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MCB 3020, Spring 2005 Chapter 15: Microorganisms in the Environment 98 1 98 2 Today: I. Microbial impact on environment II. Photosynthesis III. Methanogenesis IV. Nitrogen fixation I. Microbial Impact on the 98 Environment 3 Some examples: Photosynthesis Biodegradation wastewater treatment landfill and toxic waste degradation Methane production: sewage treatment, landfills; cow rumen; greenhouse gas Nitrogen fixation (N2 --> NH3) Nitrification, denitrification 98 Interaction of organisms on earth Solar energy (ultimate source of energy) 4 Primary producers (plants, photosynthetic microbes) Decomposers (nonphotosynthetic bacteria, fungi) Consumers (herbivores, carnivores) II. Photosynthesis 98 5 The synthesis of chemical compounds like glucose using energy from light. hv 6 CO2 + 6 H2O C6H12O6 + 6 O2 98 6 A. Overview of photosynthesis • occurs in plants, algae (eukaryotic), and cyanobacteria (prokaryotic) • makes organic carbon (also called reduced carbon or “fixed” carbon) • makes ATP and NAD(P)H (reductant) to synthesize organic carbon Two sets of reactions are involved in photosynthesis 98 7 • Light reactions: light energy is converted to chemical energy in the form of ATP and reductant [NAD(P)H] • Dark reactions (light-independent): chemical energy is used to reduce CO2, usually to the level of a sugar Light reactions generate ATP and NADPH. Dark reactions use ATP and NADPH to reduce CO2 to carbohydrate (glucose). 98 8 Oxygenic photosynthesis 6 H2O + hv Light reactions 6 O2 12 H+ + 12 e- ATP, reductant 6 CO2 Dark reactions C6H12O6 B. Light reactions of photosynthesis How do plants and microbes capture the energy of light? alga cyanobacterium Using pigments like chlorophyll 98 9 Chlorophyll 99 0 The main pigment for harvesting light energy by photosynthesis Located in photosynthetic membranes 99 1 Chlorophyll R N N Mg N N N pyrrole R R O porphyrin or “magnesium tetrapyrrole” [cf. cytochromes (Fe), vitamin B12 (Co)] TB Arrangement of chlorophyll in membranes 99 2 200-300 light harvesting chlorophyll molecules reaction center (RC) photosynthetic membrane TB Anoxygenic photosynthesis • does not produce O2 • “purple” and “green” bacteria Oxygenic Photosynthesis hv 6 CO2 + 6 H2O C6H12O6 + 6 O2 • cyanobacteria (prokaryotic) • photosynthetic algae (eukaryotic) • plants (eukaryotic) 99 3 C. Anoxygenic photosynthesis 1. overview 2. components 3. electron flow 4. membrane arrangement and ATP synthesis 99 4 TB 1. Overview (anoxygenic PHS) Used by purple and green bacteria Light 99 5 PMF + ATP + reductant Photophosphorylation use of light energy to make proton gradient for ATP synthesisTB 2. Components Reaction center Chlorophyll Bacteriopheophytin (Bph) Quinones (Q) 99 6 Quinone pool Cytochromes (Cyt) TB 3. Electron flow in anoxygenic PHS Midpoint potential -1.0V P870* light energy Bph QA QB reaction center (RC) NAD(P)+ Q pool cyt. bc1 +0.5V 99 7 P870 cyt. c2 NAD(P)H H2S, SO TB Sometimes reductant (i.e.NAD(P)H) is made using some of the electron carriers of anoxygenic photosynthesis. In this case, electrons must be supplied by an outside source like H2S (but not water!) 99 8 TB 99 9 4. Membrane arrangement + H c2 LH TB RC c2 QQ QQ Bph Q H+ H+ bc1 ATP H+ Pi ADP How is ATP made by photophosphorylation? • proton motive force (PMF) for ATP synthesis is generated when electrons are transferred from the Q pool to cytbf. • ATP is made when PMF is dissipated using ATP synthase. 10 00 D. Oxygenic photosynthesis 1. overview 2. components 3. electron flow 4. photophosphorylation 10 01 TB 1. Oxygenic photosynthesis: 10 overview 02 Algae, cyanobacteria, higher plants Light + H2 O + + NAD(P) TB ATP + PMF + NAD(P)H + O2 2. Components Photosystem II (P680) Photosystem I (P700) Plastocyanin (PC) Quinone pool Cytochromes (Cyt) 10 03 TB 3. Electron flow in oxygenic PHS P680* Ph P680 +1V FeS Fd cyclic QA QB electron flow Q pool Cyt bf 10 04 -1V P700* PC 2e- + 2 H+ + 1/2 O2 NADP+ NADPH P700 H2O water-splitting reaction TB 4. Photophosphorylation use of light energy to make proton gradient for ATP synthesis 10 05 Note: The membrane organization and ATP synthesis are generally similar to anoxygenic photosynthesis 10 photophosphorylation 06 In eukaryotes, occurs in the chloroplast ADP + Pi H+ H+ H+ ATP stroma thylakoid E. Dark reactions of photosynthesis (light-independent reactions) CO2 reduction (CO2 fixation) to form organic matter uses ATP and NADPH made in light reactions to reduce CO2 Dark reactions can occur in the light, but do not require light. 10 07 Autotrophs Organisms that use CO2 as their primary carbon source. Many are primary producers in ecosystems. 10 08 Calvin cycle reductive pentose phosphate pathway uses ATP and NADPH to fix CO2 10 09 6 CO2 + 12 NADPH + 18 ATP + fructose 6-P + 12 NADP + 18 ADP + 17 Pi TB 10 10 Key enzyme of the Calvin cycle: Ribulose bisphosphate carboxylase (RubisCo) first enzyme in the Calvin cycle CO2 + ribulose bisphosphate two 3-phosphoglyceric acids TB Subsequent reactions (after 10 RubisCo):11 In a series of reactions requiring ATP, NADPH, and molecular rearrangements, fructose 6-phosphate is produced from phosphoglyceric acid ultimately, glucose can be made TB 10 12 Photosynthesis review • energy from sunlight • chlorophyll (captures light energy) • ATP made by photophosphorylation • NADPH • CO2 reduced to carbohydrates via RubisCo and Calvin cycle Result: sugar from light, water, air III. Methanogenic Archaea 10 13 a diverse group of strict anaerobes that produce methane as a catabolic end-product. CH4 A. Methanogenic ecosystems wastewater treatment facilities, landfills, sediments, rumen, digestive tracts, anaerobic microenvironments B. Methanogenic growth substrates H2 + CO2 formate, methanol, methylamines, acetate 10 14 The anaerobic food chain (landfill, rumen, etc.)10 Polymers 15 (polysaccharides, lipids, proteins) polymer degrading microbes Monomers (sugars, fatty acids, amino acids) fermentation by microbes acetate H2 + CO2 methanogens CH4+ CO2 CH4 TB C. The unusual coenzymes of methanogenesis. 1. Methanofuran R OCH2 CH2NH2 O a formyl group carrier 10 16 10 17 2. Methanopterin O HN NH2 N H N N H R HN CH CH3 CH3 C1 carrier functionally analogous to folate Carriers C1 groups at several oxidation states 3. Factor F430 10 18 Ni a nickel tetrapyrrol Methyl carrier 10 19 4. Factor F420 R HO N N O NH O a 5-deazaflavin functions as an electron carrier 5. Coenzyme B = = O CH3 O HOPOCHCHNHCCH2CH2CH2CH2CH2CH2SH HO COOH an electron carrier with an active sulfhydryl group 10 20 6. Coenzyme M HS-CH2-CH2 SO3– a methyl carrier CH3-S-CH2-CH2 SO3– methyl-CoM 10 21 D. The pathway of methanogenesis from CO2 10 H2 H2 F420 CO2 22 MF-CHO MF = methanofuran MP-CHO MP = methanopterin MP-CH2OH H2 F420 CoB-SH MP-CH3 CoM-CH3 CoB-SS-CoM + CH4 H2 CoB-SH + HS-CoM 10 23 IV. 10 Nitrogen fixation 24 • Use of nitrogen gas (N2) as a nitrogen source. • Occurs in prokaryotes only • Some prokaryotes enter into symbiotic relationships with leguminous plants TB A. Nitrogenase 10 25 Enzyme that catalyzes the reduction of N2 to NH3. Fe protein MoFe protein 1. Overall reaction of nitrogenase 10 26 N2 + 8H+ + 8e- +16 ATP Nitrogenase 2NH3 + H2 + 16 ADP +16 Pi The formation of H2 is a by-reaction TB The microbial nitrogen cycle nitrogen fixation 10 27 N2 denitrification NO3 NH3 nitrification - Study objectives 10 28 1. Know the details of photosynthesis and be able to compare and contrast oxygenic and anoxygenic photosynthesis. 2. Compare and contrast photophosphorylation, oxidative phosphorylation, and substrate level phosphorylation. How is the proton motive force made? Where does photosynthesis occur in eukaryotes? 3. What is the role of water in oxygenic photosynthesis? Does water play the same role in anoxygenic photosynthesis? 4. Define autotroph. What is the purpose of the Calvin cycle? What types of organisms use this cycle? Know the reaction catalyzed by Rubisco. How is glucose made in the dark reactions of photosynthesis? 5. Be able to describe how a photosynthetic cell makes sugar from air, water, and light. What is the purpose of the ATP and NADPH? How are they made? How are they used in the production of sugars from CO2? 6. What are methanogenic Archaea? Where are they found? What are the substrates for methanogenesis? 7. Understand the role of methanogens in the anaerobic food chains of rumen, landfills, wastewater treatment facilities, and other anaerobic ecosystems. 8. 9. 10 Name the unusual coenzymes of methanogenesis and their general functions. 29 Define nitrogen fixation. What organisms are capable of nitrogen fixation? What is the reaction of nitrogenase? Note that it requires reductant and ATP. 10. Distinguish between nitrification and denitrification. (See last slide.) MCB 3020 Spring 2004 Chapter 11: Industrial and Environmental Microbiology 10 30 10 Industrial and Environmental Microbiology31 I. Industrial production of antibiotics II. Other microbial products III. Biodegradation A. Wastewater treatment B. Landfills C. Bioremediation I. Antibiotic production A. Genera known for production B. Discovery C. Production 10 32 A. Genera known for production 1. Streptomyces 2. Penicillium 3. Bacillus 10 33 10 B. Discovery 34 1. spread petri dish with soil dilution 2. overlay with indicator organism 3. incubate bacterial colonies zones of inhibition 4. isolate the organism 5. purify the antibiotic 6. eliminate known antibiotics 7. assign structure 8. improve yield 9. improve purification 10. animal testing 11. clinical trials 10 35 C. Production of antibiotics 1. Penicillin a. natural penicillin b. semi-synthetic c. biosynthetic 10 36 a. natural penicillin grow cells in large fermentor remove cells extract antibiotic crystallize 10 37 10 b. semi-synthetic penicillin 38 Remove R-group and add new side-chains by chemical synthesis. R H N S CH3 H O N natural penicillin R = H CH3 COOCH2-CO- c. biosynthetic penicillins Add excess R-group precursor to the fermentor. 10 39 10 40 2. Streptomycin production A-factor is added to the fermentor A-factor is an inducer of streptomycin biosynthetic genes OH O O O CH3 CH3 3. Tetracycline production 10 41 Avoid glucose in the growth medium Use low phosphate growth medium II. Other microbial products A. Vitamins B. Amino acids C. Cortisone D. Enzymes E. Vinegar F. Citric acid 10 42 G. Yeast H. Beer and Wine I. Distilled beverages J. Commodity ethanol K. Food A. Vitamins 1. Vitamin B12 2. Riboflavin 10 43 B. Amino acids glutamate aspartate phenylalanine lysine 10 44 C. Cortisone (steroid) 10 45 Produced by bioconversion Bioconversion: the use of microbes to catalyze specific chemical reactions 10 46 CH3 C O progesterone O bioconversion HO O CH3 C O hydroxy-progesterone chemical synthesis cortisone D. Enzymes 1. Proteases laundry detergents 2. Glucose isomerase fructose production 10 47 extremozymes 10 48 enzymes resistant to extreme conditions extremophiles organisms that grow in extreme environments extremophiles are the source of extremozymes 10 49 E. Vinegar Produced mainly from wine and cider Acetic acid bacteria ethanol acetaldehyde acetic acid (vinegar) F. Citric acid 10 50 Used to acidify foods and add tartness especially soft drinks Produced by Aspergillus niger (fungus) A. niger uses citrate to obtain iron from low-iron environments A. niger TCA 3+ Fe citrate citrate citrate 10 51 Fe3+ citrate 3+ Fe chelation (strong noncovalent binding) iron limitation increases citrate production G. Yeast Saccharomyces cerevisiae Grow aerobically Collect cells Baker's yeast Nutritional yeast 10 52 H. Beer and wine Saccharomyces spp. anaerobic growth 10 53 Wine fermented grapes Beer fermented malt (made from germinated barley) I. Distilled alcoholic beverages distillation Fermented malt Wine Fermented molasses Fermented potatoes Fermented grain and juniper berries 10 54 Whiskey Brandy Rum Vodka Gin J. Commodity ethanol Solvent Gasohol 90% gasoline 10% ethanol 10 55 K. Food from microorganisms 1. single-celled organisms yeast for protein 2. mushrooms fungal fruiting bodies 10 56 Microbial Impact on the Environment 10 57 Some examples: Biodegradation wastewater treatment landfill and toxic waste degradation Methane production: sewage treatment, landfill in cows; greenhouse gas Photosynthesis Nitrogen fixation (N2 --> NH3) Nitrification, denitrification III. Biodegradation biological degradation of wastes or pollutants A. Wastewater treatment B. Landfills C. Bioremediation 10 58 10 A. Waste water treatment 59 1. Treatment stages 2. Details of secondary treatment 3. Overview of treatment TB 1. Treatment stages Primary treatment removal of sediment and debris Secondary treatment removes organic matter 10 60 Tertiary treatment removes inorganic compounds TB 2. Details of secondary treatment a. anaerobic sludge digestor 10 61 b. aerobic trickling filter activated sludge treatment aerobic sludge digestor TB 10 62 a. Anaerobic wastewater anaerobic sludge digestor (closed tank) cells + CH4 + CO2 recalcitrant solids TB Inside the anaerobic digestor Polymers 10 63 (polysaccharides, lipids, proteins) polymer degrading microbes Monomers (sugars, fatty acids, amino acids) fermentation by microbes acetate H2 + CO2 methanogens CH4+ CO2 CH4 TB b. Aerobic secondary treatment i. Trickling filter wastewater 10 64 open tank containing crushed rocks cells + CO2 recalcitrant solids TB Inside the trickling filter 10 65 Microbes attached to rocks grow by consuming the organic matter in the wastewater. "biological solids" are shed from the rocks TB ii. activated sludge treatment wastewater activated sludge (flocs) 10 66 open, aerated, tank flocs wastewater is held for a short time TB Aerobic sludge digestor 10 67 Place flocs in an aerobic tank for a longer time period. TB 10 organic 68 Flocs consist of microbes and matter Organic matter and microbes in the wastewater bind to the flocs. About 10% of flocs is Zoogloea ramigera Zoogloea produces a slime that is the glue of the flocs. TB 10 3. Overview 69 activated sludge, or trickling filter solids anaerobic or aerobic sludge digestor drying, composting, pasteurization, irradiation spread on land, add to landfill, dump in ocean,or incinerate TB B. Landfills: anaerobic and aerobic 10 70 biodegradation Polymers (polysaccharides, lipids, proteins) polymer degrading microbes Monomers aerobic (sugars, fatty acids, amino acids) degradation CO2 fermentation by microbes organic acids; acetate H2 + CO2 anaerobic methanogens aerobic degradation CH4+ CO2 CH4 degradation C. Bioremediation 10 71 use of microorganisms to enhance the removal or detoxification of unwanted chemicals in the environment e.g. petroleum spills chlorinated solvents pesticides heavy metals 1. Some strategies for enhancing biodegradation in nature: 10 72 a. identify organisms that naturally degrade pollutants b. add whatever nutrient is the "limiting factor" for biodegradation c. genetically engineer better organisms (?) 10 73 2. Example: bioremediation of chlorinated solvents Cl Cl C=C Cl What organisms degrade TCE? H Trichloroethylene (TCE) sample • chlorinated solvent • common contaminant in drinking water • suspected carcinogen ? Trichloroethylene can be degraded aerobically or anaerobically. Cl aerobic degradation (e.g. ammoniaoxidizing bacteria) CO2, 3 Cl-, Cl O2 H 2O 10 74 Cl C=C H O2 3 anaerobic degradation (methanogens) Cl , H2C=CH2 In contrast, tetrachloroethylene (PCE) can be degraded ONLY by anaerobic organisms. Cl Cl C=C How might you enhance the biodegradation of TCE and PCE in polluted environments? 10 75 Cl Cl O2 4 anaerobic degradation (methanogens) Cl , H2C=CH2