Two dimensional NMR spectroscopy for molecular characterization of soil organic matter: Application to boreal soils and litter

Accepted Manuscript Two dimensional NMR spectroscopy for molecular characterization of soil organic matter: Application to boreal soils and litter Laure N. Soucémarianadin, Björn Erhagen, Mats B. Nilsson, Mats G. Öquist, Peter Immerzeel, Jürgen Schleucher PII: DOI: Reference:

S0146-6380(16)30308-4 http://dx.doi.org/10.1016/j.orggeochem.2017.06.019 OG 3580

To appear in:

Organic Geochemistry

Received Date: Revised Date: Accepted Date:

4 November 2016 31 May 2017 8 June 2017

Please cite this article as: Soucémarianadin, L.N., Erhagen, B., Nilsson, M.B., Öquist, M.G., Immerzeel, P., Schleucher, J., Two dimensional NMR spectroscopy for molecular characterization of soil organic matter: Application to boreal soils and litter, Organic Geochemistry (2017), doi: http://dx.doi.org/10.1016/j.orggeochem. 2017.06.019

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Two dimensional NM R spect roscopy for molecular char acter izat ion of soil or ganic mat t er : Applicat ion to boreal soils and lit ter

L aur e N. Soucémar ianadin a,* ,1, Björ n Er hagen b,2, M at s B. Nilsson b, M at s G. Öquist b, Pet er I mmer zeel a, Jür gen Schleucher a

a

Depar tment of M edical Biochemistr y and Biophysics, Umeå Univer sity, SE -901

87 Umeå, Sweden b

Depar tment of For est Ecology and M anagement, SLU, SE-901 83 Umeå, Sweden

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Cur r ent addr ess: CNRS, L abor at oir e de Géologie, Ecole Nor male Supér ieur e,

75005 Par is, Fr ance 2

Cur r ent addr ess: Depar t ment of Ecology and Envir onment al Sciences, Umeå

Univer sit y, SE-901 87 Umeå, Sweden

* Cor r espondence aut hor . Tel.: +46705494664.

E mail : soucmar i@ualber t a.ca (L aur e Soucémar ianadin).

K eywor ds: soil or ganic mat t er ; molecular char act er izat ion; DMSO-extracts; 2D NM R; H SQC; car bon input s; lignin; polysacchar ides.

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ABSTRACT Or ganic soils in bor eal ecosyst ems and peat lands r epr esent a huge global car bon pool and t heir composit ion st r ongly affect s soil pr oper t ies. Never t heless, t he char act er izat ion of soil or ganic mat t er (SOM ) molecular composit ion, which is essent ial for elucidat ing soil car bon pr ocesses and t ur nover , is not easily achieved, and fur t her advances in t he ar ea ar e gr eat ly n eeded. Two dimensional (2D) liquid st at e 1H –13C nuclear magnet ic r esonance (NM R) spect r oscopy has been used on dimet hyl sulfoxide (DM SO) ext r act s of SOM t o achieve molecular level char act er izat ion, wit h signals fr om many ident ifiable molecular gr oups obser vable. H er e we show t hat a simple and fast sample pr epar at ion allows t o acquir e 2D 1H – 13

C NM R spect r a on ext r act s of plant lit t er and or ganic layer s in bor eal ecosyst ems,

wit h fast dat a acquisit ion. Our 2D NM R spect r a r evealed sever al differ ences in t he DM SO-ext r act s of differ ent t r ee lit t er samples, O-hor izons of for est soil, peat for ming moss (Sphagnum ) and peat . The r esult s mir r or est ablished differ ences bet ween OM in soils and lit t er of differ ent for est ecosyst ems (e.g. bet ween deciduous and conifer ous lit t er ) but also pr ovide indicat ions for r esear ch t o unt angle pr eviously conflict ing r esult s (e.g. cut in degr adat ion in soil or car bohydr at e degr adat ion in peat ). Thus, combinat ion of 2D NM R met hods can gr eat ly impr ove analysis of lit t er composit ion and SOM composit ion, t her eby facilit at ing t he elucidat ion of t heir r oles in biogeochemical and ecological pr ocesses t hat ar e cr it ical for for eseeing feedback mechanisms on SOM t ur nover as a r esult of global envir onment al change.

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1. I nt r oduct i on Soil or ganic mat t er (SOM ) pools hold ca. 2300 Pg of car bon (C; I nt er gover nment al Panel on Climat e Change, 2007), which is appr oximat ely 3x t han for t he at mospher e (Jobbágy and Jackson, 2000). Pr edict ed war ming may change t he size and t ur nover r at e of soil C pools, pot ent i ally t r igger ing fur t her r ises in at mospher ic CO2 and war ming (Knor r et al., 2005; von L üt zow et al., 2006). Thus, it is essent ial t o elucidat e int er act ion bet ween climat ic and SOM dynamics. SOM can be r oughly divided int o act ive (labile) and passive (r ecal cit r ant ) pools for modeling but , in act ualit y, t he r esidence t imes of molecular moiet ies for m a cont inuum due t o high het er ogeneit y (Bat jes, 1996). The var iat ion in SOM composit ion st r ongly affect s it s decomposit ion and miner alizat ion, which ar e cr it ical pr ocesses for soil for mat ion, nut r ient r elease and t he net ecosyst em car bon balance (Swift et al., 1979; Tr umbor e, 2000; K leber , 2010; Schmidt et al., 2011; Er hagen et al., 2013). Thus, SOM composit ion and t ur nover ar e cr ucial ecologically, agr icult ur ally and silvicult ur ally, but t he int er act ion bet ween SOM composit ion and it s decomposit ion is not fully under st ood due t o difficult ies in char act er izing SOM in t er ms of t he moiet ies t ar get ed by t he enzymes of micr obial decomposer s, which st r ongly affect micr obi al met abolism, t ur nover r at e and associat ed ecological pr ocesses (Sollins et al., 1996). A molecular moiet y is a basic st r uct ur e par t of a mor e complex or ganic st r uct ur e, e.g. a glucose molecule in st ar ch or cellulose or a phenol unit in lignin or t annin. Even if t hese moiet ies ar e ident ical, t heir var ious chemical envir onment s make

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t hem mor e or less accessible t o micr obial enzymes and so t hey exper ience var ied t ur nover r at e values. SOM has been mainly char act er ized using wet chemist r y met hods or var ious t echniques, including solid st at e 13C NM R nuclear magnet ic r esonance (NM R) spect r oscopy (Pr est on et al., 1989; Baldock et al., 1992; K ögel K nabner , 1997; Cook, 2004), Four ier t r ansfor m infr ar ed spect r oscopy and pyr olysis gas chr omat ogr aphy-mass spect r omet r y (Py-GC-M S; Schnit zer et al., 2006; Vancampenhout et al., 2009; Yassir and Buur man, 2012; Tivet et al., 2013). These met hods ar e eit her dest r uct ive, lack sufficient r esolut ion and/or have ot her dr awbacks. For example, secondar y r eact ions in Py-GC-M S analyses may cr eat e new pyr olysis pr oduct s, which complicat e dat a int er pr et at ion (Der enne and Nguyen Tu, 2014). Solid st at e 13C NM R spect r oscopy is not dest r uct ive but only pr ovides dat a r egar ding eight br oad st r uct ur al gr oups, each of which may include moiet ies wit h subst ant ially differ ing sensit ivit y t o degr adat ion. Thus, t hese analyt ical met hods pr ovide scant moiet y-level infor mat ion (Feng and Simpson, 2011). H owever , besides 1D cr oss-polarizat ion (CP), t he most commonly used exper iment in SOM char act er izat ion, several solid-st at e exper iment s can give deep insight s on SOM composit ion (e.g., K eeler and M aciel, 2000; M ao et al., 2002; M ao et al., 2017; Schmidt -Rohr and M ao, 2002). Solut ion st at e NM R spect r oscopy offer s shar per signals, mult idimensional analyt ical capacit y and, t hus achieve higher r esolut ion mor e easily t han solid st at e NM R. This t echnique could t her efor e pr ovide ver y useful complement ar y infor mat ion t o 13C CP exper iment s and deliver a det ailed char act er izat ion of SOM molecular moiet ies. I n t wo dimensional (2D)

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t echniques, t he use of t wo chemical shift s, e.g. 1H and 13C, r educes signal over lap, allowing ident ificat ion of many specific moiet ies. Thus, 2D NM R has been used t o char act er ize dissolved or ganic car bon (H er t kor n et al., 2006; L am et al., 2007) and bot h t he composit ion and st r uct ur e of cell walls in var ious plant mat er ials (K im et al., 2008; K im and Ralph, 2010; Sun et al., 2012; K im and Ralph, 2014). Ext r act ions and dissolut ion of SOM in solvent like dimet hyl sulfoxide (DM SO) have been per for med in t he past t o st udy r esist ant fr act ions (e.g., Song et al., 2011). M or eover , soluble SOM , and specifically humic subst ances, have also been ext ensively char act er ized using a wide ar r ay of mult idimensional NM R exper iment s, including Diffusion Or der ed Spect r oscopy (DOSY; M or r is et al., 1999; Simpson et al., 2001b), Cor r elat ion spect r oscopy (COSY) and H et er onuclear M ult iple Quant um Coher ence (H M QC; H aiber et al., 1999), Tot al Cor r elat ion Spect r oscopy (TOCSY) and H et er onuclear Single Quant um Coher ence (HSQC; K elleher and Simpson, 2006) or TOCSY and H M QC (K inger y et al., 2000) or a combinat ion of t he above exper iment s (Simpson et al. 2002; Simpson et al., 2007), including wit h t he addit ion of H et er onuclear M ult iple Bond Cor r elat ion (H M BC; Cook et al., 2003). This also includes using H igh Resolut ion M agic Angle Spinning (H R-M AS) spect r oscopy t o st udy soil component s dissolved in DMSO (Simpson et al., 2001a; K elleher et al., 2006; Far ooq et al., 2013). To assess t he suit abilit y of 2D 1H –13C NM R for high r esol ut ion char act er izat ion of bulk OM samples, we used it t o analyze samples of plant lit t er and or ganic soil layer s fr om bor eal sit es because bor eal for est soils and peat lands hold massive C

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st ocks (near ly 300 Pg and 500 Pg, r espect ively) and t his st or age is pot ent ially highly sensit ive t o climat e change (Yu et al., 2010; Pan et al., 2011). The purpose of t his paper and t he dat a we pr esent ed is t o illust r at e t he pot ent ial of t his met hod, applied t o bor eal soils.

2. M at er i al and M et hods

2.1. Sample selection We sampled 0–3 cm (“super ficial”) and 5–10 cm (“deep”) layer s of or ganic hor izons and leaf or needle lit t er fr om t he dominant t r ee species at four bor eal sit es in nor t her n Sweden, which had been descr ibed and designat ed as sit es 2, 4, 6 and 7 (Table A.1; Er hagen et al., 2013). Sit es 2, 4 and 6 suppor t ed a mixed conifer ous st and of Nor way spr uce (Picea abies, L .) and Scot s pine (Pinus sylvestr is, L .), a Nor way spr uce st and, and a deciduous st and of silver bir ch ( Betula pendula, Rot h), r espect ively. Sit e 7 was r ecent ly clear -cut and cover ed by hair y gr ass [ Deschampsia flexuosa (L .) Tr in.]. Samples fr om sit es 4 and 6 ar e r efer r ed t o as “spruce” and “bir ch” lit t er and soil, r espect ively. Samples of t he peat moss (Sphagnum fuscum L .) wer e collect ed at an oligot r ophi c miner ogenic mir e, a major t ype of high lat it ude mir e (Nilsson et al., 2008), and t wo peat samples wer e t aken fr om cor es (at 150–155 and 205–210 cm dept h) fr om a lawn communit y in anot her oligot r ophic miner ogenic mir e in t he same ar ea (Table A.1).

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2.2. 2D NM R spectr oscopy 2.2.1. Sample pr epar ation Samples wer e pr epar ed using a pr ocedur e developed for char act er izing plant cell walls (K im et al., 2008; K im and Ralph, 2014). Br iefly, 200 mg of mat er ial was gr ound in a Pulver iset t e 7 Planet ar y ball mill (Fr it sch, I dar -Ober st ein, Ger many) for 5 × 10 min at 500 r pm wit h 10 min br eaks. The gr ound mat er ial (40 mg) fr om each sample was t r ansfer r ed t o an NM R t ube and dissolved in 500 μl fully deut er at ed dimet hyl sulfoxide (DM SO-d6). Samples wer e vor t ex-mixed t hor oughly befor e analysis t o maximize dissolut ion.

2.2.2. HSQC experiments 1

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The t echnique applied was H – C het er onuclear single quant um coher ence (H SQC), which cor r elat es chemical shift s (in par t s per million, ppm) of pr ot ons ( 1H at oms) wit h t heir dir ect ly bonded 13C at oms using t he one-bond J coupling bet ween 13

C and 1H . Regions and specific cr oss peaks ar e designat ed as δ C/δ H ppm. Spect r a

wer e acquir ed wit h a 600 M H z Br uker Avance I I I H D spect r omet er equipped wit h a t r iple r esonance cr yopr obe, cooled wit h heli um, using t he hsqcet gpsisp2.2 pulse sequence (Schleucher et al., 1994) fr om t he Br uker pulse sequence libr ar y. Spect r a wer e collect ed at r oom t emper at ur e (300 K ) wit h 20 (lit t er ) t o 36 (soil) t r ansient s and 16 dummy scans. The r ecycle delay was 1 s. The complex dat a point s collect ed in t he dir ect (1H ; F2) and indir ect (13C; F1) dimensions number ed 1486 and 320 wit h spect r al widt h of 12 ppm (7211 H z) and 180 ppm (27166 H z), r espect ively.

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The exper iment s wer e opt imized for a one-bond pr ot on-car bon coupling const ant of 145 H z. Spect r al pr ocessing included zer o filling (1024 and 512 complex dat a point s in F2 and F1, r espect ively) and squar ed sine-bell apodizat ion in F2 and F1 (bot h shift ed by π/2) befor e Four ier t r ansfor mat ion. Spect r a wer e manually phase- and baselinecor r ect ed, and r efer enced using t he DMSO peak at 39.50/2.49 ppm. Dat a wer e pr ocessed wit h Topspin 3.2 (Bruker BioSpin Cor por at ion, Biller ica, USA). When compar ing H SQC spect r a of t he DM SO-ext r act s, we only displayed cont our levels wit h high (t ypi cally > 1.0% of maximum) int ensit y for visibilit y r easons, but signals as low as 0.1% of t he maximum int ensit y could be det ect ed. H ence, t he r esult s r eflect only t he var iat ion in t he st r ongest signals. I n mult iple displays, t he spect r a wer e scaled, t aking int o account t he r espect ive number of scans, t ot al int egr als, amount (%) of sample dissolved and car bon cont ent of sample. For each compar ison pr esent ed her eaft er , we specified t he scaling fact or t o account for all t hese par amet er s.

2.3. Dissolution efficiency Even aft er car eful gr inding, t he samples could not be dissolved complet ely in DM SO and a pellet r emained. To est imat e t he ext r act ion efficiency and assess possible fr act ionat ion dur ing dissolut ion, we pr epar ed r eplicat e samples t o det er mine t he dissolved mat er ial and undissolved pellet mass, followed by compar ison of t he composit ion of t he pellet s wit h t he “or iginal” SOM samples (i.e.

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dir ect ly aft er milling) fr om quant ifiable dir ect polar izat ion 13C NM R spect r a in a fir st subset of samples (set 1, n = 12; Table A.1). For a second subset , we compar ed t he composit ion of t he or iginal SOM sample wit h t he dissolved fr act ion using PyGC-M S) (set 2, n = 6; Table A.1). We scaled up t he dissolut ion st ep by st ar t ing wit h 400 mg lit t er or soil sample dissolved in 5 ml DMSO in a Falcon t ube. For set 1, t he pellet (undissolved mat er ial aft er adding DM SO) was isolat ed using cent r ifugat ion, followed by vacuum filt r at ion and r emoval of as much DM SO as possible by r insing wit h M e2CO (3x) and Et OH (3x). Then, t he pellet was oven dr ied at 60 °C unt il no change in mass could be measur ed. For set 2, pellet s and dissolved samples wer e r epeat edly suspended in wat er and fr eeze-dr ied unt il no change in mass could be measur ed. For each subset , t he mass of t he pellet was r ecor ded.

2.3.1. NM R exper iments 13

Solid st at e C NM R spect r a wit h dir ect polar izat ion and magic angle spinning (DP/M AS) wer e acquir ed using a Br uker 500 M H z Avance I I I spect r omet er equipped wit h a M AS pr obe. Spect r a wer e acquir ed at r oom t emper at ur e wit h samples packed in a 4 mm zir conia r ot or and spun at 10 kH z. Scans (7200 + 4 dummy scans) wer e acquir ed using a 50 s r ecycle delay and a 90° 13C pulse wit h a 4 μs dur at ion at a fr equency of 125.8 M H z. Chemical shift s wer e ext er nally r efer enced t o t he high-fr equency peak of adamant ane at 38.56 ppm. Spect r a wer e pr ocessed using 20–30 H z line-br oadening and baseline cor r ect ion.

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Bot h t he pellet s and cor r esponding or iginal samples wer e analyzed wit h solid st at e 13

C DP/M AS NM R under similar condit ions. Because we wer e compar ing pair s of

spect r a, we assumed t hat t he backgr ound signal or iginat ing fr om t he r ot or and coil in t he DP exper iment s would be t he same for all spect r a and t hus did not cor r ect t he acquir ed spect r a by subt r act ing t he spect r um of an empt y r ot or . Typical spect r al r egions wer e used t o t est differ ences in composit ion bet ween each pellet and cor r esponding or iginal sample (K ögel -Knabner , 1997): alkyl C (0–45 ppm), N alkyl C and met hoxy C (45–60 ppm), O-alkyl C (60–90 ppm), di -O-alkyl C (90–110 ppm), ar omat ic C (110–160 ppm), and car boxyl C (160–220 ppm). We used t he r egions wit h high chemical shift (250–190 ppm) t o cor r ect for t he spinning sidebands (e.g., Smer nik et al., 2008).

2.3.2. Py-GC-M S The dissolved fr act ions and cor r esponding or iginal samples wer e analyzed wit h PyGC-M S under similar condit ions. Tr iplicat es of 80 ± 10 µg of each sample wer e weighed and t r ansfer r ed t o aut osampler cont ainer s. We used similar Py-GC-M S set t ings t o t hose descr ibed by Ger ber et al. (2012). Br iefly, samples wer e pyr olyzed for 20 s at 450 °C in a pyr olyzer wit h an aut osampler (PY-2020iD and AS-1020E, Fr ont ier L abs, Japan) connect ed t o a GC-MS syst em (Agilent 7890A/5975C, Agilent Technologies AB, Sweden). The Py-GC int erface and GC inject or wer e bot h at 320 °C. The inject or had a split r at io of 16:1 and H e was t he car r ier gas. The pyr olysat e was separ at ed on a DB-5M S column (30 m × 0.25 mm i.d., 0.25 µm film t hickness; J& W, Agilent Technologies AB, Sweden). The GC t emper at ur e pr ogr am was: 40 °C

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t o 100 °C at 32 °C/min, t o 120 °C at 6 °C/min, t o 250 °C at 15 °C/min and finally t o 320 °C (held 3 min) at 32 °C/min. The t ot al r un t ime was ca. 19 min. The mass spect r omet er was oper at ed at unit mass r esolut ion, and scanning fr om m/z 35–250 at 6.2 scan/s. For each sample, a t ot al of 88 peaks wer e int egr at ed on t he pyr ogr ams and assigned t o t he following classes: car bohydr at e or igin; guaiacyl; syr ingyl; p-hydr oxyphenol; gener ic phenolic; unknown (Ger ber et al., 2012).

2.3.3. Statistical analysis I n t he fir st subset (13C NM R), t o t est for possible differ ences in t he r elat ive int ensit ies bet ween pellet s and t he cor r esponding or iginal samples, t hese samples wer e compar ed in each spect r al r egion using pair ed t -t est s. No spr uce deep soil (sit e 4) sample was included as t he NM R spect r um of it s pellet pr esent ed evidence of r esidual DM SO (t ot al n = 11). A cor r elat ion t est bet ween t he pellet cont r ibut ion and SOM cont ent of t he sample was per formed t o t est if dissolut ion efficiency might be syst emat ically influenced by SOM cont ent . All st at ist ical analysis was per for med wit h M ATL AB 8.2 (R2013b; M at hWor ks, Nat ick, M assachuset t s, USA) wit h an  value of 0.01. A pr incipal component analysis (PCA) of t he 88 cent er ed and scaled Py-GC-M S par amet er s (peak intensit y) was per for med t o summar ize t he var iance in SOM composit ion in t he second subset of samples (or iginal samples and t heir dissolved fr act ion; n = 6) on a single fact or ial map. The PCA was per for med wit h t he R envir onment soft war e v.3.2.0 (R Cor e Team, 2016) using t he fact oext r a R package (Kassambar a and M undt , 2016).

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3. Resul t s and di scussi on

3.1. Dissolution efficiency and method limitations We det er mined t he fr act ion of each pellet r elat ive t o t he cor r esponding or iginal sample. The pellet s account ed for ca. 50–81% of t he mass of t he samples (Table A.1). For t he soil samples, t he SOM cont ent was not r elat ed t o pellet cont r ibut ion (r = −0.33; p = 0.58), indicat ing t hat t r aces of miner al soil did not seem t o significant ly incr ease t he pr opor t ion of t he pellet . Compar ison of t he 1D NM R M AS DP spect r a of t he pellet s and t heir cor r esponding or iginal samples showed no differ ence in t he dist r ibut ion of t ot al int ensit y among t he var ious spect r al r egions (Table B.1) except for t he alkyl r egion (Fig. B.1). Ther e was a small (13%) r elat i ve decr ease in alkyl signal int ensit y in t he pellet vs. t he or iginal sample (Table B.1). This pr esumably r eflect ed t he high solubility of aliphat ic compounds (such as lipids and waxes) in DM SO, while solid component s would be par t ly shielded fr om dissolut i on. The pr efer ent ial dissolut ion of aliphat ic compounds was expect ed, but fr act ionat ion among aliphat ic compounds is unlikely, and t he lower dissolut ion of ot her component s pr obably r eflect ed lar ger sample gr ains simply being physically pr ot ect ed fr om dissolut ion. We focused t he Py-GC-M S analysis on t he car bohydr at es and lignin component s t o ver ify whet her fr act ionat ion affect ed t he molecular fr agment s in t hese macr omolecules; fr act ionat ion of aliphat ic compounds could be consider ed unlikely because t hese ar e t he most soluble. No significant differ ences in t he molecular fr agment s could be det ect ed bet ween t he or iginal and dissolved fr act ion samples, as shown by t he r esult s of t he PCA (Fig.

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B.2; Table B.2). On t he biplot s pr esent ing t he fir st four PCs, which cumulat ively explained 75.7% of t he var iance, t he ellipses, indicat ing wher e 98% of samples in t he same cat egor y wer e expect ed t o occur , significant ly over lap for t he t wo gr oups of samples. I t is impor t ant t o highlight t hat only 88 peaks wer e obt ained in t he pyr ogr ams and t hey likely do not cover all t he moiet ies pr esent in t hese OM samples. M or eover because only par t of t he samples dissolved in DM SO, t he dissolved par t may not be fully r epr esent at ive of t he t ot al sample. Ther efor e t he r esult s have t o be int er pr et ed wit h car e. This should be consider ed as a semi -quant it at ive met hod, and conclusions can be dr awn only in t er ms of t he DM SO-ext r act s composit ion.

3.2 Enhanced str uctur al infor mation in 2D H SQC spectr a The 1D 13C spect r um of t he spr uce needles displayed 7–8 br oad signal envelopes (Fig. 1). I nt egr als of 0–45, 45–60, 60-90, 90–110, 110–160 and 160–220 ppm envelopes, r espect ively, indicat e t he alkyl C, N -alkyl C and met hoxy C, O-alkyl C, di-O-alkyl C, ar omat ic C and car boxyl/car bonyl cont ent (K ögel-Knabner , 1997). Unlike 1D NM R, t he 2D spect r um r esolved signals fr om numer ous moiet ies wit hin t hese envelopes (Fig. 1). The 2D spect r um displays 1H and 13C chemical shift s along t he hor izont al and ver t ical axes, r espect ively. “Cr oss peaks” r epr esent ing CH gr oups can be displayed by cont our lines, and t he volume (int egr al) of each signal r eflect s t he amount of t he cor r esponding moiet y in t he sample. The H SQC spect r um could be divided int o t he following r egions (Fig. 1): alkyl C (δ C/δ H 10–

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50/0.5–3.0 ppm); car bohydr at es (50–110/2.5–5.5 ppm); and ar omat ic C (105– 150/6.0–8.5 ppm). H er e, we r efer t o t he over lapping 1D met hoxy, N -alkyl and Oalkyl spect r al r egions as t he non-anomer ic car bohydr at e r egion (50–90/2.5–5.5 ppm) and t he 1D di -O-alkyl r egion as t he anomer ic car bohydr at e r egion (90– 110/4–5.5 ppm; Fig. 1). Wit hin each of t hese spect r al r egions in t he H SQC spect r um, dozens of individual cr oss peaks could be isolat ed, each r eflect ing one or mor e moiet ies (Fig. 1). H SQC NM R analysis cannot det ect quat er nar y car bons (e.g. car boxyl gr oups), but some moiet ies car r ying quat er nar y car bons can st ill be ident ified fr om t heir r espect ive CH gr oups (e.g. lignin unit s). H owever , t he pr esence of par amagnet ic ions in miner al soil samples would br oaden t he NM R signals, r educing sensit ivit y and r esolut ion. Ther efor e, soil samples r ich in heavy met als would r equir e pr e-t r eat ment .

3.3. Signal assignments I n conjunct ion wit h r epor t ed dat a for plant macr omolecules (e.g. lipids, hemicelluloses, cellulose, and lignin fr agment s), many signals in t he 2D H SQC spect r a wer e t ent at ively assigned t o specific molecular moiet ies, as illust r at ed for t he super ficial spr uce soil sample in Fig. 2. All assignment s ar e compiled in Table 1. The alkyl r egion was dominat ed by signals fr om CH 2 unit s of molecular fr agment s fr om lipids, pept ides, fr ee fat t y acids, waxes and st er oids (Table 1; Fig. 2a). I n addit ion, t wo ot her t ypes of signal wer e det ect ed as follows: acet yl gr oups fr om hemicelluloses and lignin, and non-specific t er minal met hyls. Signals fr om

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fr agment s assigned t o var ious polysacchar ides (most ly cellulose and hemicelluloses) wer e found in t he car bohydrat e r egions (Table 1; Fig. 2b,c). For t hese polysacchar ides, given t he r esolut ion, it is possible t o det er mine glycosidic linkage t ypes and t he posit ions of t he sugar unit s, i.e. int er nal, r educing end (R) or non-r educing end (NR). I n t he ar omat ic r egion, signals likely or iginat ing fr om lignin, suber in or t annin monomer s and unsat ur at ed compounds could be r esolved. These cr oss peaks wer e t ent at ively assigned t o C2/H 2, C5/H5, and C6/H6 of t he guaiacyl unit , C3/H 3 + C5/H 5 and C8/H 8 of t he p-coumar yl unit , and C2/H 2 + C6/H 6 of t he p-hydr oxyphenyl unit (Table 1; Fig. 2d). The definit e assignment of specific cr oss-peaks in var ious OM samples would r equir e complement ar y (2D) exper iment s like TOCSY, COSY, and H M BC. Once t his is done, infor mat ion in t he 2D spect r a can be used t o t r ack specific molecular moiet ies in SOM and follow t heir incor por at ion int o var ious polymer s t hat may have differ ing pr oper t ies for soil dynamics (for mat ion and degr adat ion).

3.4 Compar ison of DM SO-extr acts of bir ch leaf litter and spruce needle li tter To account for t he differ ences in dissolut ion efficiency (Table A.1) and C cont ent bet ween t he bir ch leaf lit t er and spr uce needle lit t er , t he spect r um of t he bir ch leaf lit t er ext r act was scaled down by a fact or 0.941 for t he compar ison (Fig. 3). The 2D spect r a r evealed numer ous differ ences bet ween t hese ext r act s in specific moiet ies (Fig. 3). For example, t he 2D spect r um of t he spr uce needles ext r act included signals assigned t o side chains of hemicelluloses and lignin (Fig. 3a) t hat wer e not

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pr esent in t he spect r um of t he bir ch lit t er ext r act . Bir ch lignin is known t o cont ain fewer β-5 linkages t han spruce lignin (Adler, 1977), t his mat ches our obser vat ion of weaker signals fr om β-5 linkages in phenylcoumar an moiet ies in t he bir ch lit t er ext r act (Fig. 3a). Ot her est ablished differ ences bet ween t hese t wo lit t er t ypes like t he lower lignin cont ent and var iabilit y in lignin moiet ies in t he deciduous lit t er vs. t he conifer ous (Nur mi, 1993; Nur mi, 1997; K ang et al., 2009) explain some of t he cont r ast ed signals we obser ved in our spect r a compar ison. I n t he ar omat ic r egion, signals associat ed wit h lignin wer e gener ally less int ense in t he spect r um of bir ch leaf ext r act t han in t he spect r um of spr uce needle ext r act (Fig. 3b). Specifically, t he spect r um of bir ch leaf ext r act showed weaker signals fr om guaiacyl unit s (t he cr oss peaks assigned t o C2/H 2 and C6/H 6 wer e ver y weak) and much weaker cont r ibut ions fr om p-hydr oxybenzoat e and p-hydr oxyphenyl unit s (Fig. 3b).

3.5. Chemical differ ences in OM extr acts at differ ent depths of the or ganic hor izon As leaf and needle lit t er pr ovides a k ey C input for soil, st r ongly influencing SOM composit ion, we compar ed needle lit t er and soil samples fr om a spr uce dominat ed sit e. To account for t he differ ences in dissolut ion efficiency (Table A.1) and C cont ent bet ween t he t wo samples, t he soil ext r act spect r um was scaled up by a fact or 1.269 for t he compar ison (Fig. 4). I n the 2D spect r a, most of t he major st r uct ur es pr esent in t he lit t er ext r act wer e also det ect ed in t he soil ext r act (Fig. 4), but some wer e missing. Par t icular ly, t he int ensit ies of lignin signals (guaiacyl,

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p-hydr oxybenzoat e, p-hydr oxyphenyl) wer e weaker in t he soil ext r act spect r um (Fig. 4a). L ignin degr adat ion involves side chain oxidat ion and demet hylat ion/demet hoxylat ion (Feng et al., 2008): t his explains t hat signals fr om lignin side chains in t he non-anomer ic C r egion wer e weaker in t he soil ext r act spect r um, and signals fr om met hoxy gr oups and acet yl gr oups in t he alkyl r egion wer e slight ly weaker (dat a not shown). M oreover , t he signals pr esent in t he soil ext r act , but not in t he lit t er ext r act (Fig. 4a), mat ched t hose of oxidized lignin unit s (Azar pir a et al., 2014), cor r obor at ing t he par t ial degr adat ion of t he lignin unit s or iginally pr esent in t he spr uce needles. Car bohydr at e signals in t he anomer ic r egion wer e qualit at ively similar but t her e wer e fewer signals in t he spect r um of t he soil ext r act t han in t hat of t he lit t er ext r act (Fig. 4b). Fur t her mor e, t he signal r at io of hexoses (glucose, mannose and galact ose) t o xylose was higher in t he spect r um of t he soil ext r act , in accor d wit h r epor t s t hat hemicelluloses ar e degr aded mor e r apidly t han cellulose dur ing lit t er decomposit ion (Ber g and M cClaugher t y, 2013). H owever , secondar y for mat ion of hexoses in t he soil also cont r ibut es (Baldock et al., 1990), as indicat ed by signals associat ed wit h fungal st r uct ur es pr esent in t he spect r um of t he soil ext r act (Fig. 4b), specifically st r uct ur es cont aining glycosidic linkages in fungal β-glucans (K im et al., 2000; L owman et al., 2011; Rut hes et al., 2013) and (put at ively) t he chit in N acet yl gr oup (Fig. 2a). An incr ease in galact ose and mannose (β-D-Galp, α-D and βD-M anp moiet ies) compar ed wit h xylan signals in t he soil ext r act (Fig. 4b) cor r obor at es r epor t s t hat micr obes cont r ibut e t o SOM dynamics by degr ading plant

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mat er ial and synt hesizing new biomass and pr oduct s t hat play key r oles in st able C pools (Baldock et al., 1990, 1992; Six et al., 2006; Der r ien et al., 2007). I n t he spect r um of t he soil ext r act , while t he G5, G2 and G6 signals of t he guaiacyl unit s ar e expect ed t o have a cer t ain r at io, t he r elat ive int egr al r at ios of t hese signals appear ed t o be skewed in favor of G5 her e (Fig. 4a), indicat ing t hat signals fr om anot her moiet y wer e over lapping. This could be due t o an incr ease in p-coumar yl unit s (pCA3/5; pCA8) linked t o t he pr esence of fungal melanin (Sjöber g et al., 2004). This highlight s anot her advant age of 2D spect r a, namely t hat t he pr esence of sever al signals for individual moiet ies enables t heir det ect ion despit e par t ial over lap. Finally, plant r oot s, which ar e abundant in t he or ganic layer s, ar e char act er ized by a high suber in cont ent (K ögel -K nabner , 2002), t his could explain t hat signals assigned t o suber in (vinylic gr oups and glycer ol; Table 1) wer e found in t he spect r um of t he soil ext r act but not in t he lit t er ext r act . The r esult s collect ively show t hat full elucidat ion of t he chemical composit ion of SOM r equir es analysis of not only above-gr ound lit t er but all significant OM input s, not ably woody mat er ial, r oot s and micr obial biomass (Baldock et al., 1990; von Lüt zow et al., 2007). The following examples illust r at e how 2D NM R can pr ovide addit ional infor mat ion t o r esolve conflict ing r esult s and/or unr esolved obser vat ions. Cut in signals wer e st r onger in lit t er t han in soil ext r act s (Fig. 4a; Table 1). Pot ent ial degr adat ion of cut in has been r epor t ed (H illi et al., 2012), but wit h t he appr opr iat e samples, t hese conflict ing obser vat ions could be easily set t led using a 2D NM R exper iment . The

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2D spect r a of t he t wo peat ext r act s wer e ver y similar (not shown), which agr ees wit h pr evious r esult s t hat showed t hat t he decomposit ion of bulk mat er ial was ver y limit ed over 700–1000 yr (cor r esponding t o t he 50 cm dept h differ ence; L ar sson et al., 2016). Even t he chemical differ ences bet ween t he ext r act s of t he

Sphagnum fuscum moss and t he fir st peat layer (150 cm dept h) appear ed t o be limit ed. To account for t he differ ences in dissolut ion efficiency (Table A.1) and C cont ent bet ween t he t wo samples, t he peat ext r act spect r um was scaled down by a fact or 0.652 for t he compar ison (Fig. 5). H owever , t he 2D spect r a r esolved specific changes. I n t he non-anomer ic car bohydr at e r egion, signal int ensit ies wer e lower in t he peat ext r act spect r um t han in t he Sphagnum moss ext r act spect r um (Fig. 5; Table 1). I n t he anomer ic car bohydr at e r egion, t her e was also a gener al r educt ion in signal int ensit y, and signals assigned t o some st r uct ur es (α- and β-L ar abinofur anosyl r esidues and oligomer s of bot h α-glucose and β-Dgalact opyr anose) (Fig. 5) had disappear ed. This pr ovides oppor t unit ies t o addr ess challenging issues, par t icular ly why car bohydr at e polymer s ar e gener ally easily degr adable under anoxic condit ions (Ber gman et al., 1999) but t hose of Sphagnum ar e highly r esist ant t o micr obial degr adat ion and accumulat e in peat (Bohlin et al., 1989; Hájek et al., 2011). Not ably, 2D NM R met hods could be used t o follow changes in car bohydr at e polymer s wit h dept h in peat lands, t her eby helping t o explain t he pr esence and accumulat ion of supposedly easily degr adable car bohydr at es at consider able soil dept hs (Dungait et al., 2012) and t o elucidat e humificat ion and peat for mat ion pr ocesses. Complement ar y NM R t echniques, such

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as CM P-NM R (Cour t ier -M ur ias et al., 2012) which have been r ecent ly applied t o whole soil by M asoom et al. (2016), will aid t he under st anding of complex int er act ions bet ween wat er and SOM , fur t her compr ehension of SOM dynamics, as well as pr ovide addit ional det ailed st r uct ur al i nfor mat ion in-situ .

4. Concl usi ons The met hod combines simple sample pr epar at ion, r easonable dat a acquisit ion t imes, r elat ively simple dat a analysis and spect r al compar ison. Wit h 2D NM R spect r oscopy, a wide r ange of st r uct ur al component s can be ident ified in SOM . The higher r esolut ion pr ovided by (2D) 1H –13C N M R r evealed det ailed species-r elat ed chemical differ ences bet ween t he DMSO-extr act s of lit t er s. H ence, it has gr eat pot ent ial t o compar e SOM in differ ent ecosyst ems or under differ ent exper iment al t r eat ment s, facilit at ing effor t s t o elucidat e lit t er and SOM decomposit ion dynamics under nat ur al and exper iment al condit ions (e.g., Talbot et al., 2012). Twodimensional NM R spect r oscopy also moves beyond t he shor t comings of many ot her t echniques by pr ovi ding t he r esolut ion r equir ed for mechanist ic st udies of SOM t ur nover . Using H R-M AS spect r oscopy, 2D exper iment s have for inst ance allowed t o follow t he evolut ion of t he molecular composit ion of SOM t hr ough decomposit ion (K elleher et al. 2006). Using specific monomer s and linkage t ypes as biomar ker s, it can indeed non-dest r uct ively det ect newly for med polymer s, and t her efor e ident ify biochemical pr ocesses r esponsible for SOM for mat ion and t ur nover . Thus, it is a pot ent t ool for det er mining and t r acking st r uct ur al moiet ies of micr obial or igin, i.e.

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bact er ial and fungal input s, in soil and peat samples wher e fungi cont r ibut e t o t he decomposit ion pr oduct s (K ļ avi ņš et al., 2008). Similar ly, using H R-M AS spect r oscopy, Spence et al. (2011) car r ied 2D exper iment s on 13C and 15N labeled soil micr obial biomass and leachat e t o t r ack t heir degr adat ion over t ime. Thus, 2D NM R can r elat e var iat ion in miner alizat ion r at e t o differ ences in molecular composit ion. Using chr onosequences, it could also be applied t o unr avel SOM t ur nover over t ime. Anot her possible applicat ion is t o follow pollut ant s/cont aminant s in soils and t heir int er act ions wit h SOM similar ly t o what was done by M asoom et al. (2016). I n conclusion, because st r uct ur al moiet ies ar e t he unit s r ecognized by enzymes involved in br eakdown, 2D NM R spect r oscopy is suit able t o ident ify compounds and biochemical pat hways as well as t o unr avel ecologically r elevant quest ions, such as t he r esponse of SOM t o climat e change. Or ganic soils r epr esent a huge C pool wit h a significant cont r ibut ion t o t he global C cycle, specifically in t he cont ext of climat e change. H er e we pr ovided an example of t he t echnique’s applicat ions and fur t her st udies should be r un t o ensur e t hat it could be used in a quant it at ive way, specifically for miner al -r ich samples.

Ack nowl edgment s The aut hor s acknowledge assist ance fr om M . H edenst r öm and T. Spar r man in t he acquisit ion of t he NM R spect r a, t he “NM R for L ife” infrast r uct ur e suppor t ed by t he 22

Wallenber g foundat ions, t he K empe foundat ion for financially suppor t ing L .S., and t he Swedish Resear ch Council for suppor t ing J.S. and M .N.. L ast ly, t he aut hor s acknowledge t wo anonymous r eviewer s for their t ime and valuable comment s on t he manuscr ipt . Refer ences Adler , E., 1977. L ignin chemist r y: past , pr esent and fut ur e. Wood Science and Technology 11, 169-218. Azar pir a, A., Ralph, J., L u, F., 2014. Cat alyt ic alkaline oxidat ion of lignin and it s model compounds: a pat hway t o ar omat ic biochemicals. BioEner gy Resear ch 7, 7886. Baldock, J.A., Oades, J.M ., Vassallo, A.M ., Wilson, M .A., 1990. Significance of micr obial act ivit y in soils as demonst r at ed by solid-st at e 13C NM R. Envir onment al Science & Technology 24, 527-530. Baldock, J.A., Oades, J.M ., Wat er s, A.G., Peng, X., Vassallo, A.M ., Wilson, M .A., 1992. Aspect s of t he chemical st r uct ur e of soil or ganic mat er ials as r evealed by solid-st at e 13C NM R spect r oscopy. Biogeochemist r y 16, 1-42. Bat jes, N.H ., 1996. Tot al car bon and nit r ogen in t he soils of t he world. Eur opean Jour nal of Soil Science 47, 151-163. Ber g, B., M cClaugher t y, C., 2013. Plant L it t er : Decomposit ion, H umus For mat ion, Car bon Sequest r at ion, 3r d Edit ion. Spr inger , Ber lin, Ger many.

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Feng, X., Simpson, A.J., Wilson, K .P., Williams, D.D., Simpson, M .J., 2008. I ncr eased cut icular car bon sequest r at ion and ligni n oxidat ion in r esponse t o soil war ming. Nat ur e Geoscience 1, 836-839. Feng, X., Simpson, M .J., 2011. M olecular -level met hods for monit or ing soil or ganic mat t er r esponses t o global climat e change. Jour nal of Envir onment al M onit or ing 13, 1246-1254. Fer r eir a, R., Gar cia, H ., Sousa, A.F., Pet kovic, M ., L amosa, P., Fr eir e, C.S.R., Silvest r e, A.J.D., Rebelo, L .P.N., Per eir a, C.S., 2012. Suber in isolat ion fr om cor k using ionic liquids: char act er isat ion of ensuing pr oduct s. New Jour nal of Chemist r y 36, 2014-2024. Ger ber , L ., Eliasson, M ., Tr ygg, J., M or it z, T., Sundber g, B., 2012. Mult ivar iat e cur ve r esolut ion pr ovides a high-t hr oughput dat a pr ocessing pipeline for pyr olysisgas chr omat ogr aphy/mass spect r omet r y. Jour nal of Analyt ical and Applied Pyr olysis 95, 95-100. H aiber , S., Bur ba, P., H er zog, H., L amber t , J., 1999. Elucidat ion of aquat ic humic par t ial st r uct ur es by mult ist age ult r afilt r at ion and t wo-dimensional nuclear magnet ic r esonance spect r omet r y. Fr esenius' jour nal of analyt ical chemist r y 364, 215-218. H ájek, T., Ballance, S., L impens, J., Zijlst r a, M ., Ver hoeven, J.A., 2011. Cell -wall polysacchar ides play an impor t ant r ole in decay r esist ance of Sphagnum and act ively depr essed decomposit ion in vit r o. Biogeochemist r y 103, 45-57.

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H edenst r öm, M ., Wiklund-L indst r öm, S., Öman, T., L u, F., Ger ber , L ., Schat z, P., Sundber g, B., Ralph, J., 2009. I dent ificat ion of lignin and polysacchar ide modificat ions in Populus wood by chemomet r ic analysis of 2D NM R spect r a fr om dissolved cell walls. M olecular Plant 2, 933-942. H er t kor n, N., Per min, A., Per minova, I ., K ovalevskii, D., Yudov, M ., Pet r osyan, V., K et t r up, A., 2002. Compar at ive analysis of par t ial st r uct ur es of a peat humic and fulvic acid using one- and t wo-dimensional nuclear magnet ic r esonance spect r oscopy. Jour nal of envir onment al qualit y 31, 375-387. H er t kor n, N., Benner , R., Fr ommber ger , M ., Schmit t -K opplin, P., Wit t , M ., K aiser , K ., K et t r up, A., H edges, J.I ., 2006. Char act er izat ion of a major r efr act or y component of mar ine dissolved or ganic mat t er . Geochimica et Cosmochimica Act a 70, 2990-3010. H illi, S., St ar k, S., Willför , S., Smeds, A., Reunanen, M ., H aut ajär vi, R., 2012. What is t he composit ion of AI R? Pyr olysis-GC/M S char act er izat ion of acidinsoluble r esidue fr om fr esh lit t er and or ganic hor izon s under bor eal for est s in sout her n Finland. Geoder ma 179-180, 63-72. I nt er gover nment al Panel on Climat e Change, 2007. Climat e Change 2007: The Physical Science Basis, Cont r ibut ion of Working Gr oup I t o t he Four t h Assessment Repor t of t he I PCC, Cambr idge Univer sit y Pr ess, Cambr idge, UK . Jobbágy, E.G., Jackson, R.B., 2000. The vert ical dist r ibut ion of soil or ganic car bon and it s r elat ion t o climat e and veget at ion. Ecological Applicat ions 10, 423-436.

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K ang, H., Ber g, B., L iu, C., West man, C., 2009. Var iat ion i n mass-loss r at e of foliar lit t er in r elat ion t o climat e and lit t er quality in Eur asian for est s: differ ences among funct ional gr oups of lit t er . Silva Fennica 43, 549-575. K assambar a, A., M undt , F., 2016. fact oext r a: Ext r act and Visualize t he Result s of M ul t ivar iat e Dat a Analyses. K eeler , C., M aciel, G.E., 2000. 13C NM R spect r al edit ing of humic mat er ial. Jour nal of M olecular St r uct ur e 550–551, 297-305. K elleher , B.P., Simpson, A.J., 2006. Humic subst ances in soils: ar e t hey r eally chemically dist inct ? Envi r onment al science & t echnology 40, 4605-4611. K elleher , B. P., Simpson, M . J., Simpson, A. J., 2006. Assessing t he fat e and t r ansfor mat ions of plant r esidues in t he t er r est r ial envir onment using H R-M AS NM R spect r oscopy. Geochimica et Cosmochimica Act a 70, 4080-4094. K im, H ., Ralph, J., Akiyama, T., 2008. Solut ion -st at e 2D NM R of ball-milled plant cell wall gels in DM SO-d6. BioEner gy Resear ch 1, 56-66. K im, H ., Ralph, J., 2010. Solut ion-st at e 2D NM R of ball-milled plant cell wall gels in DM SO-d6/pyr idine-d5. Or ganic & Biomolecular Chemist r y 8, 576-591. K im, H ., Ralph, J., 2014. A gel -st at e 2D-NM R met hod for plant cell wall pr ofiling and analysis: a model st udy wit h t he amor phous cellulose and xylan fr om ball milled cot t on lint er s. RSC Advances 4, 7549-7560. K im, Y., K im, E., Cheong, C., Williams, D.L ., K im, C., L im, S., 2000. St r uct ur al char act er izat ion of β-d-(1→3, 1→6)--linked glucans using NM R spect r oscopy. Car bohydr at e r esear ch 328, 331-341.

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von L üt zow, M ., K ögel-K nabner , I ., Ekschmit t , K ., M at zner , E., Guggenber ger , G., M ar schner , B., Flessa, H ., 2006. St abilizat ion of or ganic mat t er in t emper at e soils: mechanisms and t heir r elevance under differ ent soil condit ions - a r eview. Eur opean Jour nal of Soil Science 57, 426-445. von L üt zow, M ., K ögel-K nabner , I ., Ekschmit t , K ., Flessa, H ., Guggenber ger , G., M at zner , E., M ar schner , B., 2007. SOM fr act ionat ion met hods: Relevance t o funct ional pools and t o st abilizat ion mechanisms. Soil Biology and Biochemist r y 39, 2183-2207. Wishar t , D.S., Bigam, C.G., H olm, A., H odges, R.S., Sykes, B.D., 1995. 1H , 13C and 15N r andom coil NM R chemical shift s of t he common amino acids. I . I nvest igat ions of near est -neighbor effect s. Jour nal of Biomolecular NM R 5, 67-81. Yassir , I ., Buur man, P., 2012. Soil or ganic mat t er chemist r y changes upon secondar y succession in I mper ata Gr asslands, I ndonesia: A pyr olysis-GC/M S st udy. Geoder ma 173-174, 94-103. Yu, Z., L oisel, J., Br osseau, D.P., Beilman, D.W., H unt , S.J., 2010. Global peat land dynamics since t he L ast Glacial M aximum. Geophysical Resear ch L et t er s 37, L 13402. Zhong, J., Sleight er , R.L ., Salmon, E., M cK ee, G.A., H at ch er , P.G., 2011. Combining advanced NM R t echniques wit h ult r ahigh r esolut ion mass spect r omet r y: A new st r at egy for molecular scale char act er izat ion of macr omolecular component s of soil and sediment ar y or ganic mat t er . Or ganic

36

Geochemist r y; Applicat ions and development s of magnet ic r esonance t echniques in Geosciences 42, 903-916.

Fi gur e capt i ons

Fi g. 1. 2D 1H -13C HSQC spect r um of DM SO-ext r act of a super ficial spr uce soil and cor r esponding solid st at e 1D 13C CP spect r um of t he soil (see Er hagen et al., 2013 for det ails). The common spect r al r egions consider ed in 13C NM R spect r a (K ögel K nabner , 1997) ar e indicat ed on t he left , and t he four common 2D spect r al r egions ar e indicat ed by r ect angles. I t is impor t ant t o emphasize t hat solid-st at e NM R and H SQC do not obser ve exact ly t he same component s: 1H-13C HSQC shows only pr ot onat ed car bons and only t he fr act ion br ought int o solut ion. The over lay is simply t o demonst r at e t he higher r esolut ion t hat can be obt ained fr om HSQC vs solid-st at e N M R and t o show how t he t echnique could significant ly cont r ibut e t o t he field.

Fi g. 2. Signal assignment s for t he four spect r al r egions (see Fig. 1) of t he H SQC spect r um of t he super ficial spr uce soil DM SO-ext r act . R, r educing end; NR, nonr educing end uni t s; see Table 1 for ot her abbr eviat ions. Assignment s labeled in gr ay ar e t ent at ive.

37

Fi g. 3. Expansion of (a) non-anomer ic car bohydr at e and (b) ar omat ic r egions of t he H SQC spect r a of spr uce needle (black) lit t er and bir ch leaf (r ed) lit t er DM SOext r act s. See Table 1 for abbr eviat ions. Assignment s labeled in gr ay ar e t ent at ive.

Fi g. 4. Expansions of (a) ar omat ic and (b) car bohydr at e r egions of t he HSQC spect r a of spr uce needle lit t er (black) and super ficial spr uce soil (r ed) DM SOext r act s fr om sit e 4. See Table 1 for abbr eviat ions. Assignment s labeled in gr ay ar e t ent at ive; t hose labeled in bold indicat e st r uct ur es of fungal or igin.

Fi g. 5. Det ails of car bohydr at e r egion of t he H SQC spect r a of DMSO-ext r act s of

Sphagnum fuscum (in black) and peat (150–155 cm; in r ed). See Table 1 for abbr eviat ions.

38

39

40

41

42

43

Table 1 Tentative assignments of HSQC signals to moieties in litter, soil and peat samples. Abbreviation DMSO

δC (ppm) 39.5

δH (ppm) 2.49

Referencesa

Acetyl groups

20.4

2.02

25.7

2.00

Methylene units α

31.4–31.6 33.2

1.91–1.97 2.18–2.25

Methylene units in aliphatic chains β to acid or ester

Methylene units β

23.2–24.2 22.9

1.49 1.62

Methylene units in aliphatic chains γ to acid or ester Methylene β in a primary alcohol chain in waxes Methylene β in mid-chain alcohols in waxes

Methylene units γ

18.2

1.05

31.9–33.2

1.37–1.43

36.8

1.27

Terminal methyl groups (unspecific)

Terminal CH3

13.6

0.85

Methyl groups from amino acids in peptides / proteins, fatty acids, lipids, hemicelluloses Methyl groups in waxes

CH3 from peptides + lipids

19.1–22.1

0.81–0.84

(Simpson et al., 2007; Kim & Ralph, 2010) (Dais & Spyros, 2012) (Fang et al., 2001; Simpson et al., 2003; Simpson et al., 2007; Dais & Spyros, 2012) (Deshmukh et al., 2003; Simpson et al., 2003; Simpson et al., 2007; Kim & Ralph, 2010) (Simpson et al., 2007) (Deshmukh et al., 2003) (Deshmukh et al., 2003; Simpson et al., 2003) (Samuel et al., 2011a; Dais & Spyros, 2012) (Simpson et al., 2007)

11.2

0.82

Methylene and methine groups in aliphatic chains: polymethylene chains mainly from lipids/fatty acids and waxes

(CH2)n and CH in aliphatic chains

21.8–37.0

1.23–1.25

Moiety Dimethyl sulfoxide (solvent)b Alkyl region Acetyl groups of hemicellulose and lignin (N-acetyl group from peptidoglycan) Methylene units adjacent to alkene Methylene units α to carboxyl acid or carbonyl of an ester

44

(Kim et al., 2008)

(Fang et al., 2001) (Fang et al., 2001; Simpson et al., 2003; Simpson et al., 2007; Zhong et

al., 2011; Dais & Spyros, 2012)

Non-anomeric C C2/H2 of cellulose

Cellulose Cellulose C2/H2

C2/H2 of reducing end (β) of cellulose C5/H5 of cellulose C3/H3 of internal cellulose

72.3

3.24

Cellulose Rβ C2/H2 Cellulose C5/H5

74.3

3.13

76.1

3.35

74.1

3.57

77.0

3.65

69.9

3.21

C6/H6 of glucan

Cellulose int. C3/H3 Cellulose int. C4/H4 Cellulose NR C4/H4 Cellulose int. C6/H6 Glucan C6/H6

60.4 60.5 59.5

3.45 3.63 3.53

C2/H2 of α-arabinofuranose

Hemicelluloses α-L-Araf C2/H2 80.1

3.97

C5/H5 of α-arabinofuranose

α-L-Araf C5/H5

61.4

3.50; 3.56

Methoxy group of 4-O-methyl-αD-glucuronic acid C4/H4 of internal 4-O-methyl-αD-glucuronic acid linked O-2 to (1→4)-α-D- xylopyranose C2/H2 of xylan ((1→4)-β-Dxylopyranose) C3/H3 of non-reducing end xylan

4-O-MeGlcA (methoxy) Xmga int. C4/H4

58.7

3.32

77.3

3.63

Xylan C2/H2

71.9

3.04

Xylan NR C3/H3

75.5

3.25

C3/H3 of internal xylan

Xylan int. C3/H3

73.4

3.35

C4/H4 of non-reducing end xylan

Xylan NR C4/H4

69.3

3.37

C4/H4 of internal xylan

Xylan int. C4/H4

75.2

3.56

C5/H5 of internal xylan

Xylan int. C5/H5

C2/H2 of 2-O-acetylated xylopyranose

2-O-Ac-β-D-Xylp C2/H2

63.1 63.5 73.6

3.37 3.97 4.58

C2/H2 of 2-O-acetylated

2-O-Ac-β-D-

70.4–70.5

5.26–5.27

C4/H4 of internal cellulose C4/H4 of non-reducing end of cellulose C6/H6 of internal cellulose

45

(Kim & Ralph, 2014) (Kim & Ralph, 2014) (Kim & Ralph, 2014) (Kim & Ralph, 2014) (Kim & Ralph, 2014) (Kim & Ralph, 2014) (Kim & Ralph, 2014) (Çetinkol et al., 2012) (Sun et al., 2012) (Sun et al., 2012) (Sun et al., 2012) (Kim & Ralph, 2014) (Çetinkol et al., 2012) (Kim & Ralph, 2014) (Kim & Ralph, 2014) (Kim & Ralph, 2014) (Çetinkol et al., 2012; Kim & Ralph, 2014) (Çetinkol et al., 2012) (Martínez et al., 2011; Samuel et al., 2011b) (Martínez et al., 2011)

mannopyranose C2/H2 of the β-Dgalactopyranose C3/H3 of the β-Dgalactopyranose β-aryl ether linkages of the lignin units (α = Cα/Hα) β-aryl ether linkages of the lignin units (γ = Cγ/Hγ) Phenylcoumaran (Cβ/Hβ)

Methoxy group of the lignin units

Methylene α in a primary alcohol chain Methylene directly attached to singly bonded ester O Methine group α in mid-chain alcohols Glycerol

Methoxy

Manp C2/H2 β-D-Galp C2/H2

70.1

3.54

(Bubb, 2003)

β-D-Galp C3/H3

72.2

3.63

(Bubb, 2003)

Lignin β-O-4’ (α)

71.0

4.72

β-O-4’ (γ)

59.7

3.74

β-5

51.9 (51.4) 3.62

Methoxy (lignin)

55.6

(Martínez et al., 2011) (Martínez et al., 2011) (Hedenström et al., 2009; Cheng et al., 2013) (Hedenström et al., 2009; Kim & Ralph, 2010; Samuel et al., 2011a; Mao et al., 2013)

Cutin and suberin CH2 α to OH 60.4 (primary chain) CH2OCOR 63.3 CH α to OH (mid- 70.6 chain) 65.4 65.5 74.9 55.6

3.72–3.76

3.37 3.98 3.33

(Deshmukh et al., 2003) (Deshmukh et al., 2003) (Deshmukh et al., 2003) (Ferreira et al., 2012)

3.55 3.64 3.77 3.72–3.76 (Ferreira et al., 2012)

Cα/Hα in amino acids

Proteins/amino acids 51.9/54 Cα/Hα in amino 57.7–57.8 acids

Anomeric C Reducing-end of (1→4)-α-Dgalactopyranose and (1→4)-αD-xylopyranose

α-D-Galp(R) + αD-Xylp(R)

Reducing-end of (1→4)-β-Dgalactopyranose and (1→4)-βD-xylopyranose Reducing-end of β-D-

4.28/4.43 4.12–4.21

(Wishart et al., 1995; Hertkorn et al., 2002)

91.9–92.3

4.90

β-D-Galp(R) + βD-Xylp(R)

96.5–96.8

4.25–4.27

β-D-Glcp(R)

97.2

4.78

(Kim & Ralph, 2010; Çetinkol et al., 2012; Cheng et al., 2013) (Kim & Ralph, 2010; Çetinkol et al., 2012; Cheng et al., 2013) (Hertkorn et al., 2006)

46

glucopyranose Reducing-end of α-Dmannopyranose (1→4)-β-D-mannopyranose

α-D-Manp(R)

93.2

4.88

β-D-Manp

100.1

4.55

Mannans

100.8

4.67

Starch

99.4

5.11

C1/H1 of β-D-galactopyranose α-L-fucopyranosyl

β-D-Galp C1/H1 α-L-fucop

102.5 100.1

4.43 5.10

C1/H1 of cellulose

Cellulose Cellulose C1/H1

102.4

4.32 4.24

C1/H1 of xylans

cellulose (NR) 103.0 C1/H1 Hemicelluloses xylans C1/H1 102.1

C1/H1 of 4-O-methyl-α-Dglucuronic acid

4-O-MeGlcA C1/H1

97.1

5.08

C1/H1 of α-arabinofuranosyl residues with glycosidic linkages to xylose units β-L-arabinofuranose

α-L-Araf 1+2

107.7 106.6

4.77 4.91

β-L-Araf

102.9

5.14

2-O-acetylated mannopyranose

2-O-Ac-β-DManp

98.2

4.71

3-O-acetylated mannopyranose

99.1

4.72

2-O-acetylated xylopyranose

3-O-Ac-β-DManp 2-O-Ac-β-D-Xylp

99.4

4.59

3-O-acetylated xylopyranose

3-O-Ac-β-D-Xylp

101.9

4.25

C1/H1 of non-reducing end of cellulose

Reducing terminal C1/H1 units in α of (1→3, 1→6)-linked β-Dglucans Internal C1/H1 units in α of (1→3, 1→6)-linked β-Dglucans Glycosidic linkages of glucans in 3-O- and 3,6-di-O-substituted residues (C1/H1)

Fungal signals α-C1/H1 (β93.5 glucans)

4.29

(Kim & Ralph, 2010) (Martínez et al., 2011; Cheng et al., 2013) (Kim & Ralph, 2010) (Cheng et al., 2013) (Bubb, 2003) (Kim & Ralph, 2010) (Kim & Ralph, 2010) (Kim & Ralph, 2010) (Sun et al., 2012) (Martínez et al., 2011; Çetinkol et al., 2012) (Kim & Ralph, 2010; Cheng et al., 2013) (Kim & Ralph, 2010) (Kim & Ralph, 2010; Çetinkol et al., 2012) (Kim & Ralph, 2010) (Kim & Ralph, 2010) (Çetinkol et al., 2012)

4.87

(Kim et al., 2000)

internal C1/H1 (β-glucans)

103.0

4.48

(Kim et al., 2000)

3-O- and 3,6-diO-substituted residues (β-

102.9

4.52

(Ruthes et al., 2013)

47

glucans) Aromatic region C2 of guaiacyl unit (L+S)

Lignin (L), tannin (T) and suberin (S) G2 110.7

6.97

C5 of guaiacyl unit (L+S)

G5

115.2

6.58–6.93

C6 of guaiacyl unit (L+S)

G6

118.6

6.76

C3/C5 of p-coumaryl unit (L) C8 of p-coumaryl unit (L) C2/C6 of p-hydroxyphenyl unit (L+S+T)

pCA3/5 pCA8 H2/6

116.0 114.2 128.9

6.61; 6.75 6.51 7.24

C2/C6 of p-hydroxybenzoate unit (L) Oxidized lignin units (L)

PB2/6

130.3

7.82

Vinylic groups (S)

(not shown)

121.7 116.4 114.0 110.5 129.4

7.26 7.32 7.21 7.38 5.31

a

(Lopes et al., 2000; Mattinen et al., 2009; Kim & Ralph, 2010; Martínez et al., 2011; Samuel et al., 2011a; Cheng et al., 2013) (Lopes et al., 2000; Mattinen et al., 2009; Martínez et al., 2011; Samuel et al., 2011a; Cheng et al., 2013) (Lopes et al., 2000; Mattinen et al., 2009; Martínez et al., 2011; Samuel et al., 2011a; Cheng et al., 2013; Mao et al., 2013) (Moon et al., 2005; Mattinen et al., 2009; Kim & Ralph, 2010; Samuel et al., 2011a; Cheng et al., 2013; Mao et al., 2013) (Hedenström et al., 2009) (Azarpira et al., 2014)

(Ferreira et al., 2012)

Spectra of some of these references acquired with a different solvent from DMSO; b used as a

reference for spectra alignment. 48

49

H i ghl i ght s • 2D NM R allowed r eady char act er izat ion of composit ion of or ganic soils ext r act s.• High r esolut ion allowed a wide r ange of st r uct ur al component s t o be obser ved.• Composit ion differ ences due t o var ying above and below gr ound input wer e obser ved.• 2D NM R can help det er mine sour ce and t ur nover of individual molecular moiet ies.