Differences lie in microorganisms with respect to their growth and production of products. Hence, the microorganisms are cultured in different types of vessels in various ways. Therefore, to get the desired product, microorganisms are grown as batch, fed-batch or continuous cultures.
Batch culture is the simplest method. A desired microbe is grown in a closed culture system on a limited amount of medium of microbial culture. The laboratory grown microorganisms in ordinary flask is basically a batch culture.
After incubation of medium, the microbe (yeast, bacteria or fungal inoculums) passes through a number of growth stages such as lag phase, log (or exponential growth) phases, deceleration phase, stationary phase and death phase.
Due to gradual consumption of medium and production of metabolites the growing environment goes on changing. This influences the growing cells. Therefore, fresh medium is used in each batch.
After inoculation, the microbe adapts the new environment and does not show active growth. This period is called lag phase. Then the cells use nutrients of the growth medium and grow exponentially until the nutrients of medium are not depleted. The period of exponentially dividing cells is called log phase or exponential growth phase.
During log phase there occurs an increase in cell mass and cell number with gradual decline in nutrients. At deceleration phase, there is no microbial growth at all.
Because the growth environment gets changed due to gradual consumption of nutrients and accumulation of metabolites in fermentor.
At stationary phase microbial growth gets decreased to zero. Accumulation of metabolites exceeds and no sufficient nutrients are left in the fermentor. The cells gradually start to die. This stage is called death phase.
Microbial Growth Kinetics and Specific Growth Rate:
During log phase when cells utilise nutrients and grow to increase biomass, the growth behaves similar to autocatalytic reaction. At this phase growth rate is proportional to cell mass of that period. During this time the rate of ‘cell mass increase’ (dx/dt) is equal to the specific growth rate (µ) and cell concentration.
The specific growth rate (µ) acts as an index of rate of cell growth in that very environment. If you plot a graph between dx/dt and x, and determine the slope of straight line, you can calculate the specific growth rate.
The value of specific growth rate can be converted into doubling time (i.e. time required by a cell to divide and double its number). It gives a better appreciation of the meaning of these values.
The µ represents the capacity of the microbial culture to grow fn a specific environment. The H of a microbial culture is measured during log phase of growth during which balanced cell growth occurs. In a batch culture the values of varies having maximum value at exponential (log) phase of growth.
The environmental factors (e.g. pH, temperature, medium composition, aeration, etc.) that affect microbial growth also affect the specific growth rate of the culture. Representative values of µmax of some microorganisms are given in.
Fed-batch culture is basically a batch culture which is continuously fed with nutrient medium in the fermentor without removing the growth culture or growth products. Consequently volume of the medium is increased. The nutrients should be added at the same rate as they are consumed by the growing cells. Therefore, excess of nutrient addition should be avoided.
In batch culture when high concentration of substrate inhibits microbial growth, the fed-batch culture is preferred over the former. Hence, in a fed-batch culture substrate is fed at such a concentration that remains below the toxic level. This activity accelerates the cell growth. A high cell density is achieved in fed-batch culture as compared to fed-batch culture.
Fed-batch culture is an ideal process for production of intracellular metabolites in maximum amount. For example, alkaline protease used in biological detergents is produced by the species of Bacillus. Batch feeding of nitrogen sources (e.g. ammonia, ammonium ions and amino acids) keeps these substrates at low concentration and induces protease synthesis.
Continuous culture is an open process in which microbial cultures also grow continuously in log phase. One of the nutrients of culture medium is kept limited. Hence, at log phase the cell growth stops as the nutrients of limited quantity is exhausted. In continuous culture, fermented medium is continuously removed from the fermentor.
Therefore, to keep the culture always in log phase, fresh medium is added continuously to the fermentor (before diminishing the nutrients) at the time of removal of medium. Here the rate of supply of nutrients in the form of raw material and removal of products/cells should be volumetrically the same i.e. volume added is equal to volume removed.
It means that volume of the medium always remains constant. This should be optimised with different microbial cultures and different growth media. If the working volume of the fermentor is V m3, and the rate of flow in and out is F m3h_1, then the dilution rate (D) will be
D = F/V
Or F = DV …(8)
The unit of D is per hour (h”1).
The output of biomass from a continuous culture system is given by the rate at which medium passes out of the out flow (i.e. the flow rate, F) multiplied by the concentration of biomass in that out flow (i.e. X).
Thus, output = FX …(9)
Putting the value of F of equation (8) in equation (9), we get
Output = DVX …(10)
The productivity of this system (output per unit volume) is thus as below:
DVX =DX … (11)
Productivity = DVX/V = DX
In continuous culture cells are grown at a particular growth rate. Then it is maintained for a long time. Most often the continuous culture is used for production of biomass of metabolites. Besides, liquid wastes are treated by using continuous culture. Microorganisms utilise organic materials of liquid wastes.
Thus microbial biomass is produced in high amount. When such system is in equilibrium, cell number and nutrient status remain constant. At this stage the system is said to be in steady state.
(a) The Chemostat:
The Chemostat is the most common type of continuous culture device. It controls both population density (i.e. cell density) as well as growth rate of the culture. The chemostat is controlled by two elements, the dilution rate and the concentration of limiting nutrient e.g. carbon or nitrogen sources. The growth rate is reduced at a very low concentration of a given nutrient.
In chemostat cell density (number of cells/ml) is controlled by regulating the concentration of limiting nutrients. If the concentration of nutrient in medium is raised (with the constant dilution rate), the cell density will increase and growth rate will remain the same. Consequently, the steady state concentration of the nutrients in the culture vessel will be zero.
A variety of cell densities growing at a variety of growth rates can be maintained by adjusting the dilution rate and nutrient concentration. If medium is fed to such a culture at a suitable rate, eventually a steady state is obtained.
When steady state is maintained both cell growth and substrate consumption take place at a fixed rate. Therefore, during steady state, growth rate of cells remains constant i.e. cell density, metabolites and nutrients inside the vessel are constant. It means that the loss of culture from the vessel balances the formation of new biomass by the microbial culture.
(b) The Turbidostat:
It is another type of continuous culture system. It consists of a photocell which measures the turbidity of the culture. The flow rate of medium through vessel is automatically regulated to maintain turbidity.
The turbidostat differs from chemostat in several ways. In turbidostat, the dilution rate varies rather than remaining constant. Its culture medium lacks limiting nutrients. The turbidostat operates best at high dilution rates, while chemostat is effective stable at low dilution rates.