Neglecting diurnal variations leads to uncertainties in terrestrial nitrous oxide emissions

1 Scien i ic RepoR s | 6:25739 | DOI: 10.1038/s ep25739 www.na u e.com/scien i ic epo s Neglec ing diu nal a ia ions leads o unce ain ies in e es ial ni ous oxide emissions Na asinha J. Shu pali1, Ülla Rannik2, Simo Jokinen1, Saa a Lind1, Ch is ina Biasi1, I an Mamma ella2, Olli Pel ola2, Ma i Pihla ie2,3, Niina Hy önen1, Ma i Rä y4, Sami Haapanala2, Ma k Zahnise 5, Pe u Vi kajä i4, Timo Vesala2,6,7 & Pe i J. Ma ikainen1 Ni ous oxide (N2O) is an impo an g eenhouse gas p oduced in soil and aqua ic ecosys ems. I s wa ming po en ial is 296 imes highe han ha o CO2. Mos N2O emission measu emen s made so a a e limi ed in empo al and spa ial esolu ion causing unce ain ies in he global N2O budge . Recen ad ances in lase spec oscopic echniques p o ide an excellen ool o a ea-in eg a ed, di ec and con inuous ield measu emen s o N2O luxes using he eddy co a iance me hod. By employing his echnique on an ag icul u al si e wi h ou lase -based analyse s, we show he e ha N2O exchange exhibi s con as ing diu nal beha iou depending upon soil ni ogen a ailabili y. When soil N was high due o e ilize applica ion, N2O emissions we e highe du ing day ime han du ing he nigh . Howe e , when soil N became limi ed, emissions we e highe du ing he nigh han du ing he day. These e e se diu nal pa e ns suppo ed by iso opic analyses may indica e a dominan ole o plan s on mic obial p ocesses associa ed wi h N2O exchange. This s udy highligh s he po en ial o new echnologies in imp o ing es ima es o global N2O sou ces. Ni ous oxide (N2O) is among he mos impo an g eenhouse gases (GHGs). I is p oduced bo h in na u al and managed soils, wi h ag icul u al soils being he la ges an h opogenic sou ce1,2. Despi e he ecen p og ess in quan i ying he di e se N2O sou ces, he ange o he global es ima e is s ill la ge a ying om 8.1–30.7 Tg N (N2O) a−1 (ac oss all na u al and an h opogenic sou ces) owing p ima ily o he s agge ing spa io empo al a - ia ion in he luxes3. Recen ly, a emp s ha e been made o p esen a comp ehensi e es ima e o he global N2O emissions using obse a ions, bo om-up and in e sion models4. Despi e he u iliza ion o all cu en ly a ailable global da a on N2O emissions om a ious sou ce sec o s, he o e a ching goal o educing he la ge unce ain y in global N2O budge s ill emains a o midable ask. Addi ionally, ou cu en p ocess le el unde s anding o he ac o s egula ing N2O emissions a a gi en si e is a om comple e5,6. As a gene al me hod, s a ic chambe s a e widely used o measu e soil N2O luxes expe imen ally7–11. Mos N2O exchange measu emen s a e made using manual chambe s leading o spa se empo al esolu ion in he da a. To o e come hese disc epancies, mic ome eo ological me hods (such as he eddy co a iance (EC) echnique) a e being inc easingly applied o con inuous measu emen s o N2O exchange12–14. This me hod o e s he ad an age o in eg a ing N2O luxes o e la ge a eas. Un il ecen ly, howe e , he lack o app op ia e gas analyse s wi h a su icien ime esponse and p ecision has been a majo limi a ion. Wi h he ecen de elopmen o lase -based spec oscopic echniques, as and accu a e measu emen s in conjunc ion wi h he EC o au oma ic chambe echniques a e now possible14–18 allowing accu a e s udies on he empo al a ia ion o N2O luxes. As a pa o he In eg a ed Ca bon Obse a ion Sys em (ICOS), a Eu opean in as uc u e p og am dedica ed o high p ecision moni o ing o g eenhouse gases, we conduc ed an EC based in e -compa ison ield campaign18 u ilizing ou di e en N2O analyze s. Ni ous oxide lux measu emen s we e made o e an ag icul u al ield si e 1Biogeochemis y esea ch g oup, Depa men o En i onmen al and Biological Sciences, Uni e si y o Eas e n Finland, Yliopis o an a 1D E, PO Box 1627, Kuopio campus, FI-70211 Finland. 2Depa men o Physics, P.O. Box 48, Uni e si y o Helsinki, 00014 Finland. 3Depa men o Food and En i onmen al Sciences, Di ision o Mic obiology and Bio echnology, P.O. Box 56, Uni e si y o Helsinki, FI-00014 Finland. 4Na u al Resou ces Ins i u e Finland, G een echnology, Halolan ie 31A Maaninka FI-71750, Finland. 5Ae odyne Resea ch, Inc., 45 Manning Road Bille ica, MA 01821-3976, USA. 6Viikki Plan Science Cen e, Uni e si y o Helsinki, P.O. Box 27, FI-00014 Finland. 7Depa men o Fo es Sciences, P. O. Box 27, Uni e si y o Helsinki, 00014 Finland. Co espondence and eques s o ma e ials should be add essed o N.J.S. (email: na asinha.shu pali@ue . i) ecei ed: 19 Ma ch 2015 Accep ed: 21 Ap il 2016 Published: 09 May 2016 OPEN www.na u e.com/scien i ic epo s/ 2 Scien i ic RepoR s | 6:25739 | DOI: 10.1038/s ep25739 (in eas e n Finland) cul i a ed wi h eed cana y g ass (RCG, Phala is a undinaceae, L.), a pe ennial c op, om Ap il un il No embe in 2011 (Supplemen a y In o ma ion). The soil mois u e and empe a u e du ing he pe iod (Supplemen a y Fig. S1) we e ypical o condi ions du - ing la e sp ing, summe and ea ly au umn in his ecosys em. The mean olume ic wa e con en in he soil p o ile (0–100 cm deep) du ing ea ly May was high (0.63 ± 0.1 cm3 cm−3) owing o he mel ing o snow since Ap il. Wi h soil p o ile empe a u es s eadily inc easing om 2.5 °C (wi h a s anda d de ia ion o 2.5 °C) in ea ly May o 11.6 °C ( ± 2.7 °C) by ea ly June, mois u e con en in all soil laye s showed a s eady decline. Soil mois u e con en emained s able a 0.25 cm3 cm−3 ( ± 0.04 cm3 cm−3) du ing June o mid-Sep embe . The luc ua ions in soil mois- u e con en we e less p onounced wi h soil dep h. Soil empe a u es peaked a 15.7 °C ( ± 1.6 °C) du ing he sec- ond week o July. Towa ds he end o he s udy pe iod, soil mois u e con en was high and soil empe a u es we e low. The O2 concen a ions in he i s 30 cm o he soil p o ile s ayed high (be ween 15 and 21%) h oughou he obse a ion pe iod (Supplemen a y Fig. S1). Howe e , O2 concen a ions we e low and a ied widely a deepe soil laye s indica ing oxygen limi a ions in hese laye s. Wa e illed po e space (WFPS) which egula es soil gas di usion a e, a ied be ween 60–100% du ing he ea ly pa , 20–60% du ing he middle pa o he season and emained abo e 60% du ing he la e pa o he s udy pe iod (Supplemen a y Fig. S1) wi h he su ace soil laye s gene ally exhibi ing highe a ia ion in WFPS h oughou he season. An emission pulse (up o 33 mg m−2 d−1) ha occu ed wi hin 24 hou s o he applica ion o an N-P-K-S e ilize con aining 76 kg N ha−1 cha ac e ized he seasonal pa e n o N2O exchange om he RCG cul i a ion sys em. This high N2O emission ac i i y las ed abou 15 days (days 143–158, e e ed o he ea e as he high emission pe iod, Fig.1A). Following his pe iod, emissions declined o backg ound le els o abou 1 mg m−2 d−1 (low emission pe iod). All ope a ional N2O gas analyze s showed a simila seasonal pa e n o N2O exchange. I is ele an he e o dwell on he seasonal plan g ow h pa e n in conjunc ion wi h he abo e-indica ed seasonal end in N2O exchange. Soil mois u e le els we e op imal and he daily p ecipi a ion was well dis ibu ed du ing Figu e 1. Seasonal dis ibu ion o (A) daily a e aged N2O emissions (mg m−2 d−1) as measu ed by 4 di e en lase based gas analyse s (ARC – con inuous wa e lase spec ome e , Ae odyne Resea ch Inc., Bille ica, MA, USA; TGA – ace gas analyse , Campbell Scien i ic Inc., Logan, UT, USA; LGR - con inuous wa e lase spec ome e , Los Ga os Resea ch Inc., Moun ain View, CA, USA; ARP – pulsed quan um cascade lase spec ome e , Ae odyne Resea ch Inc., Bille ica, MA, USA) (B) daily a e aged olume ic soil mois u e con en (m3 m−3) measu ed a 2.5 cm below he soil su ace and daily sum o p ecipi a ion (mm) and (C) daily sums o g oss ecosys em p oduc i i y (GEP, g C m−2 d−1) du ing he N2O ins umen in e -compa ison o ganised du ing Ap il – No embe 2011 a a mine al soil si e a Maaninka in eas e n Finland. The si e was cul i a ed wi h eed cana y g ass (RCG, Phala is a undinaceae, L.), a pe ennial bioene gy c op. www.na u e.com/scien i ic epo s/ 3 Scien i ic RepoR s | 6:25739 | DOI: 10.1038/s ep25739 he ea ly pa o he g owing season (Fig.1B). This esul ed in a sha p inc ease in he c op g ow h ( ep esen ed in Fig.1C as g oss ecosys em p oduc i i y, GEP) wi h GEP eaching i s peak abou h ee weeks ollowing he e ilize applica ion and he la ges N2O elease o he a mosphe e du ing he season. As he EC me hod o e s unique possibili ies o insigh s in o N2O luxes a high empo al esolu ion6, we ocused he e pa icula ly on diu nal a ia ion in N2O lux as measu ed by one o he analyze s (conside ed o be he bes pe o ming N2O analyze in an in e -compa ison s udy)18. Du ing he high lux pe iod shown in Fig.2A, N2O emissions exhibi ed a diu nal a ia ion, wi h he highes luxes obse ed du ing he day ime and he lowes a nigh ime on h ee consecu i e days. All analyze s ope a ional du ing his ime exhibi ed a simila pa e n. The day ime emissions we e 2.7–4.2 imes highe han hose measu ed du ing nigh (Fig.2A). A mean diu nal pa e n o he en i e high- lux pe iod (n = 16) is shown in Fig.2B. The subsequen , much longe low lux pe iod (day 161– day 273) e ealed some peculia i ies in he empo al N2O dynamics. A close look a he diu nal pa - e n o N2O emissions du ing his N de icien low lux pe iod indica ed ha N2O emissions we e, on an a e age, abou 50% highe du ing nigh han du ing he day (Fig.3A). This is in con as o he diu nal end obse ed du ing he high- lux pe iod (Fig.2B). Impo an ly, all analyze s ope a ional du ing he ime we e consis en in eco ding such a phenomenon. The seasonal (Ap il – No embe 2011) sum o N2O emissions amoun ed o 2.8 kg N2O ha−1 wi h emissions om he sho high lux pe iod accoun ing o abou 55% o he seasonal sum. As discussed abo e, he diu nal N2O exchange du ing and ou side he high lux pe iod we e in con as . To al emissions om he high lux pe iod calcula ed only om day ime measu ed luxes (and he eby neglec ing he diu nal a ia ions) we e 23% highe compa ed o he sum calcula ed by conside ing he diu nal pa e n. In con as o his o e es ima ion, he o al emissions du ing N de icien condi ions we e 16% lowe compa ed o he sum ob ained by accoun ing o he comple e diu nal pa e n o N2O exchange. We ca ied ou an independen p ocess s udy; a 15N labelling (15NH415NO3) ield campaign du ing mid-July in 2011 ( he low- lux pe iod) on plo s cul i a ed wi h a mix u e o imo hy (Phleum p a ense L.) and meadow escue (Fes uca p a ensis Huds.) and loca ed a he same s udy si e. Repea ed measu emen s o 15N a om % abundance o accumula ed N2O in he headspace o opaque and anspa en chambe s (simula ing nigh and day- ime condi- ions) we e consis en ly and signi ican ly di e en . While he 15N a om % alues o N2O om anspa en cham- be s emained abou cons an du ing he chambe closu e pe iod, hose om he opaque chambe s inc eased linea ly o e he 35 minu es o chambe closu e (Fig.4). The slope o he build-up o he hea ie iso ope (15N) in he da k chambe headspace was an o de o magni ude highe (0.001 a om % min−1) compa ed o ha in he anspa en chambe headspace (0.0001 a om % min−1). In e es ial ecosys ems, N2O is p oduced ia a wide ange o mic obiological N cycling p ocesses such as chemoli ho ophic NH4 + oxida ion19, deni i ica ion20,21, ni i ie deni i ica ion22, codeni i ica ion23, dissimi- la o y ni a e educ ion (DNRA)24, he e o ophic ni i ica ion25,26, chemodeni i ica ion and abio ic decompo- si ion o ammonium ni a e27. The ac o s ha egula e N cycling p ocesses o deni i ica ion and ni i ica ion in soils ha e been ca ego ized as p oximal and dis al con ols28. Fo deni i ying mic obes he key p oximal ac- o s di ec ly con olling he ac i i y a e O2 concen a ion, soil NO3− and C a ailabili y and empe a u e29,30. Fo Figu e 2. E olu ion o 30-minu e alues o (A) N2O exchange in μ g m−2 h −1 ( ed solid ci cles, he downwa d a ow indica es he day o e iliza ion wi h ammonium ni a e), (B) mean diu nal lux pa e n du ing he high lux pe iod ( ed solid ci cles; he black line is a endline i ed o he mean diu nal lux da a in μ g m−2 h −1 wi h he quad a ic leas squa es i ). Measu emen s we e made a a mine al soil si e loca ed in Maaninka in eas e n Finland du ing Ap il – No embe 2011. The si e was cul i a ed wi h eed cana y g ass (RCG, Phala is a undinaceae, L.), a pe ennial bioene gy c op. www.na u e.com/scien i ic epo s/ 4 Scien i ic RepoR s | 6:25739 | DOI: 10.1038/s ep25739 ni i ica ion, a ailabili y o O2 and NH4 + and empe a u e a e impo an p oximal con ols31. Dis al ac o s such as plan g ow h, managemen p ac ices, soil ex u e and wa e a ailabili y, egula e indi ec ly deni i ica ion/ ni i ica ion ac i i ies by a ec ing he p oximal con olling ac o s28–30. We obse ed he la ges N2O emissions a he s udy si e immedia ely a e he applica ion o an ino ganic N e ilize (Fig.1A). Du ing his high emission pe iod wi h he day ime peaks in N2O emissions, a ailabili y o mine al N (NH4 + and NO3−) was high (Supplemen a y Fig. S2). I is no ewo hy a his junc u e ha he pe - ennial bioene gy c op (RCG) om he p e ious season was le on he si e o o e win e . RCG possesses a la ge amoun o nons uc u al ca bohyd a e (NSC) ese es in i s oo s as a coping mechanism o o e come he ha sh o e win e ing en i onmen . The concen a ion o NSCs in he RCG oo s has been epo ed o be abou h ee imes highe han ha in he abo eg ound plan pa s32. The si e was e ilized wi hin a mon h a e he c op om he p e ious season was ha es ed. We ing e en s and he p esence o RCG li e le o e on he soil su ace a e c op ha es ing as well as high ac i i y o RCG oo s may ha e aided he de elopmen o anae obic mic osi es in he su ace laye s33. Lack o oxygen (Suppo ing Fig.2), high soil SOC and high a ailabili y o soil N may ha e igge ed enhanced a es o N2O p oduc ion h ough deni i ica ion. Figu e 3. Mean (day 206– day 280) diu nal pa e n o (A) N2O exchange in μ g m−2 h −1 ( ed solid ci cles) and pho osyn he ically ac i e adia ion (PAR in μ mol m−2 s−1, blue solid ci cles whe e solid black lines ep esen a e age N2O emission a es du ing di e en pe iods o he day, ligh g ey lines abo e and below he black line ep esen he con idence band wi h ± 1 s anda d e o o he mean), (B) g oss ecosys em p oduc i i y (GEP, mg CO2 m−2 h −1, g een solid ci cles) du ing he low lux pe iod (day 206–280) in 2011 g owing season a a mine al soil si e in Maaninka in eas e n Finland cul i a ed wi h a pe ennial c op, (C) Low lux pe iod N2O emissions (μ g N2O m−2 h −1) i ed as a linea unc ion o he concu en ly measu ed g oss ecosys em p oduc i i y (mg CO2 m−2 h −1). The solid line ep esen s he linea leas squa es i o he da a, alues o he slope, in e cep , and he coe icien o de e mina ion esul ing om his i a e shown in he panel. www.na u e.com/scien i ic epo s/ 5 Scien i ic RepoR s | 6:25739 | DOI: 10.1038/s ep25739 Unde condi ions o high mine al N a ailabili y, he a ia ion in empe a u e likely was among he key ac o o he diu nal a ia ion in he mic obial ac i i ies associa ed wi h N2O emissions. Diu nal a iabili y wi h a day ime peak in N2O luxes was epo ed o he i s ime in he la e 1970s34,35. The i s s udy36 ha speci ically examined easons o he diu nal a iabili y a ibu ed 90% o he obse ed diu nal a ia ion o changes in soil empe a u e and associa ed changes in N2O solubili y. Ano he s udy37 conside s ha he ole o he plan pho- osyn he ic ac i i y in he N2O lux diu nal a iabili y is caused by supplying C o soil mic oo ganisms, ia oo exuda ion, since days wi h la ge diu nal a iabili y in N2O luxes had high adia ion. This could lead also o lowe oxygen con en in he soil su ace du ing he day ime. Such a day-nigh pa e n in oxygen con en was no iced a ou s udy si e (Supplemen a y Fig. 2). A s udy conduc ed in 201038 obse ed diu nal a ia ion in N2O lux du ing a b ie pe iod o high N2O lux ollowing he cul i a ion o a na i e empe a e g assland soil. Based on iso opologue da a, he a o emen ioned s udy epo ed an inc ease in N2O de i ed om bac e ial deni i ica ion om mo ning o a e noon hou s. As indica ed abo e, he high lux pe iod ollowing he e ilize applica ion las ed abou 15 days. The ea e , he soil mine al N s a us was consis en ly low (Supplemen a y Fig. S2), while he RCG c op was ac i ely pho o- syn hesizing (Fig.3B). Once he e ec o he applied N subsides, soils gene ally emi N2O a small a es, o en desc ibed as he ‘backg ound’ emissions. Du ing his ‘backg ound’ emission phase las ing un il he end o he plan pho osyn he ic ac i i y, a diu nal pa e n wi h lowe day ime ne emissions compa ed o hose du ing he nigh ime was consis en ly e iden (Fig.3A). Such obse a ions wi h low ne emissions o e en ne soil N2O up ake du ing day ime ha e been epo ed p e iously39,40. Howe e , his is one o he ew s udies ha epo such con inuous eco ds o his phenomenon wi h such high p ecision in a pe ennial g assland ecosys em unde No dic condi ions. A numbe o s udies ha e epo ed a sha p inc ease in N2O concen a ion wi h dep h25,41. In hese s udies, N2O concen a ions ha e been as high as 20–30 imes he ambien concen a ion in he a mosphe e a he deepes subsoil sampling poin s, sugges ing N2O p oduc ion a e in subsoil is su icien o main ain a s eep N2O concen- a ion g adien h ough he soil p o ile. Based on he soil mois u e con en , O2 concen a ion and WFPS pa e ns a ou si e, i is in ui i e o assume ha he e is a con inuous N2O p oduc ion deepe in he soil p o ile h oughou he g owing season. Howe e , based on he da a we ha e, i is no clea how his deep N2O could con ibu e o he con as ing diu nal a ia ion in he N2O emissions wi hin he g owing season, since he diu nal a ia ions in all en i onmen al a iables such as soil oxygen and wa e con en , empe a u e, especially a deepe laye s, a e small (Supplemen a y Fig. 2). Du ing ac i e pho osyn hesis, plan oo s discha ge eadily a ailable C42. The oo exuda ion p ocess is epo ed o ollow diu nal hy hms, wi h exuda ion inc easing du ing ligh pe iods43. Such obse a ions sugges ha he ans e o C compounds om assimila ing lea es o oo s and soil mic obes occu s as , wi hin hou s, as has been shown o RCG by using 13CO2 pulse-chase labeling44, demons a ing he igh coupling o soil mic obial ac i i y o plan pho osyn hesis45. This ex a ca bon s imula es he ac i i y o he e o ophic mic obes and immo- biliza ion o ino ganic N. Addi ionally, soil a ailable N is aken up by he plan s du ing day ime46. This leads o a sho age o + NH4 ha limi s he ac i i y o ammonium oxidizing mic obes and hei N2O p oduc ion. Wi h mo e ca bon a ailable o he deni i ie s, enhanced a es o N2O educ ion o N2 a e suppo ed unde poo N a aila- bili y and lowe oxygen con en in he soil p o ile owing o inc eased he e o ophic and au o ophic espi a ion47. The e o e, i is logical o hink ha he amoun o N2O ha di uses om he deepe laye s du ing day ime is likely o be consumed by deni i ie s mo e e icien ly hus lowe ing he o e all N2O emissions. The ela i ely highe educ ion o N2O o N2 may help explain he nega i e ela ionship be ween he g oss ecosys em p oduc i i y and ne N2O emissions (Fig.3C). Figu e 4. Measu emen s o a om % abundance ollowing he addi ion o labelled 15N e ilize o imo hy and meadow escue g ass plo s loca ed on a mine al si e cul i a ed wi h a pe ennial bioene gy c op in eas e n Finland. The blue do s ep esen measu emen s made wi h a anspa en chambe and he ed do s wi h a da k chambe . www.na u e.com/scien i ic epo s/ 6 Scien i ic RepoR s | 6:25739 | DOI: 10.1038/s ep25739 The soil oxygen concen a ions in he su ace laye s exhibi ed diu nal a ia ions in he su ace laye s (0–20 cm dep hs) du ing May h ough Augus (Supplemen a y Fig. 2). This is he pe iod when he plan s a e pho osyn he i- cally mos ac i e. The a ia ions we e dampened du ing he mon h o Sep embe when he plan s we e unde go- ing senescence. Addi ionally, he e we e no consis en diu nal a ia ions in oxygen concen a ions deepe in he soil laye (80–100 cm) sugges ing ha hese deepe laye s a e decoupled wi h espec o he plan oo ac i i y in he uppe laye s. Ou assump ion ha he plan s ha e a ole o play in C and N dynamics inds addi ional suppo in he esul s om an independen 15N labelling campaign (Fig.4), men ioned abo e. A e adding labelled e i- lize o he soil, he label was acked in N2O o a g ea e ex en unde da k han unde ligh condi ions. While he ecosys em scale EC measu emen s ep esen ed N2O emissions om RCG, a pe ennial g ass species wi h ae en- chyma ous issue, he plo scale iso opic analyses we e pe o med on imo hy and meadow escue, pe ennial g ass species wi h no such issue. These common obse a ions om his s udy highligh ing ligh -dependen plan and soil mic obial in e ac ions om s uc u ally di e en plan species sugges ha he N dynamics pe haps ollow a simila pa e n unde N de iciency ac oss ege a ion ypes in his ecosys em. Mos o he pas s udies on N cycling in ag icul u al soils ha e ocused hei a en ion p ima ily on plan a ail- able N in soluble ino ganic (NO3−, NH4 + , NO2−) o m. Recen ly, howe e , he hough ha plan s may ely on o ganic N sou ces when he e ec o he added N e ilize subsides is gaining impo ance48. In iew o his, he o iginal hole in he pipe model wi h only wo N sou ces is now sugges ed o be modi ied o include o ganic N as an addi ional N sou ce in he pipe49. The pool o soluble o ganic N, especially ee amino acids, has been obse ed in soils a concen a ions compa able o o g ea e han ha o ino ganic N50. Soil o ganic ma e u no e , plan , and mic obial N up ake p ocesses ope a e apidly and a a ying a es, causing soil amino acid composi ion o be highly dynamic in space and ime51. A s udy on soil N lux dynamics in e ilized and un e ilized bo eal o es soils has shown ha soil amino acids can a y o e sho , diu nal imescales52. In iew o such obse a ions, i is possible ha he dynamic na u e o soil o ganic N plays a ole in he diu nal egula ion o N2O emissions om ou s udy si e, especially unde condi ions o low soil N a ailabili y. Howe e , e ec s o en i onmen al change on soil amino acid concen a ions and composi ion and hei ole in soil N ans o ma ions a e no ye well unde s ood. Whe e does he N2O emi ed du ing he high and low lux pe iods o igina e om ? Wha a e he dominan p ocesses ha accoun o he con as ing diu nal pa e ns o N2O emissions ? These ob ious ques ions need u - he s udies on soil physical, chemical and biological p ope ies and plan -mic obial p ocesses. Ou obse a ions highligh ha he accu a e ne N2O exchange ac oss he soil–a mosphe e in e ace, signi ican ly imp o ed by mode n analy ical me hods such as he con inuous wa e lase spec oscopy, can now be measu ed o e eal con- as ing sho - e m exchange pa e ns unde high and low soil N a ailabili y. Such s udies need o be ideally com- bined wi h smalle scale p ocess s udies on a highe spa ial and empo al esolu ion o elucida e he soil p ocesses and d i e s o simul aneous N2O p oduc ion, consump ion and anspo wi hin he hizosphe e. In iew o he dynamic sho - e m a ia ions in N2O emissions, con inuous au oma ed chambe - and eddy co a iance-based lux measu emen s a e necessa y o a be e unde s anding o he in ima e, empo al coupling be ween plan s and soil mic obial communi ies and hei esou ce demands in e es ial ecosys ems. Me hods The s udy si e. The eddy co a iance measu emen s o N2O exchange we e made on an ag icul u al ield si e, 6.3 ha in a ea. The si e is loca ed in he u al dis ic o Maaninka, Eas e n Finland (63° 9′ 48.69″ N, 27° 14′ 3.29″ E). Long- e m ( e e ence pe iod 1981–2010) annual ai empe a u e in he egion is 3.2 °C and he annual p ecipi a ion is 612 mm53. The soil ype a ied be ween loam and clay loam. The ield was sown in June 2009 wi h a pe ennial bioene gy c op, eed cana y g ass (RCG, Phala is a undinaceae, L. c . Pala on). A e he i s yea o c op es ablishmen , he si e was e ilized a he s a o e e y g owing season (la e May) wi h an N-P-K-S e ilize con aining 76 kg N ha−1 (NO3-N : NH4-N = 47:53). The canopy heigh de eloped h oughou he 2011 g owing season om abou 10 cm in mid-May o 1.7 m by la e June wi h a slowe inc ease o a maximum heigh o 1.9 m by ea ly July (see Supplemen a y In o ma ion o u he de ails abou he si e). Eddy co a iance (EC) measu emen s o N2O and CO2 exchange. The EC measu emen s we e made as a pa o he ICOS (In eg a ed Ca bon Obse a ion Sys em) Finland p og am du ing Ap il o No embe 201118. The main objec i e behind hese measu emen s was o measu e accu a ely he ni ous oxide exchange om an ag icul u al si e and o compa e he pe o mance o ou di e en lase -based comme cial gas analyz- e s. The EC sys em consis ed o ou di e en N2O gas analyze s: ARC – con inuous wa e lase spec ome e , Ae odyne Resea ch Inc., Bille ica, MA, USA; TGA – ace gas analyse , Campbell Scien i ic Inc., Logan, UT, USA; con inuous wa e lase spec ome e , Los Ga os Resea ch Inc., Moun ain View, CA, USA; ARP – pulsed quan um cascade lase spec ome e , Ae odyne Resea ch Inc., Bille ica, MA, USA (see Supplemen a y In o ma ion o de ails). While TGA is a legacy ace gas analyse , ARP ep esen s he in e media e le el and ARC and LGR ep- esen he highes le els o ad ancemen wi h espec o hei abili y o de ec N2O signals a a p ecision necessa y o EC measu emen s. Ca e ul calib a ion and co ec ions we e pe o med in o de o mee he objec i es o he campaign. In addi ion, wo sonic anemome e s (RS-50, Gill Solen L d., UK and USA-1, Me ek Ge many GMBH) o measu ing u bulen wind componen s, an in a ed gas analyse (LI7000/LI6262 – Li-Co Inc., Lincoln, NE, USA) o de ec ing high equency CO2 and wa e apou concen a ions we e used in he s udy. A wea he s a- ion loca ed a he si e moni o ed con inuously se e al mic ome eo ological obse a ions such as ai empe a u e and humidi y, a mosphe ic p essu e, wind speed and di ec ion, adia ion balance componen s, pho osyn he i- cally ac i e adia ion (PAR), p o iles o soil empe a u e and mois u e e c. G ea e de ails on EC da a acquisi- ion, adop ed da a p ocessing p ocedu es, applied lux co ec ions and ins umen de ails a e desc ibed in he Supplemen a y In o ma ion. www.na u e.com/scien i ic epo s/ 7 Scien i ic RepoR s | 6:25739 | DOI: 10.1038/s ep25739 Iso opic analyses o 15N. Chambe echnique based ni ous oxide luxes we e measu ed on 8 July 2011 om p e-ins alled aluminium colla s (0.36 m−2 basal a ea) wi h anspa en chambe and opaque chambe (wi h a chambe heigh o 0.33 m and 0.15 m, espec i ely) om 3 plo s (9 m2 each). The plo s we e es ablished in June 2009 wi h a o age g ass mix u e consis ing o imo hy (Phleum p a ense L. ,c . Tuukka), and meadow escue (Fes uca p a ensis Huds., c An i). G ass ield had been ha es ed wo weeks ea lie (on 22 June 2011) and a second e ilize dose o 15N-en iched Ammonium Ni a e (15NH415NO3, 10 AT-% (a om pe cen abundance) in bo h N o ms a a a e o 100 kg N ha−1) was applied a day p io o he chambe lux measu emen s. The objec i e o his ield campaign was o es ima e he ac ion o N2O de i ed om he 15N-en iched N- e ilize . Each N2O lux measu emen las ed 35 min. Gas samples o AT-% o 15N-N2O analysis we e collec ed a 5, 10, 15, 25 and 35 min in e als om he chambe headspace. Gas samples we e aken wi h a 160 ml sy inge om which he sample was immedia ely injec ed in o a p e-e acua ed 120 ml ials. Fo anspa en chambe measu emen s, as he g ass species ixed CO2 om he chambe head space unde ligh , concen a ion inside he chambe was moni o ed con inuously wi h an in a ed gas analyse (LI6400, Li-Co Inc., Lincoln, NE, USA) o e he 35 min chambe deploymen du a ion. CO2 le els inside he anspa en chambe we e con inuously adjus ed du ing his pe iod by adding CO2 gas in o he chambe ai om a s anda d CO2 gas bo le so ha he CO2 con- cen a ion wi hin he chambe emained close o he ambien le el o e he measu emen pe iod. Addi ionally, a cooling uni was used in he anspa en chambe measu emen o keep he chambe ai empe a u e close o he ou side ai empe a u e. Du ing he opaque chambe measu emen , howe e , no chambe ai cooling o CO2 le el adjus men was done. The 35 min a e age empe a u es om anspa en and da k chambe headspaces a ied wi hin 5 °C. The collec ed gas samples we e analysed o he AT -% 15N-N2O wi h an iso ope a io mass spec ome- e (IRMS; The mo Finnigan Del a V plus wi h ConFlo IV, The mo Fishe Scien i ic) coupled o a ace-gas p e-concen a ion uni (P eCon, The mo Fishe Scien i ic) and a gas ch oma og aph. Wa e and CO2 gas we e emo ed om he sample gas by a chemical ap (con aining NaOH o CO2 and Mg(ClO4)2 o H2O). Re e ences 1. Myh e, G. e al. An h opogenic and Na u al Radia i e Fo cing. Chap e 8 (Camb idge, Uni ed Kingdom and New Yo k, NY, USA, 2013). 2. Reay, D. S. e al. Global ag icul u e and ni ous oxide emissions. Na . Clim. Change 2, 410–416 (2012). 3. Ciais, P. e al. Ca bon and O he Biogeochemical Cycles. Chap e 6 (Camb idge Uni e si y P ess, Camb idge, Uni ed Kingdom and New Yo k, NY, USA, 2013). 4. Saikawa, E. e al. Global and egional emissions es ima es o N2O. A mos. Chem. Phys. 14, 4617–4641, doi: 10.5194/acp-14-4617- 2014 (2014). 5. Came on, K. C., Di, H. J. & Moi , J. L. Ni ogen losses om he soil/plan sys em: a e iew. Ann. App. Biol. 162, 145–173, doi: 10.1111/aab.12014 (2013). 6. Rees, R. M. e al. Ni ous oxide emissions om Eu opean ag icul u e - an analysis o a iabili y and d i e s o emissions om ield expe imen s. Biogeosciences 10, 2671–2682, doi: 10.5194/bg-10-2671-2013 (2013). 7. Bouwman, A. F., Boumans, L. J. M. & Ba jes, N. H. Emissions o N2O and NO om e ilized ields: Summa y o a ailable measu emen da a. Glob. Biogeochem. Cy. 16, 6-1–6-13, doi: 10.1029/2001gb001811 (2002). 8. Chadwick, D. R. e al. Op imizing chambe me hods o measu ing ni ous oxide emissions om plo -based ag icul u al expe imen s. Eu . J. Soil Sci. 65, 295–307, doi: 10.1111/ejss.12117 (2014). 9. Ku zbach, L. e al. CO2 lux de e mina ion by closed-chambe me hods can be se iously biased by inapp op ia e applica ion o linea eg ession. Biogeosciences 4, 1005–1025, doi: 10.5194/bg-4-1005-2007 (2007). 10. Maljanen, M. e al. A o es a ion does no necessa ily educe ni ous oxide emissions om managed bo eal pea soils. Biogeochemis y 108, 199–218, doi: 10.1007/s10533-011-9591-1 (2012). 11. Li ings on, G. & Hu chinson, G. Enclosu e-based measu emen o ace gas exchange: applica ions and sou ces o e o . In Biogenic ace gases: measu ing emissions om soil and wa e (eds P. A. Ma son & R. C. Ha is) 14–51 (Blackwell Sci., Malden, Mass., 1995). 12. Pa ey, E. e al. Towa ds s anda ds o measu ing g eenhouse gas luxes om ag icul u al ields using ins umen ed owe s. Can. J. Soil Sci. 86, 373–400, doi: 10.4141/S05-100 (2006). 13. Baldocchi, D. Measu ing luxes o ace gases and ene gy be ween ecosys ems and he a mosphe e – he s a e and u u e o he eddy co a iance me hod. Glob. Change Biol. 20, 3600–3609, doi: 10.1111/gcb.12649 (2014). 14. Nicolini, G., Cas aldi, S., F a ini, G. & Valen ini, R. A li e a u e o e iew o mic ome eo ological CH4 and N2O lux measu emen s in e es ial ecosys ems. A mos. En i on. 81, 311–319, doi: 10.1016/j.a mosen .2013.09.030 (2013). 15. Pihla ie, M. K. e al. Compa ison o s a ic chambe s o measu e CH4 emissions om soils. Ag . Fo es Me eo ol. 171–172, 124–136, doi: 10.1016/j.ag o me .2012.11.008 (2013). 16. Sa age, K., Phillips, R. & Da idson, E. High empo al equency measu emen s o g eenhouse gas emissions om soils. Biogeosciences 11, 2709–2720, doi: 10.5194/bg-11-2709-2014 (2014). 17. We le, P. Accu acy and p ecision o lase spec ome e s o ace gas sensing in he p esence o op ical inges and a mosphe ic u bulence. App. Phy. B 102, 313–329 (2011). 18. Rannik, Ü. e al. In e compa ison o as esponse comme cial gas analyse s o ni ous oxide lux measu emen s unde ield condi ions. Biogeosciences 12, 415–432, doi: 10.5194/bg-12-415-2015 (2015). 19. Bu e bach-Bahl, K., Baggs, E. M., Dannenmann, M., Kiese, R. & Zechmeis e -Bol ens e n, S. Ni ous oxide emissions om soils: how well do we unde s and he p ocesses and hei con ols ? Philos. T ans. R. Soc. Lond. B Biol. Sci. 368, 20130122, doi: 10.1098/ s b.2013.0122 (2013). 20. W age, N., G oenigen, J. W. ., Oenema, O. & Baggs, E. M. A no el dual-iso ope labelling me hod o dis inguishing be ween soil sou ces o N2O. Rapid Commun. Mass Sp. 19, 3298–3306, doi: 10.1002/ cm.2191 (2005). 21. B ake , G. & Con ad, R. Di e si y, s uc u e, and size o N(2)O-p oducing mic obial communi ies in soils–wha ma e s o hei unc ioning? Ad . Appl. Mic obiol. 75, 33–70, doi: 10.1016/b978-0-12-387046-9.00002-5 (2011). 22. Philippo , L. e al. Mapping ield-scale spa ial pa e ns o size and ac i i y o he deni i ie communi y. En i on. Mic obiol. 11, 1518–1526, doi: 10.1111/j.1462-2920.2009.01879.x (2009). 23. Kool, D. M. e al. Ni i ie deni i ica ion can be a sou ce o N2O om soil: a e ised app oach o he dual-iso ope labelling me hod. Eu . J. Soil Sci. 61, 759–772, doi: 10.1111/j.1365-2389.2010.01270.x (2010). 24. Spo , O., Russow, R. & S ange, C. F. Fo ma ion o hyb id N2O and hyb id N2 due o codeni i ica ion: Fi s e iew o a ba ely conside ed p ocess o mic obially media ed N-ni osa ion. Soil Biol. Biochem. 43, 1995–2011, doi: 10.1016/j.soilbio.2011.06.014 (2011). 25. Laughlin, R. J. & S e ens, R. J. E idence o Fungal Dominance o Deni i ica ion and Codeni i ica ion in a G assland. Soil. Soil Sci. Soc. Am. J. 66, 1540–1548, doi: 10.2136/sssaj2002.1540 (2002). www.na u e.com/scien i ic epo s/ 8 Scien i ic RepoR s | 6:25739 | DOI: 10.1038/s ep25739 26. Mülle , C., Laughlin, R. J., Spo , O. & Rü ing, T. Quan i ica ion o N2O emission pa hways ia a 15N acing model. Soil Biol. Biochem. 72, 44–54, doi: 10.1016/j.soilbio.2014.01.013 (2014). 27. S ange, C. F. e al. Use o he in e se abundance app oach o iden i y he sou ces o NO and N2O elease om Spanish o es soils unde oxic and hypoxic condi ions. Soil Biol. Biochem. 57, 451–458, doi: 10.1016/j.soilbio.2012.10.006 (2013). 28. Cleempu , O. & Sama e , A. H. Ni i e in soils: accumula ion and ole in he o ma ion o gaseous N compounds. Fe . Res. 45, 81–89, doi: 10.1007/b 00749884 (1995). 29. Wallens ein, M. D., My old, D. D., Fi es one, M. & Voy ek, M. En i onmen al con ols on deni i ying communi ies and deni i ica ion a es: Insigh s om molecula me hods. Ecol. Appl. 16, 2143–2152, doi: 10.1890/1051-0761(2006)016[2143:ECODCA]2.0.CO;2 (2006). 30. Tiedje, J. M. Ecology o deni i ica ion and dissimila o y ni a e educ ion o ammonium. In En i onmen al Mic obiology (ed A. J. B. Zehnde ) 179–244 (John Wiley and Sons, New Yo k, 1988). 31. Mosie , A. R., Mo gan, J. A., King, J. Y., LeCain, D. & Milchunas, D. G. Soil-a mosphe e exchange o CH4, CO2, NOx, and N2O in he Colo ado sho g ass s eppe unde ele a ed CO2. Plan Soil 240, 201–211, doi: 10.1023/A:1015783801324 (2002). 32. Kowalchuk, G. A. & S ephen, J. R. Ammonia-oxidizing bac e ia: a model o molecula mic obial ecology. Annu. Re . Mic obiol. 55, 485–529, doi: 10.1146/annu e .mic o.55.1.485 (2001). 33. Tamu a, Y. & Mo iyama, M. Nons uc u al Ca bohyd a e Rese es in Roo s and he Abili y o Tempe a e Pe ennial G asses o O e win e in Ea ly G ow h S ages. Plan P od. Sci. 4, 56–61, doi: 10.1626/pps.4.56 (2001). 34. Kusel, K. & D ake, H. L. Anae obic capaci ies o lea li e . Appl. En i on. Mic ob. 62, 4216–4219 (1996). 35. Denmead, O., T., J.R., F. & J.R., S. S udies o ni ous oxide emission om a g ass swa d. Soil Sci. Soc. Am. J. 43, 726–728 (1979). 36. Ryden, J. C., Lund, L. J. & Foch , D. D. Di ec in- ield measu emen o ni ous oxide lux om soils. Soil Sci. So.c Am. J. 42, 731–737 (1978). 37. Blackme , A. M., Robbins, S. G. & B emne , J. M. Diu nal a iabili y in a e o emission o ni ous oxide om soils. Soil Sci. Soc. Am. J. 46, 937–942 (1982). 38. Ch is ensen, S. Ni ous oxide emission om he soil su ace: Con inuous measu emen by gas ch oma og aphy. Soil Biol. Biochem. 15, 481–483, doi: 10.1016/0038-0717(83)90015-9 (1983). 39. Os om, N. E. e al. Iso opologue da a e eal bac e ial deni i ica ion as he p ima y sou ce o N2O du ing a high lux e en ollowing cul i a ion o a na i e empe a e g assland. Soil Biol. Biochem. 42, 499–506, doi: 10.1016/j.soilbio.2009.12.003 (2010). 40. Ne el, A. e al. Expe imen al assessmen o N2O backg ound luxes in g assland sys ems. Tellus B 59, 470–482, doi: 10.1111/j.1600- 0889.2007.00273.x (2007). 41. Zona, D. e al. N2O luxes o a bio-ene gy popla plan a ion du ing a wo yea s o a ion pe iod. Glob. Change Biol. Bioene gy 5, 536–547, doi: 10.1111/gcbb.12019 (2013). 42. G oenigen, J. W. . e al. Subsoil 15N-N2O concen a ions in a sandy soil p o ile a e applica ion o 15N- e ilize . Nu . Cycl. Ag oecosys. 72, 13–25 (2005). 43. Mu ay, P. J. e al. Deni i ica ion po en ial in a g assland subsoil: e ec o ca bon subs a es. Soil Biol. Biochem. 36, 545–547, doi: 10.1016/j.soilbio.2003.10.020 (2004). 44. Hughes, M., Donnelly, C., C ozie , A. & Wheele , C. T. E ec s o he exposu e o oo s o Alnus glu inosa o ligh on la onoids and nodula ion. Can. J. Bo . 77, 1311–1315, doi: 10.1139/b99-077 (1999). 45. Ta i, N. M. e al. Linking mic obial communi y s uc u e and alloca ion o plan -de i ed ca bon in an o ganic ag icul u al soil using 13CO2 pulse-chase labelling combined wi h 13C-PLFA p o iling. Soil Biol. Biochem. 58, 207–215, doi: 10.1016/j.soilbio.2012.11.013 (2013). 46. Dene , K. e al. Communi y shi s and ca bon ansloca ion wi hin me abolically-ac i e hizosphe e mic oo ganisms in g asslands unde ele a ed CO2. Biogeosciences 4, 769–779, doi: 10.5194/bg-4-769-2007 (2007). 47. Macdu , J. H. & Bakken, A. K. Diu nal a ia ion in up ake and xylem con en s o ino ganic and assimila ed N unde con inuous and in e up ed N supply o Phleum p a ense and Fes uca p a ensis. J. Exp. Bo . 54, 431–444 (2003). 48. Del G osso, S. J. e al. Gene al model o N2O and N2 gas emissions om soils due o den i ica ion. Glob. Biogeochem. Cy. 14, 1045–1060, doi: 10.1029/1999GB001225 (2000). 49. Inselsbache , E. e al. Sho - e m compe i ion be ween c op plan s and soil mic obes o ino ganic N e ilize . Soil Biol. Biochem. 42, 360–372, doi: 10.1016/j.soilbio.2009.11.019 (2010). 50. Zhang, J., Mülle , C. & Cai, Z. He e o ophic ni i ica ion o o ganic N and i s con ibu ion o ni ous oxide emissions in soils. Soil Biol. Biochem. 84, 199–209, doi: 10.1016/j.soilbio.2015.02.028 (2015). 51. Inselsbache , E. & Näsholm, T. A no el me hod o measu e he e ec o empe a u e on di usion o plan -a ailable ni ogen in soil. Plan Soil 354, 251–257, doi: 10.1007/s11104-011-1061-y (2011). 52. We din-P is e e , N. R., Kielland, K. & Boone, R. D. Soil amino acid composi ion ac oss a bo eal o es successional sequence. Soil Biol. Biochem. 41, 1210–1220, doi: 10.1016/j.soilbio.2009.03.001 (2009). 53. Inselsbache , E., Oyewole, O. A. & Näsholm, T. Ea ly season dynamics o soil ni ogen luxes in e ilized and un e ilized bo eal o es s. Soil Biol. Biochem. 74, 167–176, doi: 10.1016/j.soilbio.2014.03.012 (2014). 54. Pi inen, P. e al. Tilas oja Suomen ilmas os a 1981–2010. (Finnish Me eo ological Ins i u e, Helsinki, Finland, Helsinki, Finland, 2012). Acknowledgemen s This wo k was suppo ed by he Academy o Finland (p ojec nos 118780, 127456, 1118615 and 263858). ICOS (271878), ICOSFinland (281255) and ICOS-ERIC (281250), DEFROST No dic Cen e o Excellence and InGOS EU a e g a e ully acknowledged o unding his wo k. This wo k was also suppo ed by ins i u ional esea ch unding (IUT20-11) o he Es onian Minis y o Educa ion and Resea ch. The UEF pa o he esea ch wo k was suppo ed by he unding om he UEF in as uc u e unding, Academy o Finland FidiP o p og amme (PIs – P o s Pe i Ma ikainen and Seppo Kellomäki) and he Minis y o Ag icul u e and Fo es y, Finland and MTT Ag i ood Resea ch Finland (MTT) s a egic unding (p ojec no. 21030028). Maa- ja esi ekniikan uki y and Jenny and An i Wihu i Founda ion a e hanked o esea ch unding g an ed o Simo Jokinen. Au ho Con ibu ions N.J.S., Ü.R., P.J.M., T.V. and M.P. concei ed he idea o he pape . Ü.R., I.M., O.P., N.J.S. and S.L. analysed he eddy co a iance da a o N2O and CO2 exchange om he s udy si e. S.H., S.L., N.J.S. and N.H. collec ed he ield expe imen al da a on N2O and CO2 exchange. The da a used in his analysis a e om he N2O lase spec ome e p o ided by M.Z. who ensu ed ha N2O analyse s (manu ac u ed by Ae odyne Resea ch Inc.) a e p ope ly ins alled in he ield and ha good quali y da ase s can be collec ed om hem. P.V. and M.R. we e esponsible o he si e main enance, c op husband y and es ablishmen o ield expe imen a ion o he iso opic analyses needed in he s udy. C.B. and S.J. we e esponsible o he iso opic analysis pa o he s udy. N.J.S., P.J.M. and C.B. w o e he pape and all au ho s commen ed on ea lie e sions o he manusc ip . www.na u e.com/scien i ic epo s/ 9 Scien i ic RepoR s | 6:25739 | DOI: 10.1038/s ep25739 Addi ional In o ma ion Supplemen a y in o ma ion accompanies his pape a h p://www.na u e.com/s ep Compe ing inancial in e es s: The au ho s decla e no compe ing inancial in e es s. How o ci e his a icle: Shu pali, N. J. e al. Neglec ing diu nal a ia ions leads o unce ain ies in e es ial ni ous oxide emissions. Sci. Rep. 6, 25739; doi: 10.1038/s ep25739 (2016). This wo k is licensed unde a C ea i e Commons A ibu ion 4.0 In e na ional License. The images o o he hi d pa y ma e ial in his a icle a e included in he a icle’s C ea i e Commons license, unless indica ed o he wise in he c edi line; i he ma e ial is no included unde he C ea i e Commons license, use s will need o ob ain pe mission om he license holde o ep oduce he ma e ial. 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