Nitrogen biogeochemistry Chlorophyll a, mg m‐3 0.01 0.1 1 10 Nitrogen species in nature Oxidation state ‐ 5nitrate, NO3 4nitrogen dioxide, NO2; N2O4 ‐ 3nitrite, NO2 2nitricoxide, NO 1 nitrous oxide, N2O 0nitrogen, N2 ‐1 hydroxylamine, NH2OH ‐2hydrazine, N2H4 + + ‐3 ammonium, NH4 , amines, R‐NH3 etc. Lam & Kuypers, Ann. Rev. Mar. Sci. 2011 The classical nitrogen cycle, 1934 Organic N Industrial and microbial N fixation Aerobic nitrification (ammonium oxidation and nitrite oxidation) Anaerobic Nitrate reduction denitrification to ammonium L.G.M. Baas Becking: Geobiologie (1934) Biogeochemical significance of nitrogen • Nitrogen is a nutrient – e.g., phytoplankton ≈ C106H175O42N16P… => assimilation/mineralization cycle • Nitrogen is used in microbial energy metabolism: ‐ ‐ NO3 is e acceptor for anaerobes, e.g. denitrification + ‐ NH4 is e donor for lithotrophs, e.g. nitrification => redox cycle • N2O is a greenhouse gas and ozone destroyer ‐ ‐ • NO3 /NO2 buffer H2S and, possibly, CH4 Nitrogen is essential for all life Trees: ~0.1% N Herbs: ~1% N Biomass N, e.g. in phytoplankton: 93% protein 7% nucleic acids Animals: ~10% N Phytoplankton: 5‐10% N Nitrogen in biomass Phytoplankton biomass Biomass N: 54.4% protein w. 16.3% N 93% protein 25% carbohydrate* 7% nucleic acids 16.1% lipid X% amino sugars 4% nucleic acids w. 16.6% N * bacteria have amino sugars (N‐acetyl‐glucose/galactose amine) in cell walls, crustaceans have amino sugar (chitin) in exoskeleton. Mineralization of C and N Anaerobic mineralization in closed 2008 incubation of sediment: Dalsgaard and Thamdrup Models of (aerobic) mineralization with microbial growth: al.2005 Canfield et Time (days) Time (days) Biogeochemical significance of nitrogen • Nitrogen is a nutrient – e.g., phytoplankton ≈ C106H175O42N16P… => assimilation/mineralization cycle • Nitrogen is used in microbial energy metabolism: ‐ ‐ NO3 is e acceptor for anaerobes, e.g. denitrification + ‐ NH4 is e donor for lithotrophs, e.g. nitrification => redox cycle • N2O is a greenhouse gas and ozone destroyer ‐ ‐ • NO3 /NO2 buffer H2S and, possibly, CH4 The nitrogen cycle is • Largely (micro‐)biological –exceptions: – atmospheric N fixation by lightning –NOx formations from combustion – atmospheric NOx transformations – Haber‐Bosch process –chemo‐denitrification • Largely restricted to biosphere and atmosphere –rock N important in some terrestrial ecosystems Fixed vs. free nitrogen (”water, water, every where, nor any drop to drink”) Nitrogen in the atmosphere 3.9 x 1021 g Microbial NN N fixation Microbial + ‐ NH4 , NO3 , Norg, etc. denitrification To help protect your privacy, PowerPoint has blocked automatic download o… Bioavailable / reactive / fixed N: 1.4 x 1018 g Redox chemistry N2 is unstable in oxic environments: 1 ‐ NO3 ‐ + N2 + 2.5O2 + H2O => 2NO3 + 2H ∆G0’ = –65 kJ/mol! 0.5 V , 0 N2 E 5 10 and in reduced sediments: 0 + N2 + 1.5CH2O + 3H2O + 0.5H => + ‐ 2NH4 + 1.5HCO3 ‐0.5 + NH4 ∆G0’ = –78 kJ/mol! pH Oceanic reactive nitrogen budget 160 Turn‐over time of oceanic nitrate 80 w.r.t. denitrification ~2000 y 0 1 ‐ y N input input Burial ‐80 Tg Diazotrophy Riverine denitrification ‐160 denitrification Atmospheric ‐240 Pelagic Benthic ‐320 Middelburg et al. (1996), Gruber and Sarmiento 2002, Brandes and Devol (2002), Codispoti et al. (2007) It all starts with N2 OXIC fixation N2 oxidation + NH4 Norg N2 fixation + NH4 N2 reduction ANOXIC oxidation state: ‐3 ‐2 ‐1 0 1 2 3 5 Microbial N fixation by nitrogenase + − N2 + 8H + 16MgATP + 8e → 2NH3 + H2 + 16MgADP + 16Pi Metal requirements: 4 Fe in Fe protein 30 Fe, 2 Mo in MoFe protein Synthesis involves ≈ 20 enzymes Irreversible inhibition by O2 Seefeldt et al. 2009, Ann. Rev. Biochem. Large diversity of diazotrophs Carpenter and Capone 2008, in Nitrogen Cycling in the Marine Environment Symbiotic N2 fixation in root nodules of legumes Root of soy bean with nodules Rhizobium in vesicles N fixation in soils is predominantly (75 – 95%) symbiotic N‐fixing cyanobacteria symbiotic Anabaena in water fern, Azolla toxic Nodularia N‐fixing cyanobacteria in the ocean Trichodesmium sp. mpi‐bremen.de ”Candidatus Atelocyanobacterium thalassa” a.k.a UCYN‐A inside Braarudosphaera sp. haptophyte ”Sawdust of the sea” Richelia sp. inside Hemiaulus sp. diatom www.whoi.edu Where is N2 fixed? Oligotrophic lake ≈0 Plankton Mesotrophic lake Benthos Eutrophic lake Rice paddy Saltmarsh Mangrove Seagrass bed Estuaries 2005 ≈90% of marine N fixation is pelagic N. Atlantic/N. Pacific al. ≈0 Open ocean Trichodesmium ≈0 0,1 1 10 100 1000 10000 ‐2 ‐1 N2 fixed, µmol m d after Canfield et High N2 fixation in dark sediments? ‐2 ‐1 N2 flux, Narragansett Bay, µmol m h Fulweiler and Heiss 2014, Oceanography Experimental quantification of N2 fixation Acetylene reduction assay: rate 15 Gross N2 incorporation 15 15 - 15 N N + 6e => 2R- NH2 rate Direct N2 flux measurements (mostly via N2/Ar) Net Where is N2 fixed? – natural terrestrial systems: µmol N m‐2 d‐1 180 135 Cleveland 90 et al. 2001 45 High evapotranspiration => high N2 fixation 0 rates corrected x 0.25 according to Vitousek et al. 2013 leguminous crops: 250 ‐ 5000 µmol N m‐2 y‐1 (Peoples et al. 1995) N fixation by the Haber‐Bosch process ≈ 2% of the world’s energy consumption Sutton et al. 2013 Global fixed nitrogen budget Anthropogenic N fixation Canfield et al. 2010 Nitrogen and marine eutrophication Global terrestrial fixed N budget DIN input vs. PP in estuaries 12 and coastal ecosystems Denitrification ? Denitrification Seitzinger and Harrison 2008 Nixon et al. 1996, Paerl and Piehler 2008 Atmospheric N deposition Galloway et al. 2008 Next step: N2 OXIC fixation N2 N2O oxidation Assimilation Ammonium Nitrite oxidation oxidation + NH OH ‐ ‐ NH4 2 NO2 NO3 Norg N2 fixation + NH4 N2 reduction ANOXIC oxidation state: ‐3 ‐2 ‐1 0 1 2 3 5 Nitrification 1) Ammonium oxidation 2) Nitrite oxidation + ‐ + ‐ ‐ NH4 + 1.5O2 NO2 + H2O + 2H NO2 + 0.5O2 NO3 ∆G0’ = ‐275 kJ mol‐1 ∆G0’ = ‐74 kJ mol‐1 Nitroso‐monas, ‐spira, etc. Nitro‐bacter, ‐spira, etc. Km(O2) ~ 10 µM Km(O2) ~ 50 µM • Obligately aerobic processes • Performed by specialized, autotrophic organisms • Efficiency of energy conservation < 10% => large turn‐over with small biomass • Ammonium oxidation generally rate limiting except at low PO2 • Results in acidification and leaching of nitrogen from soils • Releases N2O, particularly at low O2 levels Ammonium oxidation + ‐ + NH4 + 1.5O2 NO2 + H2O + 2H Nitrosomonas sp. + Nitrosopumilus sp. NH4 ‐oxidizing bacterium + NH4 ‐oxidizing thaumarchaeon –discovered~10 years ago + –Km(NH4 ) 2 µM –knownfor > 100 years + + – dominates in NH4 ‐poor systems –Km(NH4 ) 100 µM –7 mM + (e.g., unfertilized soil, open ocean) – dominates in NH4 ‐rich systems (e.g., wastewater treatment) Close coupling of ammonium and nitrite oxidation in a riverine sediment nmol cm-2 h-1 ‐ ‐ ‐1 NO3 , NO2 (µmol l ) Meyer et al. 2005, Appl. Environ. Microbiol. + High NH4 affinity – but slow growth. Nitropira inopiata dominates in, e.g., ground water sand filters Kitts et al. (2015) Anaerobic nitrification? + + ‐ NH4 N2 NH4 NO2 400 0 1 2468 ‐ ) + 4 200 NH ‐100 2468 (mol 0 kJ ‐200 G, ∆ ‐ ‐200 ‐300 NO2 N2 pH pH ‐ ”Feammox”: Feammox to NO2 is exergonic at pH>4! + + 2+ ‐ 6Fe(OH)3 + NH4 + 10H 6Fe + NO2 + 16H2O No robust evidence of either Fe(III) or + + 2+ Mn(IV)‐dependent NH4+ oxidation in 3Fe(OH)3 + NH4 + 5H 3Fe + N2 + 9H2O aquatic systems ‐ ‐ ‐ 2+ 2+ 2‐ [NO2 ], [HS ]: 1 µM; [NO3 ], [Mn ], [Fe ]: 10 µM; [SO4 ]: 10 mM, PN2: 1 atm Back to N2: N2 OXIC fixation N2 N2O oxidation Assimilation Ammonium Nitrite oxidation oxidation + NH OH ‐ ‐ NH4 2 NO2 NO3 Norg N2 fixation Denitrification + ‐ ‐ NH4 N2 N2O NO NO2 NO3 reduction ANOXIC oxidation state: ‐3 ‐2 ‐1 0 1 2 3 5 N2 OXIC fixation N2 N2O oxidation Assimilation Ammonium Nitrite oxidation oxidation + NH OH ‐ ‐ NH4 2 NO2 NO3 Norg N2 fixation Denitrification + ‐ ‐ NH4 N2 N2O NO NO2 NO3 N H NO 2 4 Anammox reduction ANOXIC DNRA oxidation state: ‐3 ‐2 ‐1 0 1 2 3 5 Denitrification ‐ ‐ + 5CH2O + 4NO3 2N2 +5 HCO3 + 2H2O + H • Many different facultative anaerobes ‐ • Four‐step reduction of NO3 : ‐ ‐ NO3 NO2 NO N2O N2 Nar/Nap Nir Nor Noz • Inhibited by O2 ≥ 2% air saturation • Electron donors organic C, H2, H2S or Fe2+ Denitrification as a modular process in permeable sediments Marchant et al. (2018) Environ. Microbiol. Coupled nitrification–denitrification in a riverine sediment nmol cm-2 h-1 ‐ ‐ ‐1 NO3 , NO2 (µmol l ) Coupled nitrification‐denitrification 10.8/13.5 = 80% of total Meyer et al. 2005, Appl. Environ. Microbiol. Relative contributions of N‐reducing pathways Skagerrak Aarhus Bay sediment sediment Denitrification Anammox Mae Klong DNRA OMZ Peru Estuary sed. water col. Thamdrup and Dalsgaard 2002, Lam et al. 2009, Dong et al. 2011 Anammox bacteria ”Candidatus Scalindua sp.” NO ‐ + NH + N + 2H O Hydrazine 2 4 2 2 ∆G°’ = ‐357 (N2H4) KJ/molCandidate genera: Kuenenia (1 species) Sludge, Brocadia (3 species) 2008 wastewater, al. soils, Anammoxosome Anammoxoglobus (1 species) freshwater Jettenia (1 species) Niftrik et Marine van Scalindua (4 species) environments div. freshwater habitats Ladderane Obligate anaerobes, anammoxidizers, lipids 0.2 µm and autotrophs Doubling time 1 –3 weeks! Active in freshwater and marine sediments, aquifers, sea ice, hydrothermal systems Relative importance of anammox in sediments Rivers/estuaries/marshes Nicholls & Trimmer, n=40 100 Freshwater Temperate coastal Arctic 80 Open ocean OMZ Peru Mn-rich production 2 60 Intact cores 40 2 20 9 % Ananmmox, % of N 0 0,1 1 10 100 1000 10000 Water depth, m Thamdrup (2012), amended Rates vs. depth Mean anammox contribution: 28% Trimmer & Engström 2010, in Nitrification Anammox in the global N cycle Total N2 production % anammox N2 from anammox Tg y‐1 Tg y‐1 Marine sediments 126 – 300 28 35 –84 Oxygen minimum zones 65 – 150 28 – 100 18 – 150 Marine total 191 – 450 28 –52 53 – 234 Soils 124 5? 6 Groundwater 44 25? 11 Rivers 35 10? 3.5 Lakes 30 15? 4.5 Land total 234 11? 26 Global total 425 – 684 19 –38 81 – 260 9 Global N2 inventory 3.9 x 10 Tg 6 Turn‐over time w.r.t.