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Nitrogen

Chlorophyll a, mg m‐3 0.01 0.1 1 10 species in nature Oxidation state ‐ 5nitrate, NO3

4nitrogen dioxide, NO2; N2O4 ‐ 3nitrite, NO2 2nitricoxide, NO

1 , N2O

0nitrogen, N2

‐1 hydroxylamine, NH2OH

‐2hydrazine, N2H4 + + ‐3 , NH4 , amines, R‐NH3 etc.

Lam & Kuypers, Ann. Rev. Mar. Sci. 2011 The classical , 1934

Organic N

Industrial and microbial N fixation Aerobic (ammonium oxidation and oxidation) Anaerobic reduction to ammonium

L.G.M. Baas Becking: Geobiologie (1934) Biogeochemical significance of nitrogen

• Nitrogen is a nutrient –

e.g., ≈ C106H175O42N16P… => /mineralization cycle

• Nitrogen is used in microbial energy : ‐ ‐ NO3 is e acceptor for anaerobes, e.g. denitrification + ‐ NH4 is e donor for , e.g. nitrification => cycle

• N2O is a and 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% * 7% nucleic acids 16.1% lipid X% amino sugars 4% nucleic acids w. 16.6% N

* 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

–NOx formations from – atmospheric NOx transformations – Haber‐Bosch process –chemo‐denitrification

• Largely restricted to and –rock N important in some terrestrial Fixed vs. free nitrogen (”water, water, every where, nor any drop to drink”)

Nitrogen in the atmosphere 3.9 x 1021 g

Microbial NN 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 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

+ − 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

Irreversible inhibition by O2

Seefeldt et al. 2009, Ann. Rev. Biochem. Large diversity of

Carpenter and Capone 2008, in Nitrogen Cycling in the Marine Environment Symbiotic N2 fixation in root nodules of

Root of soy bean with nodules in vesicles

N fixation in soils is predominantly (75 – 95%) symbiotic N‐fixing

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

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

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 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

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 ” 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 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 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 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