical production is chiefly confined to Japan.
Batch Culture It is a closed culture system, which contains limited amount of nutrient medium. After inoculation, the culture enters lag phase, during which there is increase in the size of the cells and not in their number. The culture then enters lag phase or exponential growth phase during which cells divide at a maximal rate and their generation time reaches minimum. The increasing population of bacterial cells, after sometime, enters into a stationary-phase due to depletion of the nutrients and the accumulation of inhibitory end products in the medium. Eventually, the stationary, phase of bacterial population culminates into death-phase when the viable bacterial cells begin to die. If we collect data of the increase in cell number at various intervals of time and plot this data in two ways (logarithm of number of bacteria and arithmetic number of bacteria versus time), we find a characteristic growth curve. This typical growth curve is only obtained in a batch culture.
Fed-Batch Culture: When a batch culture is subsequently fed with fresh nutrient medium without removing the growing microbial culture, it is called fed-batch culture. Fed-batch culture allows one to supplement the medium with such nutrients that are depleted or that may be needed for the terminal stages of the culture, e.g., production of secondary metabolites. Therefore, the volume of a fed- batch culture increases with time. Fed-batch cultures achieve higher cell densities than batch cultures. It is used when high substrate concentration causes growth inhibition. It allows the substrate to be used at lower non-toxic levels, followed by subsequent feeding. It allows the maximum production of cellular melabolities by the culture.
Continuous Culture: Contrary to the batch culture where the exponential growth of microbial population is restricted only for a few generations, it is often desirable to maintain prolonged exponential growth of microbial population in industrial processes. This condition is obtained by growing microbes in a continuous culture, a culture in which nutrients are supplied and end products are continuously removed. In continuous culture, the growth of bacterial population can be maintained in a steady state over a long period of time and is thus advantageous over batch and fed-batch culture. Mechanical stirrers are used and the agitation is achieved by the air bubbles generated by the air supply. Generally, these bioreactors are of closed or batch types, but continuous flow reactors are also used; such reactors provide a continuous source of cells and are also suitable for product generation when the product is released into the medium.
Immobilized Cell Bioreactors
Bioreactors of this type are based on immobilized cells.
Cell immobilization is advantageous when:
(i) the enzymes of interest are intracellular,
(ii) extracted enzymes are unstable,
(iii) the cells do not have interfering enzymes or such enzymes are easily inactivated/removed and
(iv) the products are low molecular weight compounds released into the medium.
Under these conditions, immobilized cells offer the following advantages over enzyme immobilization:
(i) enzyme purification is not needed,
(ii) high activity of even unstable enzymes,
(iii) high operational stability,
(iv) lower cost
(v) possibility of application in multistep enzyme reactions.
In addition, immobilization permits continuous operation of bioreactor, which reduces the reactor volume and consequently, pollution problems. Obviously, immobilized cells are used for such bio-transformations of compounds, which require action of a single enzyme.
Cell immobilization may be achieved in one of the following ways:
(1) Cells may be directly bound to water insoluble carriers, e.g., cellulose, dextran, ion-exchange resins, porous glass, brick, sand, etc., by adsorption, ionic bonds or covalent bonds.
(2) They can be cross-linked to bi- or multifunctional reagents, e.g., glutaraldehyde, etc.
(3) Polymer matrices may be used for entrapping cells; such matrices are polyacrylamide gel, K-Carrageenan (a polysaccharide isolated from a seaweed), calcium alginate (alginate is extracted from a seaweed), polyglycol oligomers, etc.
Out of these approaches, calcium alginate immobilization is the most commonly used since it can be used for even very sensitive cells, e.g., plant cells; K-Carrageenan is also a useful entrapping agent. Cell immobilization has been used for commercial production of amino acids, e.g., E. coli cells entrapped in polyacrylamide gel for the production of L-aspartic acid, L-alanine production using a mixture of E. coli and Pseudomonas dacunhae immobilized in K-Carrageenan, organic acids, e.g., L-malic acid from fumaric acid using Brevibacterium ammoniagenes cells immobilized in polyacrylamide gel/K-Carrageenan (subsequently, B. flavum was used in the place of B. ammoniagenes), NADP production by B. ammoniagenes and yeast (Saccharomyces cerevisiae) cells immobilized together in polyacrylamide gel.
Immobilized Enzyme Bioreactors
Continuous flow reactors are based on immobilized enzymes.
They offer the following advantages:
(i) greater productivity per unit amount of enzyme,
(ii) suitable for substrates having low solubility and
(iii) for uniform product quality, i.e., lack of batch to batch variation.
These reactors of three types:
(i) continuous flow stirred tank reactor,
(ii) packed bed reactor and
(iii) fluidized bed reactor.
In anaerobic fermentation, a provision for aeration is usually not needed. But in some cases, aeration may be needed initially for inoculum build-up. In most cases, a mixing device is also unnecessary, but in some cases initial mixing of the inoculum is necessary. Once the fermentation begins, the gas produced in the process generates sufficient mixing. The air present in the headspace of the fermenter should be replaced by CO2, H2, N2 or a suitable mixture of these; this is particularly important for obligate anaerobes like Clostridium. The fermentation usually liberates CO2 and H2, which are collected and used, e.g., CO2 for making dry ice and methanol and for bubbling into freshly inoculated fermenters. In case of acetogens and other gas utilizing bacteria, O2-free sterile CO2 or other gases are bubbled through the medium. Acetogens have been cultured in 400 1 fermenters by bubbling sterile CO2 and 3kg cells could be harvested in each run. Recovery of products from anaerobic fermenters does not require anaerobic conditions. But many enzymes of such organisms are highly O2 sensitive. Therefore, when recovery of such enzymes is the objective, cells must be harvested under strictly anaerobic conditions.
The main feature of aerobic fermentation is the provision for adequate aeration; in some cases, the amount of air needed per hour is about 60-times the medium volume. Therefore, bioreactors used for aerobic fermentation have a provision for adequate supply of sterile air, which is generally sparged into the medium. In addition, these fermenters may have a mechanism for stirring and mixing of the medium and cells.
Aerobic fermenters may be either of the
(i) stirred-tank type in which mechanical motor-driven stirrers are provided or
(ii) of air-lift type in which no