Published on February 7, 2008
ESM 219: F07: ESM 219: F07 N cycle Nitrogen Cycling: Nitrogen Cycling N2 fixation 85% biological (60% terrestrial, 40% marine) N2 + 8H+ + 8e- 2 NH3 + H2 nitrogenase Organisms Free-living (bacteria, many types) Symbiotic, e.g. (many types) Rhizobia in legumes Frankia in trees Slide4: N2 fixation: pathway and stoichiometry. Azospirillum brasilense: a nitrogen fixing bacterium that lives in the soil rhizosphere (image = 7 microns): Azospirillum brasilense: a nitrogen fixing bacterium that lives in the soil rhizosphere (image = 7 microns) Azotobacter sp.: free living N2 fixer in soil (image width = 2 microns). Here we see an X-section of a cyst, the resting stage analogous to an endospore: Azotobacter sp.: free living N2 fixer in soil (image width = 2 microns). Here we see an X-section of a cyst, the resting stage analogous to an endospore Rhizobium trifolii (image width 2 microns) : Rhizobium trifolii (image width 2 microns) Note capsular material around this free living soil organism. Nitrogen fixation (continued): Nitrogen fixation (continued) Nodules –will have some in the lab next week……. A few slides from our text (following) that show the process of bacteroid formation and the genes involved Rhizobium trifolii on root tip(image width 12 microns): Rhizobium trifolii on root tip (image width 12 microns) --a clover symbiont Rhizobium trifolii (image width 8 microns)--microfibrils formed preceding root invasion: Rhizobium trifolii (image width 8 microns) --microfibrils formed preceding root invasion Rhizobial bacteroids in NeptuniaTEM Image: width = 4.7 microns: Rhizobial bacteroids in Neptunia TEM Image: width = 4.7 microns Slide15: The nod gene cluster on the Sym plasmid of Rhizobium leguminosarum biovar viciae (nodulates peas). Slide16: The acetylene reduction assay for quantifying rates of dinitrogen fixation. Nitrogen Cycling (cont.): Nitrogen Cycling (cont.) Immobilization = uptake of NH4+ Ammonification = mineralization of organic N to ammonia O || urease NH2-C-NH2 + H2O 2NH3 + CO2 Observations in immobilization / mineralization C/N < 20 net ammonia production (ammonification) C/N > 20 net immobilization (plants and microbes) C/N bacteria = 4 to 5 C/N fungi = 15 C/N biomass in soil = 8 predation results in ammonium release Nitrification: Nitrification = 2 steps, 2 populations Ammonia oxidation Nitrite oxidation Step 1 ammonia oxidation: : Step 1 ammonia oxidation: -271 kJ/mol NH3 NH3 + O2 + 2H+ + 2e- NH2OH + H2O ammonia monooxygenase (inhibited by acetylene) NH2OH + H2O NO2- + 5H+ + 4e- hydroxylamine oxidoreductase Overall, NH3 + 1.5 O2 NO2- + H+ + H2O Bacteria need CO2 and oxygen byproducts: NO, N2O (low O2), acidity (lowers pH) Nitrosococcus oceanus, Nitrosomonas, Nitrosolobus, Nitrosospira, strict autotrophs Relatively large populations Step 2 nitrite oxidation:: Step 2 nitrite oxidation: -77 kJ/mol NO2- NO2- + ½ O2 NO3- nitrite oxidoreductase blocked by ClO4- Nitrobacter, Nitrospira Smaller populations relative to ammonia oxidizers Bacteria can be autotrophic or heterotrophic can use organic C or CO2 Occurs under oxic or anoxic conditions O2 or other e- acceptors Nitrosomonas europaea: with complex internal membrane structure involved in ammonia oxidization: Nitrosomonas europaea: with complex internal membrane structure involved in ammonia oxidization Nitrosomonas sp. (3 micron width TEM) : an ammonia oxidizer (NH4+ to NO2-): Nitrosomonas sp. (3 micron width TEM) : an ammonia oxidizer (NH4+ to NO2-) Slide24: Oxidation of ammonia and electron flow in ammonia-oxidizing bacteria: focus on the respiratory chain. Q = ubiquinone Slide25: Oxidation of nitrite and electron flow in nitrite-oxidizing bacteria: focus on the respiratory chain. Slide26: Nitrifier growth kinetic expression: ammonium limiting mmax for nitrifiers: much lower than for aerobic heterotrophs 0.006 to 0.035 hr-1 compared to 0.18 to 0.38 hr-1 Slide27: Nitrifier growth kinetic expression: ammonium and oxygen limiting Slide29: Nitrifier growth kinetics: ammonium and oxygen limiting, temperature and pH dependencies Anammox: anoxic NH4+ oxidation: Anammox: anoxic NH4+ oxidation NH4+ + NO2- N2 + 2H20 nitrite from aerobic ammonia oxidizers e.g. Brocadia anamoxidans Autotrophic More recently discovered Dissimilatory Nitrate Reduction: Dissimilatory Nitrate Reduction Denitrification (dissimilatory nitrate reduction) 2NO3- + 5H2 + 2 H+ N2 + 6 H2O nitrate reductase Up to 5% of soil bacteria Most need organic C (e.g. methanol in waste treatment), some use CO2 DNAR (dissimilatory nitrate reduction to NH4+) NO3- + 4H2 + 2H+ NH4+ + 3H2O Sediments with lots of C Slide32: A denitrifier growth kinetic expression --based on nitrate and C-source (methanol, here) limiting. Assimilatory Nitrate Reduction: Assimilatory Nitrate Reduction Denitrification: also by assimilatory nitrate reduction Nitrate reduced to ammonium and incorporated into the cell immediately for biosynthesis Only that needed for biosynthesis is reduced Occurs widely in Bacteria, Archaea, Eukarya (fungi and higher plants) Similar concept to assimilation of SO42- and CO2 Denitrification “Potential’: Denitrification “Potential’ Potential for denitrification “potential” Estimates capacity for the process Determined by all influences on kinetics Catalyst population size (mainly) Catalysts (i.e. induced already) Substrate concentration Determined in lab by acetylene block C2H2 blocks conversion of N2O to N2, thus N2O accumulates in headspace N2O measured against a low background Establishing the right acetylene concentration.: Establishing the right acetylene concentration. Kaspar et al. AEM Slide36: Potential measurements versus in situ estimates. Kaspar et al. AEM Finding the kinetic parameters, vmax and Km: Finding the kinetic parameters, vmax and Km Kaspar et al. AEM Slide38: Denitrification pathway Nitrogen Oxides as Pollutants: Nitrogen Oxides as Pollutants NO = nitric oxide, NO2 = nitrogen dioxide, together = NOx Nitric oxide reacts with oxygen to made nitrogen dioxide 2NO(g) + O2(g) -----> 2NO2(g) Nitrogen dioxide reacts in sunlight to make nitric oxide and singlet oxygen sunlight NO2(g) ---------->NO(g)+O(g) With oxygen, this makes ozone: O + O2 -----> O3 Nitric oxide can remove ozone: NO(g) + O3(g) -----> NO2(g) + O2(g) Ozone generation happens at high levels of nitrogen dioxide relative to nitric oxide (ratio of 3). Slide40: Want to learn more? http://www.fao.org/documents/show_cdr.asp?url_file=/DOCREP/004/Y2780E/y2780e02.htm Hole in the Pipe Model REF: Davidson, E.A. 1991. Fluxes of Nitrous Oxide and Nitric Oxide From Terrestrial Ecosystems. In Rogers, J.E. and Whitman, W. B. (eds) Microbial Production and Consumption of Greenhouse Gases: Methane, Nitrogen Oxides and Halomethanes. ASM Press. 298 pp. ORIGINALLY from Firestone and Davidson, 1989. (see Davidson for complete Ref). From Matson et al. 1998: From Matson et al. 1998 Slide42: Ref: Matson et al. 1998 Science v280 p112.