Different Systems or Modes of Microbial Cultures!
Microbial growth is defined as the increase in quantity of cellular constituents and structures and is followed by an increase in size or number of cells or both. It is the division of the microorganisms into two daughter cells. In the case of bacteria it is called binary fission. The time of interval required for the division of microbial cell called generation time. The population growth is studied by analyzing the growth curve of a microbial culture in different systems.
These are different system or mode of microbial cultures: 1. Closed System – Batch Culture 2. Fed Batch Culture 3. Continuous Culture (Open System) 4. Synchronous Culture.
Microbial cells growing in a tube or flask of liquid medium are said to be in a closed system. Here no nutrients are added to the system and no metabolic waste products are removed. When nutrients are added to the system, the cells initially divide by binary fission and the cell number increase for a period of time. Once the nutrients are over in the medium the growth eventually stops and certain metabolic waste products are accumulated in the medium (Fig. 3.1).
A culture of microorganisms produced by inoculating a closed vessel containing a single batch of medium is called a batch culture. Because no fresh medium is provided during incubation, nutrient concentration decreases and concentration of waste products increases. The growth of microorganisms reproducing by binary fission can be plotted on the logarithm of the number of viable cells versus incubation time.
The resulting curve has four distinct phases:
It is period in which bacteria adapt themselves to growth conditions. In this phase no increase in cell number, only cell size will increase. Cells physiologically active and synthesizing new enzymes and other molecules to adapt new environment.
2. Logarithmic Phase (Exponential/ Growth Phase/ Log Phase):
This period is characterized by cell doubling, i.e., the number of cells and the rate of population increase with respect to time period. Here the growth rate is maximum and constant. If we are plotting the natural logarithm of cell number against time produces a straight line. The slope of this line is the specific growth rate of the organism, which is a measure of the number of division per unit time.
During this phase the growth rate slows as a result of nutrient depletion and accumulation of toxic products. Some cells die while others grow and divide. This phase is a constant value on the rate of bacterial growth is equal to the rate of bacterial death.
In this phase the growth rate is negative due to the accumulation of inhibitory metabolic products and depletion of essential nutrient results the reduction of viable cell. Depending on the organisms, a very few cells may persist in to the trail of the curve, forming what may be called a senescent phase. Typically all cells normally die within days to months.
Microbial growth kinetics describes how the microbes grow in the fermentor, this information is important to determine optimal batch time. The relationship between the specific growth rate (μ) of the microbial population and substrate concentration are termed as microbial growth kinetics. Batch culture is a closed system which contains an initial limited amount of nutrients. The inoculated culture will pass through a number of phases- lag phase, log phase (exponential), stationary phase and decline phase.
After inoculating the culture, there is a period in which no growth takes place and it is called lag phase and may be considered as a time of adaptation. In a commercial process the length of the lag phase should be reduced as much as possible by using suitable inoculum. In the log phase the growth rate of the cells gradually increases and the cell grows at a constant, maximum rate.
The exponential phase can be described by the equation:
1n Xt =1n X0 + μt … 3
Thus, a plot of the natural log of biomass concentration against time should yield a straight line, the slope of which is equal to μ. During the exponential phase nutrients are excess and the organism is growing at its maximum specific growth rate μ max.
In general, the biochemical reaction of a fermentation process –
Substrate + oxygen + other nutrients → Carbon-dioxide + ammonia + new biomass + other end products
The concentration of biomass, X increases as a function of time due to conversion of substrate into biomass,
That is, the rate of increase in biomass is correlated with the specific growth rate μ, and the biomass concentration X, where rate of increase in cell number is correlated with μ and density N
Effect of Substrate Concentration on Growth Rate Constant:
This equation is generally called Monads equation since the relationship was first expressed by Jacques Monad. The constant Ks is the substrate concentration at which half maximum specific
The biomass production of a fermentation reaction expressed by the equation:
The substrate utilization by microorganism is a typical fermentation reaction is given by:
2. Fed Batch Culture:
In fed batch culture we add the growth limiting nutrient substrate to the culture. The fed batch process is used in bio-industrial process to reach a high cell density in the bioreactor. Controlled addition of the nutrient directly affects the growth rate of the culture and allows avoiding overflow metabolism (formation of side metabolite such as acetate, lactic acid, etc.).
The substrate limitation offers the possibility to control the reaction rates to avoid technological limitations connected to the cooling of the reactor and oxygen transfer. Substrate limitation also allows the metabolic control, to avoid osmotic effects, catabolite repression and metabolism of side products.
Continuous culture system is a culture system with constant environmental condition maintained through continuous supply of nutrients and removal of waste. Here, the cell volume and cell concentration are kept constant by adding fresh, sterile medium. In industry, it helps to maintain the active logarithmic growth phase which helps to generate maximum volume of desired products. The rate at which new cells are produced in the culture vessel is balanced by the rate at which cell are being removed.
In continuous culture microbial population can be maintained in exponential growth phase and at a constant biomass concentration for extended period.
Two major types of continuous culture system are commonly used:
A chemostat (chemical environment is static) is a bioreactor to which fresh medium is continuously added, while culture liquid is continuously removed to keep the volume of the culture constant. The culture mediums for chemostat possess essential nutrients, (e.g. amino acid) in limiting quantities.
It is the second type of continuous culture system which consists of a photocell that measures the absorbance or turbidity of the culture in growth vessel. The flow rate of media through the vessel is automatically regulated to maintain a predetermined turbidity on cell density.
The turbidostat differ from the chemostat –
1. The dilution rate in a turbidostat varies rather remaining constant, and its culture medium contains all the nutrients.
2. The dilution rate is high, but in chemostat it is low.
Generally, all cells in a microbial culture do not divide at the same time. In a traditional batch culture the microbial cells divided at random manner, i.e., the straight line that characterizes the log phase of microbial culture in batch culture is due to random cell division. There are certain laboratory techniques that manipulate the growth of cultures so that all cells divide at the same time or grow synchronously.
Growth in a cell population in which all cells divide at same time is called synchronous growth and such culture is called synchronous culture. A population can be synchronized by changing the physical environment or chemical composition of the medium. If cells are inoculated into a medium at a suboptimal temperature and kept that temperature, they will metabolize slowly but will not divide.
When the temperature is rapidly increased at optimal level the cells undergo synchronised division. Another method to obtain synchronised growth uses differential filtration or centrifugation. When cells are separated by size, all cells are synchronised by each other. But the synchronous culture lasts only a few generations.