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1. Contents Lecture -3 Bacterial growth and fermentation tools Classification and reproduction of biotechnologically important bacterial system Bacterial growth Bacterial Growth Curve Environmental factors affecting bacterial growth

2. Bacterial growth and fermentation tools Microbes are the tools of fermentation because they produce enzymes, amino acids, vitamins, bio gums, other valuable recombinant proteins, and organic acids. The biotechnology focus on the growth of unicellular bacteria As they are ideal objects for study of the growth process Current scale-up process for the manufacture of industrial products Many aspects of food biotechnology Negative aspects of microorganisms are also the most common causes of food-borne illness and food spoilage and thus the detection of pathogens

3. Fermentation technology is becoming increasingly important in the production of various bulk chemicals, fine chemicals, and pharmaceuticals. the fermentative production process is a very promising technology to produce enantiomer pure chemicals with low environmental burden. High conversion efficiencies are often achieved in fermentative production processes. it is possible to convert abundant renewable raw materials or waste materials to produce high-value products.

4. Classification and reproduction of biotechnologically important bacterial system Bacterial system is very simple and is classified based on artificial criteria such as Structure, shape, motility, nutrition, propagation and immunological reactions Tables1 and 2 summarize the most important bacterial species that are involved in biotechnology processes on the basis of the classification in Bergey’s Manual of Systematic Bacteriology.

5. Bacterial growth Living organisms grow and reproduce. The growth indicates that an organism is in active metabolism. Cells are the most fundamental units of life . All living organisms are made of one or more . Growth common refers to increase in size but with microorganisms particularly bacteria, this term refers to changes in total population rather than increase in size or mass of individual organisms .

6. Cells reproduce by copying their genetic material and then dividing—a parent cell giving rise to daughter cells. Types of Cell Division: 1 ) Binary Fission 2 ) Mitosis & Meiosis It refers to an increase in cell numbers, not in cell size . The Bacterial division/reproduction 1- Bacteria normally reproduce by a method called binary fission 2- What is Binary Fission .? Binary Fission is the process of bacteria reproduction where one cell become Tw

7. The reproduction of prokaryotic cells (bacteria and bacteria-like Archaea) is accomplished through binary fission . A bacterial cell that is ready to divide first copies its genetic material, called the nucleoid—a single, circular chromosome of DNA (deoxyribonucleic acid). The two chromosomes, each attached to the plasma membrane, move apart as the cell elongates.

8. Once the two copies of genetic instructions are separated, the cell divides, laying down new cell wall and membrane between the two chromosomes . Binary fission is essentially cloning . Barring mutations that may have occurred when the genetic material was copied (a process called replication), the two resulting daughter cells are identical to the parent cell .

9. Bacterial Growth Curve Illustrates the dynamics of growth Phases of growth Lag phase 2. Exponential or 3. logarithmic (log) phase 4 . Stationary phase 5. Death phase (decline phase )

10. Lag phase During this phase, bacteria are growing in size, but they are not undergoing binary fission . Hence , there is no increase in cell number. The bacteria are adapting to the new environment and are synthesizing cellular components such as ribosomes, enzymes, and other proteins . Bacteria have the maximum cell size towards the end of the lag phase . This phase is also referred to as the exponential phase because there is a logarithmic increase in cell number. This exponential growth is expressed as the bacteria’s generation time. During this phase, the conditions are optimal for growth and binary fission occurs. In the log phase, cells are smaller and stain uniformly.

11. Log (Exponential) Phase Also called log phase Rate of growth is constant Population number of cells undergoing binary fission doubles at a constant interval called generation time Continue as long as cells have adequate nutrients & good environmen

12. Stationary phase There is no net increase or decrease in cell number in this stage. In other words, cell growth (division) equals cell death . The birth rate decreases due to limited nutrients, lack of space, and the build up of secondary metabolic products (e.g. toxins ). The insufficient supply of nutrients also causes some bacteria to form spores during this phase . Cells frequently are gram variable and show irregular staining due to the presence of intracellular storage granules.

13. Phase of decline This phase is characterized by an exponential death of cells . When the media runs out of nutrients and there are too many toxins, cells begin to die at a faster rate . Involution forms are common in the phase of decline.

14. In the bacteria, which use the organic substrate as the sole source of carbon and energy, the growth yield can be measured in terms of the organic substrate and biomass resulting. Many microorganisms utilizing sugars as the sole source of carbon reveal that the ratio of the sugars to cellular carbon varies between20%and50%. The microbes usually use about half the carbon source to make cells and metabolize the otherhalftoCO2 or other by-products. The differences in conversion of efficiency probably reflect differences in the efficiency of generating ATP through catabolism of the substrate. In batch cultures discussed so far, nutrients are not renewed and growth remains exponential for only a few generations.

15. Thus, the physiological state of cells in 1. Batch cultures varies continuously throughout the growth cycle . Although exponentially growing cells in batch cultures may suffice for some studies, many studies on microbial physiology require a cell that is not constantly changing. A batch fermentation can be extended by feeding, either intermittently or continuously, nutrients containing a substrate that limits cell growth.

16. 2. Continuous cultures cells can be maintained in a steady physiological state for long periods of time by adding fresh medium continuously and removing equal amounts of spent medium. Continuous culture systems offer a few valuable features: 1 . They provide a constant source of cells in an exponential growth phase. 2 . They allow cultures to be grown continuously at extremely low concentrations of substrate, which is valuable in studies on the regulation of synthesis or catabolism of the limiting substrate, or in the selection of various classes of mutants. 3 . They offer an increase (over batch or fed-batch systems) in productivity per unit of product manufactured and a reduction of scale-up and capital costs.

17. Continuous culture is not widely used as an industrial process, mainly because of 1- The problems of chance contamination. 2- The danger of strain degeneration by spontaneous mutation, which produces a new strain of low product formation.

18. Environmental factors affecting bacterial growth The growth of microorganisms is influenced by various factors, including nutrients, which have already been discussed, and the interactions between the microbial cell and its environment.

19. Solutes Transport mechanisms play two essential roles in cellular function. First, they maintain the intracellular concentration of all metabolites at levels high enough to operate both catabolic and anabolic pathways at near-maximal rates. Second, transport mechanisms function in osmoregulation, which maintain the solutes (principally small molecules and ions) at levels optimal for metabolic activity.

20. Bacteria vary widely in their osmotic requirements. Microorganisms that can grow in solutions of high osmolarity are called osmophiles. Osmotic pressure is the minimum pressure which needs to be applied to a solution to prevent the inward flow of water across a SPM. Types of solution : 1. Hypotonic 2. Isotonic 3 . Hypertonic If the internal osmotic pressure of the cell falls below the external osmotic pressure, water leaves the cell and the cytoplasmic volume decreases with accompanying damage to the membrane.

21. Temperature is the most important factor that determines the rate of growth, multiplication, survival, and death of all living organisms . High temperatures damage microbes by denaturing enzymes, transport carriers, and other proteins . Microbial membrane are disrupted by temperature extremes . At very low temperatures membranes also solidify and enzymes also do not function properly 2. Temperature

22. Psychrophiles : bacteria which grow below 20°C, e.g. soil and water saprophytes. Up to -7°C reported . Esophiles :bacteria which grow between 20-40°C. e.g. Most pathogenic bacteria are mesophiles. Wide range e.g. Pseudomonas 5-43°C, narrow range e.g. Gonococcus 30-39°C . Thermophiles : bacteria which grow at higher temperature i.e. 60-80°C. e.g. Bacillus stearothermophilus. Up to 250°C reported . Thermal Death Point : The lowest temperature that kills a bacterium under standard conditions in a given time. Under moist conditions most vegetative, mesophilic bacteria have a thermal death point 50 to 65°C and most spores between 100 and 120°C

23. 3. Oxygen The oxygen requirements among bacteria are remarkably variable, and the fermentation conditions are decisively affected by whether the organism is aerobic or anaerobic . Obligate Anaerobe - only anaerobic growth, growth ceases in presence of oxygen, growth occurs only when there is no oxygen Aerotolerate Anaerobe - only anaerobic growth, but continues in presence of oxygen, oxygen has no effect Microaerophiles - only aerobic growth, oxygen required in low concentration, growth occurs only where a low concentration of oxygen has diffused into medium

24. pH refers to negative logarithm of hydrogen ion concentration . Affect the activity & integrity of enzymes & structural components of a cell 4. pH

25. Microbial growth is strongly affected by the pH of the medium . Drastic variations in cytoplasmic pH disrupt the plasma membrane or inhibit the activity of enzymes and membrane transport protein . Acidophiles Grow between pH 0 and 5.5. Examples: Ferroplasma, Thiobacillus thioxidans, Sulfolobus acidocaldarius, etc . Alkalophiles Grow between pH range of 7.5 to 14 . Examples: Thermococcus alcaliphilus, etc . Neutrophiles Grow between pH 5.5 to 8.0 Examples: Lactobacillus acidophillus, E. coli, Pseudomonas aerunginosa, etc.