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
1 ‐
d 2013 2 180 135 90 45 0
‐ 1 ‐ m al.
1995) y N
al. 2
‐ et
µmol m
Vitousek et
N
(Peoples according to µmol
fixation
2 0.25
5000 high N
‐ corrected x
rates 250
– natural terrestrial systems: fixed? 2 evapotranspiration =>
N
High leguminous crops:
Where is Cleveland et al. 2001 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. microbial N2 production 6 –9 x 10 years Isotope pairing in intact cores 15 ‐ NO3
15 ‐ 44 45 46 NO3 N2O N2O N2O 14 ‐ NO3 O2
28 29 30 N2 N2 N2 Denitrification: (1‐q)2 2(1‐q)q q2 q = [15NO ‐]/[NO ‐] Anammox: (1‐q) q 0 3 3
14 + NH4
Methods: Nielsen 1992, Trimmer et al. 2006 DNRA as nitrate/nitrite sink
DNRA, % of nitrite reduction 050100 Increased importance of DNRA at
Hythe GB Estuaries, Alresford GB Brightlingsea GB • High organic loading Etang du Prevost F Bassin d'Arcachon F • Sulfidic pore waters
Mae Klong TH lagoons, Cisadane ID Vundawa‐Rewa ID • Nitrate limitation Norsminde Fjord DK
E Matagorda Bay US • High temperature Nueces Estuary US 5 etc. Laguna Madre US Baffin Bay US
Fish farm DK 3000 – Potential causes: Horsens Fjord DK ‐ Irish Sea 6 • ∆G/mol NO3 : Denitrification > DNRA
Irish Sea 4 m ‐ Irish Sea 5 ∆G/mol e :DNRA > Denitrification
Celtic Sea 3 depth Skagerrak S9 • Low energetic efficiency of Celtic Sea 2 Celtic Sea 1 denitrification (Strohm et al. 2008) Peru OMZ 2 • Microbial specifics? Peru OMZ 4 Peru OMZ 7 Pelagic Chile OMZ 3 Chile OMZ 5 DNRA in sulfidic sediments
In situ measurements during oxygenation Effect of Beggiatoa in Tokyo Bay sediment experiment in Byfjorden, Sweden
Oxic After oxygenation Anoxic DEN+DNRA Before vs. oxygenation +B. ‐B. denitrification
%
De Brabandere et al. 2015 Sayama et al. 2005
Are high DNRA contributions accounted for by filamentous S bacteria in sulfidic sediments? DNRA linked to Fe2+ in Yarra Estuary
A
D B C E
Control ‐ ‐ NO3 added NO2 added Fe2+ added Denitrification DNRA
ABCDE ABCDE Fresh Marine Station Station Robertson et al. (2016) Limnol. Oceanogr. Fe2+‐DNRA stoichiometry and kinetics
‐ Fe:NO2 = 5.3
‐ Yarra Fe:NO3 = 10 Lake Almind
Robertson et al. 2016 Robertson & Thamdrup 2017
Fe2+ oxidation largely accounts for DNRA in a wide range of non‐sulfidic sediments • Organisms? 2+ ‐ • Most Fe oxidizing NO3 reducers in culture are denitrifiers! Summary
• The N cycle is not a cycle but a network of processes including assimilation, mineralization, and dissimilation • Microbial N cycling (N‐fixation vs. denitrification + anammox) determines the availability of fixed N and excerts important control on primary production on land and in the oceans • New pathways and players in the N cycle are still being discovered The microbial nitrogen cycle v. 2018.0 beta
N2 OXIC fixation N2 N2O oxidation
Assimilation
Ammonium Nitrite oxidation oxidation transport Intracellular + NH OH ‐ ‐ NH4 2 NO2 NO3
Methane denitrification Phototrophic Norg N2 nitrite oxidation fixation NO Denitrification + ‐ ‐ NH4 N2 N2O NO NO2 NO3
N H NO 2 4 Anammox
Anaerobic Mn(IV)/Fe(IIII) nitrification? reduction ANOXIC Dissimilatory nitrate reduction to ammonium, DNRA oxidation state: ‐3 ‐2 ‐1 0 1 2 3 5