1.3. Bacterial Growth and Nutrition-2024.pptx

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1. NUTRITION and Growth OF BACTERIA For their optimal growth bacteria have well defined requirements of proper nutrients , oxygen, PH and temperature. 1. Types of nutrients a. Macro-nutrients Needed in large quantities: CHONPS Major elements takes 96% of Dry Weight Mineral salts such as Ca+2, Fe+3, Mg+2, K+ b. Micronutrients (trace elements) Micronutrients or trace elements – required in small amounts; involved in enzyme function & maintenance of protein structure manganese, zinc, nickel

2. 2. Temperature The minimum and max. T⁰ at which a microorganism can grow is different in the different species of bacteria. This is known as T⁰ tolerance rang. Based on temperature requirement bacteria are classified as. Psycrophylic - are those bacteria, which grow in the range of -5 to 30 C with an optimum of 10-20 C. These are a bacteria include those which cause spoilages of food at refrigeration temperature (2-8 o c). Mesophilic - are those bacteria, which grow at 20-45 C and show optimum growth at 37 o C. And all medically important bacteria (pathogenic bacteria) belong to this group. Thermophilic – are those organisms which prefer high temperature (45-80 C) for their growth and show maximum growth at 50- 60 C. Hyperthermophile or extremophile – are those organisms which prefer high temperature (~ >80 C) for their growth.

3. 3. Oxygen requirement The need of oxygen for particular bacterium reflects its mechanism to meet the requirement of energy. On the basis of this requirement, bacteria have been divided in to: Obligate Anaerobes - these grow only in the environment devoid of oxygen e.g. clostridium Facultative anaerobes - these can grow under both aerobic and anaerobic conditions, e.g. Enterobacteriaceae Obligate aerobes - these cannot grow unless oxygen is present in the medium e.g. pseudomonas Microaerophilic - these organisms can grow under conditions with low oxygen tension e.g. Campylobacter Aerotolerant anaerobes – These bacteria oxidize nutrient substrates without using elemental oxygen. Although, unlike obligate anaerobes, they can tolerate it.

4. O 2 requirement Oxygen is very important in metabolism (final electron acceptor) in ATP production  aerobic bacteria Oxygen forms very reactive free radicals ( superoxides , peroxides, hydroxyl ions and hydrogen peroxide) that destroy cell components  anaerobic bacteria

5. Oxygen requirements 6

6. 4. pH requirement Most pathogenic bacteria require a pH of 7.2-7.6 for their optimal growth. Based on pH requirement bacteria can be classified as Neutrophilic :- bacteria grow best at neutral pH (pH=7) Most pathogenic micro-organism best grow at neutral pH (pH=7) Acidophilic Bacterial grow best at acidic pH (pH<7)   E.g. Lactobacilli, fungi and yeast Alkalophilic Bacterial grow best at Alkaline pH (pH>7) E.g. Vibrio cholerae grow at a pH of 8.6

7. 5. Salinity (salt concentration) In most case bacteria need small amount of salt concentration to grow. Halophiles are bacteria which thrive in high concentration of salt for their growth. Such organisms may be extemophiles E.g. Most archae , yeast and fungi Staphylococcus aureous

8. Bacterial growth Bacteria divides by binary fission Growth in bacteria is increase in number Fig. Bacterial binary fission

9. Generation time or population doubling time. The interval of time between two cell division, or the time required for a bacterium to give rise to two daughter cells under optimum conditions The generation time of bacteria ranges from as little as 20 minutes for E.coli to more than 20 hrs for Mycobacterium tuberculosis . The generation time varies not only with the species but also with the amount of nutrients, the temperature, the pH, and other environmental factors.

10. Bacterial growth curve The growth cycle of bacteria has four major phases. The Lag phase The log phase (exponential phase) The stationary phase The decline phase If a small number of bacteria are inoculated into a nutrient medium and the bacteria are counted at frequent interval, the typical phase of a standard growth curve can be demonstrated. B = number of bacteria at the beginning of a time interval b = number of bacteria at the end of the time interval n = number of generations (number of times the cell population doubles during the time interval) b = B x 2 n   (This equation is an expression of growth by binary fission)

11. Fig. Bacterial growth curve

12. 1. The Lag Phase this phase is of short duration in which bacteria adapt themselves to new environment in such away that the bacterial machinery brings itself in conformity with the nutrition available. This is a period of active macro molecular synthesis like DNA, RNA, various enzymes and other structural components It is the preparation time for division. No increase in cell number occurs, however, vigorous metabolic activity occurs. This can last for a few minutes up to many hours. The duration of lag phases varies with the species, nature of culture medium, temperature of incubation etc.

13. 2. The log, logarithmic, or exponential phase During this phase, the population can double approximately every 30 minutes with fast growing bacteria It has limited duration because of:- Exhaustion of nutrients Accumulation of toxic metabolic end products Rise in cell density Change in pH and Decrease in oxygen tension (in case of aerobic organisms)

14. The number of bacteria during log phase growth can be calculated by the following equation N t = N o x 2 t/d N t = is the number of bacteria after time (t), t/d = is the amount of time divided by the doubling time N o = the initial number of bacteria; Example: Bacillus cereus divides every 30 minutes. You inoculate a culture medium with exactly 100 bacterial cells. After 6 hours, how many bacteria are present? In 6 hours, B. cereus will divide 12 times. Therefore, t/d = 12. 2 12 = 4096 100 x 4096 = 409,600 cells

15. 3. Stationary Phase Occur when nutrients depletion or toxic products cause growth to slow until the number of new cells produced balances the number of cells that die resulting in a steady state The number of viable cell remain constant There is almost a balance between the bacterial reproduction and bacterial death 4. The death or decline phase Due to severe depletion of nutrients and accumulation of toxic end products the number of bacteria dying is much more than those dividing and hence there is gradual decline in the total number of organism. There is drastic decline in viable cells Assignment – Continuous culture

16. Detecting Growth a. Nutrient broth (liquid media) 1) Turbidity: growth well dispersed due to cloudy appearance. 2) Sediment: growth at tube's bottom. 3) Pellicle: growth at surface of broth 4) Appearance of colored pigment in broth b. Nutrient Agar(solid media) 1) Appearance of visible colony 2) Change of the color of the media

17. Some Methods used to measure bacterial growth Method Application Comments Direct microscopic count Enumeration of bacteria in milk or cellular vaccines Cannot distinguish living from nonliving cells Viable cell count (colony counts) Enumeration of bacteria in milk, foods, soil, water, laboratory cultures, etc. Very sensitive if plating conditions are optimal Turbidity measurement Estimations of large numbers of bacteria in clear liquid media and broths Fast and nondestructive, but cannot detect cell densities less than 10 7 cells per ml

18. Bacterial Metabolism Metabolism Sum up all the chemical processes that occur within a cell Anabolism : Synthesis of more complex compounds and use of energy Catabolism : Break down a substrate and capture energy

19. Catabolism The metabolic degradation (breakdown) of organic compounds that results in the production of energy and smaller molecules. Catabolic reactions involve the breaking of bonds ( energy is released). Anabolism Refers to those biosynthetic processes that use energy for the synthesis of protoplasmic materials needed for growth, maintenance, and other cellular functions. Anabolic reaction involves the creation of bonds ; it takes energy to create chemical bonds. Smaller molecules are bonded together to create large molecules

20. From a nutritional, or metabolic, viewpoint 3 major physiologic types of bacteria exist: the autotrophs (or chemolithotrophs ), the photosynthetic mos (or phototrophs ), and the heterotrophs (or chemoorganotrophs ),

21. Heterotrophic Metabolism Heterotrophic bacteria, which include all pathogens , obtain energy from oxidation of organic compounds . Carbohydrates (particularly glucose ), lipids, and protein are the most commonly oxidized compounds. Biologic oxidation of these organic compounds by bacteria results in synthesis of ATP and generation of simpler organic compounds (precursor molecules). All heterotrophic bacteria require preformed organic compounds. There are 3 energy generating Mechanism from Glucose Aerobic respiration Anaerobic respiration Fermentation

22. Respiration (Aerobic) a type of heterotrophic metabolism that uses oxygen and in which 38 moles of ATP are derived from the oxidation of 1 mole of glucose, yielding 380,000 cal. (An additional 308,000 cal is lost as heat.) In aerobic respiration, molecular 2 serves as the terminal acceptor of electrons. Most aerobic organisms oxidize glucose completely by the following reaction equation:

23. The complete oxidation of glucose may involve three fundamental biochemical pathways. The Glycolytic or Embden - Meyerhof- Parnas pathway (EMP), The Krebs cycle (also called the citric acid cycle or tricarboxylic acid cycle), and The series of membrane-bound electron transport oxidations coupled to oxidative phosphorylation .

24. Fermentation (Anaerobic) another type of heterotrophic metabolism, an organic compound rather than oxygen is the terminal electron (or hydrogen) acceptor. Less energy is generated from this incomplete form of glucose oxidation, but the process supports anaerobic growth.

25. Overview of fermentation products formed from pyruvic acid by different bacteria Organic compound is electron acceptor (NAD) End products are used for identification

26. The pyruvic acid formed during glycolysis is broken down to lactic acid and energy is released (which is used to form ATP). Glucose → Pyruvic acid → Lactic acid + energy

27. Glucose → Pyruvic acid → alcohol + carbon dioxide + energy

28. Glucose 2 Pyruvate 2 NAD + 2 NADH 2 ADP 2 ATP 2 Ethanol 2 Acetylaldehyde 2 CO 2 ALCOHOL FERMENTATION OCCURS IN YEAST

29. Anaerobic Respiration Some prokaryotes are able to carry out anaerobic respiration , respiration in which an inorganic molecule other than oxygen (O 2 ) is the final electron acceptor. For example, some bacteria, called nitrate reducers, can transfer electrons to nitrate (NO 3 - ) reducing it to nitrite (NO 2 - ). Less efficient: usually 30-34 ATPs per glucose molecule.

30. Metabolic strategies Pathways involved Final e- acceptor ATP yield Aerobic respiration Glycolysis, TCA, ET O 2 38 Anaerobic respiration Glycolysis , TCA, ET NO 3 - , So 4 -2 , CO 3 -3 Variable (34-30) Fermentation Glycolysis Organic molecules 2 33