Cultures of Microorganisms
Microorganisms isolated from different sources (soil, water and air) or genetic manipulation are cultured on growth media.
Fig. 14.1. Diagram of an aerated fermenter.
The growth media are supplemented with sources of carbon, nitrogen, phosphorus, amino acids, trace-elements, etc, sterilized and inoculated with specific micro organism for the specific products. Some of the microbial cultures are described below:
Solid or Semi-solid Culture
Varying amount of agar amended with nutrient media gives solid or semi-solid phase. For the research purpose, this type of media are used but they are generally avoided for microbial products, because such media occupy space and create difficulty during harvesting. However, for the production of amylase from Aspergillus oryzae
such media are used. After the growth of A. oryzae
for several days at 30°C mycelia are harvested, dried and ground which results in crude preparation of amylase.
This is the simplest type of culture in which micro-organisms grow in a vessel, known as fermenter (Fig. 14.1) or bioreactor. A fermenter is a vessel designed to carry out fermentation process i.e.
biological reactions under the controlled conditions. Hence it is also known as bio-reactor; while designing a fermenter, several criteria which help in maximizing the yield, are taken into account. These are (a)
long term operation in aseptic condition, (b)
adequate aeration and agitation, (c)
pH control system, (d)
sampling facility, (e)
minimum labor in operation, harvesting, cleaning and maintenance, (f) temperature control system, (g) minimum evaporation losses from fermenter, (h)
suitable for a variety of processes. Fermenter is provided with limited amount of medium containing all the nutrients at optium environmental conditions.
In batch culture, growth phase of microorganisms passes through many stages. A microbe grows in the medium until the nutrients are exhausted or toxic metabolites secreted by it reach to inhibitory level. From the beginning of inoculation to the end, microbial culture passes through several stages (Fig. 14.2). After inoculation, the microbe takes some time to adjust in the new environment according to size of fermenter, and hence does not grow in the medium. Thus, the time taken for adaptation before to come to its active growth is known as *log phase'. The microorganism grows luxuriently till nutrients are present.
Therefore, nutrient dependent logarithmic or exponential active growth and, thereby, increase in biomass is known as 'log' or 'exponential phase'. As soon as the levels of nutrients decrease, growth of culture is gradually slow down. The stage of retarding the growth to reach to stationary phase is known as 'deceleration phase'. However, during stationary phase micro-organisms do not grow and thus, fail to increase their biomass. Ultimately the number of microbial cells declines due to accumulation of toxic metabolites. This stage is known as 'death phase'. At the end, the amount of biomass depends upon the nutritional components and ability of micro-organisms to utilize the substrates and convert into biomass.
Fig. 14.2. Sigmoidal growth curve of a batch culture; DeP-deceleratiori phase; SP-stationary phase : DP-death phase.
Bu' Lock et al
(1965) have proposed different terminology for these growth stages. They used 'trophophase' for the log phase and 'idiophase' for the stationary phase of batch culture. This typical growth curve is known as 'sigmoidal growth curve'.
During the log phase of culture, growth rate of the micro-organisms reaches to its maximum (max). However, after depletion of a substrate, growth rate decreases and finally is ceased. Monod (1942) has demonstrated the relationship of growth rate and concentration of the rate-limiting substrates by the following formula :
m = Growth rate constant,
[s] = Concentration of limiting substrate,
= Saturation constant value of limiting substrate [s] at which the growth rate is half of the maximum growth rate (m max).
A continuous culture is that where a steady exponential phase for growth of culture retards due to depletion of nutrients, rather than by accumulation of toxic products; it is prevented by addition of fresh medium to the fermenter and removal of spent medium and microbial biomass from it as a result of which the exponontial phase of culture is prolonged.Fed-batch Culture
Basically it is the batch culture which is fed continuously with fresh medium without removal of the original culture medium from the fermenter. It results in continuous increase in volume of medium in the fermenter.