Lignin dimers: Structures, distribution, and potential ... - Science Direct

Gee&mica

a Cosmochimica Acla

~i6-7037/92~5.~ + ,oO

Vol.56,pp.4025-4043

Copyright5 1992PergamonRess Ltd.~nl~in~.S.A.

Lignin dimers: Structures, distribution, and potential geochemical applications MIGUEL A. Go~I* and JOHN I. HEDGES School of Oceanography, WB- 10, University of Washington, Seattle, WA 98 195 USA (Received OctoberI, 199 1; accepted in revisedform May 6, 1992)

Abstract-An extensive suite of thirty lignin-derived phenolic dimers and fourteen additional monomers has been identilied among the CuO reaction products of ~enty-four different vascular plant tissues. The various lignin dimers are characterized by five different types of linkages between phenolic units, including direct 5,5’-ring-ring bonding, as well as P,l-diketone, cr,l-monoketone, a,%monoketone, and tu,2-methyl sidechain-ring couplings. The new lignin-derived monomeric CuO reaction products include vanillyl and syringyl glyoxalic acids and vanillyl phenols with formyl and carboxyl functional groups attached at various ring positions. The distribution of all these novel compounds in twenty-four different vascular plant tissues indicates important differences in the structure and chemical composition of the lignin macromolecule among these sources. The abundances of these compounds in a selected set of sedimentary samples suggest that the lignin dimers and novel lignin monomers can characterize the ultrastructure, sources, and diagenetic state of sedimentary lignin in ways not possible from the routinely utilized lignin monomers alone. present in marine waters (MEYERS-SCHULTEand HEDGES, 1986; MORAN et al., 1991). A second application of the li~in~e~ved CuO reaction products, to draw inferences about types and amounts of different vascular plant tissues (HADDAD and MARTENS, 1987; HAMILTONand HEDGES, 1988; HEDGESet al., 1988b), is much more challenging since it requires relating sedimentary compositions back to those of fresh tissues. For example, the vanillyl, syringyl, and cinnamyl phenols used to differentiate vascular plant tissue types (HEDGES and MANN, 1979a) have variable diagenetic reactivities in different tissue types (HEDGES et al., 1988a; HEDGES and WELIKY, 1989; BENNERet al., 1990, 1991) that make the quantification of plant sources in sedimentary mixtures very difficult without a measure of the diagenetic state of the vascular plant debris present in the sample. The vanillyl acidlaldehyde ratio, [Ad/ Al], , which is elevated with fungal degradation (HEDGES et al., 1988a), can provide a qualitative estimate of diagenetic state in some samples ( ERTEL et al., 1984, HEDGES et al., 1988c). However, vanillic acid ester-bound to polysaccharides in some nonwoody vascular plant tissues (WHITEHIEADet al., 198 1; YAMAMOTOet al., 1989) can cause high [Ad/Al Iv ratios in undegraded samples (GoRI, 1992), thereby complicating the use of this parameter as a diagenetic indicator. Because of the diversity of sources and the potential for selective diagenesis, the challenge of characterizing vascular plant remains in sedimentary mixtures can be met only by having multiple biomarkers of different specificity and lability that can supply concordant information. Our strategy has been to maximize the CuO procedure by recognizing reaction products, in addition to the usual eight phenolic monomers, that with little additional effort can yield increased geochemical information about the sources and diagenetic state of the organic matter in the sample. Among these, a suite of cutinderived hydroxy fatty acids with the ability to distinguish several broad types of nonwoody vascular plant tissues in ways not previously possible has been identified (Go& and HEDGES, 1990a,b ) . Because of their similar overall reactivity,

INTRODUCTION VASCULARLAND PLANTS ACMUNT for over half of the global primary productivity (OLSON et al., 1985; MARTIN et al., 1987) and, together with vascular plant-derived soil humus, represent over 75% of the active organic carbon (OC) reservoirs on earth (HEDGES, 1992 ) . Hence, vascular plant-derived organic matter may potentially be an important component of the OC preserved in soils and marine sediments and may therefore be a major source of atmospheric O2 (ROBINSON, 1990; BERNER, 1989) and a sink for COZ ( BERNER, 1982, 1991; ROMANKEVICH, 1984; EMERSON and HEDGES, 1988). There are a variety of unique biochemicals, such as nalkanes (BARRINGTONand TRIPP, 1977), diterpenoid acids ~SIMONEI~,1977), and sterols (GAGOSIANet al., 1983), that can be used as molecular tracers, or biomarkers, to sensitively detect and characterize vascular plant remains and land-derived organic matter in soils and sediments. Another of these terrestrial biomarkers is lignin, an abundant and stable phenolic macromolecule uniquely found in the cell walls of vascular land plants ( SARKANEN and LUDWIG, 197 1). Upon alkaline CuO oxidation, lignin yields a suite of eight major vanillyl, syringyl, and cinnamyl phenols (HEDGESand ERTEL, 1982), in patterns that can distinguish various broad types of vascular plant tissues (HEDGES and MANN, 1979a). Total c~~n-no~~~ed yields of these predomin~t lignin monomer have been used to estimate the relative amount of terrestrial organic matter in marine sediments. This application, which relies primarily on little in-situ diagenesis of the lignin afier deposition, has been independently substantiated vs. 613C (HEDGES and PARKER, 1976; HEDGES and MANN, 1979b) and C/N (HEDGES et al., 1986,1988b) measurements. This approach has also been used to estimate the fraction of dissolved humic substances of terrestrial origin

* Author to whom correspondence should be addressed. 4025

4026

M. A. Got% and J. I. Hedges

cutin acids can characterize various sources of cutin-bearing vascular plant tissues in sedimentary mixtures, even after moderate &genesis (Go& and HEDGES, 1990~). Hence, the

use of lignin-derived phenols together with the cutin-derived reaction products can give more specific characterizations of vascular plant sources than are possible with either of the two compound types alone (GOUGH et al., 1992; PRAHL et al., 1992). As a continuation of this study on previously unknown CuO reaction products, we report here an extensive and structurally diverse suite of thirty lignin phenol dimers and fourteen additional lignin monomers which provide complementary, and sometimes unique, information on the origin and diagenetic history of vascular plant tissues in soils and sedimentary deposits. In particular, the diphenolic products reflect the ultrastructure of the parent lignin polymer and, in contrast to phenolic acids ester-linked to polysaccharides, have essentially no other origin. In the following sections, we discuss the structural characteristics of these dimeric products and related phenolic monomers and investigate their potential as complementary biomarkers of vascular plant sources. To illustrate geochemical applications, lignin dimer and monomer yields from a selected suite of sedimentary samples are presented and, based on the fresh tissue data, used to draw inferences about the ultrastructures, sources, and diagenetic states of the lignins in these materials. In a following paper ( Go~~I et al., 1993)) the CuO oxidation results from a variety of degraded woods will be used to assess the effects of diagenesis on the distribution of lignin dimers and to investigate further the processes responsible for the observed sedimentary compositions. METHODS

Sample Collection and Handling Gymnosperm woods (Thuja plicata, Tsuga canadensis, Pseudotsuga menziensii and Picea glauca) and woods from several temperate (Betula papyrifera, Acer saccharum. Quercus rubra, and Abuts rubra) and tropical (Zanthoxylum compactum and Nectrandra amazonium) angiosperm species were received from a variety of collaborators, including R. Blanchette (University of Minnesota, St. Paul). Gymnosperm needles ( T. plicata and P. menziensii), cones (P. menziensii), and barks ( T. plicafa and P. menziensii) and angiosperm leaves (Acer macrophyllum and Q. rubra) and barks (A. macrophyllum and Q. rubra) were collected from mature trees at the University of Washington Arboretum (Seattle). Temperate monocot species, including a sedge (Carex sp.) and grasses, were collected near Dabob Bay, Washington (Agrostis alba), and from the vicinity of Woods Hole, Massachusetts (Spartina alterniflora), and analyzed whole. All samples were oven dried at 50°C for 2-3 days, ground with a Wiley mill to pass a 42-mesh (350-pm) sieve, and stored at room temperature until analysis. Surface sediments (top 12 cm) from two sites, Crab Creek and Long Lake, in the Columbia River were collected with a 0.025 m2 van Veen grab sampler (HEDGES et al., 1984). Surface sediments from three locations throughout the Washington margin were collected with a box core, and the top O-2 cm horizons used for analysis (PFUHL and CARPENTER,1984). The three sites chosen for this study include Willapa Bay (46”30.0’N, 124”23.O’W, 90 m depth) plus continental shelf (46”49.7’N, 124”26.O’W, 73 m depth) and slope (46”44.8’N, 125”0.7’W. 700 m depth) locations. Dabob Bav sediments were collected with a 50-cm long box core from a 11O-m deep station in the northern bay (HEDGES et al., 1988b) and the O-l cm, lo- 12 cm, 28-30 cm, and 48-50 cm horizons were analyzed for this paper. All sedimentary samples were frozen after collection, oven dried at 50°C for several days, and stored at room temperature prior

to analyses. Details about location and compositional characteristics of these samples are available in the cited references. Elemental Analyses Plant tissue OC and nitrogen compositions were determined in duplicate without acidification using a Carlo Erba 1106 CHN analyzer according to HEDGESand STERN( 1984). Sedimentary OC and nitrogen determinations were carried out with vapor phase acidification also by this method. The sample mean deviation of these measurements was +2%. CuO Oxidation The CuO oxidation procedure was similar to that outlined by HEDGESand ERTEL(1982) but included some variations. About 25 mg of plant material, or 250 mg of sediment, was oxidized with CuO under alkaline conditions (8 wt% NaOH) along with 100 mg of Fe( N&)*( Sod)* - 6Hz0 for 3 h in a stainless steel minibomb. Four of such reaction chambers were loaded in a large bomb (Parr Co.), which contained additional base and was fitted in an insulated heating sleeve of new design. The heating rate and final temperature were controlled by a 02 155~series platinum RTD temperature controller (Cole Parmer); but, unlike HEDGESand ERTEL( 1982), the inside, as well as the outside, temperature of the large bomb was constantly monitored with two 100-W platinum RTD probes (Cole Parmer). After extensive experimentation with the heating sleeve, we concluded that the best settings, in terms of comparison with preexisting data collected with the old setup, were a final internal temperature of 155°C and heating rate of 30 min from room to final temperature (S. de Villiers et al., unpubl. data). Similarly to HEDGESand ERTEL ( 1982 ) , radio-labelled 14C-p-hydroxyacetophenone was added prior to bombing as a recovery standard. Following the oxidation, samples were acidified with HCl (pH 1), extracted with ethyl ether, dried under NZ, and stored frozen until analysis. Gas Chromatography Dry extracts of the CuO reaction mixture were dissolved in pyridine and l/2 to l/5 splits were used for GC analysis; the remaining extracts were dried and stored frozen. Internal standards, ethylvanillin and cinnamic acid, were added to the dissolved sample and a small amount ( l-2 pL) of this mixture was drawn in a gas-tight GC syringe (Hamilton Co.). An equal volume of Regisil (BSFTA reagent, Regis Chem. Co.) was drawn into the syringe and mixed thoroughly by pumping the plunger to ensure complete derivatization. The syringe was placed on a heating block at 60°C for 10 min, after which the sample was injected into the GC. Experiments with several sample types (Goml, 1992) showed in-syringe derivatization to give the same results as the previous in-vial derivatization ( HEIXZS and ERTEL,1982), which requires IO-100 times more silylating reagent. In-syringe derivatization also allows analysis of freshly derivatized samples in each injection and minimizes sample and reagent use when perdeuterated trimethylsilyl or methoxylamine derivatives are formed for structural determination purposes. Gas chromatography was carried out with a Hewlett-Packard 5730 GC fitted with a dual-column injection port connected to 30 m by 0.25 mm i.d. DB- 1 and DB-170 1 capillary columns (J & W Scientific), each attached to a separate flame ionization detector (FID). The injector and two detectors were maintained at 300°C. The oven was initially at lOO”C,then heated at 4”C/min to 27O’C and held at that temperature for 17.5 min (total run time equals 60 min). The retention times of all CuO reaction products were determined and expressed in Kovatts-type format relative to coinjected fatty acid standards (Table 1) . Lignin dimers eluting from each column were quantified based on their FID responses as determined by two Hewlett-Packard model 3900A integrators using ethylvanillin as a GC internal standard. Due to the lack of sufficient standards, relative response factors of only a few lignin dimer standards (synthesized by I. Pearl and donated by J. Obst of the Wood Chemistry Laboratory in Madison, Wisconsin) could be measured and varied widely from I .5-3. These responses seemed exceedingly high relative to ethylvanillin, possibly due to

Organic geochemistry of lignin

4027

Table 1. Liginin-derived CuO reaction prwIuct8.

NW%3

Symbol GcIi

DB-I RRT

MSFmgmenta

R

446LQ.l.357 460,~.371,3S7 474@&3? 1 534Jl9.44S&$j 548,533,445~ 622.6@7445 %2$547441,351 576,561 a.311 650.635.XW45 416&&,321 430.411.34 1,327 504.489322

H H

R

R”

SJ*-dlmvr

Debydmdivanillin DehydrovaniUinacetovanillcme DehydrodiacetovaniUonc ~yd~v~v~c acid ~yd~~v~~ev~c acid Dehydmdivanillic acid DehydrovaniUittvnniUylglyoxalic acid DeJtydroacctovanUovanillylglyoxalic acid DehydrovaniUicvanillylglyoxalic acid Dehydrovanillin-p-hydroxybakzaldehyde DehydmvaniEin-p-hydroxyacetophenone ~~~v~-~byd~y~~c acid VJmiUil vaniuosyringil Sytigil vulillovanillone Vanillosytingcfle sy~g~y~g~e ~Hyd~x~ylv~~e 5-V&ttilloVanUill s-vanilloacetovaniuone !&VatdlovaniUic acid 5-Syringovaniuin S-SyTingoacetovanillone 5-SyringovaniEc acid 2-Vanillylsyzingealdehyde 2-Vanillylacetisyringone 2-Vaoillylsyringic acid 2-Syringylsytigealdehyde 2Syringylacetosyfingone 2-Syriqylsy~ingic acid S-F~ylv~ S-Formyiacetovanillone 5-Fonnyhwillic acid

VI-VI Vl-vn Vn-Vn VI-Vd Vn-Vd Vd-Vd Vl-vg Vn-Vg VI-Ve

26 2173 28 2212 30 2241 32 2289 33 2319 40 2382 48 2466 49 2493 52 2548 VI-PI- 23 2037 Vl-Pn 24 2081 W-W 27 2186 &l-dlk@oae dknera VoVo 42 2403 voso 50 2493 &so 53 2593 a&monokeUmc dinwra VVo 29 2230 VSo 36 2345 SSo 47 2456 2@?5 PVO 25 u$-momketone timers VoSVl 31 2286 vo5vn 35 2393 2436 Vo5Vd 45 2366 Sowi 38 So5Vn 43 2411 So5Vd 51 2500 a&3nethyl dimera 2327 Vm2Sl 34 2366 vm2Sn 37 2427 Vm2Sd 44 2376 Sm2Sl 39 23% Sm2Sn 41 2427 Sm2Sd46 5.fermyl RtmloXners 1301 SM 5 1359 5fVn 7 1519 SfVd 13

446.43L222 476,461 .a,223 506,491&J ql(L,403.388,373,358 @&,433,418,403,388 ~,~,~8,433,418 ~,3~~58,343,3~

cH3 H CH 02 H CH 02 H H H H H OcH,

H a3 CH Otl OH CZH COOH COOH H

O=G H

-3

rnH3 -3 OCH,

-3H H H

20 Ii -, O? H

H H

-3 -3 OCH, -3

q H

n... n.a. ILL -3 OCH, Ot=, H n.a. n.a. ll... o... n.a. I).*.

446,431,357 460445,351 534s.357 476,=,387 &O&&,387 564.&@387

H CH OII H CH OF!

462&,417 476,m,43 1 550.~,505 492.=,447 506&,461 580&&535

H CH 02 H CH OII

252.m.221 266,=,221 340&L&221

H CH Od

r&e n.a. n.a.

n.L n.a. n.a.

H CH O$

n.a. n-a. n.a.

n.r. n.a. n.a.

H CH OI?

n.a. a.*. n-a.

n.r. 11.1. n.a,

H H H z$ a3

r&a. n.a. n.a. n.r. n.a. ns.

5.carboxy monomers

5-Carboxyvanuin S-CarboxyacetovaniUone 5cSrboxyvanillic acid 6carboxyvanillin 6-Cwboxyacetovanillone 6Ca&oxyvaniUic acid 2-catboxyvanillin 2Carboxyacetovanillone 2-Cuboxysyzingeaidehyde VaGllbl Acetovanillone VsniUic acid Vanillylglyoxalic acid Syringealdehyde Acetosyringone Syxingic acid S~gy~yox~c acid floumaric acid Femlic acid

340~,252,221 1487 5cVl 10 354~,251,22zzl 1550 5cVn 15 428912251,221 5cVd 22 16% &carboxymmloInera 1500 340,325,&Q 6cVl 11 35462(3251 6cVn 14 1546 428.413= 6cVd 21 1674 2.carboxy monomers 2cV1 16 1565 340~281,251 2cVn 19 1650 354&E! m355.297.281 2&l 20 1661 pheaoUe monomers VI 1 1056 224,2@u+I 238.223.208~ Vn 2 1140 6 130’7 312=282,261,252 Vd vg 12 1509 34&325?265,2X! 3 1221 S 254,239a Sn 4 1291 268253.228,223 Sd 8 1438 34=312,297,282 Sg 18 1636 370355~95,~ CA 9 1481 308m249.219 FA 17 1632 j&323.308,293

H =f, H H CH Od COOH H CH OII CUOH * *

n.r. n.a. it.*. H H H O&l as OcH, OcH, H H

Abbmviations: GC#, peak numbers in Figs. 1 and 2; RRT. r~~~tiat timcn ml&e to fatty acid standards on DB-1 liquid pbaae; n.a., au applicable. MS fragment in italics mpmsenta a molecular ion not often observed; underlined fragment indicates the base peak in the mass spectrum; R groups are according to Fig. 3.

M. A. Gofii and J. I. Hedges

4028

impurities in the dimer standards. For this reason, all lignin dimers were arbitrarily assigned the same response factor as ethylvanillin. Thus the concentrations presented here should be regarded as conservative estimates, the absolute values of which can be refined once the appropriate dimer standards become available. Yields of the more abundant lignin dimers obtained from duplicate analyses of the same samples had sample mean deviations that ranged from 2-20% and varied inversely with the amount of product. Less predominant dimers, with yields of co.005 mg/ 100 mg OC displayed higher deviations ranging from 30-50%. Among the major lignin monomers (i.e., vanillyl, syringyl, and cinnamyl phenols), sample mean deviations averaged 3% and ranged from l-IO%. The newly reported monomers (e.g., carboxyvanillyl phenols) were measured with deviations of I- 15%, depending on the amount of compound. Gas Chromatography-Mass Spectrometry

Gas chromatography-mass spectrometry (C&/MS) was carried out accordingto GORIand HEDGES ( 1990a,b) with a Hewlett-Packard 5890 GC linked to a Hewlett-Packard 5970B Mass Selective Detector. The oven settings and run times were similar to the GC procedure. Mass spectra were taken at an electron ionization energy of 70 eV. Ions were scanned from 50-550 amu for the first 40 min of the run and from 150650 amu thereafter. In most cases, the GC/MS analyses were made on the same splits analyzed previously by GC. In addition to trimethylsilyl (TMS) derivatives, perdeuterated counterparts (DTMS) were formed with deuterated Regisil (DBSTFA reagent, Regis Chem. Co) and used in the same manner to elucidate structures and fragmentation mechanisms. Similarly, methoxylamine derivatives of carbonyl-containing compounds were formed by adding 2-4 mg of MOX reagent (Aldrich) to a sample split dissolved in pyridine and heating for l-3 h. After cooling, l-2 pL of solution were drawn in the syringe and derivatized with equal volumes of TMS or DTMS reagent. Fractions of coeluting compounds (Table 1) were estimated by GC/MS, based on the relative abundances of their respective characteristic ions. RESULTS In the first part of this section, the identities and apparent structural precursors of the various lignin dimer CuO reaction

Retention

Time

products are presented. Details of the various techniques used to determine the actual structures (when standards were available) or structural features of the most important dimeric products are presented elsewhere ( GoRI, 1992) and are available upon request. The second part of this section reports the results of a survey of lignin dimer products from a variety of plant tissues (including angiosperm and gymnosperm woods, leaves, grasses, needles, and barks) and from a selected set of sedimentary samples. Lignin Dimeric Products The lignin macromolecule is made of several types ( l-3) of phenolic units linked together through a wide variety of carbon-oxygen and carbon-carbon bonds ( SARKANEN and LUDWIG, 197 1). During CuO oxidation, most ether bonds between units are hydrolyzed, followed by the oxidation of sidechains through one-electron transfers ( CHANG and AL LAN, 197 1). Representative gas chromatographic traces of CuO reaction products from an angiosperm wood and a Washington shelf sediment sample are shown in Figs. 1 and 2. The traces are dominated by early eluting (retention times of 6-30 min) lignin-derived phenolic monomers, the larger of which have been utilized widely by previous authors as source indicators (e.g., HEDGESand MANN, 1979a; HEDGES et al., 1988b). These chromatographic traces also contain smaller but much more numerous later eluting peaks (see expanded inserts in Figs. 1 and 2) corresponding to a suite of carbon-linked ring-ring and sidechain-ring phenolic dimers, whose tentative structures are illustrated in Fig. 3. The names, elution order, and characteristic mass fragments of these lignin-derived dimeric and monomeric CuO reaction products are presented in Table 1. Mass spectra and fragmentation patterns, from which the preliminary structural identifications

(mins.)

FIG. 1. Complete gas chromatogram and high retention time section of the CuO reaction products from birch wood analyzed on a DB-1 capillary column. Peak numbers correspond to assignments in Table 1.

.1

Organic geochemistry of lignin

4029

z

40-

,

20-

40

RoteZion

Tim?

1

50

(mins.)

FIG. 2. Complete gas chromatogram and high retention time section of the CuO reaction products from the Washington continental shelf surface (O-2 cm) sediment analyzed on a DB-1 capillary column. Peak numbers correspond to assignments in Table 1. Asterisks (*) mark cutin-derived products in the mixture. of compounds without available standards were based, are presented elsewhere ( Got;lr, 1992). The possibility that these specific dimers might be formed from condensation reactions of monomeric intermediates during the CuO reaction was tested by similarly reacting different mixtures of vanillyl (guaicyl ) , syringyl, and p-hydroxy phenols with aldehydic, ketonic, and acidic sidechains, as well as p-coumaric and ferulic acids. These experiments yielded only small amounts (~2% of monomer conversion) of a few high retention time compounds, some of which were identified by GC/MS and coinjection with standards as chalcones derived from aldol-condensation reactions ( CHANGand ALLAN, 197 1). None of these condensation products was observed among the reaction product suites of the analyzed tissues and sediments. The different dimers in Table 1, therefore, appear to retain linkages intrinsic to the parent lignin polymers and are not artifacts produced in the course of the CuO reaction (see also CHANG and ALLAN, 197 1; SJOSTROM, 1981). Twelve different ring-ring dimers comprised of vanillylvanillyl (guaicyl-guaicyl ) and p-hydroxyphenyl-vanillyl phenols linked at the 55’ positions were identified among the CuO products. These products exhibited sidechain substitutions with aldehyde, ketone, acid, and glyoxalic acid groups (Table 1; Fig. 3). The precursors of the 5,5’vanillyl-vanillyl (55 bv) reaction products are thought to be structural units with directly bonded rings, which have been described previously as part of the lignin ultrastructure ( SARKANENand LUDWIG, 1971; SJ~STR~M, 1981; FENGEL and WEGENER, 1984). These structural units are oxidized to yield aldehydes, ketones, and acids during the CuO reaction, depending in their initial sidechain structure (PEARL and DICKEY, 1952; CHANG and ALLAN, 197 1) . Dehydrodivanillin and dehydrodivanillic acid have been reported as lignin CuO reaction products (PEARL and DICKEY, 1952). The other ten ring-ring dimers in Table 1

have not been previously recognized and were identified based on their mass fragmentation patterns as compared to those of the two standards and monomeric counterparts. Notably, 5,5’p-hydroxyphenyl-vanillyl dimers (55 bv) provide concrete molecular evidence in support of NMR studies ( NIMZ et al., 198 1) and monomeric compositions (HEDGES and MANN, 1979a; ANDERSSONet al., 1973 ), indicating that p-hydroxy phenyl units are structural components of some types of lignin. Nineteen different sidechain-ring dimers were also observed among the phenol product suites obtained from the CuO reaction of vascular plant tissues and sediments. These carbon-linked products include p, I-diketone (@l ) and (Y,lmonoketone (a 1) dimers with the sidechain linked to the C, ring carbon and a variable number (O-2) of methoxy groups substituted at each ring (Table 1; Fig. 3). The p-hydroxyphenyl (Y,I-monoketone, p-hydroxyphenylvanillone (Fig. 3) was preliminarily identified among the CuO reaction products of a number of plant tissues (Table 2), providing further evidence in support of the existence of “p-hydroxyphenyl lignin” in these plant types ( NIMZ et al,, 198 1). The vanillyl-vanillyl p, 1-diketone, and (Y,1-monoketone dimers have been previously identified among the CuO oxidation products of lignin (PEARLand DICKEY, 1952). Coinjection and mass spectral analyses of standards confirmed the identities of these products and were used to infer the structures of the other 81 and cul dimers. The 61 dimers are thought to be produced from the oxidation of p-1 dilignol structures present in the parent lignin molecule (PEARLand DICKEY, 1952; PEARL and BEYER, 1954a,b; PEARL, 1956), while (Y,I-monoketone dimers could be oxidation products of their diketone counterparts (PEARL and DICKEY, 1952) or may be directly derived from separate macromolecular precursors. The other two types of sidechain-ring dimers, cy,5-monoketone (015) and cu,2-methyl ((~2) compounds, have been

M. A. Goiii and J. I. Hedges

4030

Dimers

Monomers

/” o\c/R’ Ot..

dH

dH

dH

ghydroxyphenolic

5,s'dimers

monomers

'R p,1diketone dimers

R"

'R Q,1-monoketone dimers

&,

dH

S-carboxy monomers

.. a.S-monoketone

dimers OH 6-carboxy monomers

a,Zmethyl

dH dimers

R' dH 2-carboxy monomers

FIG. 3. Structures of dimeric and monomeric CuO reaction products. Compound symbols and substitution patterns are according to Table 1. The bracketed cY,2-methyldimer structure is preliminary and at this point cannot be distinguished from that of 4-O-5’ biphenyl ether dimers (SJOSTROM,1981; CHEN and CHANG, 1985).

preliminarily identified based on their mass spectra. Each dimer class is composed of six different individual compounds which vary in sidechain structure and in the number of methoxy groups substituted on the rings (Table 1; Fig. 3). Although, the identities of these compounds are presently ten-

tative because of the lack of standards, we have identified 5formyl and 5-, 6-, and 2-carboxy phenolic monomers (Table 1; Fig. 3). These monomers have been reported by previous authors to arise likely from the oxidation of carbon sidechain linkages at C,, CS, and Cs ring carbons ( LAI and SARKANEN,

4031

Organic geochemistry of lignin Table 2. Average yields (mg/lOO mg GC) of lignin dimers from different types of vascular plant tissues. -PC-J

G

g

Gb

Gc

A

tA

a

a’

Ab

%oc

49.3 0.14 4

50.8 0.52 2

52.9 0.36 2

49.1 0.58 1

47.0 0.22 4

46.3

46.8 1.87 2

42.4 0.81 3

45.6 0.52 2

48.0 0.93 2

0.3O7 0.180 0.033 0.305 0.052 0038 0.042 0.014 0.044 l.OlM.26

0.423 0.205 0039 0.185 0.064 0.035 0.054 0.011 0.059 1.08M.00

0.162 0.058 0022 0.050 0.008

0.372 0.156 om9 0.160 0.03 1 0.019

0.110 0.109 0.028 0.056 0.018 0.006 0.026

0.116 0.161 0.026 0.073 0.008 0.012 O.o22

0.233 0.156 OK?9 0.133 0.033 O.O25 0.O2.5

0.195 0.093 0.020 0.087 0.018 0.012

0.3&o.o5

0.037 0.67M.M)

0.030 0.45M.15

0.016

u

0.026 0.006

0.026 u

O.Mfo..oo

0.078

%N ”

55’~ dimers 0.868 VI-VI 0.372 VI-Vn O.O45 VII-VII 0.355 M-VI 0.083 Vn-Vd 0.058 Vd-Vd 0.086 Vl-vg 0.016 vu-vg 0.078 Vd-Vg TOTAL 1.96tO.25

El Vl-b

dimerg0.045 v

VI-W

TOTAL

0.05iO.C0

piOtOberg voso so.90 TOTAL

0.201 0.2OSJ.02

al diiers WO

VSO sso PVO TOTAL

0.083 0.012 O.liO.03

aSw dimers 0.127 VOSVI vosvn 0023

VoNd

TOTAL

0.061

0.21f0.M

0.149 0.060 0.044 0.056 o.u23 O.Mn 0.015 o.o20 u 0.3w.08

0.026 0.011 tf O.OhO.CXZ

0.040

O.IJ26 tr 0.03ti.02

0.071

0.o4ko.01

0.o7ko.01

0.0&0.00

2

-

tAb

0.024 0.8QkO.03

0.046 0.4ctto.m

u tr

1r

0.010 O.OO6 0.007 0.O2kO.00

0.03a.03

0.04zo.01

O.O3?&.OO

OJY&l.CXl

0.041 o.m5 0.086 0.16ko.06

0.079 0.055 0.060 0.19ko.01

0.023 0.070 0.017 0.1 lM.03

0.035 0.036 0.015 0.ORtO.M)

0.051 0023 0.019 0.o9nI.01

0.037 0.019 O.fJZ.0 0.08ia02

0.036 0.066 0.095 0.006 0.2oko.02

0.039 0.037 0.042

0.064 0.056 0.047

0.035 0.033 0.041

0.038 0.010 v tf O.OktO.00

0.042

0.071

0.015 0.042 0.129

0.o4M.01

0.O7ko.00

0.19ko.w

.o.o30 0.036 0.016 0.08+o.00

0.119 0.093 0.056 0.27M.07

0.082 0.029 0.037 0.15M.c.a

0.021

0.087 0.042

OS06

0.04M.00

0.02&J

0.085 0.044 0.13cto.06

0.4ao.07

0.029 0.006

0.1&05

0.103 0.084 0.039 0.008 0.23io.01

0.1 io.01

0.1 lio.01

0.018 0.023 0.032 O.O7fo.(n

0.033 0.020 0.020 0.07fo.01

0.045 0.037 0.025 O.llM.O1

0.064 0.03 1 0.016 0.1 lM.01

O.MO

0.016

0.017

0.037 -

0.038 0.O6kO.03

O.OZtO.00

0.008 0.o3ko.01

0.019 O.O&O.O4

0.041 0.009 0.033 O.MktoU2

0.036 O.OO8 0.007 o.osM.o1

0.053

C&V

dimers 0.014 .%Lw

0.026

0.017

O.OlM.00

O.O3kXI

0.035 0.05io.o4

0.13io.00

0%~ dimers 0.097 Vm2sl 0.014 Vm2Sn 0.030 Vm2Sd TOTAL O.l4fo.@3

O.a)8 tr tr 0.01fo.00

0.045 0.010 0.074 0.13io.00

O.O6O 0.008 0.019 0.O9kO.00

o.o28iao17

0020

so5vn So5Vd TOTAL

c&s

diiers sm2Sl sm2sn tr sm2sd -

TOTAL

tr

TOT. DIM. 2.68fo.33

-

0.022 k

0.075 0.010

o.a21 v

0.017 1r

O.MM.00

0.C9S.M)

0.03M.00

O.OBl.00

0.184 0.038 0.051 0.27ko.02

0.055 0.015 O.OOO 0.07fo.03

0.081 0.030 0.017 0.13io.01

0.038 O.llfo.o2

0.08 0.020 0.017 0.1oio.01

1.82k0.14

0.85fo.11

1.02io.05

1.2pfo.10

0.99kO.24

tr

O.O3~*M

0.O2S.00

0.135 0.021 0.025 0.18ZKl.03

0.64M.06

1.63M.27

1.63fo.00

0.91fo.19

tr

0.089 0.006 0.019 0.11*.00

0.015

Abbnviuian~:G.~ymnospem woods; g. gym. needler;Gb.gytn.buks;Gc, gym.caper;A. tauperateansiosperm wocdr;tA. m+d mg. wood:; a, mp. mg. leave: I’. temp. mmw; Ab. temp. mg. barks; tAb, uop.utg.b&s. %GC,weight percent organic carbon; kN. weight percent tud tiwat; II, ember of

-, notdetectable; U. trace yieldr of < 0.003 mg/lOO mg OC all ok avenges f tbcir sample mean deviuions.

tissuesdyzd;

197 1;CHANG and ALLAN, 197 1) . Hence, the macromolecular precursors of (~5 and (~2 dimeric reaction products must be carbon-bridged structural units at the Cs and C2 ring locations, some of which have been proposed before (FENGEL and WEGENER, 1984; NIMZ, 1974; ADLER, 1977). At this point,

ccqamd

qmbols as given in Table 1 md the ~QL All ‘KlTALs am

however, a 4-O-5’ ether-linked structure similar to that proposed by CHEN and CHANG ( 1985 ) cannot be ruled out as an alternate to the postulated cy,2-methyl dimeric structure. Nevertheless, all the findings presented below still apply regardless of which is the actual structure.

4032

M. A. Goiii and J. I. Hedges

Several other studies using different degradative techniques, such as alkaline nitrobenzene oxidation of spruce wood ( CHANC and ALLAN, 197 1) and treatment with NaOH of graminaceous cell walls (HARTLEY and FORD, 1989), have yielded $5 dimers among the lignin-derived products, although none of the sidechain dimers identified in this study have been reported. Analyses of lignin-rich samples by inion source platinum filament pyrolysis have revealed several ions that have been tentatively identified as arising from sidechain-linked dimeric structures ( HEMPFLINGand SCHULTEN, 1988), but no ring-ring dimers were identified. More work is needed in order to better compare lignin dimer yields among various analytical techniques and sample types. Lignin Dimer and Monomer Distributions in Plant Tissues In this section, we present the lignin dimer and corresponding monomer compositions obtained from the CuO

oxidation of twenty-four different vascular plant tissues, including woods, barks, cones, needles, leaves, and grasses from species important in the northwestern region of North America, as well as tissues (woods and barks) from two tropical species. The yields of cutin acids from many of these same tissues have been described previously ( GOKIIand HEDGES, 1990b). Rather than being an exhaustive survey, the analyzed plant samples represent a first cursory characterization of the dimeric phenol CuO reaction products of selected species and tissue types, some of which may be major contributors to soil and sedimentary organic matter. The average carbon-normalized yields of individual ligninderived dimeric reaction products from the various tissue types are presented in Table 2. Yield data from the individual plant species are tabulated elsewhere (GoIQ, 1992) and available upon request. Total average yields f their sample mean deviations for each dimer structural class are also given in Table 2 as a measure of the variability among different

Table 3. Average yields (mg/lOOmg OC) of lignin monomers from different types of vascular plant tissues. G

canpound

Sf monomers 5fVl 0.054 sfvn 5fVd TOTAL

0.045 0.012 0.016 0.U7kO.00

0.047 0.006 0.065 0.12fO.M

0.051

0.216 0.023 0.026 0.27M.01

A

a’

0.048 o.zl3

0.050 0.026 0.036

0.028

Ab

tAb

0.009 0.04M.01

0.155 0.077 0.080 0.3lM.01

0.164 0.052 0.061 o.28kO.m

0.096 0.008 0.019 0.12&0.03

0.244 0.036 0.067 0.35iO.08

0.075 0.030 0.027 0.13M.02

0.085 0.021 0.037 0.14M.01

0.132 0.044 0.041 0.22tO.00

0.178 0.051 0.05 1 0.28iO.08

0.052 0.033 0.05 1 0.14fo.05

0.014 0.039 0.070 0.12Hm2

0.025 0.043 0.058 0.13M.00

0.018 0.008 0.010 0.O4iO.01

0.026 0.013 0.019 O.OktO.02

0.018 0.062 0.015 0.09kO.05

0.015 0.012 tr 0.03&0.01

o.u!o 0.038 0.033 O.O%O.CKl

0.024 0.030 0.037 0.09ko.01

0.278 0.007 0.28ztO.17 0.010

0.025 0.012 0.04iO.01 0.025

0.033 0.015 0.05iO.00 0.043

0.021 0.008 0.03M.01 O.K?OZt

0.048 0.010 0.06iO.M 0.048

0.100 0.011 0.1 lf0.04 0.018

0.013 0.012 0.03M.01 0.015

0.026 0.026 0.0sM.00 0.040

0.03 1 0.048 0.08ctO.06 0.030

1.36 0.349 0.439 0.184 2.33M.13

2.85 0.783 0.936 0.391 4.96k0.31

3.24 0.754 0.908 0.401 5.3OkO.00

3.92 0.628 0.590 0.186 5.33M.99

6.17 1.04 1.23 0.362 8.80t1.37

1.92 0.449 0.465 0.196 3.03f1.37

2.99 0.666 0.566 0.294 4.52fo.65

3.87 0.799 0.823 0.322 5.81M. 19

4.83 0.892 1.00 0.349 7.wcm9

0.104 0.024 0.014

0.05 1 0.021 0.03 1

O.M2 0.013 0.013

0.055 0.015 0.006

0.14!0.13

O.lQm.06

0.08ztO.00

0.08&0.00

13.7 262 1.80 0.315 18.5k1.45

8.74 1.77 1.66 0.218 124fo.26

1.85 0.546 0.378 0.090 2.87H.76

2.31 1.28 0.625 0.076 4.2W.17

2.45 0.584 0.538 0.101 3.67M.37

3.33 0.803 0.744 0.115 5.m1.46

0.055 0.042 0.13iO.M

V momomers

10.1 1.99 1.94 0.981 15.OiO.69

S monomers Sl Sn Sd

S8

a

0.1 lf0.04

2c monomers 2cV 0.060 2CVtt 0.021 0.08kO.02 TOTAL 2cs 0.049

TOTAL

tA

0.06s.01

6c monomers 0.032 6cvl

m-k

Gc

o.:o 0.07fo.00

Oil7 0.07iO.03

TOTAL 0.5Qto.07

VI VII Vd VI?

Gb

0.021 0.006 0.030 O.W.04

5c monomers 5cvI 0.339 SCVtl 0.071 5cVd 0.088

6CVtl 6cVd TOTAL

g

C monomers

0.080 O&7 0.O9iO.02

CA FA TOTAL

0.214 0.124 0.34iO.32

0.486 0.153 0.64ko.17

0.026 0.557 0.58f0.37

0.050 0.116 0.17fo.al

O.&O 0.09iO.M

0.008 0.102 0.1 lfo.02

0.495 0.306 0.8Ozt1.40

1.39 1.48 2.87kO.40

0.024 0.354 0.38kO.M

0.068 0.378 0.45iO.05

TOT.MON

16.2iO.61

3.83M.25

6.21M.06

6.07fo.00

24.lkOo.94

21.8k1.78

7.15zt4.42

ll.pfz.19

10.3fo.23

13.1ti.65

Abbreviatims:

All canpound and plant type symbols PR in Tables 1 md 2. All mmotner ck:s TOTALS me werages f their sample mcul &viUionr.

Organic geochemistry of lignin

4033

$5’ dimers ( 55tv), which constitute about 75% of the total dimer yield (Fig. 4a), followed by vanillyl-vanillyl (~,5monoketone ( a5vv) and /3,1-diketone (81) dimers (each averaging 8% of total dimer yields). In contrast, p-hydroxyphenyl-vanillyl 5,5’ dimers (55bv) only account for 2% of the total 5,5 ’ dimer yield ( Table 2 ) . Dehydrodivanillin is the predominant vanillyl-vanillyl ring-ring dimer (44% of 55bv). The individual products that dominate the two other major dimer classes are 5-vanillovanillin (60% of aSvv) and vanillil ( 100% of p 1) . This dominance of aldehydes and vanillyl-substituted compounds is also observed in most of the other dimeric (Table 2) and monomeric (Table 3 ) products of gymnosperm woods. Among the lignin-derived monomeric products derived from gymnosperm woods, simple vanillyl phenols (V) are the most abundant and together account for 92% of the total monomer yield (Fig. 5a). The next most important monomer class is the carboxy vanillyl phenols ( cV), which are defined here to include 5-formyl vanillyl phenols in addition to 5-, 6-, and 2-carboxy vanillyl phenols, and represent 5% of the average gymnosperm wood monomer yield (Fig. 5a). Although lignin dimer and monomer yields from the rest of the analyzed gymnosperm tissues are notably lower ( 150% ) than from wood (Tables 2 and 3 ) , in most cases, the relative

CdlmbiaR

WadI. Mmgm

SEDIMENT TYPE

FIG. 4. Relative abundances of the major dimer classes in (a) temperate vascular plant tissues and (b) selected sediment samples. Symbols: G, gymnosperm woods; g, gymnosperm needles; Gb, gymnosperm barks; Cc, gymnosperm cones; A, angiosperm woods; a, angiosperm leaves; Ab, angiosperm barks; and a’, angiosperm monocots. The four sedimentary horizons from Dabob Bay are indicated in Fig. 4b. In addition, the following symbols represent other sediment types (see Table 4). CC, Crab Creek; LL, Long Lake; WP, Willapa Bay; SH, Washington nental slope.

continental

shelf; and SL, Washington

-m

conti-

b) , tissue types. The average yields of each monomeric phenol product, including the additional fourteen monomers, are presented in Table 3 for the same plant categories. In order to facilitate the presentation of the yield results, the relative abundances of the major dimer and monomer classes for the tested plant groups are illustrated in Figs. 4a and 5a. Most of the following discussion focuses on patterns in the average yields of the various classes of lignin dimers from different plant and tissue types, although related trends in lignin-derived monomeric products are also discussed where pertinent. Differences among tissue types are discussed only when their means are separated by *two sample mean deviations ( -95% confidence interval). Gymnosperm woods give the highest carbon-normalized yields, -3.0 mg/ 100 mg OC, of total lignin dimers of all tissue types and yield relatively large amounts

of monomers,

16 mg/ 100 mg OC (Tables 2 and 3). The predominant dimeric product class of gymnosperm woods is vanillyl-vanillyl

0.1

10-12 ‘28-30

Anslorpmna

PLANT TYPE

48-50

m&Y

LL

cdmlt4aR

WP

SH

SL

Wada. Mqm

SEDIMENT TYPE

mcv

Iv

ms

Emc

FIG. 5. Relative abundances of the major monomer classes in (a) temperate vascular plant tissues and (b) selected sediment samples. All tissue and sediment symbols are as in Fig. 4.

4034

M. A. Goiii and J. I. Hedges

distributions of each product class closely resemble those obtained from the woods (Figs. 4a and 5a). However, there are some compositional differences that suggest the chemical structure of the lignin in these various tissue types is somewhat different. For example, the relative abundance of (~5 dimers in reaction product mixtures from all other gymnosperm tissues is significantly (95% confidence interval) higher than those measured in woods (Fig. 4a). In fact, the sidechainring dimers in all gymnosperm tissues but wood are characteristically dominated by linkages at positions other than the C, carbon (C, and C,). The importance of cu5 and ru2 sidechain-linked dimers in needles, barks, and cones is also reflected in their elevated yields of carboxyvanillyl phenol monomers ( cV) , which are significantly higher than obtained from gymnosperm woods (Fig. 5a). The distributions of individual compounds within their dimer classes also characterize various types of gymnosperm tissues. For example, gymnosperm barks are distinguished from the rest of the vascular plant tissues by their unusually elevated abundance of dehydrovanillinvanillic acid, which accounts for an average of 30% of the total 55;” yield vs. < 18% for other tissue types (Table 2). Other differences between gymnosperm tissues include the relative concentrations of individual cul and a5vv constituents (Table 2). For example, gymnosperm needles and barks yield elevated amounts of 5-vanilloacetovanillone, which account for -40% of the total ~r5vv yields in these tissues relative to gymnosperm woods and cones, where this compound only represents 15% of total a5vv yields. Among the phenolic monomers, the relative high yields of cinnamyl phenols from needles and barks are consistent with previously published data (e.g., HEDGES and MANN, 1979a) and clearly differentiate them from the rest of the gymnosperm tissues. Gymnosperm needles also produce considerable yields, 0.4 mg/ 100 mg OC, of 55’pv dimers (Table 2 ) . Angiosperm woods are characterized by low yields of lignin dimers (- 1.O mg/ 100 mg OC) vs. monomers ( -25 mg/ 100 mg OC; Tables 2 and 3). Other angiosperm tissues, especially leaves and barks, differ from the woods by their higher dimer yields relative to monomers. All angiosperm plant tissues analyzed are characterized by significantly lower abundances of 5,5’-ring-ring dimers, and the consequential enhanced importance of the sidechain dimers, relative to their gymnosperm counterparts (Fig. 4a). As expected, syringyl-containing units in /31, (~1, 015,and ~y2dimers are significantly more important in the product suites obtained from angiosperms than they are in those from gymnosperms (Table 2). For example, in angiosperm tissues, 40-80% of (~1 and 60-90% of (~2 dimers contain syringylsubstituted rings. The monomeric CuO reaction products also display such behavior with syringyl phenols accounting for anywhere from 40 to almost 80% of the total monomer yields in angiosperm tissues, as compared to abundances of less than 5% for gymnosperm tissues (Fig. 5a). There is a large degree of variability among plant tissues in the relative abundances of syringyl- vs. vanillyl-substituted rings in the various dimer classes. In the discussion section, we will present several dimeric S/V ratios that might be able to discriminate among tissue types and complement the monomeric S/V and C/V parameters used traditionally

(HEDGESand MANN, 1979a). Besides the dimeric S/V ratios, there are other unique dimeric compositional features that have the potential to characterize specific lignin sources. For example, temperate monocot species (and temperate angiosperm leaves to a lesser degree) give yields of dehydrovanillinacetovanillone relative to dehydrodivanillin that are significantly elevated from the rest of the plant tissues studied. This trend of low aldehyde abundance is also seen in the distribution of a5vv and cu5sv dimers from nonwoody angiosperm tissues (Table 2). As expected (HEDGESand MANN, 1979a), nonwoody angiosperm tissues (especially leaves and monocots) yield elevated amounts of cinnamyl phenols that clearly distinguish them from their woody counterparts (Fig. 5a). Monocots are also characterized by relatively high yields of 55bv dimers, which average about 0.04 mg/ 100 mg OC (Table 2). In general, the lignin-derived CuO reaction products of both the tropical angiosperm woods and barks resemble quite closely those from their temperate counterparts, although there are some subtle differences between them (Table 2). Because of the small number of tropical species analyzed, the observed dissimilarities might be species-specific and will not be treated further. Lignin Dimer and Monomer Distributions

in Sediments

Carbon-normalized yields of the major lignin dimeric and monomeric classes for the nine analyzed sediment samples from the US Pacific Northwest are presented in Table 4. Yields of individual compounds are tabulated elsewhere (Go@ 1992). The sedimentary lignin dimer yields vary widely from 3.0 mg/ 100 mg OC in Willapa Bay to 0.3 mg/ 100 mg OC in Long Lake. The dimer yields in Dabob Bay sediments are rather constant, averaging about 1.4 mg/ 100 mg OC throughout the core. Total yields of lignin monomers parallel those of the dimers and are highest in Willapa Bay sediments (9 mg/ 100 mg OC) and lowest in Long Lake (0.9 mg/ 100 mg OC; Table 4). The relative abundances of the major lignin dimer and monomer classes in the sediment reaction product mixtures are illustrated in Figs. 4b and 5b, respectively. Overall, all sediments display similar distributions of dimer types, in which 55 Gv compounds clearly predominate, accounting for 80-90% of the total dimer yield (Fig. 4b). The Columbia River sediments are characterized by their low content of a5 dimers, which only account for ~1% of dimer yields from Long Lake and Crab Creek sediments. For the most part, the sedimentary dimeric distributions resemble those obtained from gymnosperm woods. However, all sediments yield at least 15% of syringyl monomers, which in Crab Creek sediments account for as high as 40% of the total monomer yield (Fig. 5b). Such relatively high syringyl contents are also seen in some of the lignin dimer classes, such as (Y1 and (~2dimers (Table 4), and are not consistent with a mostly gymnosperm wood origin. In addition, the abundance of carboxyvanillyl phenols relative to vanillyl phenols is also higher than those measured in any tissue type except gymnosperm needles. DISCUSSION The distributions of the lignin phenol dimers and the additional lignin-derived monomers indicate that previously

Organic geochemistry of lignin

4035

Table 4. Yields (mg.000 mg OC) of the various lignin dimer and monomer classes from selected sediment samples. Dabob Bay Horizons

Columbia River

Compound

O-km

lo-12cm

2%30m

48-5Ocm

%OC %N

2.54

2.57 0.25

2.36 0.22

2.14 0.20

1.92 0.19

o.ct24 0.033 0.083 0.010 0.061 0.030 1.42

1.19 0.012 0.019 0.066 0.105 0.028 0.062 0.069 1.55

1.02 0.006 0.016 0.031 0.049 O.CXJ.3 O&3.5

1.51 0.008 0.011 0.075 0.018 0.001 0.024 0.038 1.69

0.26

Washington Margin Willapa B.

Shelf

Slope

3.25 0.26

1.20 0.088

1.00 0.093

3.00 0.36

0.279

0.010 0.006 0.311

0.113 0.052 3.02

1.20 0.016 0.86 0.032 0.070 OXXX 0.061 0.031 1.45

0.317

0.015 0.001

2.57 0.020 0.074 0.049 0.135

Crab Cr. Long L.

Lignin Dimers 1.07 0.008

55’W 55’W

ii:

0.026 0.041 0.082 0.003 0.060

05W

a&v a&s &%IM.

Oi!i!f

0.005 0.016 0.016 0,005 0.019 0.018 0.395

Lignin Monomers 0.071 0.217 0.079 0.028 2.48 0.713 0.155 3.75

0.080 ZS

0.075 0.259

q:

0.094 0.032 2.36 0.575

S WI?

0.312 0.139 0.030 3.03 0.651 0.141 4.38

MON. “;!::

Abbmiaticns:

0.049 0.069 0.051 au27 1.12 1.33 0.829 3.47

0.201 0.084 0.024 2.35 0.698 0.147 3.58

0.089 0.354 0.105 0.049 6.64 1.61 0.240 9.09

0.056 0.192 0.087 0.021 3.28 0.681 0.116 4.44

0.008 0.572 0.203 0.056 1.04

All plant symbols arid pammeters a= as in Table 2 and in&e &XL.

Most of these parameters are based on sums of individual dimer and monomer yields within structural families and therefore minimize the variability in measured yields of individual compounds, which are more subject to species-specific differences and the challenge of quantifying small chromato~aphic peaks.

unrecognized structural differences in the lignin biopolymer are present in the various types of vascular plant tissues analyzed. In this section, we summarize these tissue-specific differences in a set of parameters (Table 5) that can be applied to sedimentary mixtures to characterize the structure, sources, and diagenetic state of the lignin present in these samples.

Table 5. Selected lignin dimer and monomer parameters for the various types of vascular plant tissues. A

tA

a

a’

Ab

tAb

0.27

0.04

0.10

0.05

0.08

0.12

0.09

0.12

0.08

0.06

0.15

0.05

0.07

0.59

0.49

0.08

2.05

1.21

1.12

1.37

0.83

0.74

0.24

1.07

0.39

1.28

0.67

1.00

1.25

0.83

0.63

G

g

Gb

Gc

DIM

0.17

0.11

0.26

CV/v

0.05

0.33

0.12

0.33

0.55

@l+al)/(a2+uS)

0.83

Ligaln sourres IW0,

Parameter Llgnln Structure

0.01

0.0s

0.02

0.02

3.84

1.47

0.98

1.02

0.65

0.73

KM,

0m

0.29

0.13

0.03

0.02

0.01

0.28

0.66

0.07

0.08

[S/VI& P/V, P/VI,

0.07 0.03 0.00

0.38

0.25

0.87

1.62

1.07

0.80

0.22

0.23

0.47

0.25

0.21

0.23

7.64

3.20

2.68

5.70

1.30

1.98

0.12

0.00

0.00

9.16

2.73

1.10

0.40

0.72

1.16

[=$I

0.00

0.00

0.00

0.00

2.12

0.76

0.77

0.48

0.38

0.55

0.24 0.07 1.77

0.19 0.10 0.55

0.21 0.11 0.57

0.21 0.08 0.27

Lignin Dkgenetk Xndkators

[Ad/AU,,

0.19 0.06 0.49

0.32 0.05 0.59

0.34 0.14 0.54

0.28 0.08 0.4s

0.15 0.00 0.10

0.20 0.M 0.00

[Ad/Al]~,,,

0.00

0.23

1.57

0.00

0.00

0.21

1.88

O&Q

0.36

0.36

[Ad/A&_

0.32

a57

1.85

0.32

0.00

0.00

0.00

O.oo

0.7-l

0.20

[Ad/Al],.

-

-

-

-

0.18

0.28

0.00

0.22

0.69

0.31

[Ad/NV [Ad/Al],,w

Abbreviations: All data are avenges. AU plant symbols and parameters are as in Tabla 2 and in the text.

4036

M. A. Go% and J. I. Hedges

Structure of the Lignin Molecule Similar to the lignin-derived phenolic monomers widely utilized by previous investigators (previous references), the lignin dimers and additional monomers identified in this study have the potential to serve as source and diagenetic indicators of vascular plant debris in sedimentary mixtures (see discussion below). However, besides confirming the monomer data, these novel lignin-derived CuO reaction products can also be used to estimate the macromolecular structure of the lignin polymer in sedimentary mixtures. Ultimately, the ultrastructure of lignin affects its reactivity and, in sediments, reflects its various sources and degradative pathways. Hence, an understanding of the degree and manner in which the various phenolic units in sedimentary lignin are linked together, relative to lignin from fresh vascular tissues, may help elucidate the processes that are responsible for the cycling of these ubiquitous organic macromolecules. Extent of cross-linking The novel set of lignin-derived CuO reaction products provides two independent ways of estimating the degree of cross-linking in the lignin polymer. The ratio of dimer to monomer yields reflects the fraction of lignin phenolic units joined through carbon-carbon bonds relative to those linked through ether bonds (which are hydrolyzed during CuO oxidation). Another potential indicator of cross-linking is the ratio of carboxyvanillyl phenols (including 5-formyl as well as 5, 6-, and 2-carboxyvanillyl phenols) to vanillyl phenols. Since carboxyvanillyl phenols are likely produced from the oxidation of carbon bridges between phenolic units ( LAI and

SARKANEN, 1971; CHANG and ALLAN, 1971), whereas vanillyl phenols are the products mostly of ether-linked precursors, the CV/ V ratio should increase with elevated degrees of carbon cross-linking. The same should be true for the dimer-to-monomer ratio. The values of these two parameters for the various sources of vascular plant matter that are relevant to northwestern sediments are presented in Table 5. These data show that there is a high degree of variability among tissue types, with angiosperm woods displaying the lowest extent of cross-linking as defined by these ratios ( D/M and CV/ V values < 0.1) , while gymnosperm tissues, especially needles and barks, typically exhibit higher values. This distribution is not surprising, since lignin from angiosperm woods is considerably enriched in syringyl phenols, which lack the ability to cross-link at the favored C5 ring position because of methoxy group substitution at that site ( SARKANENand LUDWIG, 197 1). The sedimentary dimer / monomer and carboxyvanillyl / vanillyl ratios (Table 6) are plotted along with the corresponding ranges of different major plant types in Fig. 6. In general, the sedimentary lignin compositions most resemble those of gymnosperm tissues, a pattern consistent with regional vegetation. All sedimentary D/M ratios, however, are greater than 0.3 and exceed the ratios found for any plant tissue analyzed. In most cases, the sedimentary cV/V values range from 0.1-0.2; but the Washington slope and Long Lake sediments display much higher values of 0.4-0.5, which far exceed values for all tissues except gymnosperm needles. These results indicate that the degree of ring cross-linking via carbon bonds in these sedimentary lignins is higher than observed in any fresh vascular plant tissues. Hence, the lignin

Table 6. Selected lignin dimer and monomer parameters for sediment samples from the U.S. Pacific Northwest. Columbia River

Dabob Bay Parameter

0-lcm

lo-12cm 28-3&m 48-5Ocm

Crab Cr. Long L.

Washington Margin WillapaB.

Shelf

Slope

Llgnin Structure D/M

0.37

0.32

0.41

0.33

0.49

0.35

0.33

0.33

0.38

cV/v

0.19

0.19

0.16

0.16

0.17

0.50

0.09

0.11

0.37

sm

0.22

0.21

0.29

0.16

0.11

0.12

0.16

0.19

0.25

(~l+al)/(a2+a5)

0.39

0.3 1

0.32

0.41

1.06

0.84

0.41

0.40

0.37

0.22 0.05 0.12

0.29 0.06 0.26

0.30 0.06 0.07

1.19 0.74

0.55

0.24 0.04

0.21

0.35 0.10

[S/VI,

0.24 0.07 0.03

WI, [S/q1

0.43 0.09

0.50 0.09

1.10 0.16

0.79 0.06

lS/vlB,

0.08

0.05

0.10

0.05

Wgnin Sources WI, [c/v],

Lignin Diegenetic Indicators IAd/MIY 0.35

0.29 0.00

0.00

0.04 0.08

0.58

0.46

0.5 1

0.78 0.13

0.29

0.29

0.22

0.96 0.14

0.04

0.05

0.19

0.60

0.04 1.56

0.35

0.30

0.33

0.33

0.70

0.30

0.37

0.12 0.65

0.11 0.33

0.11 0.52

0.00

0.09 0.45

0.15 0.72

[Ad/Al],m ]Ad/All,

0.00 0.65

0.00 0.82

0.00 0.42

0.00 0.95

0.09 0.22 0.00

0.36

[AdhUh

0.11 0.48

[Ad/Al],

0.08

0.06

O.U3

0.W

0.16

0.88 0.06

lAd/A&VY

253 0.05

Abbreviations: AU symbols and permneters PIEes in Table 2 and in the roxt.

0.00 0.90 0.06

0.28

0.29 0.26 0.00 0.85 0.04

Organic geochemistry of lignin

r

4037

LL

0.4 SL

g

I

5 0

DB

0.2 cc

0. 0

I

I

I

0.2

I

I

0.4 D/M

FIG. 6. Plot of dimer/monomer

yield ratios (D/M) vs. carboxyvanillyl/vanillyl phenol yield ratios (cV/V) from the analyzed plant tissues and sediment samples (all symbols are as in Fig. 4). The shaded area represents the compositional range (average f 2 sample mean deviations) of the four Dabob Bay sediments. Included are the average and compositional ranges for each rissue type.

in all of the sediment samples appears to have undergone structural changes that differentiate it from the lignin found in fresh vascular plant tissues. Possible explanations for these sedimentary lignin parameters could include preferential preservation of carbon-bonded structural units during diagenetic processing and/or active cross-linking during degradation. The compositional signatures and potential importance of these processes are investigated further in an up coming study ( GORI et al., 1992). Dimeric distributions The distribution of the various dimer classes also provides information on the type of carbon-carbon bonding present in the lignin macromolecule. One of the ways that the relative importance of different carbon linkages can be evaluated is from the ratio of sidechain-ring (SR) dimers (including j31, al, a5, and (r2) to ring-ring (RR) dimers (55bv + 55&) and the ratio of &linked structures (Bl + al ) to Cs- and &linked dimers ((~5 + cr2). The average values for these two parameters in the various plant tissue types analyzed are given in Table 5. The elevated SR/RR and (/31 + al)/(cr5 + cu2) ratios of angiosperm tissues, especially angiosperm woods, indicate that carbon bonding in the lignin from these tissues is mostly through sidechain linkages at the C1 ring carbon. Again, this is consistent with what is known about syringyl-rich angiosperm lignin, which does not have the ability to form bonds at substituted Cs ring carbons. On the other hand, dimeric structures in the lignin from gymnosperm tissues appear to be linked mostly through direct 5,5’-ringring bonds (Table 6). Lignin from gymnosperm woods and, to a lesser degree, needles, appears to be composed of similar fractions of Cr-linked structures and Cs- and &linked units, with (@1 + (Y1 )/ ((~5 + a2) ratios close to 1. In contrast,

sidechain-ring dimers linked at the Cs and Cz ring carbons are more abundant in gymnosperm bark and cone lignins. One potential complicating factor in using these compositional differences to characterize the ultrastructure of lignin in sedimentary mixtures is that some of these compounds might be derived from lignans rather than from extended lignin polymers. Lignans are extractive constituents formed by biosynthetically controlled oxidative couplings of two phenolic units rather than by the random free-radical pathway responsible for lignin synthesis ( SJ~STROM, 198 1) . Since, for example, 2-methyl linkages are known to occur in many lignans ( SJ~STR~M, 198 1; FENGEL and WEGENER, 1984)) tissues containing such lignans should give elevated yields of (~2dimers. Alternatively, portions of the lignin polymer itself might result from nonrandom synthesis ( FORSSand FREMER, 198 1) , which could also lead to some of the unique compositional patterns seen in certain tissues (see the following discussion). The sedimentary values of these two dimeric parameters, SR/RR and (/31 + (~l)/(c15 + o12), arelisted in Table 6 and plotted along with corresponding tissue compositional ranges in Fig. 7. All sedimentary lignin reaction mixtures are characterized by extremely low SR/RR ratios of less than 0.3, which are smaller than the values obtained from fresh vascular plant tissues other than gymnosperm woods. Both the Dabob Bay and Washington margin sediments also display (81 + (ul)/(a5 + (~2) ratios < 0.3, while the Columbia River sediments give notably higher values of about 1.O. The above compositions indicate that, regardless of their origins, the lignins present in all these sediments display unusual carboncarbon bonding patterns not found in any of the vascular plant sources analyzed. Specifically, the sedimentary lignin present in these samples appears to have suffered selective

4038

M. A. Go% and J. I. Hedges

1.5

1.0

0.5

0.0

FIG.7.Plot of the ratio of sidechain-ring dimers over ring-ring dimers (SR/RR) vs. the ratio of the sum of &Idiketone -t or,l-monoketone dimers over the sum of a,5-monoketone + a,2-methyl dimers, (81 + ~yl)/(a5 t cu2), from the analyzed plant tissues and sediment samples (all symbols as in Figs. 4 and 6). alteration of side&&n linkages, especially those linked at the C1 ring carbon. All of the parameters presented above, which can be used to characterize the macromolecular structure of the lignin polymer, might also help determine its sources and diagenetic state in sedimentary mixtures once degradative alterations are better delineated and understood (C&RI et al., 1992).

Vascular Plant Sources Traditionally, previous investigators have taken advantage of the unique distribution of syringyl phenols and cinnamyl phenols (only produced by angiosperm and nonwoody vascular plant tissues, respectively) to characterize the sources of vascular plant-derived organic matter present in a variety of sedimentary mixtures (e.g., HEDGES and MANN, 1979b; HEDGESet al., 1988b). These determinations are hampered by the fact that diagenetic processes can alter the lignin compositional signature by preferentially degrading different phenol types in complex patterns that are dependent both on the type of tissue and the degrading organism (HEDGES et al., 1988a; HEDGES and WELIKY, 1989; BENNER et al., 1990, 1991). The newly identified lignin dimers have the potential to solve this problem because they are likely to be less sensitive to diagenetic processes and might preserve the source signal better than the monomer. The independently determined monomeric [S/Q and [C/V], ratios for both the plant and sediment samples studied here (Tables 5 and 6; Fig. 8) support previous inferences that the vascular plant sources to Dabob Bay and Washington margin sediments are mostly gymnosperm tissues (HEDGES et al., 1988b, HEDGESand MANN, 1979b), while the Colum-

bia River sediments from Long Lake and Crab Creek appear to be enriched in nonwoody angiosperm tissues (HEDGES et al., 1984). Figure 8 incorporates previously unpublished compositions of other tissues such as cones and barks, the latter of which produces appreciable amounts of cinnamyl phenols. The low [C/V 1~ ratios in Dabob Bay sediments cannot be taken to necessarily mean there is little nonwoody tissue debris in these samples, since preferential in-situ degradation of cinnamyl phenols from conifer needles in these sediments is known to occur (HEDGES and WELIKY, 1989) and the degradation of barks is unstudied.

The parallel distributions of syringyl rings among the dimerit and monomeric CuO reaction products of angiosperm tissues suggest that a variety of novel dimeric [S/V] ratios could be formulated which may be more source specific and/ or less ~ageneti~~ly sensitive than the conventional monomer-based ratios. Four [S/V] ratios can be calculated for the four dimer classes which incorporate syringyl and vanillyl dimers in their structure (Table 5). For example, crSvv and cy2vs dimer yields can be ratioed to a5sv and a2* yields to give dimeric syringyl/vanillyl ratios ([S/V Jes and [ S/Vlo2, respectively). Analogous ratios can also be formulated for sy~n~l-sy~ngyl 8,1-diketone and iy, 1-monoketone dimer yields relative to their vanillyl-vanillyl counterparts, [S/Vlsl and [S/V],, (Table 5). As expected, angiosperm tissues are characterized by elevated dimeric S/V values, which in the case of woods can average as high as 9 for [S/& and [S/V], (Table 5). Other angiosperm tissues, such as leaves and barks, give uni-

4039

Organic geochemistry of lignin

1.5

I

I

I

I

I

I

I

I

I

I

I

I

I

a -t

1.0

?

VI

Ab t

LL

0.5

0.0

G$GbI

0.0

I

I3 I

I

0.2

0.4 [cM,

I

0.8

0.6

--_

FIG. 8. Blow-up plot of the monomeric cinammyl/vanillyl, [C/VIM, and syringyl/vanillyl, [S/V],, phenol ratios from the analyzed plant tissue and sediment samples (all symbols are as in Figs. 4 and 6). The shaded insert illustrates the complete compositional ranges for all tissue types. [S/V] ratios, while monocots yield low [S/V],,, [S/VJBI, and[S/V],, ratiosbut relatively high [S/ VI,, values near 6. Although gymnosperm woods yield dimerit [S/V] ratios near zero, gymnosperm cones, needles, and barks give [ S/V],5 ratios up to 0.8 and average values for [S/V],, and [S/V],, ofO.l-0.2 (Table 5). AH dimeric S/V values in sediments are quite low and seem to suggest that only a small fraction of the vascular plant debris in these samples is derived from angiosperm woods (Table 6). This result is consistent with that previously drawn from other dimeric and monomeric compositions (F@. 6, 7, and 8). In most sediments, however, the dimer S/V ratios are not zero and cannot be explained entirely by gymnosperm tissues. For example, the elevated, >0.4, [S/ VI,, ratios obtained from all sedimentary samples indicate that some angiosperm lignin is present (Fig. 9). Furthermore, an important nonwoody angiosperm tissue source is required to explain the relatively high [S/V],, and [S/V],, ratios in Crab Creek sediments (Fig. 9 ) . A similar picture arises from the sedimentary [S/V],, and [S/V]ps ratios (Table 6). In spite of their extensive cross-linking, resembling that of gymnosperm woods, the lignins from these sedimentary mixtures have retained some of the compositional characteristics of their angiosperm plant sources. Although quantification of plant sources is not warranted until the diagenetic behavior formly low dimeric

of each lignin dimer class is better known, together, the diphenols provide a qualitative indication of vascular plant sources that is consistent with and complements that obtained from the corresponding monomers (Fig. 8). Lignin Diigenetic Indicators

One of the processes that can extensively change the structure of the lignin polymer, and therefore be responsible for some of the important differences observed between sedimentary lignin compositions and those from fresh tissues, is degradation by fungi. White-rot fungi are known to increase the ratio of vanillic acid to vanillin ([Ad/Al],) in angiosperm woods during lignin degradation (HEDGES et al., 1988a); therefore, [Ad/Al]” has been used to characterize diagenetically altered lignin in a variety of geochemical samples (e.g., HEDGESet al., 1986; BENNERet al., 1990). However, certain fresh nonwoody tissues (e.g., BENNERet al., 1990), including gymnosperm barks (Table 3 ) , have high contents of vanillic acid, much of which is thought to be ester-bound as such to polysaccharides (WHITEHEAD et al., 1981;YAMAMOTO et al., 1989). These tissues display high [Ad/Al]” valuesof0.30.4 that can complicate the use of this ratio as an indicator of lignin degradation by white-rot fungi.

4040

M. A. Ciofii and J. I. Hedges

2.0 1

,

I

I

I

I

I

I

I

I

I

I

f

CC

+ Ab

DB LL SH WP

.G 0.0

0.2

0.4

0.6

0.8

1.0

FIG. 9. Blow-up plot of the dimeric S/V ratios of a,l-monoketone dimers, [S/V],, , vs. cY,2-methyl dimers, [S/ VI,,, from the analyzed plant tissue and sediment samples (all symbols are as in Figs. 4 and 6). The shaded insert illustrates the complete compositional ranges for all tissue types.

Dimeric acid aldehyde ratios

If fungal degradation affects other lignin structures similarly to those which yield vanillyl phenol monomers, lignin dimers might provide additional acid/ aldehyde ratios whose application as diagenetic indicators would not be compromised by ester-bound acids. In Table 6, the acidfaldehyde ratios for the five major lignin dimeric classes are calculated for the various analyzed vascular plant sources. In the case of the 55&v dimers, [Ad/Al]55+, was calculated as the yield ratio of dehydrodivanillic acid to dehydrodivanillin. For the four sidechain-ring dimer classes, the [Ad/Al] ratios of one or more tissue types exceed 0.5 (Table 5). For example, [Ad/ Al lnzwratios are rather low (dO.3) in most plants analyzed, but angiosperm barks exhibit values over 0.7. As mentioned before, these unique compositional patterns could be due to lignans, which may contribute to the dimeric CuO reaction products of certain tissues. Because of this high variability among plant tissues, acidlaldehyde ratios of sidechain-ring dimers cannot be unambiguously related to the diagenetic state of the lignin polymer in sedimentary mixtures. In contrast, the acid/aldehyde ratio of ring-ring 55;~ dimers displays uniformly low values in all plant tissues analyzed, ranging from an average near zero in angiosperm woods

to an average of 0.14 in gymnosperm barks (Table 5 ). In Fig. 10, the ranges of the average [Ad/AI IVand [Ad/A1]55rvv ratios of the major plant tissue types are plotted along with the values obtained from the sediments (Table 6). The inshore sediments in the Washington margin, Crab Creek sediments, and all of the Dabob Bay sedimentary horizons display somewhat elevated [Ad/Al], (0.3-0.4) ratios and low [Ad/A1ls~vvratios of less than 0.15. In contrast, the Washington slope and Long Lake sediments give rather high values greater than 0.6 for [Ad/Al], and greater than 0.3 for

[Ad/Ails+ The elevated monomeric and dimeric acid/aldehyde ratios of the Washington slope and Long Lake sediments suggest that the lignin in these sediments has undergone extensive white-rot fungal degradation. This process could be partially responsible for the high degree of cross-linking inferred from the high dimer/ monomer ratios observed in these sediments (Fig. 6). On the other hand, the moderately high [Ad/Al], ratios of the other sediments do not necessarily imply a whiterot diagenetic signature since the [ Ad/A1]55 Iw ratios are quite low. Another possibility is that tissues, such as gymnosperm needles and barks, might be important lignin sources to these sediments and selectively elevate their monomeric acid/aldehyde ratios via ester-bound acids. In spite of the low [Ad/

4041

Organic geochemistry of lignin

FIG. 10. Plot of the acid/aldehyde ratios for vanillyl-vanillyl 55 dimers, [Ad/AI] 5S,w,vs. the acid/aldehyde ratios for vanillyl monomers, [Ad/AI],, from the analyzed plant tissues and sediment samples (all symbols are as in Figs. 4 and 6).

Alh’vv ratios, which suggest a low degree of degradation, the lignin in sediments from Dabob Bay, the Washington shelf, Willapa Bay, and Crab Creek appears to have the same degree of elevated cross-linking as that from the slope and Long Lake sediments (Fig. 6). Thus, processes other than white-rot fungal degradation may have all&ted the ultmstructure of the sedimentary lignin in these locations. It is clear from this example that [ Ad/AI]55~,, ratios have the potential to complement and extend some of the interpretations about lignin diagenetic state that can be made based on monomeric acidlaldehyde ratios and, in some cases, distinguish between source and ~~eneti~ly induced compositional trends. Experiments investigating the effect of white-rot fungal degradation on the dimeric acid/aldehyde ratios are presented in GORI et al. ( 1992) and generally sup port the above interpretations. OVERSEW

Lignin dimers are challenging to measure because of their relatively low concentrations and high gas chromatographic retention times, requiring standards in that range (few of which are available) and CC/MS for proper identification. However, unlike some of the eight conventionally used monomeric phenols, the more numerous and s~cturally diverse dimers are derived almost exclusively from polymeric lignin and provide a more detailed and multifaceted characterization of the ultrastructure, sources, and diagenetic state of the lignin polymer. The sedimentary distributions of lignin dimers presented in this paper suggest that, while the preserved lignin macromolecules must have undergone sub-

stantial structural changes during diagenesis and transport, complementary source information can be derived from these high-molecular-weight products. In addition, lignin-derived CuO reaction products could increase the knowledge about fungal lignin degradation by providing unique markers of liinolitic activity (TORI et al., 1993), while unusual dimers resulting from spontaneous cross-linking could be indicative of abiotic reactions during thermal alteration of lignin (BATES and HATCHER, 1989). Lignin dimers, when used together with lignin monomers and cutin acids, which are simulta-

neously obtained from CuO oxidation, provide a powerful means to better characterize and quantify terrestrially derived organic matter in geochemical

samples.

Acknowledgments-The authors are grateful to R. Blanchette for the wood samples and B. Hrutfiord for his help in the initial phases of this project. We wish to thank 1. Pearl and J. Obst for their invaluable contributions of standard compounds and critical reviews. We dedicate this paper to the memory of Dr. Kyosti Sarkanen, who was an inv~uable and enthusiastic collaborator throughout much of this project. This paper benefited greatly from perceptive comments by R. Benner and an anonymous reviewer. In addition, helpful comments from S. Opsahl, B. Bergamaschi, R. Keil, and M. McCarthy shaped the paper in its present form. This research was supported by grants OCE86-13868 and OCE90-07873 from the National Science Foundation. This is contribution number 1927 from the School of Oceanography, University of Wonton, Seattle, W~hin~on, USA. Editorial handling: S. G. Wakeham REFERENCES ADLER

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4042

M. A. Goiii and J. I. Hedges

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