2670729.pdfDecomposition Dynamics of Six Salt Marsh Halophytes as Determined by Cupric Oxide Oxidation and Direct Temperature-Resolved Mass SpectrometryAuthor

8/14/2019 2670729.pdfDecomposition Dynamics of Six Salt Marsh Halophytes as Determined by Cupric Oxide Oxidation and

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Decomposition Dynamics of Six Salt Marsh Halophytes as Determined by Cupric OxideOxidation and Direct Temperature-Resolved Mass SpectrometryAuthor(s): Vincent A. Klap, Patrick Louchouarn, Jaap J. Boon, Marten A. Hemminga and Josvan SoelenSource: Limnology and Oceanography, Vol. 44, No. 6 (Sep., 1999), pp. 1458-1476Published by: American Society of Limnology and OceanographyStable URL: http://www.jstor.org/stable/2670729 .Accessed: 03/11/2013 17:08

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Liunnol. ceanog. 44(6), 1999, 1458-1476(C 1999,by the Ariei-ican ociety f Limnology nd Oceanography, nc.

Decomposition ynamics f six salt marsh alophytes s determined y cupric xideoxidation nd direct emperature-resolved ass spectrometry

Vincent . Klap1Netherlands nstitute f Ecology, Centre of Estuarine and Coastal Ecology, P.O. Box 140, 4400 AC Yerseke,The Netherlands

Patrick ouchouarn2Research Centre n Isotope Geochemistry and Geochronology, University of Quebec at Montreal, CP 8888,Succ. A, Montreal, Quebec, Canada H3C 3P8

Jaap J. BoonFundamental Research on Matter, nstitute or Atomic and Molecular Physics, Kruislaan 407, 1098 SJ Amsterdam,The Netherlands

MartenA. Hemminga nd Jos van SoelenNetherlands nstitute f Ecology, Centre of Estuarine and Coastal Ecology, P.O. Box 140, 4400 AC Yerseke,The Netherlands

AbstractThis paper presents he results f a comparative tudy n the aerobic decomposition f six salt marsh plant

species over a period f 2 yr. n addition o ash-free ry weight AFDW) determination nd elemental nalysis Cand N), two analytic methods' ave been applied o obtain nsight nto he decomposition ynamics f ignin n thevarious lant issues. he analyticmethods re 1) cupric xide CuO) oxidation ollowed y gas chromatography-mass spectrometry GC-MS) and 2) direct emperature-resolved assspectrometry DT-MS).

AFDW lossescould generally e well described y doubleexponential elationswith ime. Carbon-to-nitrogenratios ncreased uring he nitial tages f decomposition nd decreased gain afterward. or five f the ix plantspecies, correlation etween nitial ignin ontent nd AFDW loss wasobserved. ecay dynamics f ignin enoteda rapid relative ncrease uring he first eeksof field xposure, ollowed y stabilizing ontents ver the next 2yr. CuO oxidation ata ndicate he stablishment f a stable lignin ndmember" ithin -2 months. T-MS data,on the contrary, howed ontinuousmodification f the ignin olymer hroughout heduration f the xperiment.

Evidencewas found or he ncorporation f presumably) icrobial -acetylglucosaminen the omplex esidueproduced pon decomposition. ombination f CuO oxidation nd DT-MS data suggested hat ignin egradationproducts ecame attached o the originalmacromolecular aterial nd could still be identified s lignin-derivedmaterial. he data support humification echanism ia condensation f smalldegradation roducts nstead f theselective reservation f certain iomacromoleculeslike ignin).

Lignin, a structural omponent of the cell walls of vas-cular plants and the second most abundant naturally occur-ring polymer in the biosphere (after cellulose), has a highpreservation potential Hedges and Mann 1979a; Swift et al.1979; Kirk and Shimida 1985). Its decomposition is oftenregarded as the rate-limiting tep in the biospheric cycle ofcarbon (Colberg 1988). Notwithstanding ts relatively high

' Present ddress: he FOM/AMOLF nstitute.2 Present ddress: The Marine cience nstitute, he University

of Texas at Austin, 50 Channel View Drive, Port Aransas, exas78373-1267.Acknowledgments

We thank oopNieuwenhuize nd YvonneMaas for performingthe elemental nalyses. Jos Pureveen s thanked or his assistancewith heDT-MSanalysis nd Gert ijkel for his help with hemul-tivariate nalysis.

This is publication 524 of the NIOO Centre or Estuarine ndCoastalEcology,Yerseke, he Netherlands.

recalcitrance, ignin does not behave conservatively uringdecayof plant itter. ndeed,numerous eports xist n ignindegradation with artial r complete mineralization) y bothaerobic nd anaerobic ecomposers. White-rot ungi re thebest known representatives f the first roup Amer andDrew 1980; Chen and Chang 1985; Higuchi 1985a,b; Kirkand Shimida 1985; Harvey t al. 1987; Hedges et al. 1988a;Lewis and Yamamoto 990; Goini t al. 1993; Gamble et al.1994), whereas he second group s represented y bacteria(Benner t al. 1984a,b, 1991; Young and Frazer 1987). Ow-ing to modification f the macromolecular ignin tructureduring ecay, he ignin moiety f fresh lant materialmaybe an unsuitable racer f vascular plant remnants n theenvironment.

Here we present he results f a litterbag tudy n thedecay dynamics f six different altmarsh lant pecies dur-ing 2 yr of aerobicdecomposition. he main objectives orthis tudy were 1) to find ut whether he igninmoiety fhalophytic erbaceous lants hows high resistance o bio-

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Salt marsh halophytes 1459

degradation elative o bulk organic matter nd 2) if so, toestablish chemically efined ndmember f biodegradedlignin uitable o serve as a tracer or halophytic materialexported rom alt marshes nto hemarine nvironment.

Throughout hedecomposition eriod, emaining sh-freedry weights AFDWs) have been determined, s well as or-ganic carbon nd nitrogen ontents. wo different nalyticmethodswere appliedto monitor hemical modification fthe decomposing itter. he first method, xidation f igninwith cupric oxide (CuO) under lkaline conditions, ieldsdistinct lasses of monomeric nd dimeric roducts hat anbe quantitatively nalyzed y meansof gas chromatography(Hedgesand Mann 1979a; HedgesandErtel 1982; Gonii ndHedges 1992). Ratios between hosedifferent roduct las-ses are used as indicative arameters or he diagenetic tageof the material. uO oxidationhas often been applied tostudy he dynamics f lignin decay (Haddad and Martens1987; Hedges et al. 1988a;Benner t al. 1991; Haddad etal. 1992; Gonii t al. 1993; Opsahland Benner 1993, 1995;Louchouarn t al. 1998) andto characterize nd quantify he

inputs f terrigenous atter o diverse oastal and deep-seamarine nvironments Hedges and Mann 1979b;Moran etal. 1991a,b;Gonii ndHedges 1992; Gough t al. 1993;Prahlet al. 1994; Louchouarn t al. 1998).The second methodused is direct emperature-resolved assspectrometry DT-MS), in which polymericmaterial s thermally issociatedinto fragments hat re subsequently nalyzedby means ofmassspectrometryBoon 1992).DT-MShas often eenusedfor he nalysis f ignin nd ignified fossil)plantmaterial(Genuit t al. 1987; Saiz-Jimenez t al. 1987; Stout et al.1988; Boon 1989; Scheijen and Boon 1989; Stout et al.1989;Faix et al. 1990; Pouwels and Boon 1990; Ralph andHatfield 991;Van der Hage et al. 1993;Van der Heijdenand Boon 1994).

Combination f these wo methods rovides hemeans oovercome ome of the imitations f each ndividualmethod(Van Bergen et al. 1994). Oxidationwith CuO is, for n-stance, more suitable for quantitative ignin determination,whereas tructural etailsof the polymer an better e ob-tainedwithDT-MS (Vander Hage et al. 1993). n a separatepaper on the degradation f Spartina nglica litter, t hasbeen shown hat purification f this material s required oobtainvaluableanalytic ata on the ignin raction Klap etal. 1998).The purification ethod pplied was a twofoldenzymatic digestion protease followed by polysacchari-dase), yielding residue alled milled wood enzyme ignin(MWEL).All DT-MS results resented ere concern am-ples that have undergone hispurification rocedure. uOoxidationwas conducted n material hatwas ball milled utotherwise npurified, ecause t has been reported hatmajormatrix-related ffects o not occur on the yield of lignin-derived ompounds pon CuO oxidation Youngand Frazer1987;Gofii nd Hedges 1995).

Experiment

Litterbag xperiment-Aboveground arts f the six hal-ophytic erbaceous lant pecies S. anglica, Festuca rubra,Aster ripolium, imnoniumulgare, rtemisiamaritima, nd

Suaeda maritima were harvested n the first week of Sep-tember 1992 in the Sint Annaland salt marsh 51?36'N,4?07'E). This marsh s situated n the northeastern art ofthe Oosterschelde The Netherlands), n arm of the NorthSea. By the time f harvesting, heplants were n their ar-liest stage of senescence first ellowing f the eaves). Dif-ferent ypesof tissues were selected for each of the threemajor plant groups used in this study: for the grasses S.anglica and F. rubra), he omplete boveground arts wereselected; henonwoody erbs A. tripolium nd L. vulgare)were exclusively epresented y their eaf material; nd theshrub-like erbs A. maritimna nd S. maritimna) ere repre-sented by small twigs, from which thicker ranches wereremoved. . maritima nd S. maritima lready arried eedswith iameters xceeding hemesh ize of the bags.Allplantparts except hose rom . rubra) were ut nto mall pieces-8 cm long and dried with issue paper; -25 g of this ma-terial was put nto nylon itterbags f 12 X 12 cm (1-mmmesh ize)without ny further reatment. he tissuewas ustpaper dried before t was left n the field o avoid unnaturalchanges caused by extensive rying. he bags were sewnshut, weighed, nd brought othe marsh he following ay.They were attached with hin ines to long horizontal tickswithin he canopy of the vegetation, t a distance f - 15cm above the marsh surface. The location was near theboundary etweenmarsh nd intertidal lat. he bags weresubmerged or hort imes < 1 h) during pring ideperiods.Samples were collected 6, 16, 31, 56, 196, 259, 386, and766 d after he start f the experiment.

At the start f the experiment, he weight atiobetweenfresh nd freeze-dried aterial was determined orfive n-dependent amples of each species.This ratio was used tocalculate he dry weights t t = 0 of the samples hatwereleft n the field. This indirect alculation of the starting

weightwas a consequence f our choice to fill he itterbagswith undried tarting material. ubsequently, he ash con-tents f three ubsamples f each of these freeze-dried am-ples were determined y combustion n a muffle urnace t570?Cfor 3 h. The AFDWs of the samplesat t = 0 werethus based on a total f 15 analyses.

At each collection ate, hree ags per species were akenfrom he field. Macromaterial dhering o the bags, for n-stance macroalgae,was rarely noticed, ut n the few oc-curring ases the material was removed. The bag contentswere freeze dried, without revious rinsing, nd weighed.The dry materialwas ground, nd the ash content was de-termined n triplicate or ach litterbag. he low amounts fremaining . tripoliumn aterial fter ome weeks did notallow the determination f the ash contents n triplicate ndafter 1 yr not even in singular. nstead, he material wassavedfor hemical nalyses.After ravimetric nalysis,ma-terial from hree ags was pooled and organic arbon andnitrogen ontents were determined ith a Carlo Erba NA1500 C/N analyzer. he remainingmaterialwas powderedfor 3 h (at 5?C) in a vibratory all mill Retsch MM2) andused for CuO and DT-MSanalysis r for ignin reparationwith ubsequent T-MS analysis.

Milled wood enzyme ignin reparation-MWEL prepa-ration was conducted ccording o the procedure escribed



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1460 Klap et al.

elsewhere Klap et al. 1998). Briefly, 00 mg of the ball-milledmaterial as percolated t room emperature ith th-anol: benzene (2: 1) and acetone: water 9: 1) and exten-sively washed with Milli-Q water. This extractive-freematerialwas incubated or 6 h with 0.07% (w/w)protease(Pronase E from treptonyces riseus, EC 3.4.24.4, 4 Umg-', Merck) n a 50 mMTris olution t pH 7.4 and washedwith Milli-Qwater fterward. hen t was incubated or 12h with .5% (w/w) polysaccharidaseCellulase OnozukaR-10 from Trichoderma iride, EC 3.2.1.4, >1.0 U mg-',Merck) n a 50 mM ammonium cetate olution t pH 4.5and again extensively ashedwith Milli-Qwater fterward.Both ncubations ere carried ut n the dark t 37?Cundercontinuous otation f the test tubes. Finally, ll sampleswere freeze ried o produce residue eadyfor nalysis.

Cupric oxide oxidation-Lignin-derived uO oxidationproducts LOP) were determined y the method evelopedby Hedges and coworkers Hedgesand Ertel 1982; Gonii ndHedges 1992), with light modifications Louchouarn t al.1998). Briefly, 5-50 mg of groundedmaterialwas hydro-lyzed for 3 h in a nitrogen-purged M NaOH solution nthe presence of CuO (-1 g) and Fe(NH4)2(SO4)2 6H2O(-100 mg) within stainless teel minibomb. our suchreaction hambers were oaded, under n inert tmosphere(N2), into larger omb Parr) hat ontained dditional ase.This container as heated n an insulated eating leeve, heheating ate nd final emperature f whichwere controlledby a CN76000-series emperature ontroller Omega Engi-neering).Heating rom oom temperature o a final nternaltemperature f 162?C was performed n 30 min; this finaltemperature ?2?C) was maintained or .5 h. Ethyl anillin(3-ethoxy-4-hydroxybenzaldehyde;ldrich Chemical)wasemployed s a recovery tandard nd was added directly(-20 ,ug) to the minibombs pon cooling. After xidation,samples were acidifiedwith HCI (6N) to pH 1, extractedwith freshly istilled diethyl ther, oncentrated y roto-evaporation, nd dried under N2. Dried extracts were thenstored rozen ntil nalysis.

Gas chromatography-Gas hromatographic nalysis ofsilylated xidation roductswas carried ut with Hewlett-Packard 890 GC fitted ith fused apillary olumn DB-1, 30 m by 0.25 mm .d.; J & W Scientific) ttached o aflame onization etector FID). The injector nd detectorwere maintained t 300?C,and split njections -1/30 to 1/

40) were performed ith helium s carrier as. The oventemperature as programmed o rise from 00?Cto 270?Cat a rate of 4?C min-I and was kept sothermal or 15-20min. Retention ime nd quantity f all eight major mono-meric OP studied ere V, the um of monomeric uaiacylstructures; , the um of syringyl tructures; nd C, the umof p-hydroxycinnamic cids; see below) were determinedfrom heir ID responses. dentification f all lignin-derivedmonomers nd dimerswas performed y GC-MS, and LOPresponse actorswere assigned qual to the recovery tan-dard, ethyl vanillin Goiii and Hedges 1992; Opsahl andBenner 995).

Direct temperature-resolved ass spectrometry-A ou-ble focusing B/E geometry) EOL SX-102 mass spectrom-eter was used for he analysis f -5 ,ug of material. ropsof suspended ample material were deposited on a Pt/Rhwire nd dried under acuum. During he nalysis eriod f90 s, the mass spectrometer canned mass range betweenmlz 20 and mlz 1,000 each second at a resolution f 3,000.Source temperature as 180?C.Filament urrent or pyrol-ysis was programmed rom to 1.5A in 90 s, nducing inaltemperatures f 800?C. Bothelectron mpact El, 16 eV) andchemical ionization CI, NH3, 200 eV) were employed.Sourcepressure orEl was 10-4 Pa, whereas orCI a pres-sure of 20 Pa (NH3)was maintained. ata acquisition wasperformed n a JEOLMP-7000 system. he set of ntegratedspectra f the total on current TIC) intervals etween 0and 60 s was used for factor nalysis by the FOMpyrMAPprogram, modified ersion f the Arthur ultivariate nal-ysis program Infometrix) Hoogerbrugge t al. 1983;Win-dig et al. 1983). Sampleswere analyzed n singular xceptfor A. maritima nder El conditions, n which triplicate

analysiswas performed, hus llowing iscriminant nalysis.

Nomenclature-Some ifferences n terminology xistbe-tween he two fields f lignin esearch, hich re united nthis project. n this paper the terminology f the pyrolysisfield will be applied, o the terms guaiacyl" and "p-hy-droxycinnamic cid" are used rather han "vanillyl" and"cinnamyl henol."

Results and discussion

Gravimetric nalysis-AFDWs of the different lant pe-cies are shown n Fig. 1. As outlined n the Experiment

section, he nitial dry weights f material eft n the fieldwere not directlymeasured ut were calculated n the basisof measured reshweight: ry weight atios fivefold) t t =0. As a consequence, heweight ercentages t t = 0 beara standard eviation round 100%. Decomposition ates orlitter an usually be well described y a double exponentialequation, uggesting hat itter an roughly e regarded sbeing omposed f twofractions ith istinct ecompositionrates Wieder nd Lang 1982). It is evident rom ig. 1 thatin four ut of six cases, the doubleexponentialmodelcurvefits icely ndeed o the weight-loss ata. The calculated e-composition ates nd the relative izes of the wofractions,as well as the quared orrelation oefficients f the fittings,are shown n Table 1.

S. anglica and A. maritima how similar decompositioncurves, defined y equally arge fractions f labile and re-fractory material. For both species, -35% of the initialweights emain fter yr of field decay. A very good fit sachievedfor F. rubra, haracterized y relatively mall dif-ferences n decomposition atesbetween he abile and re-fractory raction. he fact hat no AFDWs were determinedfor this species after 00 d of exposure has undoubtedlyimproved he fit f the curve r2 = 0.98).Actually, eliabledetermination f the size and the decomposition ateof therefractory raction equires ata points t prolonged xpo-sure imes. herefore, or . rubra, he alculated alues for



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Spartina nglica Festucarubra

eco b 1v5 ,,OP rr$ ?vs11111 1 1 I I _ 11111 1. 1 1I

100 - 1. . l0 -.

80 80

? 60 60 . ...

40 40

20 20

0 200 400 600 800 0 200 400 600 800

Aster ripolium Limonium ulgare

100 _ 100-

80 80 -

60- . . . 60-U-

S 40 - 40-

20 20 -

0 200 400 600 800 0 200 400 600 800

Artemisia aritima Suaedamaritima

100 100 -

80 8.. .... .. ...

6 60 - - 60 - -

40 -40 -

20 -....20 - . ....0 0

0 200 400 600 800 0 200 400 600 800

day number day number

Fig. 1. Ash-free ry weight ossesfor he ix plant peciesover period f 766 d. The standarddeviations t t = 0 are due to the experimental et up (see text). The curves represent hebest

fittingouble xponentialmodels X = Ae-ldt + (1 - A)ek2dt). Valuesfor A,

k,and k2 re given

in Table 1.

the respective ractions nd their ecomposition ates houldbe considered s indicative nly. The weight oss of A. tri-polium material ccurs o rapidly hat fter yr not enoughmaterialwas left or sh content etermination. he remark-able shift n weight oss after -20 d suggests very abilefraction omprising 75% of the organicmaterial f A. tri-polium.The uncertainty bout hefraction izes and decom-position ates s mentioned bove for . rubra s smaller orA. tripolium, lthough here re also no data after rolonged

exposure imes vailable for his pecies. The clear kink nthe curve indicates hat modeling he weight oss of thisspecies requires t least a double exponential unction. hegood fit uggests hat hematerial onsists f two fractions,one of which s extremely abile.

A peculiar decomposition urve s calculated or . mar-itima, ndicating shift rom weight oss to weight ncreaseafter 37 d. The outcome f the modelcalculation oes notindicate wo fractions f different tabilities, ut rather ne



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1462 Klap et al.

Table 1. Calculated parameters or chieving he best fit f adouble xponential unction X = Ae-kldt + (1 - A) e-k2dt) o themeasuredweight-loss alues. The accompanying urves re plottedin Fig. 1.

A k, 1-Aki (Organic (10-4 (Organic

Species (10-4 d-l) matter) d-l) matter) r2Spartina 194 0.521 5.3 0.479 0.96Festuca 260 0.276 54.9 0.739 0.98Aster 2280 0.756 35.2 0.242 0.99Li,nonium 749 0.186 12.4 0.829 0.93Artemnisia 122 0.566 4.1 0.476 0.96Suaeda 127 0.773 -13.5 0.260 0.86

decomposing raction A) and a produced fraction 1-A).This does not seem to be a realistic ituation, lthoughslightmass ncrease might ccurbecause of colonization ydecomposers. he magnitude f the pparent ncrease, ow-ever, makes colonization n unlikely rimary ause in thiscase. More probably heheterogeneous omposition f thelitterbag ontents characterized y relatively igh contentsof seeds and woody parts with deviating decompositionrates)has incidentally ed to the peculiarweight-change at-tern.

The other pecies for which he fit s not satisfactory sL. vulgare,which hows unexpected igh weight osses forthe amples ollected fter 86 and 766 d. The 766-dsampleis based on one bag only, ecausethe other ags were ostor torn y that ime the standard eviation hus qualsthatof t = 0). The unexpected igh weight oss during he aterstages of the experiment s explainedby the fact that L.vulgare eaves ose their obust tructure nd become brittleafter ome time. Particles maller han he mesh ize of thelitterbag re lost, which wrongly uggests hemicalratherthan physicaldecomposition. his is an intrinsic isadvan-tage of the itterbag ethod nd leads to an overestimationof the hemical ecomposition ate. n general, eaf tissue s

more usceptible o comminution han ther issues. or theset of plant issues sed n this tudy, he process nly eemsto be of concern or he eaves of L. vulgare.

Elemental analysis The organic carbon and nitrogencontents f both he untreated nd MWEL samples re given

in Table 2. The carbon alues do not how clear orrelationwith exposure ime, but the nitrogen ontents end to in-crease with ncreasing xposure imes, which s in accor-dance with iterature ata Buth and Voesenek 1987; Hem-minga t al. 1988;Benner t al. 1991).It s noteworthy hat,with he exception f L. vulgare, nitrogen ontents re farmore nfluenced y the enzymatic urification han arboncontents. n Fig. 2, the atomicC: N ratios or he untreatedand purified aterial re shown. t can be seen that he ang-es for hetwo types f samples differ onsiderably. he en-zymatic reatment including protease digestion) learlyremoves elatively orenitrogen han arbon, ielding igh-er C: N ratios han or heuntreated amples.Especiallyforthe MWEL samples f the wo grass pecies, he C: N ratios

reachhighvaluesof more han 00. Even higher alues havebeen reported orSpartina lterniflora issue hathad beenextracted ith neutral etergent Buchsbaum t al. 1991). ngeneral, : N patterns or alt marsh macrophytes re char-acterized by increasing alues during he leaching stage,which teadily ecrease to below the nitial value) duringthe ater tages of decomposition Buth and De Wolf 1985;Benner t al. 1991;Buchsbaum t al. 1991).In this study,the C N patterns f the MWEL preparations how betterresemblance o this general pattern han he untreated ma-terial. An exception o this ule s L. vulgare, heC: N ratioof the MWEL of which never eaches lower value than tt = 0.

Oxidation roducts In addition o the carbon nd nitro-gen contents, he measured oncentrations f lignin xida-tion products LOP) and calculated arameters re listed nTable 2. p-Hydroxyphenyl tructures, hich are produced

Untreated material MWEL

50- 130 -- S. anglica 120

Y'F. rubra 1040 - A. tripolium40 -~~~~~~X-~L.ulgare 100

-} A.maritima 90--- S. maritima

z ~~~~~~~~~~~~~800 30 - 7

E i : L ~~~~~~~ S 1~~~~-E0 v60-20-

10 - 2010

0 200 400 600 800 0 200 400 600 800


Fig. 2. AtomicC: N ratios or unpurified nd MWEL samples.



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upon alkaline oxidation of lignin Sarkanen and Ludwig1971; Hedges and Mann 1979a), are not ncluded n this istbecausenonlignin ources uch as marine lankton nd es-peciallybacteria ave been shown o be significant ourcesof these ompounds Hedges et al. 1988b; Gonii nd Hedges1995). Moreover, C-MS analyses of these compounds n-dicate that oelution with other ompounds ccurs, whichhampers eliable uantification Haddad and Martens 987).

In Fig. 3, the ignin ynamics or he different lant pe-cies are expressed y A6, the normalized um mg per 100mg OC) of the main hree uaiacyl nd three yringyl truc-tures roduced pon oxidation. his sum-parameter as beenproposed s a more reliable ndicator f total ignin n non-woodyand herbaceous lant issues han he traditional 8,in which hep-hydroxycinnamiccids are ncluded Opsahland Benner 1995). Those p-hydroxycinnamic cids (p-cou-maric nd ferulic cid) are considered o act as linkages e-tween ignin ndhemicellulosen nonwoody ndparticularlyin gramineous issues Hedges and Mann 1979a; Hartley ndHaverkamp 984; Aiken et al. 1985; Jung nd Ralph 1990)

and therefore re not an integral art f the ignin olymer.Hence, A6 is used to represent ignin n this set of grassyand herbaceous issues. igure 3 indicates hat he six plantspeciescan be roughly ivided nto hree roups: ignin-richS. anglica,A. maritima, nd S. maritima; ignin-poor . tri-poliun and L. vulgare; nd F. rubra, which s characterizedby an intermediate ignin ontent. considerable ncreasein A6 values s found or ll species during hefirst monthof the experiment, overing he leaching tage. After hisperiod, most A6 values vary somewhat round constantvalue until he end of the experiment.

The previous road lassification s confirmed y the val-ues of additional ignin-derived onomers nd dimers Table2). Ratios between dditionalmonomers nd major mono-

mers Mono: A6;Table2) remain ather onstant hroughoutthe degradation eriod t approximately .10 for ll six spe-cies, suggesting qual stabilities or both groups f mono-

10--* S. anglica

v Frubra

8 /


8/20

1464 Klap et al.

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e ooo0 oo s t c n Oc\ C t?t>Ns N

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m m w in om noto4ct ;>X?t? . . . . . . . . . . Oo It NOmmOr, *C::> Or in z z z in m m I t m -4 1 N C- N (- cl C- N C- cl N N

7W c0=vZt- En w w w m m r- z m r- m r- Z W X01 r- 00 00 Z O C, O.C m O.O2 0 N r- r- r4 1- r N 1OOOOOO4r- O- s-

=~~~~~~~~~~-t in m z -4 q in It C m C, Cm - V) r- r N C m m in O

;1 ;-4 ,r X = = p.,_ t > ~~n00 if i3>> It ?NmN0 t? >t)c) 'o ? - V) o:; w w z CN z OIt r- N It O, V m w m m m w

Y~~~~~~~~~ z It z i n W ?t m 0 It C" if z int 00 -4 -. r q vn oo C_ 00voocD7: ur ONC;~t C', 0. r- V) C) - 00 Z r- M 00 Ch n r- - O ON 0 Choo ?ti~~~~~~~~~~~~~~r6 Ct ^ o- \6 C' r- C' \6 06 r- 11 6 00 4 vi \. rt 6 C 0t \T C N 06 o ON

0 M g * YX8 m~V - m CmOC t OC f VI cf OC if) >?>XXnO

*- On t;g?=Xt C, n Z C O 4- if cl Z ) O 't r- ir > 0 't if C, 't c 00 O) Z cn O r-

t? Y E O r S X t ~m n "t 't m m "t cn t "t t "t m m m m n m4mt "t m m cn m cn m cn It It It It m

Ol Oni C,\: .Yt 00 z cn W' ON if m V' in m 0m in CN V' 00S (- - N cn r--- N N m - rqm r-



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mC t m m c c 00 cm N cN

CB >T% Cl>mm m

A mm m 00 mooo > Co O 00O Cl O

C It~c 0 CI> C" ~C mO _C _ _ _I _

Cl C oo ?OU 66666666666666666

66666666>>X666

o O Cl >0000o OCl> - C 00 c r r

00 0 C O O Co i c

IC I >t m 0>0n > n OC 0 N 0 0m It inin t In CN m Ii nn

00t C 00 l - It >t in

Cn 00) >) C

I~tC No oImt , C) in

r- c 't0 --' 00 c n 00 IZ icc C > C l~0;m cfi ci c-i ,6C,, - - 00 0 C0m 0 C,, oo

C) i C) 00 m> m 0 i

Cl , Cl~ l~4 \ -i N

o o 00Oin c t o00

~~~~~r- - N ooom m >t n rm

000inin000O 0n In00c

O 00 00 m OC 00 q r- Oc m C,,COC CDO ttn > , n m cIt ooIt m oo m m00O

tn C- OC)o oin OC tc m m

; ChmP ~~~O) OC q N oo8~~~~ in OC) m~ N It N r OCNO -i 6 4oi vm o6 -4 c. tro c- oo ? t

S \ m m ~mn in OCmc m in C, in 0mz = XNmC~~>XmmtX?m>O

U o?Cl J:

?'G0NNNNP

the alkali-extracted mounts of esterified cids producesclose to constant : V ratios n all plant issues tudied. nline with his explanation, t is observed hat nother om-mon parameter, he ratio between syringyl nd guaiacylstructures S :V), is relatively nvariant pon degradation(Table 2). Only S. maritimna hows a clear ncrease duringthe first 00 d, whereas . anglica and F. rubra how small,more or less consistent : V changes hroughout he wholeduration f the experiment. he remaining hree speciesshow no trend n their :V ratios, ndicating omparablestabilities or guaiacyl and syringyl tructures nder hoseconditions.

Elevated acid over aldehyde ratios Ad:Al) have beenused as indicators or xidative egradation f ignin Hedg-es et al. 1988a; Goni et al. 1993; Nelson et al. 1995). Dia-genetic lteration f the ide chains f ignin as always beenassociatedwith xic decomposition rocesses; however, e-cent evidencehas shown hat hese ratios an increase ub-stantially uring ong-term noxic subaqueous) degradationof nonwoody plant material Opsahl and Benner 1995).

Thus, elevated Ad: Al ratios should be attributed o bothoxic and long-term noxic degradation. hese ratios n thesix plants tudied re presented n Table 2. Contrary o theexpected ncreases n acid over ldehyde atios, he Ad: Al)vratios vanillic cid over vanillin) decreasefor ll six plantspecies during he first ear of degradation. n the secondyear; he ratio ncreases lightly or . anglica, A. tripolium,and L. vulgare. Less evident rends an be extracted romthe (Ad:Al), ratios (syringic cid over syringaldehyde).Nonetheless, ome evidence an be found or generalde-creaseduring hefirstmonth ollowed y stabilization n thesubsequent ear nd a slight ncrease uring he ast year ofdegradation. he observed eduction f Ad: Al ratios n theearly stage of degradationmight e explained by loss of

ester-bound cidic components rom he halophyte issues,as has been observed reviously or eagrasses Opsahl andBenner 1993). Subsequent ncreaseof Ad: Al ratios wouldthen ndicate ommon iagenetic lteration f ignin.

DT-MSanalysis-The DT-MS results oncern nzymati-cally purified ignin esidues MWEL).It was shown for .anglica that untreated issues yield less specific DT-MSspectra han their MWEL counterparts Klap et al. 1998).Thisphenomenon asconfirmed or he ther ive lant pe-cies. Therefore, nly results n purified amples are pre-sented. The specificity f the techniquewas also modifiedusing different onizationmethods, .e., electron mpact El)and chemical onization CI). El is the more universal on-ization echniquewith broad nalyticwindow,whereasCIhas a narrower window with high onizing efficiency orpolysaccharidesPouwels 1989). However, I has also beensuccessfully ppliedfor he onization f dehydrated ono-lignols Van der Hage et al. 1995).More nformation n thetechnique s given lsewhere Boon 1992; Klap 1997).

In general, DT-MS generates omplex spectra, ince nomolecular eparation s conducted rior o analysis.More-ovel; usually the spectra f a defined ime -temperature)window are integrated o provide one spectrum or eachsample. In order to highlight ompositional ariation e-tween ndividual amples, multivariate actor nalysis r dis-



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1466 Klap et al.

S. anglica F. rubra100 100

O~~~~~~~~~~~~~~~~~ oo

90 9080 8070 7060 6~~~~~~K~ ~ ~ ~ 6

E 50 50 \40 0030 --DW 3020 10 20130 - \0/TN10 -Lignin 10

0 , ., I, ., II . I .,I .... I

0 200 400 600 800 0 200 400 600 800

A. tripolium L. vulgare100 12090

80~~~~~~~~~~~~~00~~~~~~~~~~~~~~~~~~~~~60~ ~ ~ ~ ~ ~~~~~~~9

E 0 6040 ~ >50

7 ~~~~~~~4030 - . 3020 2010 10

0 200 400 600 800 0 200 400 600 800

A.maritima S. maritima150 -130140 120 4130 1120j

c,,100 K~~~~~~~~~~~090 K~~~~~~~~~~~980 8

Et 7070

60 5.50

40 V 3~~~~~~~~~~~030 320 2010 100 0

0 200 400 600 800 0 200 400 600 800


Fig. 4. Remaining uantities f dry weight, rganic arbon, otalnitrogen, nd ignin Alig) asa function f time, ormalized o initial mounts.

criminant nalysiswas carried ut on sets of these ntegratedspectra. The essence of this numeric nalysis method n-volves calculation f the weighted verageof all spectra nthe et nd subsequent omparison f ndividual pectra withthis average spectrum. ifferences re expressed s factorscores or (when analyzed in multiplicate) iscriminantscores. Details on the procedure an be found elsewhere(Hoogerbrugge t al. 1983; Windig t al. 1983;Boon et al.1984;Tas 1991; Klap 1997).

Figure 5 shows two average spectra, n this case the re-sults obtained fter l (Fig. 5a) and CI (Fig. Sb) ionizationofA. maritima. oth spectra howmany ignocellulose har-

acteristics see symbol esignations), lthough n differentratios. The CI results Fig. Sb) confirm he high sensitivityof sugars Pouwels and Boon 1990) and dehydrated onifer-yl and sinapyl lcohol (Van der Hage et al. 1995) for thisionizationmethod. n Figs. 6b, 7b, the scores of individualspectra n the first iscriminant r factor unction re plottedrespectively. he zero lines n these plots represent he av-erage spectra.

Sampleswere nalyzed n triplicate n the El mode;hence,discriminant nalysiswas performed Fig. 6). The first is-criminant unction ccounts for 82% of the characteristicvariance =29% of the total variance n the et).The scores



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G G

137 180100 a

SE80 - S 210

50 100 150200 250300516773

C 60 1101 G

0 8

CD ~~~~~~~~~~149> ~~~~~~~~~~G8W 0 S G

a) 19a+GEguiclnt

CD411944 272cc ~~~~~~~~~~~~~~~~~G+S

29 E E

20 -222 252 302 SE332

0

50 100 150 200 250 300 350

mlz

H0

180100b.0AC polysaccharide general)

l + Pc pentose* + H -~hexose

80 -G 0 ~~~~~~~Ei-> lignin general)163 E~~~~~0+- guaiacyl nit

SCl) 60 ~ ~ ~ ~ ~ ~ ~ ~~~1 rE + S ->syringyl unit193

P P> 40- 40- ~132150 0 Ha) H 202220

P0

20 -H

96 114 Li240 270 288 31

100 150 200 250 300 350

MlZFig. 5. AverageDT-MSspectra El andCl, [a] and [b],respectively) f all purified . mnaritimnalitterbag amples.These spectra epresent he ero ines n Figs. 6b and 7b.

in Fig. 6b illustrate he mass spectral ifferences etweenthe ndividual pectra with espect o the average pectrum.The negative DF, axis expresses compositionalmodifica-tions related o the eachingprocess, whereas he positivepart f the DF1space s representative or he decompositionprocess. t must be realized hat hediscriminant pectra fFig. 6 are calculated istributions hat o not loselyresem-

ble the ndividual pectra with he highest nd owest cores;they nly ndicate which ets of masses covary relative othe verage pectrum. he spectrum f day 766, for nstance,still contains he two main peaks of the lower spectrum,m/z 10 and 180, but heir ntensities re ess dominant hanin the spectrum f day 16.

The discriminant pectra re interpreted y comparison



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1468 Klap et al.

100 --~~~~~~~~~fm1lignin egradationroduct

so0 10 G5 178 25S0 5

150

1 8

Co,, / G

40 218~~~H272D40-

1 70 S216 241 \284-~20 -310 3260) ~~~~~~~~~~~~~~~~~~~

0

50 100 150 200 250 300 350

mlZ

3-

2 2

LL b00

0- ...1.....8.....

-1

0 100 200 300 400 500 600 700 800

daynumber

S0

210100

G

ol 0~~~~~~~~l

80E8

C: 60 73 SCD 85 P 13760 167

40 11440> ~~~~~I G31 95 G

-~20 45 124 19

0514

252 279

50 100 150 200 250 300 350

Fig. 6. DT-MS (El)results fter iscriminant nalysis f purified . maritima itterbag et.Scoreplot b) and calculatedpositive a) and negative c) discriminant pectra. ampleswere analyzedin triplicate; rror ars denote tandard eviations f the means. Variance described s 82% ofcharacteristic nd 29% of total variance. ymbols s in Fig. 5.

with peak patterns f pure compounds, olymers, nd plantmaterial r by comparisonwith Py-GC-MSdata obtainedindependently Pouwels 1989; Van der Hage et al. 1993;Klap et al. 1998). Interpretation f the DF1(-) spectrum e-veals a strong ignocellulose haracter. nterpretation f theDF1(+) spectrums muchmore omplicated, ecause t con-tains many peaks with ather igh ntensities ut withoutrecognizable ignature f bio-organicmaterial. onsideringthat his fraction s produced pondecomposition f naturalmaterial, t can probably lso be referred oas "humic ub-

stances." Assigning tructures o individualmasses n suchfull pectra s problematic, ut the imultaneous resence fthe ions m/z 208, 192, 178, 162, 150, and 120 (shadedsquares) n this pectrum trongly uggests he presence fdegraded ignin.The score pattern nd the general eaturesof the discriminant pectra nd the average spectrum revery comparable o the results f S. anglica (Klap et al.1998).Taken ogether, heseDT-MS(El) data describe setof samples with a high average content f lignocellulose(Fig.5). This ignocellulose haracter s enhanced uring he



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* 221 *


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1470 Klap et al.

S. anglica (55,5% var.)

oOj Cb C CP30-~ ~ ~~~~

lii111 I I

20 - heterogeneous rganic raction10- + lignin egradation roducts

- 0 - lignin polysaccharides-10-20 polysaccharides pentose hexose) + lignin-30

0 100200 300 400 500 600 700 800

30 F. rubra (62.6%ar.)20- heterogeneous rganic raction C16:0 FA10-]

LL 0 - .. polysaccharides lignin C16:0FA-10 j-20 polysaccharidespentose)-30-

0 100200 300400 500 600 700 800

20 A. tripolium 52,6%ar.) phenols C0210-T0 - . phenols lipids lignin

L -10-i /-20 lipids lignin guaiacyl syringyl)-30 1____________

0 100 200 300 400 500 600 700 800

L.vulgare (35.0%var.)120 T < , 1 small non-specific-ragments10

I i ........... C02 + polysaccharideshexose)+ mlz 170LL-10 + lignin C16:0 FA-20 m/z 70 + polysaccharideshexose)-30

0 100 200 300 400 500 600 700 800

25 - . maritima 43,3%var.)20-15- heterogeneous rganic raction lignin10- degradation roducts lignin imers0 -. < lignin syringyl guaiacyl) polysaccharides

-5--10-15 I ligninsyringyl) C02

0 100 200 300 400 500 600 700 800

day number

Fig. 8. El score plots of S. anglica, F. rubra, A. tripolium, . ivulgare, nd S. m71aritirnaiththe amounts f variance escribed ndicated ehind he peciesnames.The main haracteristics fthe ccompanying pectra re expressed n the right idesof the plots.For each of theseplots, heupper ine gives the characteristics f the F,(+) spectrum, he middle one those of the averagespectrum, nd the ower ne those f the (-) spectrum. he sequence f mentioned haracteristicsis the sequence f mportance n the pectrum.



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S.anglica (46.6% var.)

30- r I20 - heterogeneous rganic raction

10- (incl.m/z 21, 203, 185 & 77)-t 0 I ... . polysaccharides lignin-10 ]-20 - polysaccharides ligninguaiacyl syringyl)

0 100 200 300400 500 600 700 800

40 F.rubra (60.0%ar.)30- heterogeneous rganic raction20- -Y (incl.m/z 21, 203, 185 & 77)

LL 01 ;......... polysaccharides (some) ignin-10 j _ _ _ _ _ _ _ _ _ _ _ _-30 I polysaccharides (some) ignin

0 100200 300 400 500 600 700 800

20-A. tripolium 68.7%ar.) heterogeneous rganic raction10- (incl.m/z 21, 203,185 &77)0 -{l aritima.eeFig. 8 for explanation.

score plot for F. rubra hows a comparable attern o thatof S. anglica and A. maritima Figs. 8 and 6b, respectively).For this pecies, the undefined eterogeneous rganic rac-tion s negatively orrelated o a pentose raction nstead fa lignocellulose raction. he three emaining lots showdifferent atterns. he scores for A. tripoliumnhift within

month rom xtremely egative o positive nd remain tableafterward. his shift n scores, which orrelates ith he ap-id weight oss (Fig. 1), expresses he onversion f ipid ndlignin nto ome simple phenolic ompounds. hus, n con-trast o the previous pecies, decomposition f most A. tri-poliummaterial oes not gradually roduce complexmix-



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1472 Klap et al.

ture. he scores or . vulgare o not how consistent rendduring he decomposition rocess, nd the factor pectra renot asily nterpretable. t s clear, however, hat ignin s notan important ompoundwith regard o the decompositiondynamics f this pecies.The score plot for . maritima oesnot reveal shift uring he first ew weeks,but the generalcompositional hift rom ignin-rich aterial o an unchar-acterizedheterogeneous rganic fraction s comparable othe rends eenfor . anglica andA.maritima. he relativelyhigh abundance f syringyl nits n the ignin moiety f S.maritima ompared o S. anglica and A. maritima s in ac-cordancewith he CuO oxidation esults Table 2). Also, themainfeatures f the verage pectra not hown) onfirm hedivision f the different lant issues ccording o their ignincontent s determined y CuO oxidation.

The CI results Fig. 9) provide consistent ompositionalshift or ll speciesduring he2 yr f decomposition.n thisperiod, ignin-rich S. maritima) r lignocellulose-richallother pecies)material s changed nto a heterogeneous r-ganicfraction. nly . anglicaandF. rubra how leachingpattern uring he nitial tages of decomposition. he pre-viously mentioned markers f N-acetylglucosamine mlz221, 203, 185, and 77) are always part f the heterogeneousorganic fraction. his finding orrelates ell with hegen-erally bserved ncrease n relative itrogen ontents uringthe decomposition rocess Table 2).

General discussion and overview

Initial ignin ontent ersus decomposition ate-The de-composition ates f the different lant peciesvary onsid-erably. ince ignin s considered decay-retardingubstance,decomposition ateshave often een correlated ith nitiallignin ontents Wilson et al. 1986a,b; Buth and Voesenek

1987;Hemminga t al. 1988;Hemminga ndButh1991).Theresults fter uO oxidation nd DT-MS both denote he tis-sues of S. anglica,A.maritima, nd S. maritima s lignin ichand the issues f A. tripolium nd L. vulgare s lignin oor,and F. rubra ccupies n intermediate osition. his patterncorrelates uitewellwith he bserved weight osses Fig. 1),except or . vulgare,where low lignin ontent s not cor-relatedwith apidweight oss. This deviation ecomes venmorepronounced f t s realized hat he decomposition ateof L. vulgare s probably verestimated ecause of the ossof brittlematerial rom he itterbags. bviously, nother e-cay-retarding ubstance han ignin etermines hedecompo-sition ateof L. vulgare.As indicated n the Results ection,the ontribution f physical rocesses othe weight oss is aninherent isadvantageonnected o itterbag xperiments. orthis ample et, his roblem eems o be of concern nly forthe eavesof L. vulgare.

Lignin ynamics uring ecay-The results btained withthe CuO oxidation nd DT-MS method artly onfirm achother, ut discrepancies lso exist. Conformity xists on therapid elative ncrease f ignin uring hefirst eeksof thedecomposition rocess Figs. 3, 6-9). This increase an beexplained s the eachingof soluble and easily degradablecomponents, uch as storage ugars, mino cids,etc.,whilestructural omponents,ncluding ignin nd cellulose, emain

in the itter. t is remarkable hat he extensive xtractionsand rinses nvolved n the MWELpurification rocedure p-parently o not mask the leaching process. t seems thatconformity oes not xist on the relative ignin ontents ur-ing the postleaching eriod. The CuO results ndicate moreor ess stableLOP yields normalized o OC, Fig. 3) duringthe second stage of decomposition or most of the studiedtissues, whereas he DT-MS results ndicate gradual ossof intact ignocellulose oward he end of the experiment(Figs. 6-9).

Considering he comparative pproach f this study, weshould ddress he uestion f the xtent o which heresultsobtainedwith he wo analytic methods re comparable. otonly do the methods ave their pecific eatures, heyhavealso been appliedto different amples, .e., untreated onlyball milled) nd purified lant issues.Before omparing helignin dynamics n the postleaching eriod, hese potentialobjections re discussed n some detail.

A rough haracterization f these nalyticmethodsmightbe that he combination f untreated aterial with CuO ox-idation provides merely uantitative ata, whereas DT-MSof MWEL samplesyields a mostly ualitative iew on thelignocellulose fraction. However, the "quantitative" p-proach f the CuO method oes not meanthat o qualitativeindications re achieved;ratios f different ompounds reused as diagenetic ndicators Hedges and Mann 1979a;Goini nd Hedges 1992; Haddadet al. 1992). Accordingly,the term qualitative" hould not be taken o be too strictfor heDT-MS method.Here it indicates hat elative eakintensities f ligninmarkers n the mass spectra f the var-ious samples re compared. eak intensities re proportionalto detected on numbers nd represent emiquantitative ata(as long as the pproximate yrolysis/ionization fficienciesof the various compounds n the sample are known). Thepreviouslymentioned isagreement etween he two meth-ods should therefore e nuanced to a lack of conformitybetween he amount f lignin xidation roducts f wholesamples and mass spectral ata of thermally iberated ig-nocellulose ragments f purified amples.

If we consider hedifferences etween rude nd purifiedsamples, source of variationmight e extraction ith or-ganic solvents. his s particularly he ase since t has beenshown that xtraction f lignin by organic olvents s en-hanced by ball milling Lai and Sarkanen 1971).We havechecked hiseffect or . anglica, and it turned ut ndeedthat MWELobtained fter h of ball millingwas richer nlignin hanMWEL obtained fter 0 h of ball milling. allmilling hus nduced nhanced ignin xtraction. he risk fhigh depletion houldnot be exaggerated, owever, ecausethe ethanol-extractable ignin fraction f angiosperms socalled Brauns ignins) s generally maller han .2% of thetotal plant material Lai and Sarkanen 971). Moreover, hemilling ime has been kept short 3 h, whereas 48 h wasrecommended n the original ublication f Bj6rkman 956)in order o minimize ignin xtraction. s far s intact igninis concerned,we assume that rude and purified amplesmay be compared, lthough odata are availableon whetherthe effect f ball milling aries as a function f decompo-sition eriod.No effect f the ball millingwas found n theCuO oxidationmethod comparison etweenunmilled nd3-h-milledmaterial).



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Quantitation f the ignin xidation roducts ith he CuOmethod oes not bear arge uncertainties. ith egard o theDT-MS technique, he question s pertinent hether iffer-ences in pyrolysis/ionization fficiency etween ompoundclasses bias the resulting pectra n such way that he corepatterns btained fter iscriminant nalysis o not representprevalent hemical hanges. f, for nstance, he heteroge-neous organic raction hat s produced pon decompositionin the field would consist of compounds hat re far moreefficiently yrolyzed nd/or onized than other ompoundsin the sample, his fractionmight ause nonlinearity f they-scales n Figs. 6-9. A control xperiment ith series ofmixtures f the two A. maritima MWEL sampleswith hemost xtreme cores n Fig. 6b pointed ut that he discrim-inant cores are almost inearly elated o the relative on-centrations f the two samples results ot hown). t seemslikely hat his lso counts or he other coreplots Figs. 6-9). Overall, he woanalyticmethods rovide pecific iewson the ignin ynamics f the xposedplant issues, nd withthis n mind he resultsmay be compared.

At first e consider he pparent ontradiction hat uringthe postleaching eriod, heDT-MS results ndicate gradualconversion f ntact ignocellulose nto heterogeneous r-ganic fraction, hile the relative mount f LOP as deter-mined y CuO remains table. t is important o realize thatthe DT-MS technique llows distinguishing ompoundswithdifferent olatility r thermal tability. mall compoundswill generally esorb t low temperatures nd thus nduceshift n the thermal esorption rofile f the sample. TheMWEL residues did not show such a shift n the thermaldesorption rofile with ncreasing ecomposition or ny ofthe plant species in this study. hus, the MWEL residuesmainly onsist f polymeric ompounds hroughout he ex-periment. he reconstructed ass spectrum hat ypifies he

heterogeneous rganic material hat s produced upon de-composition ncludesmarkers f altered monolignols Fig.6a, dashed squares),which suggests hat hesechemicallymodifiedmonolignols till belong to the igninmacromole-cule. Unfortunately, t is not possible to make a thoroughquantitative omparison etween ntact ignin n the residueafter eaching nd modified ignin n the decomposed rac-tion.We conclude hat he DT-MS results ssentially ndicatethe chemical modification f lignin nd that t cannot bedecidedhow the rate of this modification elates o that fbulk organicmatter oss.The quantitative ndication rovid-ed by CuO that ignin s not better reserved hanbulk or-ganic matter uring hepostleaching hase is thus not op-posed by the DT-MSresults, s might e concluded t firstsight.Nevertheless, here emains quantitative eculiarity. ncontradiction o the MWEL samples,which onsist mainlyof macromolecular aterial, heuntreated amples till on-tain a fraction hat an be removed y the purification ro-cedure. One may wonder what the removable raction fsmallcompounds onsists f. One wouldexpect relativelylow lignin ontent or his fraction nd thus negative n-fluence n the LOP yield. However, heLOP yields n thepostleaching hase are stable nstead f decreasing. wo rea-sonableexplanations or his bservation ppear.At first heinfluence f the removable raction ay be compensated or

by relative ignin nrichment f the remaining acromolec-ular fraction. therwise, he removable raction ay s wellnot be depleted n lignin or material hat produces LOP)compared o the macromolecular raction. n the basis ofthe DT-MS results, which ndicate hat he relative ignincontent f the macromolecular raction oes not increaseduring ecomposition, e conclude that he second optionis more probable. his scenario equires hat heremovablefraction ontains mall ignin decomposition roducts hat,upon CuO oxidation, ield comparable uit of products sthe ntact ignin macromolecule. his may be the ase, sincenatural xidation s well as CuO oxidation onverts he a-carbon tom of the side chain of a phenolic ignin nit ntoa carbonyl roup nd eventually n acid group Hedges etal. 1988a; Dijkstra t al. 1998).We conclude hat mall ig-nin decomposition roducts ecome attached o the tissueremains nd can still be identified s LOP.

Another pparent isagreement etween he results b-tained with he two rnethods oncerns qualitative spect.From heCuO oxidation esults t appears hat, or ll plantspecies, the ignin moiety eaches an endmember ithinmonth. n fact, his nly relates o the rapid tabilization fthe C: V ratio ecause other ualitative arameters S: V andAd: Al) do not howconsistent rends uring he25 monthsof aerobicdecomposition. he DT-MS (El) results ndicatethat the intensities f several specific ignin degradationproducts Fig. 6a) increase upon decay for he three ignin-rich issues.According othese DT-MSresults, lignin nd-member s not yet reached fter more than 2 yr of aerobicdegradation. ctually, hese esults re not ontradictory utcomplementary, ecause the C: V ratio s better eterminedwith the CuO method, whereas first-stage lteration f thepropenolic ide chain of a monolignol s more easily den-tified sing DT-MS. Based on these results, he suitability

of lignin o monitor fflux f halophyticmaterial rom altmarshes o adjacentwater ystems s dependent n the an-alyticmethod sed. f the CuOmethod s used, there o notseem to be limitations ith egard o the tability f the first-stagediagenetic ndmember. f DT-MS is used, ypical ignincharacteristics re continuously ost andmay ventually an-ish.

Both methods eveal no clear-cut ifferences n degrada-bility f guaiacyl nd syringyl nits.The relative tabilitiesof the different onomer amilies avebeen studiedmanytimes Hedgeset al. 1985;Benner t al. 1991;Haddadet al.1992; Gonti t al. 1993; Gamble t al. 1994;Van der Heijdenand Boon 1994; Opsahl and Benner 1995),but no uniformimage ppears rom ll these tudies. ven n well-controlledlaboratory xperiments ith white-rot ungi, he observa-tions re ambiguous. he lack of uniformity, articularly n-der field onditions, eems logical because the stability fmonolignols nd oligolignols epends not only on intrinsicchemical properties, ut also on (1) the biochemical ndphysicochemical onditions uring ecomposition includingthe different egradation athways mployed by differentdecomposers),2) their ocation n the igninmacromolecule(Terashima t al. 1993),and (3) interaction ith other ellwall components.

An interesting bservation n the DT-MS results f thethree ignin-rich lant pecies S. anglica,A. maritima, nd



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1474 Klap et al.

S. maritima) s that ignin nd polysaccharide how corre-lated behavior uring ecay. The apparent omparable ta-bilities xplain why we commonly aveusedthe designation"lignocellulose" nstead f "lignin" and "polysaccharide"separately. his observation s contradictory oother eportsthat denoted cellulose as relatively abile (Hodson et al.1984; Opsahl and Benner 1999).

Geochemical mplications rom this study-Rice (1982)has exhaustively ddressed he phenomenon f increasingnitrogen ontent ith ncreasing ecomposition ime,whichwas also observed n this study Fig. 2 and Table 2). Hiswell-accepted ypothesis Wilson et al. 1986a; Hemmningaet al. 1988; Benner t al. 1991; Buchsbaum t al. 1991),which shares imilarities ith the mechanism roposed nthe previous ection, s that mall reactive ligomeric mol-ecules produced uring hefirst tage f decomposition ormreactive eomacromolecules. hese geomacromolecules ctas condensation uclei for nitrogenous icrobial xudatesand becomemore nd more efractory n the ourse of time.

The remaining lant fraction husbecomes ncreasingly n-richedwith hose nitrogen-rich eomacromolecules. ur DT-MS (CI) results ndicate gradual ncrease f N-acetylglu-cosamine for all plant species studied and thus providestrong vidence or contribution f allochthonous itrogen-rich material.

It s worthwhile orelate hepresented esults o the om-mon viewpoints n the formation f humic ubstances. wodifferent athways ave been distinguished: 1) condensa-tion of small degradation roducts, uch as monopeptidesand oligopeptides,monosaccharides nd oligosaccharides,lipids, nd phenolic ubstances, nto a refractory eteroge-neous organic olymer nd 2) condensation f only lightlymodified iogenic macromolecules Mayer 1985; Hedges1992; Ishiwatari 992; De Leeuw and Largeau 1993). Aspreviously utlined, e interpret he ombined esults f DT-MS and CuO oxidation s an indication or heoccurrenceof the first f the woproposed athways. f coursedifferentsubstances avedifferent reservation otentials, o the ec-ondpathwaywill apply o some extent. evertheless, t doesnot eem ikely hat ertain iogenicmolecules emain ntactin humus. As such, hesecondpathway might e regardedas an intermediate tage of the first athway n which de-composition f the ontributing ubstances s lessprolonged.

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Received: 7 April 1999Accepted: 13 April 1999Amended: 10 May 1999

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