Characterization of carbohydrates during early diagenesis of five vascular plant tissues

Characterization of carbohydrates during early diagenesisof ®ve vascular plant tissues

Stephen Opsahl a, Ronald Bennerb, *aEPA-Ecosystems Research Division, 960 College Station Road, Athens, GA 30605, U.S.A.

bMarine Science Institute, University of Texas at Austin, Port Aransas, TX 78373-1267, U.S.A.

Received 1 August 1997; accepted 16 September 1998

(returned to author for revision 9 March 1998)

Abstract

Long-term changes in the carbohydrate composition of 5 di�erent vascular plant tissues, including blackmangrove leaves and wood (Avicennia germinans), cypress needles and wood (Taxodium distichum) and smoothcordgrass (Spartina alterni¯ora), were measured as these tissues decomposed over a 4 yr period under sub-aqueous

conditions. Carbohydrate composition was measured using a molecular-level analysis for neutral sugars and amodi®ed version of the MBTH (3-methyl-2-benzothiazolinone hydrazone hydrochloride) method for colorimetricdetermination of total carbohydrate yields. Minimal cross contamination from non-carbohydrate vascular plantconstituents indicated the MBTH method was highly speci®c for carbohydrates. The di�erence between total

carbohydrate yields using the MBTH method and total neutral sugar yields revealed a substantial carbohydratefraction (7±23% of the total plant carbon) in fresh and senescent tissues that was not identi®ed at the molecularlevel. The molecularly uncharacterized fraction of carbohydrates probably consisted of ketoses, uronic acids and

amino sugars.The decomposition series demonstrated certain features about carbohydrate diagenesis not apparent from

previous short-term degradation studies. During the latter phase of decomposition (2±4 yr), selective carbohydrate

loss relative to bulk tissue was not evident in 2 of 3 herbaceous tissues. This indicates that carbohydrates may be ofsimilar reactivity as bulk tissue in highly decomposed particulate organic matter. The extent and timing of allcompositional changes were tissue dependent, yet certain trends emerged which were consistent with geochemical

observations. In herbaceous tissues, both glucose and xylose were selectively degraded while deoxy sugars increasedin relative abundance. These changes resulted in an increased abundance of initially minor neutral sugars and ageneral trend towards a more uniform neutral sugar composition. A clear reduction in carbohydrate yields (mgcarbohydrate carbon/100 mg organic carbon) among all tissues provided the most consistent indicator of diagenetic

status. Total carbohydrate yields, mole percentages of glucose and percent deoxy sugars in highly degradedherbaceous tissues were similar to those measured in particulate organic matter fractions of major world rivers, andprovide diagenetic parameters which link relatively fresh plant tissues to their degraded counterparts in aquatic

environments. # 1999 Elsevier Science Ltd. All rights reserved.

Keywords: Carbohydrates; neutral sugars; early diagenesis; decomposition; vascular plants; MBTH method

1. Introduction

Vascular plants represent the largest component of

living biomass on earth and play a major role in the

Organic Geochemistry 30 (1999) 83±94

0146-6380/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.

PII: S0146-6380(98 )00195-8

PERGAMON

* To whom correspondence should be addressed. Tel.: +1-

512-749-6772; fax: +1-512-749-6777; e-mail: benner@

utmsi.wtexas.edu

global carbon cycle (Olson et al., 1985). Most of the

organic matter in large river systems consists of vascu-lar plant remains that have undergone degradationduring residence in soils and groundwaters (Hedges et

al., 1994). Decomposition of plant tissues fuels mi-crobial metabolism which promotes substantial carbonremineralization (Martens et al., 1992; Moers et al.,

1994) and supports detritus-based food webs (Fencheland Jorgensen, 1977; Benner et al., 1988) in aquatic

and terrestrial ecosystems. Due to the high percentageof structural carbohydrates in vascular plant tissues,most carbon and energy ¯ow results directly from the

oxidation of carbohydrates.As plants senesce and begin to decompose, a sub-

stantial portion of organic matter is solubilized andrapidly leached from particulate material (Tukey, 1970;Godshalk and Wetzel, 1978a). However, only a few

studies have attempted to characterize carbohydratesin the leached fraction (Benner et al., 1990a,b; Opsahland Benner, 1993). Selective changes in chemical com-

position also take place during microbial degradationof particulate material. For example, cellulose is more

rapidly degraded than hemicellulose (Suberkropp etal., 1976; Valiela et al., 1984; Benner et al., 1990a).Examination of the subunits which comprise these

polymers indicates varying reactivities of speci®c sugars(Suberkropp et al., 1976; Benner and Hodson, 1985;Hedges et al., 1985; Wilson et al., 1986). However, the

extent to which such trends continue has not been ade-quately addressed due to a lack of long-term studies

which employ detailed chemical characterizations.Carbohydrates are the most abundant constituents

of vascular plant tissues because of the predominance

of structural polysaccharides including cellulose, hemi-cellulose and pectin (Aspinall, 1970). Storage carbo-hydrates such as starch and sucrose, which play critical

roles in cellular metabolism, also contribute to thetotal carbohydrate reserves in plants (Loewus and

Tanner, 1981). Additionally, certain carbohydrates areeither peripheral or integral components of othermajor compounds such as lignins and tannins

(SjùstroÈ m, 1981; Zucker, 1983). Neutral sugars com-prise the majority of carbohydrate building blocksbecause they are the primary constituents of cellulose

and hemicelluloses as well as storage metabolites(Aspinall, 1970). Of all neutral sugars, glucose is pre-

dominant in vascular plants because it is the sole con-stituent of cellulose, the single most abundant polymerin nature. The remaining neutral sugars are often

grouped and considered to be derived from hemicellu-lose, although additional sources include pectin, gumsand other polysaccharides (Aspinall, 1970).

When molecular-level analysis of speci®c neutralsugars is used for carbohydrate determinations, the

sum total of individual sugars is often taken to rep-resent the total carbohydrate content. Although neu-

tral sugar determinations are speci®c and

unambiguous, neutral sugar analyses do not measure

ketoses, amino sugars or acidic sugars, all of which

may contribute to the total carbohydrate content of

vascular plants. To date, molecular-level analyses of

acidic and amino sugars have proven di�cult because

these compounds degrade during hydrolysis, prevent-

ing quantitative recoveries. The MBTH (3-methyl-2-

benzothiazolinone hydrazone hydrochloride) method

of carbohydrate analysis (Johnson and Sieberth, 1977)

has been applied routinely to samples of marine dis-

solved and particulate organic matter. A recent modi®-

cation employing a concentrated H2SO4 pre-treatment

(Pakulski and Benner, 1992) permitted analysis of par-

ticulate samples containing hydrolysis-resistant poly-

saccharides such as cellulose. In addition to being

rapid and simple, the MBTH method is relatively

speci®c because it involves cleavage of functional

groups that are characteristic of carbohydrates.

Direct evidence for carbohydrate levels higher than

MBTH estimates has been presented in recent studies

by Benner et al. (1990a,b) and Pakulski and Benner

(1992). These authors estimated the carbohydrate con-

tent of red mangrove leaves (Rhizophora mangle) using

four independent methods. Their neutral sugar yields

were similar to gravimetric recoveries using the deter-

gent method of analysis (26±27 wt%), but their esti-

mate using the MBTH analysis was much higher

(39 wt%). The highest carbohydrate content (49 wt%)

was provided by solid-state 13C NMR. However, in

order to routinely use the MBTH method or other

non-speci®c methods for total carbohydrate determi-

nations, the possibility of cross reactions with other

plant constituents needs to be examined.

In this study, changes in the carbohydrate content

of ®ve vascular plant tissues (three herbaceous and

two woody) were examined during long-term (4 yr)

decomposition in seawater. Carbohydrates were

measured at the molecular level as neutral sugars

(Cowie and Hedges, 1984a) and colorimetrically

using a modi®ed version of the MBTH method

(Johnson and Sieberth, 1977; Pakulski and Benner,

1992). The MBTH method was also tested for cross

reactions with a variety of non-carbohydrate com-

pounds. The speci®c changes observed in carbo-

hydrate compositions among the di�erent tissues, as

well as the broader biogeochemical implications of

these changes are considered here. Complementary

information regarding speci®c molecular-level changes

in the lignin and cutin content and the comparative

kinetics of decomposition of these same tissues, is

presented elsewhere (Opsahl and Benner, 1995;

Opsahl and Benner, in preparation).

S. Opsahl, R. Benner / Organic Geochemistry 30 (1999) 83±9484

2. Materials and methods

2.1. Plant collection and litter bag experiment

A more detailed description of the plant tissues andthe litter bag experiment has been presented previously(Opsahl and Benner, 1995). Samples of green andsenescent black mangrove leaves (Avicennia germinans),

smooth cordgrass (Spartina alterni¯ora) and cypressneedles (Taxodium distichum) were collected. Woodsamples were also collected from mangrove and

cypress trees. All tissues were air dried at room tem-perature and 10±13 g of each tissue were placed in sep-arate litter bags (63 mm mesh size). Subsamples of each

tissue type were dried at 458C to make wet/dry conver-sions. Thirty bags of each tissue type were placed inlarge (500 l) ¯ow-through seawater tanks (separate

tanks for each tissue type) and incubated in the dark.Duplicate litter bags were harvested at progressivelylonger intervals over the 4 yr decomposition period.The contents of each bag were rinsed in distilled water,

dried for 3 days at 458C and ground for subsequentanalysis. Contents of litter bags were not combinedand all chemical analysis were made on the contents

from each replicate bag. Ash content of the materialfrom each litter bag was determined by combustingduplicate subsamples at 5508C for 6 h. Organic carbon

was measured on a Carlo-Erba EA 1108 CHN analy-zer. The analytical precision for duplicate analyses was22% for ash and elemental analyses.

2.2. Neutral sugars

Neutral sugars were analyzed using the method ofCowie and Hedges (1984b). Plant tissues (25 mg) werepretreated with 12 M H2SO4 for 2 h at room tempera-ture, diluted to 1.2 M H2SO4 and hydrolyzed for 3 h at

1008C. Adonitol was added to the mixture after hy-drolysis as an internal standard. The hydrolysate wasneutralized with Ba(OH)2, deionized in a bed of mixed

cation/anion exchange resins, and evaporated to dry-ness using a Savant Speed-Vac centrifugal evaporatingsystem. Samples were resuspended in pyridine and sor-

bitol was added as an absolute recovery standard. Asubsample was mixed with an equal volume of 0.4%(w/v) LiClO4 in pyridine and held at 608C for 36±48 h.The mixture was then derivatized with Sylon BFT

(Supelco) at 608C for 10 min and separated on aHewlett Packard 5890 gas chromatograph using bothnon-polar and polar capillary columns (J&W DB1 and

DB1701, respectively). The temperature programbegan at 1408C, held for 4 min, increased at a rate of68 minÿ1 to 2708C and then held for an additional

4 min. Each sugar may have from one to ®ve separateisomeric peak. The largest clearly resolved peak waschosen for quanti®cation. With few exceptions, the

DB1 column was used for all neutral sugar quanti®-cations. Analytical error for this method ranges from

5±15% and is generally lower for more abundantsugars. Neutral sugar recoveries have not beenadjusted for incomplete hydrolysis of polymers such as

cellulose, which has a hydrolysis e�ciency of approxi-mately 80% (Cowie and Hedges, 1984b).

2.3. Total carbohydrates

Total carbohydrate analysis of plant tissues wasbased on the method of Johnson and Sieberth (1977)and the modi®cations of Pakulski and Benner (1992).Samples containing about 5 mg OC were pre-treated

with 12 M H2SO4 for 2 h, diluted to 1.2 M and hydro-lyzed for 3 h at 1008C. A 1:100 dilution of the hydroly-sate mixture was neutralized with NaOH and the

monosaccharides reduced to alditols using KBH4.After reduction, this mixture was acidi®ed with HCl toremove unreacted borohydride. Aliquots (1.0 ml) for 3

sample and 2 blank determinations were then treatedwith HIO4, which cleaves vicinal diols and adjacentcarbon atoms when each contains either a hydroxyl orcarbonyl functional group. After termination of the

oxidation with NaAsO2, samples were acidi®ed withHCl and reacted with MBTH (3-methyl-2-benzothiazo-linone hydrazone hydrochloride) at 1008C for 3 min in

order to form azo compounds. Blank determinationswere made on subsamples which were prepared with-out HIO4 oxidation (both HIO4 and NaAsO2 added at

the same time). Blank absorbance was always less than10% of the sample absorbance. Samples and blankswere cooled and FeCl2 added and allowed to react for

Table 1

Carbohydrate response (mg TCOH-C/100 mg OC) from

model compounds measured using the MBTH method

Compound Responsea

Glucose 100

Gallic acid 0.68

Phloroglucinol 0.49

Tannic acid 5.64

Catechin 0.36

p-Coumaric acid 0.52

Ferulic acid 2.50

Vanillin 12.3

DHP lignin 0.20

Bovine serum albumen 0.37

Lysozyme 0.21

Apple fruit cutin 4.84

Pectin 46.0

Galacturonic acid 56.0b

a Each value represents the average of two replicate ana-

lyses.b Data from Pakulski and Benner (1992).

S. Opsahl, R. Benner / Organic Geochemistry 30 (1999) 83±94 85

Table 2

Mass losses (ash-free dry weight, AFDW) and carbohydrate yields (mg TCOH-C/100 mg OC) measured from fresh, senescent and

decomposed vascular plant tissues

%Rem Lyx Ara Rib Xyl Rha Fuc Man Gal Glc Myo Ns-tot Total Uniden.

Mangrove leaves

Green 0.06 2.23 0.05 1.87 0.98 0.03 0.40 0.82 9.33 0.04 15.80 21.44 5.64

Senescent 100.0 0.07 2.62 0.06 2.44 1.64 0.05 0.40 0.94 9.72 0.08 18.00 26.92 8.92

21 d 80.1 0.10 2.26 0.05 2.39 1.54 0.06 0.38 0.85 6.92 0.02 14.56 22.96 8.40

42 d 68.7 0.09 2.44 0.04 2.51 1.47 0.05 0.39 0.86 6.87 0.02 14.72 22.08 7.36

77 d 53.4 0.14 1.32 0.03 2.86 0.96 0.06 0.38 0.48 6.27 0.00 12.52 16.92 4.40

125 d 45.9 0.13 1.01 0.02 2.76 0.90 0.07 0.31 0.36 5.04 0.00 10.64 15.96 5.32

0.5 yr 37.9 0.14 0.86 0.02 2.55 0.95 0.08 0.31 0.34 5.08 0.00 10.32 13.84 3.52

0.7 yr 29.5 0.11 1.16 0.02 2.82 1.15 0.07 0.36 0.44 5.21 0.00 11.36 13.56 2.20

1.0 yr 19.0 0.08 1.34 0.03 2.11 1.30 0.10 0.50 0.62 4.64 0.00 10.72 12.88 2.16

1.4 yr 11.9 0.05 1.44 0.04 0.90 1.43 0.10 0.54 0.62 3.64 0.00 8.76 11.00 2.24

1.8 yr 9.1 0.03 0.97 0.05 0.40 1.41 0.12 0.50 0.60 2.56 0.00 6.64 12.04 5.40

2.3 yr 7.4 0.04 1.43 0.06 0.40 1.72 0.14 0.65 0.79 2.90 0.00 8.12 10.16 2.04

3.0 yr 3.7 0.04 1.52 0.06 0.42 1.44 0.16 0.65 0.79 2.60 0.00 7.64 9.96 2.32

4.0 yr 2.6 0.03 1.10 0.08 0.36 1.22 0.18 0.69 0.76 2.82 0.00 7.24 10.80 3.56

Smooth cordgrass

Green 0.37 1.94 0.07 15.64 0.22 0.04 0.35 0.76 27.59 0.03 47.04 54.80 7.76

Senescent 100.0 0.47 2.38 0.06 18.26 0.22 0.06 0.55 0.84 26.84 0.00 49.60 60.00 10.40

21 d 93.0 0.41 2.10 0.02 17.42 0.18 0.03 0.34 0.60 25.55 0.00 46.64 58.44 11.80

42 d 89.2 0.57 1.73 0.03 14.78 0.14 0.04 0.39 0.72 24.18 0.00 42.56 58.24 15.68

77 d 76.1 0.48 1.97 0.02 16.44 0.15 0.05 0.34 0.61 21.86 0.00 41.88 59.68 17.80

125 d 77.4 0.77 1.67 0.04 14.52 0.17 0.04 0.52 0.57 21.36 0.00 39.60 58.92 19.32

0.5 yr 77.7 0.42 1.88 0.02 17.15 0.16 0.04 0.34 0.54 23.21 0.00 43.68 57.56 13.88

1.0 yr 41.3 0.78 1.18 0.03 10.95 0.19 0.06 0.86 0.70 20.49 0.00 35.20 55.16 19.96

1.4 yr 35.7 0.75 2.70 0.03 17.23 0.68 0.04 1.58 1.08 31.14 0.00 55.20 56.40 1.20

1.8 yr 28.2 0.64 2.59 0.03 17.54 0.66 0.04 1.42 1.06 32.11 0.00 56.08 51.88 0.00

2.3 yr 27.2 0.96 2.47 0.04 15.54 0.62 0.03 1.50 0.95 27.97 0.00 50.04 46.40 0.00

3.0 yr 12.4 0.44 2.19 0.05 13.54 0.63 0.04 1.05 1.14 25.58 0.00 44.64 38.88 0.00

4.0 yr 6.7 0.18 0.92 0.04 4.28 0.30 0.09 0.81 0.87 7.03 0.00 14.52 14.56 0.04

Cypress needles

Green 0.16 2.06 0.12 1.11 0.36 0.22 2.09 2.02 13.96 0.11 22.60 29.64 7.04

Senescent 100.0 0.03 1.62 0.04 0.80 0.52 0.14 1.54 1.63 8.50 0.01 14.84 21.68 6.84

21 d 88.5 0.04 1.43 0.03 0.94 0.50 0.14 2.16 1.63 10.10 0.00 16.96 25.44 8.48

42 d 85.8 0.05 1.23 0.03 0.88 0.46 0.14 2.20 1.47 10.42 0.00 16.88 23.92 7.04

77 d 73.5 0.03 1.08 0.02 0.77 0.43 0.12 1.71 1.34 8.50 0.00 14.00 20.76 6.76

125 d 66.5 0.05 1.18 0.02 0.81 0.38 0.12 1.69 1.36 7.89 0.00 13.52 17.12 3.60

0.5 yr 55.6 0.04 1.11 0.03 0.72 0.38 0.12 1.32 1.38 6.63 0.00 11.72 15.08 3.36

0.7 yr 55.4 0.02 1.02 0.03 0.70 0.37 0.11 1.22 1.26 6.34 0.00 11.12 14.24 3.12

1.0 yr 41.7 0.03 0.77 0.03 0.43 0.31 0.10 0.68 1.04 3.59 0.00 7.00 10.88 3.88

1.4 yr 30.9 0.02 1.01 0.02 0.41 0.29 0.08 0.56 1.15 2.76 0.00 6.32 8.48 2.16

1.8 yr 26.6 0.01 0.52 0.03 0.16 0.20 0.06 0.31 0.82 1.44 0.00 3.56 6.72 3.16

2.3 yr 24.1 0.01 0.49 0.02 0.14 0.20 0.06 0.30 0.66 1.21 0.00 3.12 5.88 2.76

3.0 yr 16.7 0.01 0.58 0.02 0.14 0.21 0.06 0.30 0.69 1.02 0.00 3.04 4.64 2.32

4.0 yr 13.7 0.01 0.58 0.02 0.13 0.19 0.05 0.32 0.66 0.98 0.00 2.92 4.68 1.76

Mangrove wood

Senescent 100.0 1.00 1.63 0.04 9.03 0.61 0.06 1.38 1.00 21.25 0.00 36.00 65.48 23.40

42 d 73.6 0.77 2.65 0.05 15.98 0.71 0.05 1.50 1.06 28.71 0.00 51.48 63.40 11.92

77 d 70.7 0.21 1.94 0.03 11.02 0.70 0.05 0.66 0.71 20.65 0.00 35.72 60.56 24.84

0.5 yr 64.2 0.31 3.06 0.03 15.66 0.73 0.07 1.09 1.11 27.59 0.00 49.68 69.88 20.20

1.0 yr 55.4 0.46 2.26 0.04 13.60 0.68 0.05 1.09 1.07 24.68 0.00 43.92 62.00 18.08

S. Opsahl, R. Benner / Organic Geochemistry 30 (1999) 83±9486

30 min. Acetone (1.0 ml) was added and absorbance

was measured at 635 nm. Final absorbance was deter-mined by subtracting the absorbance of the blankfrom that of the sample. The analytical error associ-ated with the MBTH analysis of plant tissues was

found to be <15%.Each mole of a typical alditol oxidized by HIO4 pro-

duces 2 mol as of formaldehyde from the terminal car-

bon atoms and 4 mol as of formic acid from theinternal carbon atoms. However, due to the presenceof a C6 carboxyl group, only one molecule of formal-

dehyde is produced from uronic acids which results ina 50% reduction of the carbohydrate signal. MBTHanalysis of a commercially available preparation of

pectin (Sigma), a polygalturonic acid polymer, gave46% the response of glucose (Table 1). Similarly, thecarbohydrate signal from deoxy sugars is also reducedapproximately 50% because the C6 methyl group does

not yield formaldehyde upon HIO4 oxidation (Johnsonand Sieberth, 1977). Cyclitols, which are important cel-lular metabolites of vascular plants, have di�erent

stereochemistry than other alditols and are notmeasured at all using the MBTH analysis.

2.4. Cross reactions during the MBTH analysis

We used a number of compounds (tannins, lignins,

proteins and puri®ed cuticles) to test for cross-reac-tions with the MBTH because they are among themost abundant components of woody and herbaceous

plant tissues. Tannins are phenolic compounds which

range in molecular weight from 500±3,000 Da and

have the ability to complex strongly with proteins and

carbohydrates (Zucker, 1983; Porter, 1989). Highly

puri®ed tannin complexes are not available commer-

cially and many have a carbohydrate component as-

sociated with them which would complicate our

investigation of cross-reactions. Instead we used sim-

pler tannin derivatives from the two major subdivisions

of this compound class, hydrolyzable tannins (gallic

acid, phloroglucinol and tannic acid) and condensed

tannins (catechin). Tannic acid yielded the largest re-

sponse of all tannin model compounds (Table 1).

However, this signal was still very low (equivalent to

5.64 mg TCOH-C/100 mg OC), indicating that tannins

are not likely to cause substantial cross-reactions in

the MBTH analysis.

Lignin is a polyphenolic structural macromolecule

found in all herbaceous and woody vascular plants

(Sarkanen and Ludwig, 1971). The lignin content of

wood may be as high as 30%, thus lignin represents a

potentially signi®cant source of cross-reaction in the

MBTH analysis. It is very di�cult to separate and pur-

ify lignin from plant tissues without chemically modify-

ing it. In addition, lignin preparations often contain

other compounds (Zech et al., 1987; Benner et al.,

1990a), thus sources of contamination to the MBTH

analysis may still be present. Instead of using a chemi-

cal isolate of lignin, we analyzed several simple phenols

which are associated with lignin polymer and also exist

Table 2 (continued )

%Rem Lyx Ara Rib Xyl Rha Fuc Man Gal Glc Myo Ns-tot Total Uniden.

1.4 yr 52.7 0.74 2.40 0.05 14.16 0.63 0.05 1.41 0.94 24.53 0.00 44.88 59.92 15.04

2.3 yr 46.5 0.31 2.56 0.04 14.67 0.61 0.05 0.95 0.94 26.74 0.00 46.88 59.40 12.52

3.0 yr 35.1 0.30 2.80 0.03 13.96 0.66 0.07 1.00 1.00 23.76 0.00 43.60 56.76 13.16

4.0 yr 32.0 0.58 2.90 0.06 12.92 0.69 0.10 1.07 1.14 24.57 0.00 44.04 56.40 12.40

Cypress wood

Senescent 100.0 0.19 0.79 0.00 3.11 0.27 0.11 4.71 2.06 26.08 0.00 37.32 48.52 11.20

42 d 95.6 0.18 0.74 0.00 3.64 0.22 0.06 5.14 3.35 27.12 0.00 40.48 46.84 6.36

77 d 93.6 0.18 0.71 0.00 3.68 0.24 0.07 4.92 3.57 26.24 0.00 39.60 46.76 7.16

0.5 yr 88.2 0.26 0.80 0.00 3.50 0.26 0.07 5.64 3.08 26.92 0.00 40.52 47.52 7.00

1.0 yr 85.0 0.09 0.77 0.00 3.80 0.23 0.06 5.22 3.48 25.93 0.00 39.60 47.00 7.40

1.4 yr 82.2 0.24 0.70 0.00 3.36 0.22 0.06 4.94 3.81 24.36 0.00 37.68 47.00 9.32

2.3 yr 72.8 0.27 0.79 0.00 3.34 0.24 0.07 5.05 3.49 23.22 0.00 36.52 44.60 8.08

3.0 yr 69.4 0.39 0.73 0.00 2.94 0.24 0.08 5.23 3.72 22.28 0.00 35.60 43.32 7.72

4.0 yr 61.4 0.11 0.77 0.00 3.62 0.30 0.08 4.66 4.95 24.82 0.00 39.32 42.84 3.52

Deviationa (%) 3 19 7 20 8 5 8 7 6 6 5 5 4 22

a Percent mean deviation determined for replicate litter bags averaged throughout the time series and for all ®ve tissue types.

Abbreviations: %Rem., percent remaining; Lyx, lyxose; Ara, arabinose; Rib, ribose; Xyl, xylose; Rha, rhamnose; Fuc, fucose;

Man, mannose; Gal, galactose, Glc, glucose; Myo, myo-inositol; Ns-tot, sum of all neutral sugars; Total, total carbohydrates deter-

mined by modi®ed MBTH method (Johnson and Sieberth, 1977; Pakulski and Benner, 1992); Uniden, molecularly unidenti®ed

carbohydrates.

S. Opsahl, R. Benner / Organic Geochemistry 30 (1999) 83±94 87

in free form within herbaceous tissues. Although feru-

lic acid gave a higher response than p-coumaric acid(Table 1), both MBTH responses were low and rep-resent at most, trace sources of cross-contamination.

However, vanillin, an abundant phenol producedduring the chemical oxidation of lignin polymer, gavea stronger MBTH response (12.3 mg TCOH-C/100 mg

OC). We speculate that this signal was derived fromthe quantitative reaction of the aldehyde functional

group at the C-1 position on the aromatic ring of thiscompound because the MBTH signal was equivalentto about 12% of vanillin carbon. Although vanillin

does not exist in free form in plant tissues, this moresubstantial reaction from vanillin suggested the possi-bility that lignin may react during this analysis, poss-

ibly through liberation of reactive components duringthe HIO4 oxidation.

To further test for cross reactions with lignin, weused a dehydrogenative polymer (DHP lignin; providedby Ken Hammel from the USDA Forest Products

Laboratory), a carbohydrate-free synthetic lignin de-rived from the polymerization of guiacyl phenols (Kirk

et al., 1975). DHP lignin contains many of the majorintermonomeric linkages characteristic of intact ligninpolymer and is often used in structural elucidation stu-

dies of lignin. Virtually no MBTH response was pro-duced by DHP lignin (Table 1). Assuming that DHPlignin is reasonably representative of lignin in its natu-

ral state, we conclude that lignin does not substantiallycross-react in the MBTH analysis.

Johnson and Sieberth (1977) found that serine gavea substantial cross-reaction in the MBTH method. Wetested two commercially available proteins, bovine

serum albumin and lysozyme, with molecular weight of14,000 and 66,000 Da, respectively. Both of these pro-teins produced only minor MBTH responses (Table 1).

The lack of a signi®cant response and the fact thatproteins and free amino acids are minor constituents

of vascular plant tissues indicates that this compoundclass will also have an insigni®cant e�ect on totalcarbohydrate determinations using the MBTH method.

Plant cuticles contain a substantial amount of cutin,a polyester primarily composed of hydroxy and epoxy

acids (Martin and Juniper, 1970). Cutin acids maycomprise 10% or more of the total mass of certain her-baceous tissues and 50% or more of isolated cuticles,

thus representing another possible source of cross-reac-tion for the MBTH analysis. To test for cross-reactionfrom both cutin acids and the remaining associated cu-

ticular material, we obtained a sample of isolatedcuticles from English Holly leaves (Ilex aquifolium;

provided by M. Goni and J. Hedges), which had beentreated to remove associated carbohydrates (Goni andHedges, 1990). A cross-reaction of 4.84 mg TCOH-C/

100 mg OC (Table 1) was measured during the MBTHanalysis of isolated cuticles. Therefore, plant cuticles

along with their associated cutin acids will have a neg-ligible e�ect on determinations of total carbohydrates

using the MBTH method.

3. Results and discussion

3.1. Initial carbohydrate compositions

Glucose was the most abundant neutral sugar in all

®ve senescent tissues (Table 2). Glucose accounted for54±70% of the total neutral sugar yields and rep-resented 8.5±26.8% of the plant tissue carbon. Xylose,a major sugar in angiosperm tissues (Cowie and

Hedges, 1984a,b), was particularly abundant in smoothcordgrass and mangrove wood. Other more predomi-nant sugars included arabinose, rhamnose, mannose

and galactose. Lyxose, ribose, fucose and myo-inositolwere found in much lower abundance. Smooth cord-grass had the highest total neutral sugar yields

(49.6 mg TCOH-C/100 mg OC) followed closely by thetwo woods (36.0 and 37.3 mg TCHO-C/100 mg OC)which in turn had 2±3-fold higher neutral sugar yieldsthan mangrove leaves and cypress needles (18.0 and

14.8 mg TCOH-C/100 mg OC, respectively). We alsomeasured the neutral sugar composition of green tis-sues collected from the same locations as the senescent

material (Table 2). Yields and compositional patternsin green mangrove leaves and smooth cordgrass werequite similar to their senescent tissue counterparts,

with the exception of green cypress needles, which pro-duced 60% higher glucose yields. An abundance ofglucose in green tissues may be due to high concen-

trations of glucose-containing metabolites such asstarch.Total carbohydrate yields were higher than total

neutral sugars yields in all green and senescent tissues,

ranging from 21.4 mg TCOH-C/100 mg OC in greenmangrove leaves to 65.5 mg TCOH-C/100 mg OC inthe case of mangrove wood (Table 2). The large di�er-

ence between total carbohydrate and total neutralsugar yields represents a molecularly unidenti®edcarbohydrate fraction accounting for 33, 17, 32, 36

and 23% of the total carbohydrate content for senes-cent mangrove leaves, smooth cordgrass, cypress nee-dles, mangrove wood and cypress wood, respectively.Given the higher estimates of carbohydrates using the

MBTH method and the lack of signi®cant contami-nation from non-carbohydrate compounds, totalcarbohydrate determinations of vascular plants made

using the MBTH method should be considered morerepresentative of total carbohydrate yields than totalneutral sugar yields.

Uronic acids (acidic sugars) are one carbohydratesource not measured using the neutral sugar analysisthat likely contributes to higher total carbohydrate

S. Opsahl, R. Benner / Organic Geochemistry 30 (1999) 83±9488

measurements. Uronic acids are important constituentsof a variety of plant polymers, including hemicellulose

and pectin (Aspinall, 1970). Hemicellulose, an abun-dant component of vascular plant tissues, would likelybe the primary source for uronic acids in woody tissues

which contain virtually no pectin. The uronic acid resi-dues in xylans have been shown to be particularlyabundant in both angiosperm and gymnosperm woods

(Aspinall, 1970; SjùstroÈ m, 1981). In herbaceous tissues,uronic acids are found in hemicellulose and pectin.Pectin, a polymer consisting primarily of polygalac-

turonic acid, is found universally in the primary cellwalls and intercellular layers of vascular plants(Aspinall, 1970), functioning primarily as an adhesiveto attach plant cuticles to underlying tissues.

Therefore, pectin is likely to be particularly abundantin the herbaceous tissues with a large cuticle, such asmangrove leaves. In addition, other carbohydrate

classes such as amino sugars which are quantitativelyrecovered in the MBTH analysis (Pakulski andBenner, 1992) may be present. Although amino sugars

are not major structural components of vascularplants, they may be associated microbial biomass withplant detritus, particularly in more highly decomposed

material (Hicks et al., 1991).

3.2. Bulk decomposition trends

Decomposition is de®ned here as any loss of dis-solved and particulate organic matter, including losses

due to leaching, microbial mineralization and fragmen-tation, from the initial starting material placed in litterbags. A detailed presentation of the kinetics of long-

term vascular plant decomposition will be given else-where (Opsahl and Benner, in preparation) and onlytrends relevant to the present discussion are provided

here. Patterns of mass loss were typical of thosedescribed for aquatic decomposition of vascular plants(Godshalk and Wetzel, 1978b; Valiela et al., 1985).

Large early losses due to leaching were most apparentin mangrove leaves, which lost over 30% of their in-itial ash-free dry weight (AFDW) after 42 d (Table 2).In contrast, leaching of organic constituents from

cypress wood was almost negligible (<5%). Masslosses after 4 yr of decomposition were extensive (86±97%) in the three herbaceous tissues, whereas man-

grove and cypress wood lost 68 and 38% of initialmass, respectively (Table 2).

3.3. Carbohydrate dynamics during decomposition

3.3.1. Soluble carbohydratesLarge mass losses during the leaching phase of de-

composition are attributed to rapid removal of easilyextractable compounds such as cellular metabolites. Inthis study, we have assumed the leaching phase

includes all losses occurring during the ®rst 42 d of de-composition. The percentage of organic carbon loss

during leaching that can be accounted for as carbo-hydrates was estimated from TCOH-C yields (Table 2)and organic carbon measurements (Opsahl and

Benner, 1995). Approximately 45, 34, 7, 36 and 57%of the organic carbon loss during the leaching phasecould be accounted for as carbohydrate carbon in

mangrove leaves, smooth cordgrass, cypress needles,mangrove wood and cypress wood, respectively. Thus,a substantial fraction of early organic carbon loss was

accounted for as carbohydrate carbon in 4 out of 5 tis-sues. The low percentage observed for cypress needlesindicates that leachates can also consist primarily ofnon-carbohydrate compounds. The neutral sugar com-

ponent of carbohydrates removed during leaching ran-ged from 59±86% of the total carbohydrate loss.Several of the more common carbohydrates found in

soluble extracts of vascular plants include glucose,fructose, sucrose, several oligosaccharides such as ra�-nose and sedoheptulose and cyclitols (Holligan and

Drew, 1971). Most of the leached carbohydrate whichwas not neutral sugar may be derived from ketosessuch as fructose and heptoses, which are not measured

during the neutral sugar analysis but are measuredwith the MBTH method. Although cyclitols aremeasured during neutral sugar analysis but not withthe MBTH method (Johnson and Sieberth, 1977), the

cyclitol content of tissues used in this study was verylow and could not make a large contribution to massloss.

3.3.2. Neutral sugars

Beyond the leaching phase, herbaceous tissues allunderwent large decreases in the relative percentage ofglucose (Table 2). Xylose was the only other sugarwhich decreased in relative abundance in the herbac-

eous tissues. In a separate study, Opsahl and Benner(1993) reported that glucose and xylose were the twomost labile neutral sugars during seagrass decompo-

sition. The consistent pattern of selective degradationof glucose and xylose relative to all other neutralsugars in herbaceous tissues supports the observation

of a direct association between xylose residues and cel-lulose in vascular plants (Aspinall, 1970).Corresponding increases in the relative percentages ofarabinose, rhamnose, mannose and galactose occurred

as a result of the loss of glucose and xylose from her-baceous tissues. In contrast, no large change in neutralsugar composition or yields were evident in the woody

tissues.Changes in neutral sugar composition of herbaceous

tissues can be summarized in a plot of glucose plus

xylose versus all remaining neutral sugars (Fig. 1a±c).At the expense of glucose and xylose, an increase inthe relative proportions of the other neutral sugars was

S. Opsahl, R. Benner / Organic Geochemistry 30 (1999) 83±94 89

evident. This was clear for mangrove leaves and

cypress needles (Fig. 1a and c) in which the initial per-

centage of glucose + xylose (>60%) decreased to

about 40% at the end of the experiment. The pattern

in smooth cordgrass (Fig. 1b) was not as pronounced,

although the relatively large shift between 3 and 4 yr

indicates that substantial changes may occur during

the later stages of decomposition.

The relative decrease in glucose concentration wasalso evident in the woods (Table 2), and appeared to

be the most consistent trend observed for any individ-ual neutral sugar among all ®ve tissue types. However,

woods did not show large corresponding decreases in

xylose yields. Although changes in the neutral sugarcomposition of woods were less substantial than for

herbaceous tissues, more pronounced changes mayoccur as the woods approach a more advanced state of

decomposition. If glucose continues to be among themost labile of the individual sugars, then larger shifts

towards predominance of some of the initially lessabundant neutral sugars will also occur in woods after

more extensive degradation.

Mangrove leaves, smooth cordgrass and cypress nee-dles all demonstrated 2±5-fold increases in the percen-

tage of deoxy sugars (rhamnose and fucose) at the

expense of glucose, whereas changes in mangrovewood and cypress wood were negligible (Fig. 2).

Hedges et al. (1994) observed an increase in the per-centage of deoxy sugars and corresponding decreases

in glucose in progressively more degraded particulateorganic matter samples from the Amazon River,

suggesting that deoxy sugars derived from vascularplants may be relatively stable during decomposition.

Alternatively, elevated deoxy sugar levels may arise

from contributions from external sources, such as mi-crobial biomass which is enriched in deoxy sugars

(Cowie and Hedges, 1984b; Hamilton and Hedges,1988). Distinctions between selective preservation of

speci®c sugars and contributions from the external en-vironment could not be resolved in this study because

any inputs from microbial biomass were not large

enough to de®nitively demonstrate external sources. Ineither case, elevated deoxy sugar content appears to be

a useful indicator of highly degraded plant tissues.Leaching of soluble constituents and selective mi-

crobial degradation are responsible for most of the

early compositional changes in decomposing vascularplants. However, the long-term processes associated

Fig. 1. A comparison of glucose + xylose (closed circles, .)vs. other neutral sugars (open circles, w) during the decompo-

sition of mangrove leaves (a), smooth cordgrass (b) and

cypress needles (c). Values are expressed as a percentage of

the total neutral sugar yields (% NS-TOT). Other neutral

sugars represents the sum of all remaining sugars including

lyxose, arabinose, ribose, rhamnose, fucose, mannose, galac-

tose and myo-inositol.

Fig. 2. The relative proportion of deoxy sugars expressed as a

percentage of the total neutral sugar yields during decompo-

sition of mangrove leaves (w), smooth cordgrass (q) and

cypress needles (r).

S. Opsahl, R. Benner / Organic Geochemistry 30 (1999) 83±9490

with decomposition of the residual particulate material

are not well understood. Structural polysaccharides

may be protected from enzymatic degradation by as-

sociations with other biochemical constituents includ-

ing lignins, tannins and other carbohydrates. In

addition, the stability of linkages which bind individual

subunits together are important in determining the

relative degree of bioavailability. Sugars such as glu-

cose and xylose which degrade more rapidly may be

less highly cross-linked with other constituents and/or

their inter-monomeric linkages may be more suscep-

tible to enzymatic degradation. Such a phenomenon

would help explain the more rapid decomposition of

abundant sugars and the observed trend towards com-

positional uniformity of the neutral sugar fractionobserved in progressively decomposed tissues.

3.3.3. Bulk carbohydratesTotal carbohydrate yields followed the same general

pattern as neutral sugars, decreasing 2±5-fold in theherbaceous tissues and mangrove wood after 4 yr, butchanging little in cypress wood (Table 2). Total carbo-

hydrates can be subdivided into neutral sugars(measured using GC) and a fraction which cannot beidenti®ed at the molecular level (MBTH minus GC

sugars). In mangrove leaves and cypress needles, therelative abundance of neutral sugars the molecularlyuncharacterized fraction varied during decomposition,

Fig. 3. Percentages of the total neutral sugar (.) and molecularly uncharacterized carbohydrate (w) fractions in decomposing man-

grove leaves (a), smooth cordgrass (b), cypress needles (c), mangrove wood (d) and cypress wood (e).

S. Opsahl, R. Benner / Organic Geochemistry 30 (1999) 83±94 91

although percentages at 4 yr (approximately 65% neu-

tral sugar) demonstrated little net overall change frominitial values (Fig. 3a and c). In smooth cordgrass,mangrove wood and cypress wood (Fig. 3b, d and e),

the percentages of neutral sugars increased slightlyduring decomposition and at 4 yr, 80±100% of theremaining carbohydrate could be identi®ed as neutral

sugars. Tissues such as grasses and woods are compo-sitionally much simpler than tree leaves and needles,which contain additional abundant compounds includ-

ing cutin, waxes and tannins (Benner et al., 1990a).Given that most carbon ¯ux is associated with litter-fall rather than woody biomass (Olson et al., 1985), itseems likely that vascular plant-derived organic matter

in soils and sediments will have a substantial non-neu-tral sugar carbohydrate component. Future studiesshould include measurements of total carbohydrates

for comparison with neutral sugars to determine theextent to which neutral sugars represent the totalcarbohydrate fraction and how this varies among

di�erent environments.

3.4. Relative carbohydrate stability

The relative stability of carbohydrates can be exam-ined using the ratio of carbohydrate yield (mg carbo-hydrate/100 mg organic carbon; Table 1) at a giventime relative to the initial yield (relative stabili-

ty = yieldtime point/yieldinitial). When the relative stab-ility of total carbohydrates was examined during the4 yr decomposition period, the carbohydrate fraction

in all tissues was found to be labile relative to totalmass (Fig. 4; relative stability <1). However, the pat-tern and extent of carbohydrate decomposition varied

considerably among the di�erent tissues. In mangroveleaves, a progressive depletion in carbohydrates wasevident through 1 yr (about 81% mass loss), but little

further selective carbohydrate loss occurred between 1and 4 yr. Although a small carbohydrate enrichment

was initial apparent in cypress needles, the long-termtrend was similar to mangrove leaves. Selective carbo-hydrate losses did not slow until beyond 3 yr (76%

mass loss), however. In contrast, little selective carbo-hydrate depletion was apparent in smooth cordgrassthrough the ®rst 1.8 yr (72% mass loss), but much lar-

ger losses became apparent between 1 and 4 yr.Carbohydrate fractions in both woods demonstratedsmaller selective losses after 4 yr, but these tissues had

not decomposed to the same extent as herbaceous tis-sues. Although it is clear that carbohydrate depletionwas characteristic of all tissues, both extent and timingof carbohydrate loss was strongly tissue dependent.

Most litter bag decomposition studies have reportedthat carbohydrates decompose more rapidly than otherabundant plant constituents such as lignin. However,

one inherent shortcoming in the litter bag approach isthat physical loss of smaller, more highly degradedparticles from the litter bags leaves particulate material

that is not completely representative of all partiallydecomposed plant tissue. The small mesh size used inthis experiment optimized for retention of more highly

decomposed particles. In 2 of 3 tissues, selective carbo-hydrate loss did not appear to continue inde®nitely,and carbohydrates remained a relatively constant pro-portion of the remaining detritus in the latter stage of

decomposition (Fig. 4). Thus, selective changes in thechemical composition predicted from relatively short-term studies may not apply to more highly degraded

particulate organic matter such as that found in riversand estuarine environments.

3.5. Diagenetic indicator parameters from carbohydratecomposition

The biochemical composition of plant detritusobtained from litter bag studies should be similar tothat of natural riverine particulate matter because vas-

cular plant remains are the most abundant componentof riverine organic matter. Hedges et al. (1994)measured the neutral sugar composition through a size

continuum of organic matter in the Amazon Riverwhich included coarse particulate organic matter(CPOM, >63 mm), ®ne particulate organic matter(FPOM, <63 mm) and ultra®ltered dissolved organic

matter (UDOM, >1,000 Da). These authors reportedthat the percentage of deoxy sugars in CPOM andFPOM ranged from 5±15%. The percent deoxy sugars

in this study increased in all herbaceous tissues (Fig. 2).The percent deoxy sugars ranged from 3±19% in thehighly degraded (4 yr) tissues, which is similar to that

reported by Hedges et al. (1994). These authors alsoshowed a diagenetic sequence in which neutral sugaryields progressively decreased from 17 to 11 to 3 mg/

Fig. 4. Relative stability (calculated from: relative stabili-

ty = yieldtime point/yieldinitial) of the total carbohydrates vs.

time in decomposing mangrove leaves (+), smooth cordgrass

(q), cypress needles (r), mangrove wood (.) and cypress

wood (R). A relative stability of 1 indicates no selective de-

pletion or enrichment of a given constituent during decompo-

sition.

S. Opsahl, R. Benner / Organic Geochemistry 30 (1999) 83±9492

100 mg OC in CPOM, FPOM and UDOM, respect-ively. Total neutral sugar yields calculated for thehighly degraded (4 yr) herbaceous tissues in this study

were variable (3±15 mg/100 mg OC) but within therange reported for riverine organic matter. Therefore,all tissues were consistent with the proposed diagenetic

sequence because all neutral sugar yields decreasedduring decomposition. Total carbohydrate yieldsshowed a similar diagenetic trend as neutral sugar

yields, with the exception of cypress wood. Therefore,total carbohydrate yields determined using the MBTH

method may also be useful as an indicator of diage-netic status.The neutral sugar pro®le provides additional indi-

cators of diagenetic alteration (Cowie and Hedges,1994; Hernes et al., 1996). Mole percentages of glucosein fresh tissues were consistently high (54±58 mol%

glucose) relative to degraded counterparts (30±50 -mol%; Fig. 5). The highly degraded tissues had molepercentages of glucose similar to those reported for

particulate organic matter from major river systemsincluding the Amazon, Indus and Parana (Ittekkot andArain, 1986; Deptris and Kempe, 1993; Hedges et al.,

1994). Compositional data for dissolved carbohydratesin the Orinoco, Mackenzie, Amazon and WilliamsonRivers is also supportive of this proposed diagenetic

sequence (Sweet and Perdue, 1982; Deptris andKempe, 1993; Hedges et al., 1994). However, the molepercentage of glucose for the Caroni, Parana an Niger

Rivers (Deptris and Kempe, 1993) was found to be ashigh as 50%. Thus, with some exceptions, the relative

contribution of glucose strongly resembles that ofnaturally occurring riverine particulate organic matter.

Acknowledgements

We thank Miguel Goni for providing the samples ofEnglish Holly leaf cuticle and Ken Hammel for provid-ing a sample of DHP lignin. We also thank J. Hedges

for a helpful review of the manuscript. This researchwas supported by NSF grants OCE 9413843 and BSR8910766. This is contribution number 1075 from the

University of Texas at Austin Marine Science Institute.

Associate EditorÐP. Hatcher

References

Aspinall, G. O., 1970. Polysaccharides. Pergamon Press.

Benner, R., Hodson, R. E., 1985. Microbial degradation of

the leachable and lignocellulosic components of leaves and

wood from Rhizophora mangle in a tropical mangrove

swamp. Mar. Ecol. Prog. Ser. 23, 221±230.

Benner, R., Lay, J., K'nees, E., Hodson, R. E., 1988. Carbon

conversion e�ciency for bacterial growth on lignocellulose:

implications for detritus-based food webs. Limnol.

Oceanogr. 33, 1493±1513.

Benner, R., Hatcher, P. G., Hedges, J. I., 1990a. Early dia-

genesis of mangrove leaves in a tropical estuary: bulk

chemical characterization using solid-state 13-C NMR and

Fig. 5. A proposed diagenetic sequence of vascular plant-derived particulate organic matter based on the mole percentage of glu-

cose. Samples include fresh vascular plant herbaceous tissues (SC, ML, CN), the degraded (4 yr) particulate organic matter (POM)

of these tissues, and POM and DOM collected from major riverine systems. Abbreviations: IR, Indus River (data from Ittekkot

and Arain, 1986); ARC and ARF, Amazon River coarse (>63 mm) and ®ne (<63 mm) POM (data from Hedges et al., 1994); PR,

Parana River (data from Deptris and Kempe, 1993); CR, Caroni River; NR, Niger River; OR, Orinoco River; MR, Mackenzie

River (data from Ittekkot and Arain, 1986); WR, Williamson River (data from Sweet and Perdue, 1982); Other abbreviations pro-

vided in Table 2.

S. Opsahl, R. Benner / Organic Geochemistry 30 (1999) 83±94 93

elemental analysis. Geochim. Cosmochim. Acta 54, 2003±

2013.

Benner, R., Weliky, K., Hedges, J. I., 1990b. Early diagenesis

of mangrove leaves in a tropical estuary: molecular-level

analyses of neutral sugars and lignin derived phenols.

Geochim. Cosmochim. Acta 54, 1991±2001.

Cowie, G., Hedges, J., 1984a. Carbohydrate sources in a

coastal marine environment. Geochim. Cosmochim. Acta

48, 2075±2087.

Cowie, G. L., Hedges, J. I., 1984b. Determination of neutral

sugars in plankton, sediments and wood by capillary gas

chromatography of equilibrated isomeric mixtures. Anal.

Chem. 56, 497±504.

Cowie, G. L., Hedges, J. I., 1994. Biochemical indicators of

diagenetic alteration in natural organic mixtures. Nature

369, 304±307.

Deptris, P. J., Kempe, S., 1993. Carbon dynamics and sources

in the Parana River. Limnol. Oceanogr. 38, 382±395.

Fenchel, T. M., Jorgensen, B. B., 1977. Detritus Food Chains

of Aquatic Ecosystems: the Role of Bacteria. Plenum Press.

Godshalk, G. L., Wetzel, R. G., 1978a. Decomposition of

aquatic angiosperms. I. dissolved components. Aquat. Bot.

5, 281±300.

Godshalk, G. L., Wetzel, R. G., 1978b. Decomposition of

aquatic angiosperms. III. Zostera marina l. and a concep-

tual model of decomposition. Aquat. Bot. 5, 329±354.

Goni, M. A., Hedges, J. I., 1990. Cutin derived CuO reaction

products from puri®ed cuticles and tree leaves. Geochim.

Cosmochim. Acta 54, 3065±3072.

Hamilton, S. E., Hedges, J. I., 1988. The comparative geo-

chemistries of lignins and carbohydrates in an anoxic fjord.

Geochim. Cosmochim. Acta 52, 129±142.

Hedges, J. I., Cowie, G. L., Ertel, J., Barbour, R. J., Hatcher,

P. G., 1985. Degradation of carbohydrates and lignins in

buried woods. Geochim. Cosmochim. Acta 49, 701±711.

Hedges, J. I., Cowie, G. L., Richey, J. E., Quay, P. D.,

Benner, R., Strom, M., Forsberg, B. R., 1994. Origins and

processing of organic matter in the Amazon River as indi-

cated by carbohydrates and amino acids. Limnol.

Oceanogr. 39, 743±761.

Hernes, P. J., Hedges, J. I., Peterson, M. L., Wakeham, S. G.,

Lee, C., 1996. Neutral carbohydrate geochemistry of par-

ticulate material in the central equatorial Paci®c. Deep-Sea

Res. 43, 1181±1204.

Hicks, R. E., Lee, C., Marinucci, A. C., 1991. Loss and recy-

cling of amino acids and protein from smooth cordgrass

(Spartina alterni¯ora) litter. Estuaries 14, 430±439.

Holligan, P. M., Drew, E. A., 1971. Routine analysis by gas±

liquid chromatography of soluble carbohydrates in extracts

of plant tissues. N. Phytol. 70, 271±297.

Ittekkot, V., Arain, R., 1986. Nature of particulate organic

matter in the river Indus, Pakistan. Geochim. Cosmochim.

Acta 50, 1643±1653.

Johnson, K. M., Sieberth, J. M., 1977. Dissolved carbo-

hydrates in seawater. I. A precise spectrophotometric analy-

sis for monosaccharides. Mar. Chem. 5, 1±13.

Kirk, T. K., Conners, W. J., Bleam, R. D., Hackett, W. F.,

Zeikus, J. G., 1975. Preparation and microbial decompo-

sition of synthetic [14C] lignins. Proc. Natl. Acad. Sci.

U.S.A. 72, 2515±2519.

Loewus, F. A., Tanner, W. 1981. Plant Carbohydrates. I.

Intracellular Carbohydrates. Springer-Verlag, Berlin.

Martens, C. S., Haddad, R. I., Chanton, J. P., 1992. Organic

matter accumulation, remineralization, and burial in an

anoxic coastal sediment. In Organic Matter Productivity,

Accumulation and Preservation in Recent and Ancient

Sediments, eds. J. K. Whelan and J. W. Farrington, Vol.

82±98. Columbia University Press.

Martin, J. T., Juniper, B. E. 1970. The Cuticles of Plants. St.

Martin's Press.

Moers, M. E .C., De Leeuw, J. W., Baas, M., 1994. Origin

and diagenesis of carbohydrates in ancient sediments. Org.

Geochem. 21, 1093±1106.

Olson, J. S., Garrels, R. M., Berner, R. A., Armentano, T.

V., Dyer, M. I., Taalon, D. H. 1985. The Natural Carbon

Cycle.

Opsahl, S., Benner, R., 1993. Decomposition of senescent

blades of the seagrass Halodule wrightii in a subtropical

lagoon. Mar. Ecol. Prog. Ser. 94, 191±205.

Opsahl, S., Benner, R., 1995. Early diagenesis of vascular

plant tissues: lignin and cutin decomposition and biogeo-

chemical implications. Geochim. Cosmochim. Acta 59,

4889±4904.

Pakulski, J. D., Benner, R. H., 1992. An improved method

for the hydrolysis and MBTH analysis of dissolved and

particulate carbohydrates in seawater. Mar. Chem. 40, 143±

160.

Porter, L. J., 1989. Tannins. In Methods in Plant

Biochemistry, eds. P. M. Dey and J. B. Harborne, Vol. 1.

Academic Press, pp. 389±420.

Sarkanen, K. V., Ludwig, C. H. 1971. Lignins: Occurrence,

Formation, Structure, and Reactions.

SjùstroÈ m., 1981. Wood Chemistry, Fundamentals and

Applications. Academic Press.

Suberkropp, K., Godshalk, G. L., Klug, M. J., 1976. Changes

in the chemical composition of leaves during processing in

a woodland stream. Ecology 57, 720±727.

Sweet, M. S., Perdue, E. M., 1982. Concentration and specia-

tion of dissolved sugars in river water. Environ. Sci.

Technol. 16, 692±698.

Tukey, H. B., 1970. The leaching of substances from plants.

Ann. Rev. Plant Phys. 21, 305±324.

Valiela, I., Teal, J. M., Allen, S. D., Etten, R. V., Goehringer,

D., Volkman, S., 1985. Decomposition in salt marsh ecosys-

tems: the phases and major factors a�ecting disappearance

of above-ground organic matter. J. Exp. Mar. Biol. Ecol.

89, 29±54.

Valiela, I., Wilson, J., Buchsbaum, R., Rietsma, C., Bryant,

D., Foreman, K., Teal, J., 1984. Importance of chemical

composition of salt marsh litter on decay rates and feeding

by detritivores. Bull. Mar. Sci. 35, 261±269.

Wilson, J. O., Valiela, I., Swain, T., 1986. Carbohydrate

dynamics during decay of litter of Spartina alterni¯ora.

Mar. Biol. 92, 277±284.

Zech, W., Johansson, M., Haumaier, L., Malcolm, R. L.,

1987. CPMAS 13C NMR and IR spectra of spruce and

pine litter and the Klason lignin fraction at di�erent stages

of decomposition. Z. P¯anzenernaehr. Bodenkde. 150, 262±

265.

Zucker,W. V., 1983. Tannins: does structure determine function?

An ecological perspective. Am. Natural. 121, 335±365.

S. Opsahl, R. Benner / Organic Geochemistry 30 (1999) 83±9494