![Advantages :-
Cells can be maintained at a constant
physiological state because the
specific growth rate and the substrate
concertration can be set simply by
setting the dilution rate [ µ=D ] .
It can take advantage of cell
immobilization which allows the
maintenance of high concertrations of
cells in the reactor at low substrate
concertrations.](https://image.slidesharecdn.com/batchcont-241010053305-2832bad5/85/Batch-cont-synchro-cultures-classs1-ppt-22-320.jpg)
Batch cont. synchro cultures classs1.ppt
- 1. Fig. 6-1 Growth and culturing of Bacteria Bacterial growth is affected by a variety of physical and nutritional factors. Knowing these allows culture of bacteria in the laboratory and methods of growth prevention elsewhere. Growing bacteria in pure cultures is an important step in isolating and characterizing a bacterium, and diagnosing a disease. Microbial growth is defined not in terms of cell size but as the increase in the number of cells, usually binary fission or sometimes budding.
- 2. Binary fission Unlike eukaryotic cells, prokaryotic cells do not have a cell cycle with a specific period of DNA synthesis. In continuously dividing cells, DNA synthesis is continuous. Duplication of the circular chromosome is completed prior to cell division. Incomplete separation of the cells produces linear chains, tetrads, sarcinae, etc (Figure 4.2) Some bacilli always from chains, others form them only under unfavorable growth conditions. Streptococci form chains when grown on artificial media but exist as single or paired cells in a lesion.
- 3. Budding, in yeast and a few bacteria, is the development of a small, new cell from the surface of an existing cell. This eventually separates.
- 4. Batch culture technique Batch culture technique is also called as closed system of cultivation. In this technique at first nutrient solution is prepared and it is inoculated with inoculum (culture organism) and then nothing is added in the fermentation tank except aeration. In batch culture, neither fresh medium is added nor used up media is removed from the cultivation vessel. Therefore volume of culture remains same. Since fresh media is not added during the course of incubation, concentration of nutrition decreases continuously. Furthermore various toxic metabolites also accumulates in the culture vessel. Therefore batch culture technique gives characteristics growth curve with lag phase, log phase, stationary phase and decline phase. Advantage: Chance of contamination of culture is minimum in batch culture technique because it is closed system of cultivation. Disadvantage: It gives low product yield and it is not economic procedure.
- 6. Fed-batch culture technique Fed-batch culture is also called as semi-closed system of cultivation. In this technique, at first nutrient media is prepared and it is inoculated with culture organism and then incubated for particulate time. During the course of incubation a particular nutrient is added at intervals without removing the used up media. so the volume of culture increases continuously. Fed batch culture technique is applied in many types of fermentation process. In fermentation some nutrient is very essential for the process but when these nutrients are provided in higher concentration in the culture they inhibit the growth of bacteria ultimately ceasing the fermentation. Therefore such nutrients are kept in lower concentration initially and it is added slowly and continuously during the course of fermentation. Advantage: Fed batch culture gives greater product yields than batch culture technique. Disadvantage:
- 7. Fig. 6-3 Phases of Growth depend on the genetics of the particular bacteria and on the medium (mixture of substances on which the bacteria are grown). Four phases include: The Lag Phase in which the microbes are adapting to their new media and growing larger. They do not increase in number, but are metabolically active and produce large amounts of energy (ATP). The Log Phase in which the bacteria population expands exponentially or logarithmicly (by 10-fold increments). They are dividing at their fastest rate - a genetically determined interval called the generation time. Generation time is usually 20 minutes to 20 hours, but varies. Mycobacteria have a generation time much longer. Synchronous growth, when all bacteria in a population divide at the same time, is not a natural situation. In an active culture, each cell divides sometime during the generation time - nonsynchronous growth. As the number of microbes increases, nutrients decrease, wastes build up, oxygen becomes depleted, the population enters the stationary phase.
- 8. As the number of microbes increases, nutrients decrease, wastes build up, oxygen becomes depleted, the population enters the Stationary Phase. During this phase the number of new cells is equal to the number of cells that die. What will happen if you add fresh media? If the media is not replenished cell division decreases to the point that new cells are more cells die and the number of live cells decreases at a logarithmic rate (X10) - The Decline or Death Phase. Many cells undergo involution - take on various unusual shapes. This makes them hard to identify. Spore-forming organisms consist of more spores than vegetative cells. Colonies growing on a solid medium contain all phases at the same time. The colony grows rapidly at its edges and cells begin to die in the center.
- 9. Fig. 6-4 Scientists can induce synchronous growth - all bacteria in the population divide at the same moment. This is not what normally happens. Grown in media, bacteria divide nonsynchronously.
- 10. Continuous culture technique Continuous culture technique is also called as open system of cultivation. In this technique fresh sterile medium is added continuously in the vessel and used up media with bacterial culture is removed continuously at the same rate. So the volume and bacterial density remain same in the cultivation vessel.
- 11. In this technique, bacteria grow continuously in their log phase. This type of growth is known as steady state growth. The cell density in continuous culture remains constant and it is achieved by maintaining constant dilution and flow rate. Types of approach to continuous culture a. Chemostat b. Turbidostat
- 12. a. Chemostat culture technique It is the most common type of approach which controls the population density and growth of culture. Two elements are used in chemostat, the dilution rate and concentration of limiting nutrient. In continuous culture, end products do not accumulates and nutrients are not completely depleted, therefore bacteria never reach in stationary phase because fresh nutrients are supplied continuously and end products are removed continuously. In chemostat, the liquid media contain some nutrient in growth limiting concentration and the concentration of limiting nutrient determines the rate of bacterial growth.
- 13. During steady state chemostat, concentration of limiting nutrient remains constant because the rate of addition of nutrient equals the rate at which it is used by organism plus flow through outlet. To check whether there is constant cell density or not, concentration of that essential nutrient in the vessel is checked. If the concentration of that nutrient is altered then it indicates bacterial density is changing. Therefore in this case flow rate is adjusted to maintain constant cell density.
- 16. b. Turbidostate In turbidostat, a photoelectric device is used to monitor the cell density in the cultivation vessel. The optical sensing device measure the turbidity (absorbance) of the culture in the vessel. If concentration is altered, it is noticed by the photoelectric device and the flow rate is adjusted to Maintain constant cell density in the culture.
- 19. Continuous culture :- The exponential growth in batch culture can be prolonged by the addition of more substrate but it will lead to the overflowing from culture vessel . Hence , a overflowing device is fitted to the fermentor such that the added medium displace an equal volume of culture from vessel than continuous production of cells can be taken place & thus system called CONTINUOUS CULTURE .
- 20. If the medium is fed continuously to batch culture at a suitable rate , then a steady state is achieved that formation of new biomass by the culture is balanced by the loss of cell from the vessel . The flow of media in the vessel is related to volume of vessel in the form of dilution factor . D = F/V where F= flow rate V= volume of vessel D= dilution factor
- 21. Change in cell biomass concertration :- Change in cell biomass= Growth – Output dx/dt = µx – Dx Under steady state , the cell concertration remains constant that is dx/dt = 0 , so µx = Dx cut x both side µ = D Under steady state the specific growth rate is controlled by dilution rate .
- 22. Advantages :- Cells can be maintained at a constant physiological state because the specific growth rate and the substrate concertration can be set simply by setting the dilution rate [ µ=D ] . It can take advantage of cell immobilization which allows the maintenance of high concertrations of cells in the reactor at low substrate concertrations.
- 23. Most downstream processing operations operate in a continuous manner . Continuous reactors do not need to be shut down and cleaned as regularly as a batch reactor and thus have a shorter “ turn- around “ time . This also reduces costs associated with cleaning and filling of the reactor .
- 24. Disadvantages :- These are unsuitable for products which are predominantly produced when growth ceases . Such products include many antibiotics , toxins produced during endospore production by Bacillus spp. And Clostridium spp. And monoclonal antibodies . They are unsuitable for producing various fermented foods and beverges which depend on a complete batch cycle to produce full flavours . Large scale continuous cultures are still regarded as risky ventures .
- 26. Synchronous culture Synchronous cultures are composed of the population of cells that are the same stage of their life cycle. All the cells in the culture will divide at the same time, will grow for a generation time, and all will divide again at the same time. Thus the entire population is kept uniform with respect to growth and division. It is not possible to analyze a single bacterial cell to obtain the information about growth behavior. Ie. Organisation, differentiation and macromolecular synthesis. Synchronous culture provides the entire cell crop in the same stage of growth. Measurement made on such cultures are equivalent to the measurement made individual cells.
- 27. 1. Selection by Size and Age: • A population of cells is fractionated on the basis of size. The cells are filtered so that smallest cells pass through the filter. These small cells are the youngest, and must go through their whole life-cycle before dividing. Alternatively, the largest cells, which are ready to divide, may be retained or retarded by a filter. • These are then collected separately and used to obtain a synchronous culture. The most widely used method for obtaining synchronous cultures is the Helmstetter-Cummings technique. A population of cells is passed through a membrane filter of pore size small enough to trap bacteria in the filter. • The filter is then inverted, and fresh nutrient medium is allowed to flow through it (Fig. 18.29). After loosely associated bacteria are washed from the filter, the only bacterial cells in the effluent stream of the medium are those which arise through division.
- 28. If a sample of this stream is collected over a short period of time, all the cells in this sample are newly formed, and are therefore of the same age and divide synchronously.
- 29. 2. Selection by Induction Technique • A synchronous culture is also obtained by the use of shock treatments. These include variation in temperature, starvation, exposure to light (for photosynthetic organisms), drugs, and sub-lethal doses of radiation. A commonly used technique involves submitting a culture of microorganisms to single or multiple changes in temperature.
- 30. • An exponentially growing culture at 37°C is held for about 30 minutes at 20°C. The lower temperature retards cell division. During the interval of 30 minutes all the cells mature to the point of fission. However, at 20°C none divide. On sudden return of the culture to 37°C, all the cells divide synchronously. By repeating the alterations of temperature, synchrony can be maintained in the culture for several generations.