Lecture 2&3.pptx
- 1. Lecture 2 & 3 Role of microbial systems, microbial metabolism and growth kinetics – Principle and characteristics
- 2. Microbes and metabolism Metabolic pathways (Michal, 1992) are interlinked to produce what can develop into an extraordinarily complicated network, involving several levels of control Interaction of natural cycles and represent the biological element of the natural geobiological cycles Impinge on all aspects of the environment, both living and nonliving Carbon cycle as an example, carbon dioxide in the atmosphere is returned by dissolution in rainwater, and also by the process of photosynthesis to produce sugars, which are eventually metabolised to liberate the carbon once more
- 3. In addition to constant recycling through metabolic pathways, carbon is also sequestered in living and nonliving components such as in trees in the relatively short term, and deep ocean systems or ancient deposits, such as carbonaceous rocks, in the long term Cycles which involve similar principles of incorporation into biological molecules and subsequent re-release into the environment operate for nitrogen, phosphorus and sulphur. All of these overlap in some way, to produce the metabolic pathways responsible for the synthesis and degradation of biomolecules. Superimposed, is an energy cycle, ultimately driven by the sun, and involving constant consumption and release of metabolic energy.
- 4. Microbes are referred to as such, simply because they cannot be seen by the naked eye. Many are bacteria or archaea, term ‘microbe’ also encompasses some eukaryotes, including yeasts, which are unicellular fungi, as well as protozoa and unicellular plants. In addition, there are some microscopic multicellular organisms, such as rotifers, which have an essential role to play in the microsystem ecology of places such as sewage treatment plants. An individual cell of a eukaryotic multicellular organism like a higher plant or animal, is approximately 20 microns in diameter, while a yeast cell, also eukaryotic but unicellular, is about five microns in diameter. Although bacterial cells occur in a variety of shapes and sizes, depending on the species, typically a bacterial cell is rod shaped, measuring approximately one micron in width and two microns in length.
- 5. At its simplest visualisation, a cell, be it a unicellular organism, or one cell in a multicellular organism, is a bag, bounded by a membrane, containing an aqueous solution in which are all the molecules and structures required to enable its continued survival. In fact, this ‘bag’ represents a complicated infrastructure differing distinctly between prokaryotes and eukaryotes Microorganisms may live as free individuals or as communities, either as a clone of one organism, or as a mixed group. Biofilms are examples of microbial communities, the components of which may number several hundred species. Represent the structure of microbial activity in many relevant technologies such as trickling filters
- 6. A biofilm is a mixed population of microbes live in close proximity which may be mutually beneficial Such consortia can increase the habitat range, the overall tolerance to stress and metabolic diversity of individual members of the group. Recalcitrant pollutants are eventually degraded due to combined contributions of several of its members Bacterial transformation Bacterium may absorb free deoxyribonucleic acid (DNA), the macromolecule which stores genetic material, from its surroundings released by other organisms, as a result of cell death Process is dependent on the ability, or competence, of a cell to take up DNA, and concentration of DNA in the surrounding environment. This is commonly referred to as horizontal transfer as opposed to vertical transfer which refers to inherited genetic material, either by sexual or asexual reproduction. Bacterial transformation is the transfer of free DNA released from a donor bacterium into the extracellular environment that results in assimilation and usually an expression of the newly acquired trait in a recipient bacterium.
- 7. Sliminess often associated with biofilms is usually attributed to excreted molecules often protein and carbohydrate in nature, which may coat and protect the film Biofilm may proliferate at a rate to cause areas of anoxia at the furthest point from the source of oxygen, thus encouraging the growth of anaerobes Composition of the biofilm community is likely to change with time
- 8. Microbial Metabolism Energy required to carry out all cellular processes is obtained from ingested food in the case of chemotrophic cells, additionally from light in the case of phototrophs and from inorganic chemicals in lithotrophic organisms Definition All biological macromolecules contain the element carbon, a dietary source of carbon is a requirement Ingested food is therefore, at the very least, a source of energy and carbon, the chemical form of which is rearranged by passage through various routes called metabolic pathways
- 9. One purpose of this reshuffling is to produce, after addition or removal of other elements such as hydrogen, oxygen, nitrogen, phosphorous and sulphur, all the chemicals necessary for growth. Other is to produce chemical energy in the form of adenosine triphosphate (ATP), also one of the ‘building blocks’ of nucleic acids. Where an organism is unable to synthesize all its dietary requirements, it must ingest them, as they are, by definition, essential nutrients. The profile of these can be diagnostic for that organism and may be used in its identification in the laboratory. An understanding of nutritional requirements of any given microbe, can prove essential for successful remediation by bioenhancement.
- 10. Microbial Growth Kinetics Growth of a microorganism is the basis of biotechnological exploitation of microflora for production of desired product Optimization of growth of microorganism in a particular media is desirable due to economical and availability of particular growth constituent in a region Some microorganisms have specific requirement and they grow in a particular growth media Common media for growth of different microorganism Bacterial cell division Methods of measuring growth Different phase in bacterial growth and Growth kinetics
- 11. Bacterial Cell Division Binary division Binary division is the most common mode of cell division in bacteria. In this mode of cell division, a single bacteria cell grows transversely with the synthesis of chromosomal DNA. A transverse septum appears in the middle of the cell body that divides the bacterial cell into the two with a distribution of chromosomal DNA, ribosome and other cellular machinery. Budding In this mode of cell division, chromosomal DNA divides to form two copies. Sister chromosomal DNA moves to one side of the cell and this portion of the cells protrude from main body to form bud. Eventually bud grows in size and get separated from main cell to develop a new cell.
- 12. Fragmentation This mode of asexual division is more common in filamentous bacteria. In this mode, filament of the growing cell gets fragmented into small bacillary or coccoid cells, these cellular fragments eventually develop into new cell. Measuring Bacterial growth Microscopic count - Bacterial cells can be counted easily on a “petroff-hausser counting chamber” The chamber has a ruling to make square (1/400 mm2) of equivalent volume. A glass slide is placed (~1/50mm height) to make a chamber filled with bacterial cell suspension. Volume of each chamber is 1/20,000 mm3. This chamber can be used to observe bacteria with phase contrast microscope. For example, if each chamber has 8 bacteria then there are 8x20,000,000 or 1.6x108 bacteria/ml. A very high or low concentration of bacterial sample cannot be counted accurately.
- 13. Plate count method A defined amount of bacterial culture suspension is introduced onto solid support media to grow and give colonies. If number of colonies on solid media is too high, then serial dilution of original stock can be plated on solid media and number of colony can be counted with a colony counter. A manual colony counter has lamp at the bottom, a grid to divide the bacterial culture plate and a magnifying glass to visualize and count single colony. A plate with colony count of 30-300 can be used to determine the number of bacteria present in original stock Number of bacteria per ml= Number of colonies counted on plate X dilution of sample
- 14. Turbidimetric methods This method is based on light scattering principles of particulate matter such as bacteria. A bacteria cell suspension is placed in test cuvette and corresponding media in reference cuvette. The optical density or absorbance of the bacterial suspension is used to measure the number of bacteria number. This method can not distinguish between live or dead bacteria as both form contribute to the turbidity
- 15. Nitrogen content and Dry weight A bacterial cell mass can be measured by direct measurement of dry weight of culture or nitrogen content Growth cycle of bacteria The most common method of bacteria division is binary fission and by this method, one bacteria cell gives two daughter cells. The time a bacteria takes to complete one division is called as generation time and it depends on bacteria species and media properties
- 16. Glycolysis via the Embden-Meyerhof-Parnas Glycolytic Pathway Glycolysis is the almost universal pathway that converts glucose into pyruvate along with the formation of nicotinamide adenine dinucleotide (NADH) and adenosine triphosphate (ATP). It primarily occurs in the cytoplasm of the cell Under aerobic conditions, the pyruvate passes into the mitochondria where it is completely oxidized by O2 into CO2 and H2O and its chemical energy largely conserved as ATP. Pyruvate generated via aerobic glycolysis feeds into the TCA or Kreb’s Cycle In the absence of sufficient oxygen, the pyruvate is reduced by NADH via anaerobic glycolysis or fermentation to a wide range of products, routinely lactate in animals and ethanol in yeasts
- 18. Starting molecule for glycolysis is glucose, a simple and abundant sugar found in carbohydrates, which provides the energy for most cells. Carbohydrates synthesized during photosynthesis act as the main storage molecules of solar energy. When ingested, complex carbohydrates are enzymatically hydrolyzed to monosaccharides, such as starch to D(+)-glucose. Catabolism of glucose is the primary energy source for short-term requirements. Embden-Meyerhof-Parnas (EMP) pathway, name of the discoverers, Gustav Embden, Otto Meyerhof, and Jakub Karol Parnas.
- 19. 10 STEPS IN THE GLYCOLYTIC PATHWAY AND ENZYMES OF GLYCOLYSIS
- 20. Reaction Enzyme IUBMB EC Number 1. Phosphorylation of glucose. D(+)-Glucose is phosphorylated with ATP to give glucose-6-phosphate. Hexokinase EC 2.7.1.1 2. Isomerization of glucose-6-P to fructose-6-P. The isomerization of glucose-6-phosphate in the second reaction to fructose-6-phosphate occurs via ring-opening and subsequent keto-enol-tautomerization. Glucose-6- phosphate isomerase EC 5.3.1.9 3. Phosphorylation of fructose-6-P. The third reaction is another phosphorylation with ATP, whereby fructose-6-phosphate is converted to fructose-1,6-bisphosphate. 6-P-Fructokinase EC 2.7.1.11 4. Fructose-1,6-bisphosphate to glyceraldehyde phosphate and dihydroxyacetone phosphate. A key branching reaction is the fourth reaction: a ring-opening reaction of fructose-1,6-bisphosphate, which is cleaved in a retro-aldol reaction into D-glyceraldehyde-3-phosphate, and dihydroxyacetone phosphate. Fructose- bisphosphate aldolase EC 4.1.2.13 5. Isomerization of dihydroxyacetone-P to glyceraldehyde-P. The branch via dihydroxyacetonephosphate is channelled back into D- glyceraldehyde-3-phosphate in the fifth reaction by an isomerization. Triose-phosphate isomerase EC 5.3.1.9 6. Glyceraldehyde phosphate oxidation & phosphorylation to 1,3- bisphosphoglycerate. In the sixth reaction, the combined D- glyceraldehyde- 3-phosphate from both routes is oxidized at the C1 to a carboxylic acid and then phosphorylated in the 1-position to yield 1,3- bisphospho-D-glycerate. Glyceraldehyde phosphate dehydrogenase EC 1.2.1.12 7. ATP formation. This phosphate group in the 1-position is transferred in the seventh reaction from the carboxyl group to ADP to give 3-phospho- D-glycerate. Phosphoglycerate kinase EC 2.7.2.3
- 21. 8. 3-Phosphoglycerate to 2-phosphoglycerate. The eighth reaction is an isomerization of 3-phospho-D-glycerate to 2-phospho-D-glycerate. Phosphoglycerate mutase EC 5.4.2.1 9. 2-Phosphoglycerate to phosphonenolpyruvate. The next metabolite, phosphoenolpyruvate, is formed in a dehydration reaction from 2- phospho-D-glycerate. Enolase EC 4.2.1.11 10. Formation of pyruvate & ATP. The glycolysis pathway from D(+)- glucose to two molecules of pyruvate is concluded by the tenth reaction, which transfers a phosphate group from phosphoenolpyruvate to ADP, thereby giving ATP and pyruvate. Pyruvate kinase EC 2.7.1.40
- 22. TCA cycle is usually described beginning with acetyl-CoA
- 23. TCA cycle description 1. TCA cycle begins with an enzymatic aldol addition reaction of acetyl CoA to oxaloacetate, forming citrate 2. Citrate is isomerized by a dehydration-hydration sequence to yield (2R,3S)-isocitrate 3. Further enzymatic oxidation and decarboxylation gives 2-ketoglutarate 4. After another enzymatic decarboxylation and oxidation, 2-ketoglutarate is transformed into succinyl-CoA 5. Hydrolysis of this metabolite to succinate is coupled to the phosphorylation of guanosine diphosphate (GDP) to guanosine triphosphate (GTP) 6. Enzymatic desaturation by flavin adenine dinucleotide (FAD)-dependent succinate dehydrogenase yields fumarate 7. After stereospecific hydration, fumarate catalyzed by fumarase is transformed to L-malate 8. Last step of NAD-coupled oxidation of L-malate to oxaloacetate is catalyzed by malate dehydrogenase and closes the cycle.
- 24. Glyoxylate Cycle Glyoxylate cycle is an anabolic pathway that is considered a variation of the tricarboxylic acid (TCA) cycle. TCA cycle occurs in plants, bacteria and fungi and acetyl - CoA is converted into succinate Glyoxylate cycle was thought not to occur in animals due to the absence of these enzymes isocitrate lyase and malate synthase, however, this hypothesis is being explored Glyoxylate cycle occurs in glyoxysomes, which are specialized peroxisomes. There are no decarboxylation reactions in the glyoxylate cycle. Glyoxylate cycle allows cells to utilize 2 carbon units of acetate, and convert them into 4 carbon units, succinate, for energy production and biosynthesis Additionally, each turn of the cycle produces a molecule of FADH2 and NADH
- 25. Function in plants Seeds cannot carry out photosynthesis as they lack chloroplasts. However, seeds have specific peroxisomes known as glyoxysomes, where the glyoxylate cycle can occur. Glyoxylate cycle occurs in seeds during germination so that: Lipids stored in seeds can be used as an energy source for the formation of carbohydrates for the growth and development of the shoot. Acetate is converted to acetyl - CoA, which in turn is: Utilized as a source of carbon and energy Used to produce NADPH, which drives ATP synthesis in the electron transport chain
- 26. Reactions, Yield, and Energy Balance Plants, fungi and bacteria require carbohydrates for energy and cell wall synthesis (e.g., cellulose, chitin, and glycans). The glyoxylate cycle enables organisms to producecarbohydrates using acetyl - CoA from the β-oxidation of fatty acids. Reactions The pathway begins with 2 molecules of acetyl – CoA Citratesynthase converts 1 of the acetyl - CoA molecules to citrate. Citrate is converted to isocitrate by the enzyme aconitase. Isocitrate is converted to glyoxylate and succinate. Succinate is converted to fumarate by succinate dehydrogenase. The next step involves the formation of 2 molecules of malate: 1 molecule of malate is formed by the combination of acetyl - CoA and glyoxylate. The 2nd molecule is formed by the conversion of fumarate to malate in the presence of fumarase. Malate dehydrogenase converts 2 malate molecules into 2 oxaloacetate molecules. 1 molecule of oxaloacetate is converted to citrate, and 1 molecule of oxaloacetate is used for gluconeogenesis.