Decomposition Dynamics of Six Salt Marsh Halophytes as Determined by Cupric Oxide Oxidation and Direct Temperature-Resolved Mass Spectrometry

Author(s): Vincent A. Klap, Patrick Louchouarn, Jaap J. Boon, Marten A. Hemminga and Jos van Soelen Source: Limnology and Oceanography, Vol. 44, No. 6 (Sep., 1999), pp. 1458-1476 Published by: American Society of Limnology and Oceanography Stable URL: http://www.jstor.org/stable/2670729 . Accessed: 03/11/2013 17:08
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Liunnol. Oceanog. 44(6), 1999, 1458-1476 (C 1999,by theAriei-ican Societyof Limnology and Oceanography, Inc.

Decomposition dynamics of six salt marsh halophytes as determined by cupricoxide oxidation and direct temperature-resolved mass spectrometry
A. Klap1 Vincent
NetherlandsInstituteof Ecology, Centre of Estuarine and Coastal Ecology, P.O. Box 140, 4400 AC Yerseke, The Netherlands

Patrick Louchouarn2
Research Centre in Isotope Geochemistry and Geochronology, Universityof Quebec at Montreal, CP 8888, Succ. A, Montreal, Quebec, Canada H3C 3P8

Jaap J. Boon
Fundamental Research on Matter,Institutefor Atomic and Molecular Physics, Kruislaan 407, 1098 SJ Amsterdam, The Netherlands

Marten A. Hemminga and Jos van Soelen

NetherlandsInstituteof Ecology, Centre of Estuarine and Coastal Ecology, P.O. Box 140, 4400 AC Yerseke, The Netherlands

Abstract
This paper presents the resultsof a comparative of six salt marshplant studyon the aerobic decomposition to ash-free and elemental speciesover a periodof 2 yr.In addition dryweight (AFDW) determination analysis(C in the and N), two analytic methods' have been appliedto obtaininsight intothedecomposition dynamics of lignin variousplanttissues.The analytic are (1) cupricoxide (CuO) oxidation methods followed by gas chromatographymass spectrometry mass spectrometry (GC-MS) and (2) direct temperature-resolved (DT-MS). AFDW losses could generally be well described relations withtime.Carbon-to-nitrogen by double exponential ratiosincreased theinitialstagesof decomposition and decreasedagain afterward. For fiveof thesix plant during oflignin initial content and AFDW loss was observed. denoted species,a correlation between lignin Decay dynamics a rapidrelative thefirst contents over thenext2 increaseduring weeks of fieldexposure, followedby stabilizing data indicate theestablishment of a stable"ligninendmember" 1-2 months. DT-MS data, within yr.CuO oxidation on thecontrary, modification of theligninpolymer theduration of theexperiment. showedcontinuous throughout in thecomplexresidue fortheincorporation of (presumably) microbial Evidencewas found N-acetylglucosamine of CuO oxidation and DT-MS data suggested thatlignindegradation Combination produced upon decomposition. as lignin-derived became attached to the originalmacromolecular material and could stillbe identified products a humification of smalldegradation instead of the material. The data support mechanism via condensation products selective of certain biomacromolecules preservation (like lignin).

during recalcitrance, lignindoes not behave conservatively existon lignin reports decayof plantlitter. Indeed,numerous mineralization) byboth degradation (withpartial orcomplete White-rot are the fungi aerobicand anaerobicdecomposers. group (Amer and best known representatives of the first Drew 1980; Chen and Chang 1985; Higuchi 1985a,b; Kirk and Shimida1985; Harveyet al. 1987; Hedges et al. 1988a; et al. 1993; Gambleet al. Lewis and Yamamoto1990; Goini by bacteria 1994), whereasthe second groupis represented ' Present address:The FOM/AMOLF Institute. and Frazer 1987). Ow(Benner et al. 1984a,b, 1991; Young 2 The University Present address:The MarineScience Institute, ligninstructure ing to modification of the macromolecular of Texas at Austin, 750 ChannelView Drive,PortAransas, Texas of fresh plantmaterial may during decay,theligninmoiety 78373-1267. be an unsuitable in the tracerof vascularplant remnants Acknowledgments environment. We thank and YvonneMaas forperforming JoopNieuwenhuize Here we presentthe resultsof a litterbag studyon the the elemental is thanked forhis assistance analyses.Jos Pureveen six different salt marsh plant speciesdurdecay dynamics of withtheDT-MS analysisand GertEijkel forhis helpwiththemulThe mainobjectivesfor ing 2 yrof aerobicdecomposition. tivariate analysis. of theligninmoiety thisstudy were (1) to findout whether This is publication 2524 of theNIOO CentreforEstuarine and to bioThe Netherlands. herbaceousplantsshows highresistance halophytic Coastal Ecology,Yerseke,
Lignin, a structuralcomponent of the cell walls of vascular plants and the second most abundant naturallyoccurring polymer in the biosphere (after cellulose), has a high preservationpotential (Hedges and Mann 1979a; Swift et al. 1979; Kirk and Shimida 1985). Its decomposition is often regarded as the rate-limiting step in the biospheric cycle of carbon (Colberg 1988). Notwithstandingits relatively high

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Salt marshhalophytes to bulk organicmatter degradation relative and (2) if so, to of biodegraded establisha chemically definedendmember ligninsuitableto serve as a tracer forhalophytic material exported from salt marshes intothemarineenvironment. Throughout thedecomposition period, remaining ash-free as well as ordryweights (AFDWs) have been determined, contents. ganic carbonand nitrogen Two different analytic methods were appliedto monitor chemicalmodification of litter. of lignin thedecomposing The first method, oxidation withcupricoxide (CuO) underalkalineconditions, yields can distinct classes of monomeric and dimeric that products be quantitatively analyzedby meansof gas chromatography and (Hedges and Mann 1979a; Hedges and Ertel1982; Gonii clasHedges 1992). Ratios betweenthosedifferent product ses are used as indicative forthediagenetic parameters stage of the material.CuO oxidationhas oftenbeen applied to studythe dynamicsof lignindecay (Haddad and Martens 1987; Hedges et al. 1988a; Benneret al. 1991; Haddad et et al. 1993; Opsahl and Benner1993, 1995; al. 1992; Gonii Louchouarn et al. 1998) and to characterize and quantify the matter to diversecoastal and deep-sea inputsof terrigenous marineenvironments (Hedges and Mann 1979b; Moran et al. 1991a,b;Gonii andHedges 1992; Goughet al. 1993; Prahl et al. 1994; Louchouarnet al. 1998). The second method used is direct mass spectrometry temperature-resolved (DTMS), in whichpolymeric material is thermally dissociated thatare subsequently intofragments analyzedby means of mass spectrometry beenused (Boon 1992). DT-MS has often fortheanalysisof ligninand lignified (fossil)plantmaterial et al. 1987; Stoutet al. (Genuitet al. 1987; Saiz-Jimenez 1988; Boon 1989; Scheijen and Boon 1989; Stout et al. 1989; Faix et al. 1990; Pouwels and Boon 1990; Ralph and Hatfield1991; Van der Hage et al. 1993; Van der Heijden and Boon 1994). Combination of thesetwo methods themeansto provides some of thelimitations of each individual method overcome (Van Bergen et al. 1994). OxidationwithCuO is, forinstance,more suitableforquantitative lignindetermination, be obwhereasstructural detailsof the polymer can better tainedwithDT-MS (Van derHage et al. 1993). In a separate of Spartinaanglica litter, it has paper on the degradation been shownthatpurification of thismaterial to is required obtainvaluableanalytic data on theligninfraction (Klap et methodapplied was a twofold al. 1998). The purification enzymaticdigestion(protease followed by polysaccharia residuecalled milledwood enzymelignin dase), yielding here concernsam(MWEL). All DT-MS resultspresented thispurification CuO ples thathave undergone procedure. oxidation was conducted on material that was ball milledbut otherwise becauseithas beenreported unpurified, that major matrix-related effects do not occur on the yield of ligninderivedcompounds upon CuO oxidation (Young and Frazer 1987; Gofiiand Hedges 1995). Experiment Litterbag experiment-Aboveground partsof the six halherbaceous ophytic plantspecies S. anglica,Festuca rubra, Astertripolium, Artemisia and Limnonium vulgare, maritima,

1459

Suaeda maritima were harvested in the first week of September1992 in the Sint Annaland salt marsh (51?36'N, in the northeastern 4?07'E). This marshis situated partof the Oosterschelde an arm of the North (The Netherlands), Sea. By thetimeof harvesting, theplantswerein their earof theleaves). Diflieststageof senescence(first yellowing ferent typesof tissueswere selectedforeach of the three major plant groupsused in this study:for the grasses (S. anglica and F. rubra),thecomplete aboveground partswere herbs(A. tripolium and L. vulgare) selected;thenonwoody leaf material; and the were exclusively represented by their herbs(A. maritimna shrub-like and S. maritimna) were representedby small twigs,fromwhich thicker brancheswere removed. A. maritima and S. maritima already carried seeds themeshsize ofthebags. All plant withdiameters exceeding F. rubra)werecutintosmallpieces parts(exceptthosefrom -8 cm long and driedwithtissuepaper; -25 g of thismaterialwas put into nylonlitterbags of 12 X 12 cm (1-mm meshsize) without The tissuewas just treatment. anyfurther it was leftin thefieldto avoid unnatural paperdriedbefore The bags were sewn changescaused by extensivedrying. to themarshthefollowing shut, weighed,and brought day. They were attached withthinlines to long horizontal sticks within the canopy of the vegetation, at a distanceof - 15 cm above the marsh surface.The location was near the betweenmarshand intertidal flat.The bags were boundary forshort times(< 1 h) during tideperiods. submerged spring Samples were collected6, 16, 31, 56, 196, 259, 386, and the start of theexperiment. 766 d after At the start the weightratiobetween of the experiment, freshand freeze-dried material forfiveinwas determined dependent samplesof each species. This ratiowas used to calculatethe dryweights at t = 0 of the samplesthatwere left in the field. This indirect calculationof the starting was a consequenceof ourchoice to fillthelitterbags weight withundriedstarting material.Subsequently, the ash contents of three of each of thesefreeze-dried samsubsamples in a muffle furnace at ples were determined by combustion 570?C for3 h. The AFDWs of the samplesat t = 0 were thusbased on a totalof 15 analyses. At each collection date,three bags per speciesweretaken fromthe field.Macromaterial to the bags, forinadhering stancemacroalgae,was rarelynoticed,but in the few occases the material was removed.The bag contents curring were freezedried,without and weighed. previousrinsing, The drymaterial was ground, and the ash content was deof termined in triplicate foreach litterbag. The low amounts A. tripoliumn materialaftersome weeks did not remaining of theash contents allow thedetermination in triplicate and after1 yr not even in singular. Instead,the materialwas saved forchemicalanalyses.After magravimetric analysis, terialfromthreebags was pooled and organiccarbonand contents were determined with a Carlo Erba NA nitrogen The remaining 1500 C/N analyzer. material was powdered for3 h (at 5?C) in a vibratory ball mill (RetschMM2) and used forCuO and DT-MS analysisor forligninpreparation withsubsequent DT-MS analysis. Milled wood enzyme ligninpreparation-MWEL preparationwas conducted according to the procedure described

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1460

Klap et al. mass spectrometry-A douDirect temperature-resolved JEOL SX-102 mass spectromble focusing (B/E geometry) Drops eterwas used forthe analysisof -5 ,ugof material. of suspendedsample materialwere depositedon a Pt/Rh wireand driedundervacuum.Duringtheanalysisperiodof scanneda mass rangebetween 90 s, themass spectrometer of 3,000. mlz 20 and mlz 1,000 each secondat a resolution current forpyrolSource temperature was 180?C. Filament from final 0 to 1.5 A in 90 s, inducing ysiswas programmed impact (El, 16 eV) and temperatures of 800?C.Bothelectron chemical ionization (CI, NH3, 200 eV) were employed. forEl was 10-4 Pa, whereasforCI a presSource pressure was sure of 20 Pa (NH3) was maintained. Data acquisition on a JEOL MP-7000 system. The setofintegrated performed between30 (TIC) intervals spectraof the totalion current analysisby theFOMpyrMAP and 60 s was used forfactor analversion of theArthur multivariate program, a modified et al. 1983; Win(Hoogerbrugge ysis program (Infometrix) except dig et al. 1983). Samples were analyzedin singular for A. maritima on which triplicate underEl conditions, thusallowingdiscriminant analysis. analysis was performed, in terminology existbeNomenclature-Somedifferences whichare unitedin tweenthetwo fieldsof ligninresearch, of the pyrolysis thisproject.In thispaper the terminology fieldwill be applied,so the terms"guaiacyl" and "p-hyacid" are used rather than "vanillyl" and droxycinnamic "cinnamyl phenol." Results and discussion

elsewhere(Klap et al. 1998). Briefly, 200 mg of the ballmilledmaterial ethwas percolated at roomtemperature with anol: benzene (2: 1) and acetone:water (9: 1) and extensively washed with Milli-Q water. This extractive-free material was incubated for6 h with0.07% (w/w)protease (Pronase E fromStreptonyces griseus,EC 3.4.24.4, 4 U at pH 7.4 andwashed mg-', Merck)in a 50 mM Trissolution Then it was incubated for12 withMilli-Q waterafterward. h with0.5% (w/w)polysaccharidase (Cellulase OnozukaR10 fromTrichoderma viride,EC 3.2.1.4, >1.0 U mg-', Merck) in a 50 mM ammonium acetatesolutionat pH 4.5 and again extensively washedwithMilli-Q waterafterward. Both incubations outin thedarkat 37?C under werecarried continuous of the test tubes. Finally,all samples rotation werefreezedriedto producea residuereadyforanalysis. Cupric oxide oxidation-Lignin-derived CuO oxidation products (LOP) were determined by the method developed and by Hedges and coworkers (Hedges and Ertel1982; Gonii Hedges 1992), withslight modifications (Louchouarn et al. 1998). Briefly, 25-50 mg of grounded material was hydro2 M NaOH solutionin lyzed for3 h in a nitrogen-purged the presenceof CuO (-1 g) and Fe(NH4)2(SO4)2 6H2O (-100 mg) withina stainlesssteel minibomb. Four such reactionchambers were loaded, underan inertatmosphere bomb(Parr)that contained additional base. (N2), intoa larger This container was heatedin an insulated heating sleeve,the of whichwerecontrolled rateand finaltemperature heating by a CN76000-seriestemperature controller (Omega Engito a finalinternal roomtemperature neering). Heatingfrom of 162?C was performed in 30 min; thisfinal temperature for2.5 h. Ethyl vanillin temperature (?2?C) was maintained AldrichChemical) was (3-ethoxy-4-hydroxybenzaldehyde; and was added directly employedas a recoverystandard (-20 ,ug) to theminibombs upon cooling.After oxidation, withHCI (6N) to pH 1, extracted samples were acidified with freshly distilleddiethylether, concentrated by rotowere then evaporation, and driedunderN2. Dried extracts stored frozen untilanalysis.

Gravimetric plantspeanalysis-AFDWs of thedifferent cies are shown in Fig. 1. As outlinedin the Experiment leftin the field section,the initialdryweightsof material werenotdirectly measured butwerecalculatedon thebasis at t = of measured ratios(fivefold) fresh weight: dryweight the weight at t = 0 bear 0. As a consequence, percentages a standard ratesfor deviation around100%. Decomposition litter can usuallybe well described by a doubleexponential as thatlitter be regarded can roughly equation,suggesting withdistinct decomposition beingcomposedof twofractions Gas chromatography-Gas chromatographic analysisof from Fig. 1 that rates(Wiederand Lang 1982). It is evident oxidation was carried outwitha Hewlett- in fourout of six cases, thedoubleexponential silylated products modelcurve Packard5890 GC fitted witha fusedcapillary column(DBdedata.The calculated fits nicelyindeedto theweight-loss attached to a 1, 30 m by 0.25 mm i.d.; J & W Scientific) ratesand therelative sizes of thetwofractions, composition flameionizationdetector (FID). The injectorand detector as well as thesquaredcorrelation of thefittings, coefficients at 300?C, and splitinjections weremaintained (-1/30 to 1/ are shownin Table 1. withheliumas carrier 40) were performed gas. The oven show similardecomposition S. anglica and A. maritima to rise from100?C to 270?C was programmed temperature of labile and recurves,defined by equally large fractions at a rateof 4?C min-I and was keptisothermal for 15-20 material.For both species, -35% of the initial fractory timeand quantity of all eightmajormono- weights min.Retention remainafter 2 yrof fielddecay.A verygood fitis mericLOP studied here(V, thesum of monomeric guaiacyl achievedforF. rubra,characterized smalldifby relatively and C, thesum ferences structures; S, thesumof syringyl structures; in decomposition ratesbetweenthe labile and reof p-hydroxycinnamic acids; see below) were determined fractory fraction. The factthatno AFDWs weredetermined from their FID responses. of all lignin-derived for this species after200 d of exposurehas undoubtedly Identification monomers and dimers was performed by GC-MS, and LOP thefitof thecurve(r2 = 0.98). Actually, reliable improved were assignedequal to the recovery stan- determination responsefactors rateof the of the size and the decomposition dard,ethylvanillin(Goiii and Hedges 1992; Opsahl and fraction exporequiresdata pointsat prolonged refractory Benner1995). forF. rubra, thecalculatedvaluesfor suretimes.Therefore,

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Salt marshhalophytes
anglica Spartina Festucarubra

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losses forthesix plantspeciesovera periodof766 d. The standard dryweight Fig. 1. Ash-free the best set up (see text).The curvesrepresent at t = 0 are due to the experimental deviations models(X = Ae-ldt + (1 - A)ek2dt). Values forA, k, and k2are given doubleexponential fitting in Table 1.

ratesshould decomposition fractions and their therespective only.The weightloss of A. trias indicative be considered thatafter 1 yrnotenough occursso rapidly poliummaterial The remarkdetermination. material was leftforash content a verylabile able shift in weightloss after-20 d suggests of A. tri-75% of theorganicmaterial fraction comprising sizes and decomaboutthefraction polium.The uncertainty for above forF. rubrais smaller position ratesas mentioned prolonged are also no data after A. tripolium, although there

exposuretimesavailable forthisspecies. The clear kinkin the curve indicatesthatmodelingthe weightloss of this The function. species requiresat least a double exponential consistsof two fractions, thatthematerial good fitsuggests labile. one of whichis extremely curveis calculatedforS. marA peculiardecomposition loss to weight increase a shift from weight indicating itima, does not after 237 d. The outcomeof themodelcalculation butrather one of different indicatetwo fractions stabilities,

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1462

Klap et al. moresusceptible to comminution thanother tissues.For the setof planttissuesused in thisstudy, theprocessonlyseems to be of concern fortheleaves of L. vulgare.

Table 1. Calculatedparameters forachievingthe best fitof a to the doubleexponential function (X = Ae-kldt+ (1 - A) e-k2dt) values.The accompanying measured weight-loss curvesare plotted in Fig. 1. A (Organic ki (10-4 d-l) matter) k, 1-A (Organic matter)

Elemental analysis The organic carbon and nitrogen contents of boththeuntreated and MWEL samplesaregiven (10-4 in Table 2. The carbonvaluesdo notshowa clearcorrelation r2 Species d-l) tend to inwithexposuretime,but the nitrogen contents 0.96 Spartina 194 0.521 5.3 0.479 crease withincreasing exposuretimes,which is in accorFestuca 0.276 54.9 0.98 260 0.739 dance withliterature data (Buth and Voesenek 1987; Hem0.242 Aster 2280 0.756 35.2 0.99 et et al. 1991). It is noteworthy al. Benner that, minga 1988; 0.186 12.4 0.93 Li,nonium 749 0.829 are far withthe exceptionof L. vulgare,nitrogen contents 4.1 0.96 Artemnisia 122 0.566 0.476 moreinfluenced thancarbon by the enzymatic purification 127 0.86 Suaeda 0.773 -13.5 0.260 In Fig. 2, the atomicC: N ratiosfortheuntreated contents. and purified are shown.It can be seenthat therangmaterial The enes forthetwo typesof samplesdiffer considerably. fraction (1-A). decomposing (A) and a producedfraction zymatictreatment (includinga proteasedigestion)clearly a although This does not seem to be a realisticsituation, removes morenitrogen thancarbon, highrelatively yielding occurbecause of colonization by slight mass increasemight samples.Especiallyfor how- er C: N ratiosthanfortheuntreated decomposers. The magnitude of theapparent increase, ever,makes colonization an unlikely primary cause in this theMWEL samplesof thetwograssspecies,theC: N ratios valueshave the heterogeneous of the reachhighvaluesof morethan100. Even higher case. More probably composition that had been been for tissue reported Spartina alterniflora litterbag contents (characterized by relatively highcontents withneutral detergent (Buchsbaum et al. 1991). In of seeds and woody parts with deviatingdecomposition extracted are charC: N patterns forsalt marshmacrophytes led to thepeculiar pat- general, rates)has incidentally weight-change acterizedby increasingvalues duringthe leaching stage, tern. whichsteadilydecrease(to below the initialvalue) during The otherspecies forwhichthe fitis not satisfactory is (Buth and De Wolf 1985; L. vulgare,whichshowsunexpected losses for thelaterstagesof decomposition highweight thesamplescollectedafter 386 and 766 d. The 766-dsample Benneret al. 1991; Buchsbaumet al. 1991). In this study, of the MWEL preparations show better is based on one bag only,because theother bags were lost the C N patterns mato this than the untreated resemblance general pattern deviation thusequals that or tornby thattime(the standard to this L. the N ratio C: terial. An rule is exception vulgare, the of t = 0). The unexpected loss later during highweight of theMWEL of whichneverreachesa lowervalue thanat is explainedby the fact thatL. stages of the experiment robust structure and becomebrittle t = 0. vulgareleaves lose their after some time.Particles smallerthanthemeshsize of the to thecarbonand nitroare lost, whichwrongly Oxidation litterbag suggestschemicalrather products In addition the measuredconcentrations of ligninoxidaThis is an intrinsic disadvan- gen contents, thanphysicaldecomposition. are listedin and leads to an overestimation tionproducts (LOP) and calculatedparameters method tage of thelitterbag which are produced of thechemicaldecomposition rate.In general, leaftissueis Table 2. p-Hydroxyphenyl structures,
Untreated material 50-

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Salt marshhalophytes upon alkaline oxidationof lignin (Sarkanen and Ludwig 1971; Hedges and Mann 1979a), are notincludedin thislist because nonlignin sourcessuch as marine and esplankton peciallybacteria have been shownto be significant sources of thesecompounds and Hedges (Hedges et al. 1988b; Gonii in1995). Moreover, GC-MS analysesof thesecompounds dicate thatcoelutionwithothercompoundsoccurs,which hampers reliablequantification (Haddad and Martens1987). In Fig. 3, thelignindynamics forthedifferent plantspecies are expressed by A6,thenormalized sum (mg per 100 strucmg OC) of themainthree guaiacyland three syringyl tures produced uponoxidation. This sum-parameter has been of totalligninin nonproposedas a morereliableindicator woodyand herbaceous planttissuesthanthetraditional A8, in whichthep-hydroxycinnamic acids are included(Opsahl and Benner 1995). Those p-hydroxycinnamic acids (p-coumaricand ferulic to act as linkages beacid) are considered andhemicellulose in nonwoody andparticularly tweenlignin in gramineous tissues(Hedges andMann 1979a; Hartley and and Ralph 1990) Haverkamp 1984; Aikenet al. 1985; Jung and therefore are notan integral partof theligninpolymer. Hence, A6 is used to represent ligninin this set of grassy and herbaceous tissues.Figure3 indicates thatthesix plant dividedintothree speciescan be roughly groups: lignin-rich and S. maritima; A. triS. anglica,A. maritima, lignin-poor poliun and L. vulgare;and F. rubra,whichis characterized A considerable increase by an intermediate lignincontent. in A6 values is foundforall species during the first month of the experiment, coveringthe leachingstage. Afterthis period,most A6 values vary somewhatarounda constant value untiltheend of theexperiment. The previous broadclassification is confirmed by thevalues of additional monomers and dimers lignin-derived (Table monomers and major mono2). Ratios betweenadditional mers(Mono: A6; Table 2) remain rather constant throughout thedegradation 0.10 forall six speperiodat approximately forbothgroupsof monocies, suggesting equal stabilities

1463

bonds in mers.The CuO oxidation procedure cleaves ether the ligninmacromolecule efficiently, whereasmanyof the carbon-carbon bonds remainintactunderthese conditions (Chang and Allan 1971). Hence, thismethodyieldsa suite of lignin-derived dimersthatretaintheiroriginalcarboncarbon linkages.Furthermore, it is believed thata higher to ether diagenetic stability of carbon-carbon bondsrelative bondsand/or activecrosslinking degradation induces during to monomeric increasing yieldsof dimeric moieties relative ones uponlignindecay (Gonii and Hedges 1992). To testfor enrichment in lignindimersrelativeto ligninmonomers, No increase theirratios(Dim: A6; Table 2) are monitored. in thisratiois found foranyof theplantspecies,suggesting thatcarbon-carbon and etherbonds are cleaved at similar the degradation rates throughout process, an observation (Opsahl consistent withresultsobtainedforS. alternifiora and Benner 1995). For all fresh tissues analyzed, the summedcontribution of theseadditional monomers and diat -25% (w/w) mersto thetotalpool of LOP is moderate of A6. The normalizedlignincontents do not provide a clear in thelitterbags view on theremaining the during quantities In Fig. 4, theremaining percentdecomposition experiment. (A6 + moages of the summedligninoxidationproducts = ALig) are presented + dimeric together nomeric products of dryweight withtheremaining (DW), organic percentages carbon(OC), and totalnitrogen (TN). For mostspecies,the theinitialstageof thefieldexposure is loss of ligninduring retarded comparedto DW, OC, and TN. In some cases the amount ofremaining evenexceedstheinitial amounts, lignin A. maritima above 100. Especiallyfor leadingto percentages the immediate and S. maritima, increaseto values above of the lignin 100% probablyindicatesan underestimation content at t = 0. This might be related to theinitial presence of compoundsotherthanligninthatsuppressthe yield of If these compoundsare leached ligninoxidationproducts. veryrapidly, theywill not influence yieldsof ligninoxidat = 0. This explanation, is not tionproducts after however, in line withthe findings of Young and Frazer(1987), who 10 S. anglica --* on CuO oxidation minimal influence of thematrix reported v Frubra the initialleachingphase, the loss of product yields.After / <A A. tripolium 8 / lignindoes not grosslydeviatefromDW, OC, and TN, so / / \ \ - - ~~~~~~~L. vulgare .~ A. maritima that ligninthenbasicallybehaveslike bulk organicmatter. m \ the ratiobetweenp-hydroxycinthe experiment, During 1 -*-a-S. maritimna 6 6~ acids and structures namic guaiacyl (C : V) decreasesconE spicuouslyforall plantspecies exceptforF. rubra (Table evolution of that is consistent withthediagenetic 2), a trend 4 - .,... ......v 4 in studies and this ratio Weliky reported previous (Hedges o v 1989; Benneret al. 1990, 1991; Haddad et al. 1992; Opsahl and Benner1993, 1995). An interesting pointto noteis that 2thestart after thisratiostabilizes rather quickly(1-2 months) can be exof the degradation process. This phenomenon plainedby therapidloss of ester-bound p-hydroxycinnamic 0 fromthe acids. It seems thatafter loss of these structures 800 0 200 400 600 of the ligninmacromolecule, a C: V endmember periphery daynumber is producedthatresemblesmore closely the more crosslinkedinternal macromolecule. Indeed,prepartofthelignin as A6 Fig. 3. Lignindynamics during decomposition expressed vious degradation studies(Opsahl and Benner 1993, 1995) of 3 guaiacyland 3 syringyl oxidation (summations products per theyieldsof bothp-hydroxycinhave shownthatcorrecting 100 mg organiccarbon)againsttime.Otherligninparameters are namic and guaiacyl CuO reactionproducts by subtracting givenin Table 2.
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1464
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Salt marshhalophytes

1465

CB

TT% >

Cl>mm

m t ^Ct

m m m

m N c

N c 00 m c N

cN

ooo

>

Co

0O O

> m m

00

Nm > 00

Cl

Cl C

66666666666666666 6666666

oo > ?O

> C

It~c

CI>

C"

6>>X666 ~C mO _C

_ _I

Cl >0000 oo OCl> o

> C 00

00

iO

i oC

IC I m I t

>t m inin t

0>0 in

0 In C N

> C n C O 0 r I i m

N 0 I

0 I n n

C 00 Ct C, C No m I C) l in I~t00 mt o

It

Ct >

in

Cn

00 C) C) >

~~~~~r-

- N oo

om

m >t > In rm

- c C,, rC) i C) ncc i C tn o > tn , o

C 0 - 00 0 m OC,, --' 0m 00 c 00> C 't0 l> m cfi 00 l~ 00;m ci m c-i 0 m cIt >in 00O

,6

oo n 00

the alkali-extracted amountsof esterified acids produces close to constant C: V ratiosin all planttissuesstudied.In line withthisexplanation, it is observedthatanother common parameter, the ratio between syringyl and guaiacyl structures (S :V), is relatively invariant upon degradation shows a clear increaseduring (Table 2). Only S. maritimna thefirst 200 d, whereasS. anglica and F. rubrashow small, moreor less consistent S: V changesthroughout the whole durationof the experiment. The remaining threespecies show no trendin theirS :V ratios,indicating comparable for guaiacyl and syringyl stabilities underthose structures conditions. Elevated acid over aldehyderatios (Ad:Al) have been of lignin(Hedgused as indicators foroxidative degradation es et al. 1988a; Goni et al. 1993; Nelson et al. 1995). Diagenetic alteration oftheside chainsoflignin has alwaysbeen reassociatedwithoxic decomposition processes;however, centevidencehas shownthattheseratioscan increasesubstantially during long-term anoxic(subaqueous)degradation of nonwoodyplant material(Opsahl and Benner 1995). to both Thus, elevated Ad: Al ratios should be attributed oxic and long-term anoxic degradation. These ratiosin the in Table 2. Contrary six plantsstudiedare presented to the expected increases in acid overaldehyde ratios, the(Ad: Al)v ratios(vanillicacid over vanillin)decreaseforall six plant In the second species duringthe first year of degradation. forS. anglica,A. tripolium, year;theratioincreasesslightly from and L. vulgare.Less evidenttrends can be extracted the (Ad: Al), ratios (syringicacid over syringaldehyde). some evidencecan be foundfora generaldeNonetheless, in the thefirst month followed creaseduring by stabilization thelast yearof increaseduring subsequent yearand a slight of Ad: Al ratiosin the The observedreduction degradation. early stage of degradation mightbe explainedby loss of fromthe halophyte ester-bound acidic components tissues, forseagrasses(Opsahl and as has been observedpreviously Benner 1993). Subsequentincreaseof Ad: Al ratioswould of lignin. thenindicatecommondiagenetic alteration DT-MS analysis-The DT-MS resultsconcernenzymatically purified ligninresidues(MWEL). It was shownforS. tissues yield less specificDT-MS anglica that untreated spectrathantheirMWEL counterparts (Klap et al. 1998). fortheother fiveplantspeThis phenomenon was confirmed cies. Therefore, only resultson purified samples are preof the technique sented.The specificity was also modified ionization usingdifferent methods, i.e., electron impact(El) ionand chemicalionization (CI). El is the moreuniversal izationtechnique whereasCI witha broadanalytic window, for has a narrower window with high ionizingefficiency CI has also been polysaccharides (Pouwels 1989). However, of dehydrated monosuccessfully appliedfortheionization on the lignols(Van derHage et al. 1995). More information is givenelsewhere technique (Boon 1992; Klap 1997). In general,DT-MS generates complex spectra,since no is conducted molecularseparation priorto analysis.Moreovel; usuallythe spectraof a defined time (-temperature) to provide one spectrum for each window are integrated bevariation sample. In orderto highlight compositional tweenindividual samples, multivariate factor analysis or dis-

O 00 00 m OC 00 q r- Oc m C,,C mO t oo OC CD
OC tc m m -i N 0n In00c

m m00O It m oo c m o t o00

tn CCl ,

OC)o m oin Cl~Cl~ \4

000inin000O

8~~~~
O -i 6

in OC)
4 ooi vm

; ChmP T~~~O)

m~

OC q

N It
oo tr

N r OCN
oo ? o ct

oo

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S\ U o?
Cl
J:

m m ~m tn

t mc in OC

0 in C, in mm

z = XN m lC~~> XmmtX?m>O

?'G0NNNN

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1466
S. anglica 100 90 80 70 60 E 50
40

Klap et al.
F. rubra
100

90 80
70

~~~~~~K ~ ~ O~~~~~~~~~~~~~~~~~
--DW 00
-

6
6

50 oo 40 0 30 120 10

30 20
130 10

-Lignin

\0/TN

200

400

600

800

200

400

600

800

100 90

A. tripolium

120

L. vulgare

80~~~~~~~~~~~~~0 70~~~~~~~~~~~~~~~~~~~~~ 60~ ~ ~ ~ ~ ~~~~~~~9

E 0 40

30 20 10
0

60 >50

~~~~~~~40
30
20 10

, .,

I,

.,

200

II

I .,I

....

400

600

800

200

400

600

800

150 -130 140 130 120j c,,100 90 80 Et 70 60 50 40 30 20 10 0 0

A. maritima
120 4 1

S. maritima

K~~~~~~~~~~~0 K~~~~~~~~~~~9
5.
V

8
70

3~~~~~~~~~~~0 3
20 10 0 0 200 400 day number 600 800

200

400 day number

600

800

of dryweight, totalnitrogen, and lignin(Alig) as Fig. 4. Remaining quantities organiccarbon, a function of time, normalized to initialamounts.

outon setsoftheseintegrated criminant analysiswas carried spectra.The essence of this numericanalysismethodinin averageof all spectra volves calculation of theweighted ofindividual with spectra thesetand subsequent comparison are expressedas factor this average spectrum. Differences scores or (when analyzed in multiplicate)discriminant scores. Details on the procedurecan be foundelsewhere et al. 1983; Windiget al. 1983; Boon et al. (Hoogerbrugge 1984; Tas 1991; Klap 1997). in thiscase the reFigure5 shows two averagespectra, sultsobtainedafter El (Fig. 5a) and CI (Fig. Sb) ionization ofA. maritima. Bothspectra showmanylignocellulose char-

in different although acteristics (see symboldesignations), thehigh sensitivity ratios.The CI results(Fig. Sb) confirm coniferof sugars(Pouwels and Boon 1990) and dehydrated yl and sinapylalcohol (Van der Hage et al. 1995) forthis In Figs. 6b, 7b, the scoresof individual ionization method. areplotted in thefirst discriminant or factor function spectra the avThe zero lines in theseplotsrepresent respectively. erage spectra. in theEl mode;hence, Sampleswereanalyzedin triplicate dis(Fig. 6). The first discriminant analysiswas performed criminant function accountsfor 82% of the characteristic variance(=29% of thetotalvariancein theset). The scores

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Salt marsh halophytes

G G 180

1467

100

137

S
80 50 100 S 150200 35167 G

E 250300

21 0

73
1101

60

> 8W
CD

CD

0
411944

~~~~~~~~~~149

~~~~~~~~~~G S
E 252

G
272

cc
29 20 -222

~~~~~~~~~~~~~~~~~G+S E
302

332

SE

0
0

8 150 200 250 300 350

50

100

mlz a)
6a+GEguiclnt 19

100

-AC b.0

0 180

(general) polysaccharide sc
*

l
80 -G

+P
+

pentose

H -~hexose

163
Cl)

--> lignin (general) ~~~~~~~Ei unit G- guaiacyl E~~~~~0+

60

~ ~ ~ ~ ~ ~ ~ ~~~1
P P 0 H
0

rE 193

+ S ->syringyl unit

>

4-

40 40-

~132150

a)
20 -H 96 114

H 20222
P0

Li240
150 200 250

270 288

31

100

300

350

MlZ
A. mnaritimna of all purified (El and Cl, [a] and [b], respectively) Fig. 5. AverageDT-MS spectra thezero lines in Figs. 6b and 7b. represent litterbag samples.These spectra

in Fig. 6b illustrate the mass spectraldifferences between theindividual spectra withrespect to theaveragespectrum. The negativeDF, axis expressescompositional modificationsrelatedto the leachingprocess,whereasthe positive partof theDF1 space is representative forthedecomposition process.It mustbe realizedthatthediscriminant spectra of Fig. 6 are calculated distributions do notcloselyresemthat

ble theindividual spectra withthehighest and lowestscores; theyonly indicatewhichsets of masses covaryrelativeto theaveragespectrum. The spectrum of day766, forinstance, still containsthe two main peaks of the lower spectrum, than m/z210 and 180, buttheir intensities are less dominant of day 16. in the spectrum The discriminant by comparison spectraare interpreted

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1468

Klap et al.

100

150 1 8

product degradation --~~~~~~~~~fm1 lignin

/
G 241 \284 326

Co,, (D

so0 40 40
-310

70

10

~~~~H272

G5

178 25S0 218 S216

-~20 0) 0

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
350

50

100

150

200

250

300

mlZ
32 2

LL 00 -1

0
-

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

100

200

300

400

500

600

700

800

daynumber
S 0 210 G E8 73 60 85 P 114 95 137 S 167 19 G

100 80 C: CD 60 40

-~20

> ol

~~~~~I 31

45

124

0~~~~~~~~l
252
200 250

14
05 50 100 150

279
300 350

set.Score litterbag A. maritima ofpurified analysis after discriminant Fig. 6. DT-MS (El) results spectra.Samples were analyzed plot (b) and calculatedpositive(a) and negative(c) discriminant deviationsof the means. Variancedescribedis 82% of errorbars denotestandard in triplicate; and 29% of totalvariance.Symbolsas in Fig. 5. characteristic

masses in such structures to individual and plant stances." Assigning polymers, of purecompounds, withpeak patterns of presence butthesimultaneous is problematic, withPy-GC-MS data obtained fullspectra materialor by comparison the ions m/z 208, 192, 178, 162, 150, and 120 (shaded (Pouwels 1989; Van der Hage et al. 1993; independently of the presence suggests strongly squares) in thisspectrum reof theDF1(-) spectrum Klap et al. 1998). Interpretation and the generalfeatures of the degradedlignin.The score pattern character. Interpretation lignocellulose veals a strong are spectraand the average spectrum of the discriminant because it conis muchmorecomplicated, DF1(+) spectrum a but without very comparableto the resultsof S. anglica (Klap et al. highintensities tainsmanypeaks withrather a set theseDT-MS (El) data describe material. Considering 1998). Takentogether, of bio-organic signature recognizable of lignocellulose of natural of samples with a high average content upondecomposition is produced thatthisfraction the during is enhanced character (Fig. 5). This lignocellulose to as "humicsubalso be referred it can probably material,

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Salt marshhalophytes
* <- N-acetylhexosamine

1469

100l
_

151 101

185

221

0-

~~~~~~113
165 195 212a

130

C 60

40

86276

20 -233 100 150 200 250 300 350

mlz
30 -020 10

-10

-20 -

LI

ti

. .... . ... 14 100

200

300

I111

400

I 600 700 800

500

daynumber
H 100 80 C) 60 4U
_

180H GH pH 13 222 ~~~~~~~~163 ~~~~~~~~~~~208 P P 228H 20288

40 26

p 15~~~i0\
132H

CZ

20

314

342

100 in Pig 0..

150

200

250

300

350

mlZ

factor after A. mnaritimia set. Score plot Fig. 7. DT-MS (CI) results litterbag analysisof purified factor function. bars elrror (b) and spectra(a and c) of first Samples were analyzedin triplicate; deviations of themeans.Variancedescribed is 52% of totalvariance.Symbolsas denotestandard in Fig. 5.

leachingstageand is gradually substituted by a combination of degradedligninand a complex heterogeneous organic thelaterstagesof decomposition fraction during (Fig. 6). The CI results in Fig. 7 confirm the El results, although the scorepattern to Fig. 6b. The of Fig. 7b is not identical initialdecreaseoverthefirst weeksis lacking, butthegradual shift to positivescorescoinciding from negative withan increasein highlycomplexmaterial at the expense of lignocelluloseis similarto the El results.Again,mostof the mass peaks in theF1(-) spectrum (Fig. 7c) can be assigned to lignocellulose, whereasthecomplexF1(+) spectrum designatesa poorlydefined heterogeneous In organicfraction. thiscase, however, F1(+) also containsspecificadditional peaksindicative ofN-acetylglucosamine, whichappearat m/

is an important z 221, 203, 185, and 77. This aminosugar cell walls (Boon et al. 1987), fungi constituent of bacterial unitof chitin, (De Nobel et al. 1993), and the monomeric skeletons(De of arthropod the structural polysaccharide of the mentioned Leeuw and Largeau 1993). The identity with by MS-MS comparison fragment ions was confirmed pyrolysis of pure after ions of equal nominalmass resulting chitin. in abbreThe resultsforthe otherspecies are presented in Figs. 8 (El) and 9 (CI), whichshowthescore viatedform of thecorof themaincharacteristics plotswitha summary Details on thechemicalcharacterization responding spectra. elsewhere (Klap et al. 1998). For of S. anglica can be found considered.The the otherspecies, the El resultsare first

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1470
S. anglica (55,5% var.)
oOj Cb
I I

Klap et al.

30-~ 20 10 0lii I11

~ ~~~~
I

CP

-10

fraction heterogeneous organic + lignin products degradation lignin + polysaccharides (pentose> hexose) + lignin polysaccharides

-20-30

0 100 200 300 400 500 600 700 800 var.) 30 F. rubra (62.6% 20 10 -] 0 - ..

fraction + C16:0 FA organic heterogeneous + C16:0 FA + lignin polysaccharides (pentose) polysaccharides

LL

-10-j
-20-30-

0 100 200 300 400 500 600 700 800 20 A. tripolium(52,6% var.)

10-T
0-

phenols+ C02 + lignin phenols+ lipids > syringyl) + lignin (guaiacyl lipids

-10-i / -20-30 1____________

0 100 200 300 400 500 600 700 800

-L. vulgare (35.0% var.) 120 T < 10

LL-10

,1 ...........

small-non-specificfragments (hexose)+ mlz 170 C02 + polysaccharides + C16:0 FA + lignin 170 + polysaccharides (hexose) m/z

-20-30
0 100 200 300 400 500 600 700 800

0 -. -5-10 -15 0

25 -S. maritima (43,3%var.) 20 15 10 -

<
I

+ lignin organic fraction heterogeneous dimers + lignin products degradation > guaiacyl) + polysaccharides (syringyl lignin (syringyl) + C02 lignin

100 200 300 400 500 600 700 800

day number and S. m71aritirna with L. ivulgare, Fig. 8. El score plots of S. anglica, F. rubra,A. tripolium, theamounts of variancedescribed of indicated behindthespeciesnames.The maincharacteristics theaccompanying are expressed on theright sides of theplots.For each of theseplots,the spectra of the F,(+) spectrum, the middleone thoseof the average upperline gives the characteristics characteristics and thelowerone thoseoftheF(-) spectrum. The sequenceof mentioned spectrum, in thespectrum. is thesequenceof importance

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Salt marshhalophytes S. anglica

(46.6% var.)

1471

30- r 20 10 -t 0 I
-20

-10-]
-

...

heterogeneous organic fraction (incl.m/z 221, 203, 185 & 77) + lignin polysaccharides
> syringyl) + lignin polysaccharides (guaiacyl

0 100 200 300 400 500 600 700 800 var.) 40 F. rubra (60.0% 30 20 LL

-10

01

-Y (incl.m/z 221, 203, 185 & 77)

_ _ _ _

heterogeneous organic fraction

+ (some) lignin polysaccharides

;.........
_ _

-30

0 100 200 300 400 500 600 700 800

+ (some) lignin polysaccharides

var.) 20 -A. tripolium (68.7% 10 0 -{l i- -10-] -20 -30,, , ,,, -40 -,,

<..

heterogeneous organic fraction (incl. 221, 203,185 & 77) m/z polysaccharides (hexose)+ lignin polysaccharides (hexose)+ lignin (guaiacyl > syringyl)

0 100 200 300 400 500 600 700 800 var.) 15 -L. vulgare (47.5% 10 5........................................ o-

iZ -5-25

heterogeneous organic fraction (incl. m/z 221, 203, 185 & 77) polysaccharides (hexose> pentose) (hexose)+ (some) lignin polysaccharides

-205 i

-10-1

0 100 200 300 400 500 600 700 800 20 S. maritima (43.6%var.) 10 .. . U- -10I i-2o -30-, ,,, ,, ,

,/

+ heterogeneous polysaccharides organic fraction (incl.m/z 221, 203, 185 & 77) > guaiacyl) lignin (syringyl +polysaccharides > guaiacyl) lignin (syringyl

0 100 200 300 400 500 600 700 800 day number See L. vulgare,and S. m7>aritima. Fig. 9. CI score plots of S. anglica, F. rubra,A. tripolium, Fig. 8 for explanation.

score plot forF. rubrashows a comparable pattern to that of S. anglica andA. maritima (Figs. 8 and 6b, respectively). For thisspecies,theundefined heterogeneous organicfractionis negatively correlated to a pentosefraction insteadof The threeremaining a lignocellulosefraction. plots show 1 different The scoresforA. tripoliumn shift within patterns.

and remain stable month from extremely negative to positive withtherapafterward. This shift in scores,whichcorrelates of lipidand id weight loss (Fig. 1), expresses theconversion Thus,in conligninintosome simplephenoliccompounds. of mostA. tritrast to thepreviousspecies,decomposition does notgradually poliummaterial producea complexmix-

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1472

Klap et al.

ture. The scoresfor L. vulgaredo notshowa consistent trend during thedecomposition process,and thefactor spectra are noteasilyinterpretable. It is clear,however, that lignin is not an important compoundwithregardto the decomposition of thisspecies.The scoreplotforS. maritima dynamics does notreveala shift during thefirst fewweeks,butthegeneral shift fromlignin-rich to an uncharcompositional material acterizedheterogeneous is comparableto organicfraction thetrends seenforS. anglica andA. maritima. The relatively highabundance of syringyl unitsin theligninmoiety of S. maritima compared to S. anglica and A. maritima is in acwiththeCuO oxidation cordance results (Table 2). Also, the of theaveragespectra mainfeatures the (notshown)confirm ofthedifferent division to their planttissuesaccording lignin as determined content by CuO oxidation. The CI results (Fig. 9) providea consistent compositional shift the2 yrof decomposition. forall speciesduring In this period,lignin-rich or lignocellulose-rich (S. maritima) (all otherspecies) material is changedinto a heterogeneous organicfraction. OnlyS. anglica and F. rubrashowa leaching theinitialstagesof decomposition. The prepattern during markersof N-acetylglucosamine viously mentioned (mlz 221, 203, 185, and 77) are alwayspartof theheterogeneous This finding organicfraction. correlates well withthe generallyobservedincreasein relative contents nitrogen during thedecomposition process(Table 2).

in the litter. It is remarkable thatthe extensive extractions and rinsesinvolved in theMWEL purification procedure apparently do not mask the leachingprocess. It seems that conformity does notexiston therelative lignin contents during thepostleaching period.The CuO results indicatemore or less stableLOP yields(normalized to OC, Fig. 3) during the second stage of decomposition formostof the studied tissues,whereasthe DT-MS resultsindicatea gradualloss of intactlignocellulosetowardthe end of the experiment (Figs. 6-9). the comparative we Considering approachof this study, shouldaddressthequestion of theextent to whichtheresults obtained withthetwo analytic methods are comparable. Not onlydo themethods have theirspecific features, theyhave also been appliedto different samples,i.e., untreated (only ball milled)and purified the planttissues.Beforecomparing lignindynamics in the postleaching period,thesepotential objections are discussedin some detail. A roughcharacterization of theseanalytic methods might be thatthecombination of untreated material withCuO oxidationprovidesmerelyquantitative data, whereasDT-MS of MWEL samplesyieldsa mostly qualitative view on the lignocellulosefraction.However, the "quantitative"apdoes notmeanthat no qualitative proachof theCuO method indications are achieved;ratiosof different compoundsare used as diageneticindicators(Hedges and Mann 1979a; Goiniand Hedges 1992; Haddad et al. 1992). Accordingly, the term"qualitative"shouldnot be takento be too strict General discussion and overview fortheDT-MS method. Here it indicatesthatrelative peak intensities of ligninmarkers in themass spectra of thevarInitiallignincontent versusdecomposition rate-The deious samplesare compared. Peak intensities areproportional ratesof thedifferent composition plantspeciesvaryconsid- to detected ion numbers and represent data semiquantitative is considered a decay-retarding erably. Sincelignin substance, (as long as theapproximate pyrolysis/ionization efficiencies rateshave oftenbeen correlated withinitial of the various decomposition compoundsin the sample are known).The lignincontents (Wilsonet al. 1986a,b; Buth and Voesenek previously mentioned betweenthe two methdisagreement et al. 1988;Hemminga 1987;Hemminga andButh1991).The ods should therefore be nuanced to a lack of conformity results after CuO oxidation and DT-MS bothdenotethetis- betweenthe amountof ligninoxidationproducts of whole sues ofS. anglica,A. maritima, andS. maritima as lignin rich samples and mass spectraldata of thermally liberated ligand thetissuesofA. tripolium and L. vulgareas lignin poor, nocellulosefragments of purified samples. and F. rubraoccupiesan intermediate This pattern position. thedifferences If we consider between crudeand purified correlates theobserved losses (Fig. 1), quitewell with weight be extraction withorsamples,a sourceof variation might exceptforL. vulgare, wherea low lignin is notcor- ganic solvents. content thecase sinceit has been This is particularly related withrapidweight loss. This deviation becomeseven of ligninby organicsolventsis enshown thatextraction morepronounced if it is realizedthat thedecomposition rate hancedby ball milling(Lai and Sarkanen1971). We have of L. vulgareis probably overestimated because of theloss checkedthiseffect forS. anglica, and it turned out indeed of brittle material from thelitterbags. another deObviously, thatMWEL obtainedafter in 3 h of ball milling was richer thedecompo- ligninthanMWEL obtainedafter cay-retarding substance thanlignin determines 20 h of ball milling. Ball sition rateof L. vulgare. As indicated in theResultssection, milling The riskof thusinducedenhanced ligninextraction. thecontribution of physical to theweight loss is an processes shouldnotbe exaggerated, because highdepletion however, inherent connected to litterbag For disadvantage experiments. the ethanol-extractable of angiosperms ligninfraction (so thissampleset,thisproblem seemsto be of concern onlyfor called Braunslignins)is generally smaller than0.2% of the theleaves of L. vulgare. totalplantmaterial the (Lai and Sarkanen1971). Moreover, millingtimehas been kept short(3 h, whereas48 h was obtained with recommended Lignindynamics during decay-The results of Bj6rkman in theoriginal publication 1956) theCuO oxidation and DT-MS method confirm each partly in order to minimize As faras intact ligninextraction. lignin butdiscrepancies also exist.Conformity existson the is concerned, other, we assume thatcrude and purified samples increaseof ligninduring thefirst weeksof the maybe compared, rapidrelative no data are availableon whether although decomposition process(Figs. 3, 6-9). This increasecan be the effect of ball millingvaries as a function of decompoexplainedas the leachingof soluble and easily degradable sitionperiod.No effect of theball milling was foundon the suchas storage aminoacids,etc.,while CuO oxidationmethod(comparison components, sugars, betweenunmilledand structural material). components, including lignin and cellulose, remain 3-h-milled

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Salt marshhalophytes Quantitation ofthelignin oxidation with theCuO products method does notbear largeuncertainties. Withregard to the DT-MS technique, the questionis pertinent whether differences in pyrolysis/ionization efficiency betweencompound in sucha way that classes bias theresulting thescore spectra discriminant obtained after patterns analysisdo notrepresent prevalent chemicalchanges.If, forinstance, the heterogethatis produced neous organicfraction upon decomposition in the fieldwould consistof compounds thatare farmore ionized thanothercompounds efficiently pyrolyzed and/or in the sample,thisfraction cause nonlinearity of the might witha seriesof y-scalesin Figs. 6-9. A control experiment mixtures of the two A. maritima MWEL sampleswiththe mostextreme out that thediscrimscoresin Fig. 6b pointed relatedto the relative coninantscores are almostlinearly of thetwo samples(results notshown).It seems centrations likelythat thisalso countsfortheother scoreplots(Figs. 6methods views 9). Overall,thetwo analytic providespecific of theexposedplanttissues, on thelignin andwith dynamics thisin mindtheresults maybe compared. At first theapparent contradiction that we consider during thepostleaching theDT-MS results indicate a gradual period, of intact intoa heterogeneous orconversion lignocellulose ganic fraction, while the relativeamountof LOP as determinedby CuO remains stable.It is important to realizethat theDT-MS technique allows distinguishing with compounds different or thermal Small compounds volatility stability. and thusinducea will generally desorbat low temperatures in the thermal of the sample. The shift desorption profile MWEL residuesdid not show such a shift in the thermal forany of withincreasing desorption profile decomposition the plant species in this study.Thus, the MWEL residues consistof polymeric theexmainly compounds throughout The reconstructed mass spectrum thattypifies the periment. heterogeneous organicmaterialthatis producedupon deof alteredmonolignols includesmarkers composition (Fig. 6a, dashed squares),which suggeststhatthese chemically modified stillbelongto theligninmacromolemonolignols cule. Unfortunately, it is not possible to make a thorough betweenintact quantitative comparison ligninin theresidue after leachingand modified ligninin the decomposedfraction.We concludethat theDT-MS results indicate essentially the chemicalmodification of ligninand thatit cannotbe decidedhow therateof thismodification relatesto thatof bulkorganic indication matter loss. The quantitative provided by CuO thatligninis notbetter thanbulk orpreserved thepostleaching ganic matter during phase is thusnot opas might be concludedat first posed by theDT-MS results, sight. thereremainsa quantitative In Nevertheless, peculiarity. contradiction to theMWEL samples,whichconsistmainly of macromolecular theuntreated material, samplesstillcontain a fraction thatcan be removed by thepurification procedure. One may wonderwhat the removablefraction of smallcompounds of. One would expecta relatively consists low lignincontent forthisfraction and thusa negativeinfluenceon theLOP yield.However, theLOP yieldsin the of decreasing. postleaching phaseare stableinstead Two reasonableexplanations forthisobservation the appear.At first influence of theremovable fraction maybe compensated for

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by relative ligninenrichment of theremaining macromolecularfraction. Otherwise, theremovable fraction mayas well not be depletedin lignin(or materialthatproducesLOP) fraction. comparedto the macromolecular On the basis of the DT-MS results, which indicatethatthe relativelignin contentof the macromolecular fraction does not increase during decomposition, we concludethatthe second option is moreprobable.This scenariorequires thattheremovable fraction containssmall lignindecomposition products that, upon CuO oxidation, yielda comparable suitof products as theintact lignin macromolecule. This maybe thecase, since natural oxidationas well as CuO oxidation converts the acarbonatomof the side chainof a phenolicligninunitinto an acid group(Hedges et a carbonyl groupand eventually al. 1988a; Dijkstraet al. 1998). We concludethatsmalllignin decomposition become attachedto the tissue products as LOP. remains and can stillbe identified Anotherapparent betweenthe resultsobdisagreement tainedwiththe two rnethods concernsa qualitative aspect. it appearsthat, FromtheCuO oxidation results forall plant within1 species,the ligninmoietyreachesan endmember month. In fact,thisonlyrelatesto therapidstabilization of theC: V ratiobecause other qualitative parameters (S: V and Ad: Al) do notshowconsistent trends the25 months during of aerobicdecomposition. The DT-MS (El) results indicate that the intensities of several specificlignin degradation products (Fig. 6a) increaseupon decay forthethree ligninrichtissues.According to theseDT-MS results, a lignin endmember morethan2 yr of aerobic is not yetreachedafter are notcontradictory but degradation. Actually, theseresults because theC: V ratiois better determined complementary, withthe CuO method, alteration of the whereasfirst-stage side chain of a monolignol is moreeasily idenpropenolic tified the suitability using DT-MS. Based on theseresults, of ligninto monitor efflux of halophytic material fromsalt is dependent on the anmarshesto adjacentwatersystems is used,there do not used. If theCuO method alyticmethod to thestability ofthefirstseemto be limitations withregard IfDT-MS is used,typical endmember. stagediagenetic lignin characteristics arecontinuously lostandmayeventually vanish. differences in degradaBoth methods revealno clear-cut units.The relative stabilities bilityof guaiacyland syringyl of the different monomer have been studiedmany families times(Hedges et al. 1985; Benneret al. 1991; Haddad et al. et al. 1993; Gambleet al. 1994; Van derHeijden 1992; Gonti and Boon 1994; Opsahl and Benner1995), but no uniform Even in well-controlled all thesestudies. imageappearsfrom with white-rot laboratory experiments fungi,the observauntionsare ambiguous. The lack of uniformity, particularly der fieldconditions, of seems logical because the stability and oligolignols monolignols dependsnot only on intrinsic chemical properties, but also on (1) the biochemicaland conditions physicochemical during decomposition (including the different degradation pathwaysemployedby different macromolecule locationin thelignin decomposers), (2) their et al. 1993), and (3) interaction withothercell (Terashima wall components. An interesting observation in the DT-MS resultsof the threelignin-rich plantspecies (S. anglica,A. maritima, and

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of thelignin and polysaccharide biodegradation components of lignocelluloseand synthetic lignin by sedimentmicroflora. Appl. Environ. Microbiol.47: 998-1004. , S. Y. NEWELL, A. E. MACCUBBIN, AND R. E. HODSON. 1984b. Relativecontributions of bacteria and fungi to ratesof degradation of lignocellulosic detritus in salt-marsh sediments. Appl. Environ. Microbiol.48: 36-40. 5 K. WELIKY, AND J. I. HEDGES. 1990. Earlydiagenesisof mangrove leaves in a tropical estuary: Molecular-level analyses of neutral sugarsand lignin-derived phenols.Geochim.Cosmochim. Acta 54: 1991-2001. on finely BJORKMAN, A. 1956. Studies dividedwood-extraction of solvents. Sven. Papperstidn. 59: 477-485. ligninwithneutral BOON, J. J. 1989. An introduction to pyrolysis mass spectrometry of lignocellulosic material: Case studieson barleystraw, corn stemand Agropyron, p. 25-49. In A. Chesson and E. R. Orskov[eds.],Physico-chemical characterisation ofplant residues and feeduse. Elsevier. forindustrial . 1992. Analytical pyrolysis mass spectrometry: New vistas in-source PYMS. Int.J.Mass openedby temperature-resolved Ion Proc. 118/119:755-787. Spectrom. , B. BRANDT-DE BOER, G. B. EIJKEL, E. VLEGELS, L. SIJTSMA, AND J. T. M. WOUTERS. 1987. Differentiation of phage and phageresistant sensitive strains Streptococcus cremoris by mass spectrometry and discriminant pyrolysis analysisof the cell walls,p. 187-208.In E. HeinzleandM. Reuss [eds.],Mass in biotechnological spectrometry processanalysisand control. Plenum. A. TOM, B. BRANDT, G. B. EIJKEL, P. G. KISTEMAKER, F J.W. NOTTEN, AND F H. M. MIKX. 1984. Mass spectrometric and factordiscriminant analysisof complex organicmatter fromthe bacterialculture environment of Bacteroidesgingivalis. Anal. Chim.Acta 163: 193-205. BUCHSBAUM, R., I. VALIELA, T. SWAIN, M. DZIERZESKI, AND S. ALLEN. 1991. Available and refractory in detritus of nitrogen coastal vascularplantsand macroalgae. Mar. Ecol. Prog. Ser. 72: 131-143. of Spartinia BUTH, G. J.C., AND L. DE WOLF. 1985. Decomposition in a anglica, Elytrigia pungensand Halimnione portulacoides Dutch salt marshin association withfaunaland habitat influences. Vegetatio 62: 337-355. of standing , AND L.A.C.J.VOESENEK. 1987. Decomposition and fallenlitter of halophytes in a Dutch salt marsh, p. 146162. In A. H. L. Huiskes, C.W.P.M. Blom, and J. Rozema land and sea. Dr. W. Junk. between [eds.],Vegetation CHANG, H.-M., AND G. G. ALLAN. 1971. Oxidation, p. 433-485. In and C. H. Ludwig[eds.],Lignins:Occuirence, K. V. Sarkanen formation and reactions. Wiley. of ligninbioCHEN, C.-L., AND H.-M. CHANG. 1985. Chemistry and degradation, p. 535-556. Is] T. Higuchi[ed.],Biosynthesis of wood components. Academic. biodegradation microbial of cellulose, COLBERG, P. J. 1988. Anaerobic degradation and monoaromatic lignin, olilignols lignin derivatives, p. 333of 372. In A. J.B. Zehnder[ed.],Environmental microbiology anaerobes. Wiley. DE LEEUW, J. W., AND C. LARGEAU. 1993. A reviewof macromoand lecularorganic that compounds comprise livingorganisms their rolein kerogen, coal, and petroleum formation, p. 23-72. In M. H. Engel and S. A. Macko [eds.],Organic geochemistry. Plenum. DE NOBEL, J.G., AND OTHERS. 1993. Analysis of cell wall mutants mass spectrometry. of Saccharomyces cerevisiaeby pyrolysis Acta Bot. Neerl.42: 505-516. DIJKSTRA, E. F, J. J. BOON, AND J. M. VAN MOURIC. 1998. Anaof a soil profile underScots pine.Eur.J. Soil lytical pyrolysis Sci. 49: 295-304.

S. maritima) is thatligninand polysaccharide show correlated behaviorduring decay. The apparent comparable stabilities explainwhywe commonly have used thedesignation "lignocellulose"insteadof "lignin" and "polysaccharide" is contradictory to other separately. This observation reports labile (Hodson et al. thatdenotedcellulose as relatively 1984; Opsahl and Benner1999). Geochemicalimplications fromthis study-Rice (1982) of increasing addressedthe phenomenon has exhaustively nitrogen content withincreasing decomposition time, which was also observedin this study(Fig. 2 and Table 2). His well-accepted hypothesis (Wilson et al. 1986a; Hemmninga et al. 1988; Benneret al. 1991; Buchsbaumet al. 1991), which sharessimilarities withthe mechanism proposedin theprevioussection, molis thatsmall reactiveoligomeric form ecules produced thefirst during stageof decomposition reactivegeomacromolecules. These geomacromolecules act as condensation nuclei fornitrogenous microbialexudates and becomemoreand morerefractory in thecourseof time. The remaining plantfraction enthusbecomesincreasingly riched withthosenitrogen-rich OurDTgeomacromolecules. MS (CI) results indicatea gradualincreaseof N-acetylglucosamine for all plant species studied and thus provide of allochthonous strong evidencefora contribution nitrogenrichmaterial. It is worthwhile to relatethepresented to thecomresults of humicsubstances. monviewpoints on theformation Two different have been distinguished: pathways (1) condensation of small degradation such as monopeptides products, and oligopeptides, and oligosaccharides, monosaccharides into a refractory lipids,and phenolicsubstances, heterogeof onlyslightly neousorganic and (2) condensation polymer modified biogenic macromolecules (Mayer 1985; Hedges 1992; Ishiwatari1992; De Leeuw and Largeau 1993). As ofDTthecombined previously outlined, we interpret results MS and CuO oxidation fortheoccurrence as an indication ofthefirst ofthetwoproposed Of coursedifferent pathways. substances have different so thesecpreservation potentials, it does ondpathway will applyto someextent. Nevertheless, notseemlikelythat certain remain intact biogenicmolecules in humus.As such,the secondpathway be regarded might in whichdeas an intermediate stage of the first pathway of thecontributing substances is less prolonged. composition References
AIKEN, G. R., D. M. McKNIGHT, R. L. WERSHAW,AND P. MACCARTHY. 1985. An introductionto humic substances in soil, sediment,and water,p. 1-9. In G. R. Aiken, D. M. McKnight, R. L. Wershaw, and P. MacCarthy [eds.], Humic substances in

AMER,

and water. soil, sediment Wiley. G. I., AND S. W. DREW.1980. Microbiology of lignindegradation, p. 67-103. In G. T. Tsao, M. C. Flickinger, and R. K. Finn [eds.], Annual reports on fermentation processes.Academic. BENNER, R., M. L. FOGEL, AND E. KENT SPRAGUE.1991.Diagenesis of belowground biomassof Spartinaalternifiora in salt-marsh sediments. Limnol.Oceanogr. 36: 1358-1374. , A. E. MACCUBBIN, AND R. E. HODSON. 1984a. Anaerobic

This content downloaded from 196.200.142.112 on Sun, 3 Nov 2013 17:08:47 PM All use subject to JSTOR Terms and Conditions

Salt marshhalophytes
FAIX,

1475

degradation O., D. MEIER, AND I. FORTMANN. 1990. Thermal of , C. J. KOK, AND W. DE MUNCK.1988. Decomposition products of wood. Gas chromatographic separation and mass Spartinaanglica roots and rhizomesin a salt marshof the spectrometric characterization of monomericlignin-derived Westerschelde estuary. Mar.Ecol. Prog.Ser.48: 175-184. products. Holz Roh-Werkstoff. 48: 281-285. HIGUCHI, T. 1985a. Biosynthesis of lignin, p. 141-160. In T. HiguGAMBLE, G. R., A. SETHURAMAN, D. E. AKIN, AND K.-E. L. ERof wood compochi [ed.], Biosynthesis and biodegradation IKSSON. 1994. Biodegradation of lignocellulosein Bermuda nents. Academic. . 1985b.Degradative pathways of ligninmodelcompounds, Grass by whiterot fungianalyzedby solid state13C nuclear magnetic resonance. Appl.Environ. and biodegraMicrobiol. 60: 3138-3144. p. 557-578. In T Higuchi [ed.], Biosynthesis GENUIT, W., J. J. BOON, AND 0. FAIX. 1987. Characterization of dationof wood components. Academic. beech milledwood ligninby pyrolysis-gas chromatography- HODSON, R. E., R. R. CHRISTIAN, AND A. E. MACCUBBIN. 1984. mass spectrometry. Anal. Chem.59: 508-512. Lignocellulose and ligninin the salt marshgrassSpartinaalphotoionization GoNi, M. A., AND J. I. HEDGES. 1992. Lignindimers:Structures, Initialconcentrations and short-term, post-depositernifiora: distribution, and potential geochemical tionalchangesin detrital matter. Mar. Biol. 81: 1-7. applications. Geochim. HOOGERBRUGGE, R., S. J. WILLIG, AND P. G. KISTEMAKER. 1983. Acta 56: 4025-4043. Cosmochim. . 1995. Sourcesand reactivities , AND Discriminant analysis by double stage principalcomponent of marine-derivedorganicmatter in coastal sediments as determined by alanalysis.Anal. Chem.55: 1710-1712. kalineCuO oxidation. Geochim.Cosmochim. Acta 59: 2965material (humicsubstance) ISHIWATARI, R. 1992. Macromolecular 2981. in thewatercolumnand sediments. Mar. Chem.39: 151-166. comJUNG, H.-J.G., AND J. RALPH. 1990. Phenolic-carbohydrate 5 B. NELSON, R. A. BLANCHETTE, AND J. I. HEDGES. 1993. of wood lignins: on lignocellulose Fungaldegradation Geochemical plexes in plant cell walls and theireffect perspectives from CuO-derived phenolicdimersand monomers. Geochim. J. R. utilization, p. 173-182. In D. E. Akin,L. G. Ljungdahl, Acta 57: 3985-4002. Cosmochim. Wilson,and P. J. Hairis [eds.], Microbialand plantopportuGOUGH, M. A., R. FAUZI, C. MANTOURA, AND M. PRESTON. 1993. nitiesto improve utilization Elby ruminants. lignocellulose in marinesediments. Terrestrial Geochim. sevier. plantbiopolymers Acta 57: 945-964. The Cosmochim. KIRK,T. K., AND M. SHIMIDA. 1985. Ligninbiodegradation: in andthephysiology and biochemistry microorganisms involved HADDAD, R. I., AND C. S. MARTENS. 1987. Biochemical cycling an organic-rich marinebasin. 9. Sources and accumulation of degradation by white-rot fungi, p. 579-605. In T. Higuchi Cosand biodegradation of wood components. ratesofvascular plant-derived material. Geochim. [ed.], Biosynthesis organic Acta 51: 2991-3001. Academic. mochim. exchange KLAP, V. A. 1997. Biogeochemical aspectsof saltmarsh , S. Y NEWELL, C. S. MARTENS, AND R. D. FALLON. 1992. Univ. of Amprocessesin the SW Netherlands. Ph.D. thesis, Early diagenesis of lignin-associated phenolics in the salt Acta sterdam. marsh grassSpartinaalternifiora. Geochim.Cosmochim. 56: 3751-3764. , J.J.BOON, M. A. HEMMINGA, AND J. VAN SOELEN. 1998. of ligninpreparations of Spartina Chemical characterization HARTLEY, R. D., AND J. HAVERKAMP. 1984. Pyrolysis-mass specof thephenolic of plantcell walls.J.Sci. mass spectrometry. Org. Geochem.28: trometry constituents anglica by pyrolysis Food Agric.35: 14-20. 707-727. H. E. SCHOEMAKER, AND J.M. PALMER. 1987.Lignin HARVEY, P. J., LAI, Y. Z., AND K. V. SARKANEN. 1971. Isolationand structural PlantCell Environ.10: 709degradation by whiterotfungi. studies,p. 165-240. In K. V. Sarkanenand C. H. Ludwig and reactions. 714. stlucture [eds.],Lignins:Occulrence, formation, HEDGES, J. I. 1992. Global biogeochemical cycles: Progressand Wiley. bioproblems. Mar. Chem.39: 67-93. LEWIS, N. G., AND E. YAMAMOTO. 1990. Lignin:Occurrence, Annu. Rev. Plant Physiol.Plant genesis and biodegradation. , R. A. BLANCHETTE, K. WELIKY, AND A. H. DEVOL. 1988a. Effects of fungaldegradation on the CuO oxidation Mol. Biol. 41: 455-496. products of lignin:A controlled GAGNE, AND L.-F LOUCHOUARN, P.,M. LUCOTTE, R. CANUEL, J.-P. laboratory study.Geochim.Cosmochim.Acta 52: 2717-2726. RICHARD. 1998. Sources and early diagenesisof ligninand and the bulkorganicmatter in thelowerSt-Lawrence Estuary , W. A. CLARK, AND G. L. COWIE. 1988b. Organicmatter sources to thewater column and surficial sediments ofa marine SagenayFjord.Mar. Chem.58: 3-26. ofhumicsubstances in estuarine bay. Limnol.Oceanogr. 33: 1116-1136. MAYER, L. M. 1985. Geochemistry of carenvironments, p. 211-232. In G. R. Aiken,D. M. McKnight, , G. L. COWIE, AND J. R. ERTEL. 1985. Degradation in andlignins in buried woods.Geochim. Cosmochim. R. L. Wershaw, and P. MacCarthy [eds.],Humicsubstances bohydrates Acta 49: 701-711. and water. soil, sediment Wiley. of ligninby gas MORAN, M. A., L. R. POMEROY, E. S. SHEPPARD, L. P. ATKINSON, 5 AND J. R. ERTEL. 1982. Characterization AND R. E. HODSON. 1991a. Distribution derived of cupricoxide oxidation of teirestrially capillarychromatography products. Anal. Chem.54: 174-178. dissolvedorganicmatter on the southeastern U.S. continental of plant shelf.Limnol.Oceanogr. 36: 1134-1149. , AND D. C. MANN. 1979a. The characterization tissuesby theirligninoxidationproducts. Geochim.Cosmo, R. J. WICKS, AND R. E. HODSON. 1991b. Exportof disEvchim.Acta 43: 1803-1807. solvedorganic matter from a mangrove swampecosystem: . 1979b. The ligningeochemistry AND and of marine idencefrom natural dissolvedlignin phenols, fluorescence, sediments from thesouthern bacterial coast.Geochim. CosMar.Ecol. Prog.Ser.76: 175Washington secondary production. Acta 43: 1809-1818. mochim. 184. needlesin a , AND K. WELIKY. 1989. Diagenesisof conifer NELSON, B. C., M. A. GoNi,J.I. HEDGES, AND R. A. BLANCHETTE. coastal marineenvironment. Acta 53: Geochim.Cosmochim. 1995. Soft-rot of ligninin 2700 year old fungaldegradation 49: 1-10. 2659-2673. woods. Holzforschung. archaeological in HEMMINGA, M. A., AND G. J. C. BUTH. 1991. Decomposition of senescent OPSAHL, S., AND R. BENNER. 1993. Decomposition salt marsh blades of the seagrassHalodule wrightii in a subtropical laof theS.W. Netherlands: The effects of ecosystems bioticand abioticfactors. Vegetatio 92: 73-83. goon. Mar.Ecol. Prog.Ser.94: 191-205.

This content downloaded from 196.200.142.112 on Sun, 3 Nov 2013 17:08:47 PM All use subject to JSTOR Terms and Conditions

1476

Klap et al.

. 1995. Earlydiagenesis , AND of vascularplanttispositionin terrestrial ecosystems. Studiesin ecology.Blackwell. sues: Ligninand cutindecomposition and biogeochemical imTAS,A. C. 1991. Mass spectrometric fingerprinting: Softionization plications. Geochim.Cosmochim. Acta 59: 4889-4904. and pattern recognition. Ph.D. thesis, Univ.of Leiden. , AND . 1999. Characterization of carbohydrates durTERASHIMA,N., K. FUKUSHIMA,L. HE, AND K. TAKABE. 1993. ing earlydiagenesisof fivevascularplanttissues.Org. Geomodelof thelignified Comprehensive plantcell wall, p. 247chem.30: 83-94. 270. In H. G. Jung, D. R. Buxton, R. D. Hatfield, and J.Ralph POUWELS, A. 1989. Analytical pyrolysis mass spectrometry ofwood and [eds.], Forage cell wall structure digestibility. American derived polymer fractions. Ph.D. thesis, of AmsterUniversity Societyof Agronomy. dam. VAN BERGEN,P. F, M. A. GoNi,M. E. COLLINSON, P. J.BARRIE,J. , AND J. J. BOON. 1990. Analysisof Beech wood samples, S. SINNINGHE DAMSTE, AND J. W. DE LEEUW. 1994. Chemical its milledwood ligninand polysaccharide fractions by Curieand microscopic characterization of outerseed coats of fossil point and platinum filament pyrolysis-mass J. spectrometry. and extant Acta58: 3823water plants. Geochim. Cosmochim. Anal. Appl. Pyrolysis 17: 97-126. 3844. PRAHL, F G., J. R. ERTEL, M. A. GoNI, M. A. SPARROW, AND B. VAN DER HAGE, E. R. E., M. M. MULDER, AND J. J. BOON. 1993. EVERSMEYER. 1994. Terrestrial organic carboncontributions to of ligninpolymers Structural characterization by temperaturesediments on the Washington margin. Geochim.Cosmochim. resolved in-sourcepyrolysis-mass and Curiespectrometry Acta 58: 3035-3048. J. point pyrolysis-gas chromatography/mass spectrometry. RALPH, J., AND R. D. HATFIELD. 1991. Pyrolysis-GC-MS characAnal. Appl. Pyrolysis 25: 149-183. terization of forage materials. J.Agric.Food Chem.39: 1426AND J.J.BOON.1995. Ammonia chemical , T. L. WEEDING, 1437. ionization mass spectrometry of substituted phenylpropanoids andphenylalkyl ethers. J.Mass Spectrom. 30: 541-548. phenyl New observations RICE,D. L. 1982. The detritus nitrogen problem: and perspectives from Mar.Ecol. Prog. organicgeochemistry. VAN DER HEIJDEN, E., AND J. J.BOON. 1994. A combined pyrolysis Ser. 9: 153-162. and lightmicroscopic mass spectrometric studyof peatified SAIZ-JIMENEZ, C., J. J.BOON, J. I. HEDGES, J.K. C. HESSELS, AND Calluna wood from raisedbog peat deposits.Org. Geochem. J.W. DE LEEUW. 1987.Chemical characterization and ofrecent 22: 903-919. buried woods by analytical ofpyrolysis pyrolysis: Comparison R. K., AND G. E. LANG. 1982. A critique of theanalytical WIEDER, data with 3C NMR and wetchemicaldata.J.Anal. Appl. Pymethods used in examining decomposition data obtained from rolysis11: 437-450. litterbags. Ecology63: 1636-1642. SARKANEN, K. V., AND C. H. LUDWIG. 1971. Lignins:Occurrence, WILSON, J. O., R. BUCHSBAUM, I. VALIELA, AND T. SWAIN. 1986a. and reactions. structure formation, Wiley. Decomposition in salt marshecosystems: Phenolicdynamics oftobacco SCHEIJEN, M. A., AND J. J.BOON. 1989. Characterization during decayof litter of Spartinaalterniflora. Mar.Ecol. Prog. lignin preparations by curie-point pyrolysis-mass spectrometry Ser.29: 177-187. and curie-point resolution pyrolysis-high gas chromatography/ ,I. VALIELA, AND T. SWAIN. 1986b.Carbohydrate dynamics mass spectrometry. J.Anal. Appl. Pyrolysis 15: 97-120. of Spartinaalternifiora. Mar. Biol. 92: during decay of litter STOUT, S. A., J. J. BOON, AND W. SPACKMAN. 1988. Molecular 277-284. AND P. G. KISTEMAKER.1983. InterWINDIG, W., J. HAVERKAMP, aspects of the peatification and early coalification of angioanalpretationof sets of pyrolysismass spectraby discriminant Acta woods. Geochim.Cosmochim. spermand gymnosperm ysis and graphical rotation.Anal. Chem. 55: 81-88. 52: 405-414. YOUNG, L. Y., AND A. C. FRAZER. 1987. The fate of lignin and , W. SPACKMAN, J. J. BOON, P G. KISTEMAKER, AND D. F lignin-derivedcompounds in anaerobic environments.GeomiBENSLEY. 1989. Correlations betweenthe microscopicand crobiol.J.5: 261-293. chemicalchangesin wood during and earlycoalpeatification Received: 7 April 1999 A canonicalvariant ification: study. Int.J.Coal Geol. 13: 41Accepted: 13 April 1999 64. Amended: 10 May 1999 SWIFT, M. J.,0. W. HEAL, AND J. M. ANDERSON. 1979. Decom-

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