Growth of Microorganisms (With Diagram)
The growth of microorganisms is a highly complex and coordinated process, ultimately expressed by increase in cell number or cell mass. The process of growth depends on the availability of requisite nutrients and their transport into the cells, and the environmental factors such as aeration, O2 supply, temperature and pH.
Doubling time refers to the time period required for doubling the weight of the biomass while generation time represents the period for doubling the cell numbers. Doubling times normally increase with increasing cell size and complicity as given below.
Bacteria 0.30 – 1 hour
Yeasts 1 – 2 hours
Animal cells 25 -48 hours
Plant cells 20 -70 hours
In general, when all other conditions are kept ideal, growth of the microorganisms is dependent on the substrate (nutrient) supply. The microorganisms can be grown in batch, fed-batch, semi-continuous or continuous culture systems in a bioreactor.
A diagrammatic representation of microbial cell growth in relation to substrate is depicted in Fig. 19.11. In batch fermentation, the growth medium containing the substrates is inoculated with microorganisms, and the fermentation proceeds without the addition of fresh growth medium.
In fed-batch fermentation, substrates are added at short time intervals during fermentation. In batch and fed-batch fermentation, the growth of the cells is quite comparable. And in both cases, growth medium is not removed until the end of fermentation process.
In case of continuous fermentation, as the fermentation proceeds, fresh growth medium is added continuously. Simultaneously, an equal volume of spent medium containing suspended microorganisms is removed. This enables the cells to grow optimally and continuously (Fig. 19.11C).
Batch Culture or Batch Fermentation:
A batch fermentation is regarded as a closed system. The sterile nutrient culture medium in the bioreactor is inoculated with microorganisms. The incubation is carried out under optimal physiological conditions (pH, temperature, O2 supply, agitation etc.). It may be necessary to add acid or alkali to maintain pH, and anti-foam agents to minimise foam. Under optimal conditions for growth, the following six typical phases of growth are observed in batch fermentation (Fig. 19.12).
1. Lag phase
2. Acceleration phase
3. Logarithmic (log) phase (exponential phase)
4. Deceleration phase
5. Stationary phase
6. Death phase.
1. Lag phase:
The initial brief period of culturing after inoculation is referred to as lag phase. During the lag phase, the microorganisms adapt to the new environment—available nutrients, pH etc. There is no increase in the cell number, although the cellular weight may slightly increase.
The length of the lag phase is variable and is mostly determined by the new set of physiological conditions, and the phase at which the microorganisms were existing when inoculated. For instance, lag phase may not occur if the culture inoculated is at exponential phase (i.e., log phase), and growth may start immediately.
2. Acceleration phase:
This is a brief transient period during which cells start growing slowly. In fact, acceleration phase connects the lag phase and log phase.
3. Log phase:
The most active growth of microorganisms and multiplication occur during log phase. The cells undergo several doublings and the cell mass increases. When the number of cells or biomass is plotted against time on a semi logarithmic graph, a straight line is obtained, hence the term log phase.
Growth rate of microbes in log phase is independent of substrate (nutrient supply) concentration as long as excess substrate is present, and there are no growth inhibitors in the medium. In general, the specific growth rate of microorganisms for simpler substrates is greater than for long chain molecules. This is explained on the basis of extra energy needed to split long chain substrates.
Two log phases are observed when a complex nutrient medium with two substrates is used in fermentation, and this phenomenon is referred to as diauxy. This happens since one of the substrates is preferentially metabolised first which represses the breakdown of second substrate. After the first substrate is completely degraded second lag phase occurs, during which period, the enzymes for the breakdown of second substrate the synthesized. Now a second log phase occurs.
4. Deceleration phase:
As the growth rate of microorganisms during log phase decreases, they enter the deceleration phase. This phase is usually very short-lived and may not be observable.
5. Stationary phase:
As the substrate in the growth medium gets depleted, and the metabolic end products that are formed inhibit the growth, the cells enter the stationary phase. The microbial growth may either slow down or completely stop. The biomass may remain almost constant during stationary phase. This phase, however, is frequently associated with dramatic changes in the metabolism of the cells which may produce compounds (secondary metabolites) of biotechnological importance e.g. production of antibiotics.
6. Death phase:
This phase is associated with cessation of metabolic activity and depletion of energy reserves. The cells die at an exponential rate (a straight line may be obtained when the number of surviving cells are plotted against time on a semi logarithmic plot). In the commercial and industrial fermentations, the growth of the microorganisms is halted at the end of the log phase or just before the death phase begins, and the cells are harvested.
Fed-Batch Culture or Fed-Batch Fermentation:
Fed-batch fermentation (See Fig. 19.11) is an improvement of batch fermentation wherein the substrate is added in increments at different times throughout the course of fermentation (Note: In batch culture method, substrate is added only at the beginning of the fermentation). Periodical substrate addition prolongs log and stationary phases which results in an increased biomass. Consequently, production of metabolites (e.g. antibiotics) during stationary phase is very much increased.
As it is difficult to directly measure substrate concentration in fed-batch fermentation, other indicators that correlate with substrate consumption are used. The formation of organic acids, production of CO2 and changes in pH may be measured, and accordingly substrate addition carried out. In general, fed-batch fermentation requires more careful monitoring than batch fermentation, and is therefore not a preferred method by industrial biotechnologists.
Fed-batch fermentation for the production of recombinant proteins:
In recent years, fed-batch fermentation has become popular, due to very high yield, for the production of recombinant proteins. Depending on the microorganism and the nature of recombinant protein, the fed-batch fermentation can increase the product yield from 25% to 1000% compared to batch fermentation. Careful monitoring of the fermentation reaction and appropriate addition of substrates (carbon and nitrogen sources, and trace metals) substantially increases the product yield.
Limitations:
The major limitation of fed batch fermentation is that the microorganisms in the stationary phase produce proteolytic enzymes or proteases. These enzyme attack the recombinant proteins that are being produced. By carefully monitoring the fermentation, the log phase can be prolonged and the onset of stationary phase is delayed. By this way, the formation of proteases can be minimised.
Fed-batch cultures for higher organisms:
Fed-batch cultures are successfully employed for mammalian and insect cells. This is very advantageous for the production of human therapeutic proteins with good yield.
Semi-Continuous Culture or Semi-Continuous Fermentation:
Some of the products of fermentation are growth-linked, and such products are formed at the end of the log phase e.g. ethanol production. In semi-continuous fermentation, a portion of the culture medium is removed from the bioreactor and replaced by fresh medium (identical in nutrients, pH, temperature etc.).
This process of culture medium change can be repeated at appropriate intervals. In the semi-continuous fermentation, the lag phase and other nonproductive phases are very much shortened. The product output is much higher compared to batch culture systems. Semi-continuous fermentation technique has been successfully used in the industrial production of alcohol. There are however, certain disadvantages of semi-continuous fermentation. These include the technical difficulties of handling bioreactors, long culture periods that may lead to contamination, mutation and mechanical breakdown.
Continuous Culture or Continuous Fermentation:
Continuous fermentation is an open system. It involves the removal of culture medium continuously and replacement of this with a fresh sterile medium in a bioreactor. Both addition and removal are done at the same rate so that the working volume remains constant.
Further, to maintain a steady state condition in continuous process, it is advisable that the cell loss as a result of outflow is balanced by growth of the organisms. The two common types of continuous fermentation and bioreactors are described below (Fig. 19.13).
Homogeneously mixed bioreactors:
In this type, the culture solution is homogeneously mixed, and the bioreactors are of two types
Chemostat bioreactors:
The concentration of any one of the substrates (carbohydrate, nitrogen source, salts, O2) is adjusted to control the cell growth and maintain a steady state.
Turbidostat bioreactors:
In this case, turbidity measurement is used to monitor the biomass concentration. The rate of addition of nutrient solution can be appropriately adjusted to maintain a constant cell growth.
Plug flow bioreactors:
In plug flow bioreactors, the culture solution flows through a tubular reaction vessel without back mixing. The composition of the medium, the quantity of cells, O2 supply and product formation vary at different locations in the bioreactor. Microorganisms along with nutrient medium are continuously added at the entrance of the bioreactor.
Industrial applications of continuous fermentation:
Continuous fermentation processes have been used for the production of antibiotics, organic solvents, single-cell protein, beer and ethanol, besides waste-water treatment.
Advantages of continuous fermentation:
1. The size of the bioreactor and other equipment used in continuous fermentation are relatively smaller compared to batch fermentation for the production of the same quantity of product.
2. The yield of the product is more consistent since the physiological state of the cells is uniform.
3. The ‘down time’ between two successive fermentations for cleaning and preparing the bioreactor for reuse is avoided in continuous fermentation.
4. Continuous fermentation can be run in a cost-effective manner.
Disadvantages of continuous fermentation:
Despite many advantages of continuous fermentation (described above), it is not very widely used in industries. Some of the drawbacks are listed.
1. Continuous fermentation may run continuously for a period of 500 to 1,000 hours. Maintenance of sterile conditions for such a long period is difficult.
2. The recombinant cells with plasmid constructs cannot function continuously and therefore the product yield decreases.
3. It is not easy to maintain the same quality of the culture medium for all the additions. Nutrient variations will alter the growth and physiology of the cells, and consequently the product yield.
In addition to the disadvantages listed above, industrial biotechnologists are rather reluctant to switch over to continuous fermentation from the batch fermentation. However, it is expected that continuous fermentation will also become, popular in due course.
Growth Kinetics of Microorganisms:
The different types of fermentation processes- batch, fed-batch, semi-continuous and continuous are described above. The kinetics of microbial growth with special reference to log phase of batch fermentation are briefly discussed here.
After completion of lag phase, the cell enters log phase which is characterized by exponential growth (See Fig. 19.12). If the initial number of cells is N0, then
After 1st generation, the cell number will be N0 × 21.
After 2nd generation, the cell number will be N0 x 22.
After 3rd generation, N0 × 23 and so on. Thus, the number of cells after a given time (Nt) will be as follows:
Nt = N0 x 2n
where n is the number of generations.
The term doubling time (td) or mean generation time (MGT) refers to the time taken for doubling the cell number or biomass. The specific growth rate constant expressed by µ, is the direct measure of rate of growth of the organism. If N is the number of cells at a given time, then the increase in the number of cells (growth rate) with time is given by the formula.
dN/dt = µN (1)
If X is the biomass concentration at a given time, then the increase in the biomass (growth rate) with time is given by.
dX/dt = µX (2)
In general, the specific growth rate (n) is a function of the concentration of limiting substrate (S), the maximum specific growth rate (µmax) and a substrate specific constant (Ks). Their relationship was expressed by Monond by the following equation
µ = µmax S/ Ks + S (3)
Both S and Ks are expressed as concentrations e.g., in moles or grams per liter.
The growth rate (µ) of an organism is not fixed but it is variable depending on the environmental conditions such as concentration of substrate and temperature. At a low concentration, the substrate is the limiting factor for growth (Fig. 19.14A). The Fig. 19.14B represents the growth rate for a given substrate concentration (by plotting against S).
In batch culture, the substrate is initially present at a higher concentration i.e. (S) > Ks, hence the equation (3) is approximately 1.
S/ Ks + S = 1
Thus, µ = µmax.
When the substrate concentration is low, as usually occurs at the end of growth phase, then,
S/ Ks + S < 1
Hence µ < µmax.