Biogeochemical Processes That Produce 

Dissolved Organic Matter From Wheat 

Straw

By Robert L. Wershaw, David W. Rutherford, Jerry A. Leenheer,

Kay R. Kennedy, Larry G. Cox, and Donald R. Koci

Water-Resources Investigations Report 03-4213

U.S. Department of the Interior

U.S. Geological Survey
U.S. Department of the Interior

Gale A. Norton, Secretary

U.S. Geological Survey

Charles G. Groat, Director

U.S. Geological Survey, Reston, Virginia: 2003

For more information about the USGS and its products:

Telephone: 1-888-ASK-USGS

World Wide Web: http://www.usgs.gov/

Any use of trade, product, or firm names in this publication is for descriptive purposes only and does not imply 

endorsement by the U.S. Government.

Robert L. Wershaw, David W. Rutherford, Jerry A. Leenheer, Kay R. Kennedy, Larry G. Cox, and Donald R. Koci, 2003, 

Biogeochemical Processes That Produce Dissolved Organic Matter From Wheat Straw: Reston, Virginia, U.S Geological Survey, 14 pages; available online only at: http://pubs.water.usgs.gov/wri034213

Library of Congress Cataloging-in-Publication Data

Author1LastName, Author1FirstName Q.

Title of book/ Author List

p. cm. –(U.S. Geological Survey professional paper ; 0000)

Includes bibliographic references.

1. Subject heading. 2. Subject heading. 3. Subject heading. I. Main entries. 

II.Main entries. III. Main entries.

TN24.M9 M55 2002

553’.09786’6—dc21

2001051109

ISBN 0-607-97815-9 (Set)

ISBN 0-607-97815-7 (Volume 1)

ISBN 0-607-97815-5 (Volume 2)
iii

Contents

Abstract.......................................................................................................................................................... 1

Introduction ................................................................................................................................................... 1

Materials and Methods ............................................................................................................................... 2

Sample collection ................................................................................................................................ 2

Dissolved organic carbon and inorganic analyses ....................................................................... 2

Carbohydrate analysis ........................................................................................................................ 2

Fractionation......................................................................................................................................... 3

Dialysis .................................................................................................................................................. 3

Spectral analyses ................................................................................................................................ 3

Molecular weight measurements..................................................................................................... 4

Biochemical Study Results ......................................................................................................................... 4

Elemental composition........................................................................................................................ 4

Results of carbohydrate analysis...................................................................................................... 4

Ultraviolet-visible, nuclear magnetic resonance and infrared spectral results ....................... 5

Biogeochemical process implications of the spectral data......................................................... 8

Molecular weight of dissolved organic matter............................................................................... 9

Biogeochemical process implications of the molecular-weight data...................................... 11

Summary and Conclusions........................................................................................................................ 12

References Cited ........................................................................................................................................ 12
Abstract

The chemical reactions that lead to the formation of 

dissolved organic matter (DOM) in natural waters are poorly 

understood. Studies on the formation of DOM generally 

are complicated because almost all DOM isolates have been 

derived from mixtures of plant species composed of a wide 

variety of different types of precursor compounds for DOM 

formation. This report describes a study of DOM derived 

mainly from bales of wheat straw that had been left in a field 

for several years. During this period of time, black water from 

the decomposing wheat straw accumulated in pools in the 

field. The nuclear magnetic resonance and infrared spectra 

of the black water DOM indicate that it is composed almost 

entirely of lignin and carbohydrate polymeric units. Analysis 

by high-performance size-exclusion chromatography with 

multi-angle laser-light scattering detection indicates that the 

number average molecular weight of the DOM is 124,000 

daltons. The results presented in this report indicate that 

the black water DOM is composed of hemicellulose chains 

cross-linked to lignin oligomers. These types of structures 

have been shown to exist in the hemicellulose matrix of plant 

cell walls. The cross-linked lignin-hemicellulose complexes 

apparently were released from partially degraded wheat-straw 

cell walls with little alteration. In solution in the black water, 

these lignin-hemicellulose polymers fold into compact globular particles in which the nonpolar parts of the polymer form 

the interiors of the particles and the polar groups are on the 

exterior surfaces of the particles. The tightly folded, compact conformation of these particles probably renders them 

relatively resistant to microbial degradation. This should be 

especially the case for the aromatic lignin structures that will 

be buried in the interiors of the particles.

Introduction

Natural organic matter (NOM) has been studied for 

over 200 years. Most workers have attempted to elucidate 

the structures of chemical components of NOM by using the 

same methods that have been used to elucidate the chemical 

structures of pure compounds. These methods, however, are 

unsuitable for this task because NOM isolates are complex 

mixtures of compounds that form molecular aggregates in 

solution. Wershaw (2000) has proposed that a more fruitful 

approach would be to study the biochemical reactions that the 

chemical components of plant tissue undergo as they degrade 

into NOM. As plant organs (e.g. leaves) degrade in natural 

systems, soluble material is leached from the tissue. The 

organic compounds in this leachate contribute to the watersoluble component of NOM found in streams and soil waters 

which is commonly designated as dissolved organic matter 

(DOM) or dissolved organic carbon (DOC). A comparison of 

the spectral features of the DOM with those of the components 

of the plant tissue from which it is derived should give some 

clues about the chemical reactions that produced the DOM.

Generally, such studies are complicated because almost 

all DOM has been derived from mixtures of plant species. 

Due to an unusual set of circumstances, DOM derived mainly 

from wheat straw was formed in a field in southeastern Kansas 

where bales of wheat straw had been stored for use in the manufacture of particle board. The particle board venture failed, 

and the bales of straw were left in the field for several years. 

During this period of time, black water from the decomposing 

wheat straw accumulated in pools in the field.

Wheat straw is composed of approximately 70 percent 

cellulose and hemicellulose, 20 percent lignin, 5 percent 

silicates, and 5 percent ethanol-benzene extractable material 

(Mckean and Jacobs, 1997). The aromatic structures in the 

DOM therefore will be derived mainly from lignin. The lignin 

polymers in vascular plants are composed of varying amounts 

of p-hydroxyphenylpropanoid, guaiacylpropanoid and syringylpropanoid monomeric units (Douglas, 1996; Saake and 

others, 1996). The most common linkages formed during 

lignin polymerization are β-O-4 ether linkages (fig. 1), however, Saake and others (1996) have shown that other linkages 

such as α-O-4, β-β, β-5, and 5-5 also are present in lignins. 

Camarero and others (1994) have pointed out that especially in 

grasses the C-4 also may be attached to a hydroxyl group (C-4 

phenol). The enzymatic degradation of lignin by micro-organisms generally involves depolymerization and aromatic-ring 

cleavage (Crestini and others, 1998).

In a previous study Wershaw and others (1996) developed 

a procedure for the fractionation of leachates from senescent 

leaves. The nuclear magnetic resonance (NMR) spectra of the 

Biogeochemical Processes That Produce Dissolved 

Organic Matter From Wheat Straw

By Robert L. Wershaw1, David W. Rutherford1, Jerry A. Leenheer1, Kay R. Kennedy1, Larry G. Cox1,

and Donald R. Koci2

1

U.S. Geological Survey, Federal Center, Denver, Colorado 80225, USA

2

Equus Beds Groundwater Management District No. 2, Halstead, Kansas 

67056, USA
Lignin

A = lignin momomer:

CH O

CHOH

OR

Syringl

OR

Guaiacyl

OR

p-hydroxyphenyl

OCH3 CH3O OCH3

CH2OH

O

B

A

1

4

6 2

5 3

Figure 1. Lignin monomeric structural units.

2 Biogeochemical Processes That Produce Dissolved Organic Matter From Wheat Straw 

leachate fractions from different types of leaves were measured and compared with the spectra of the leaves from which 

they were derived. In general, the spectra of the leachate fractions can be correlated to different components in the leaves 

(Wershaw and other, 1996, 1998).

In this report the biogeochemical processes that produce 

DOM from wheat straw are investigated. The results of this 

investigation should provide a clearer picture of the incorporation of lignin-derived components into the DOM pool than has 

previously existed.

Materials and Methods

Sample collection

The black water from decomposing wheat straw was collected in Teflon bottles from pools in a field near Hutchinson, 

Kansas, where bales of wheat straw were stacked. The bottles 

were packed in ice and shipped overnight to a USGS laboratory in Denver, Colo., where they were immediately refrigerated. Wheat straw was purchased from a local feed dealer in 

the Denver area.

Dissolved organic carbon and inorganic analyses 

Dissolved organic carbon (DOC) measurements were 

made by persulfate wet oxidation in a gas-tight reaction vessel in an Oceanography International (OI) Model 700 carbon 

analyzer (Aiken, 1992). Samples were introduced into the 

reaction vessel through a fixed-volume sample loop. Prior to 

oxidation, the samples were acidified with phosphoric acid 

and purged with nitrogen to remove dissolved carbon dioxide.

Inorganic cation concentrations in the black water were 

measured by inductively coupled plasma-emission spectrometry. The black water was filtered through a 0.45-micrometer 

filter prior to analysis.

Carbohydrate analysis

The composition of the carbohydrate polymers in the 

black water DOM was determined by gas chromatographic 
Materials and Methods 3

analysis of the alditol acetates of the acid hydrolyzate of the 

DOM by V-Labs (Covington, Louisiana 70433) using the 

method described by Albersheim and others (1967). A dried, 

weighed sample was hydrolyzed in dilute trifluoroacetic acid 

followed by reduction with sodium borohydride and acetylation with acetic anhydride. The resulting alditol acetates were 

separated by gas-liquid chromatography and quantitated by 

comparison with calibration standards.

Free and bound carbohydrates in a sample were measured 

colorimetrically by the phenol-sulfuric acid test (Dubois and 

others, 1956). A weighed sample was first shaken vigorously 

with water, and an aliquot of the supernatant was reacted with 

the phenol-sulfuric acid reagent and analyzed. This yielded 

a measure of free carbohydrate in the sample. The bound 

carbohydrates were measured by reacting the suspension with 

the phenol-sulfuric acid mixture.

Fractionation

The DOM in the water was fractionated into four fractions of increasing polarity using a modification of the procedure described by Wershaw and others (1996). In this procedure the black water without pH adjustment was first pumped 

into a glass column with fluorocarbon fittings filled with 

150 milliliters (mL) of XAD-8 resin, and the column was then 

rinsed with 300 mL of deionized water. The adsorbed material 

(fraction 1) was eluted with 100 mL of a solution of 75 percent 

by volume acetonitrile in water. The eluate was evaporated to 

dryness in a rotary evaporator, the DOC was dissolved in water 

and then lyophilized. The combined black water and rinse 

water that passed through the column in the first step of the 

procedure was acidified to pH 2 with hydrochloric acid (HCl) 

and pumped through the column again followed by 300 mL 

of 0.01 molar (M) HCl. The fraction adsorbed (fraction 2) 

was eluted and lyophilized as above. The pH of the combined 

black water and rinse solution that passed through the column in the second step was adjusted to 5 with NaOH, and the 

volume was reduced to 500 mL by rotary evaporation. The pH 

was then lowered to 2, and the concentrated solution was again 

pumped onto the XAD-8 column. The fraction adsorbed (fraction 3) was eluted and lyophilized. The pH of the solution of 

residual DOC that did not adsorb on the XAD 8 was adjusted 

to 2 with 1 M HCl and pumped through a 100 mL XAD-4 

column. The column was eluted with 100 mL of 75 percent 

by volume acetonitrile in water, and the eluate was concentrated to 20 mL by rotary evaporation. The residual HCl 

and water were removed by repeated additions of anhydrous 

acetonitrile followed by evaporation. The dried sample (fraction 4) was dissolved in 50 mL of water and lyophilized. In 

all of the operations above a positive displacement pump with 

a stainless steel body, ceramic piston, and sapphire ball valves 

and fluorocarbon tubing were used. Therefore, the solutions 

only came in contact with glass, fluorocarbon, stainless steel, 

ceramic, and sapphire. The relative concentrations of the fractions obtained in one of the fractionation runs were: fraction 1, 

31 percent; fraction 2, 53 percent; fraction 3, 14 percent; and 

fraction 4, 2 percent.

The humic acid fraction (designated here as black water 

precipitate or BWP) was isolated by adjusting the pH of a 30 

mL aliquot of the black water to 1 with 1 M HCl and then 

centrifuging the resulting suspension. The supernatant was 

poured off and the precipitate was dispersed in 20 mL of water 

and re-centrifuged. The supernatant was discarded, and the 

precipitate was washed and centrifuged two more times; the 

supernatant was discarded after each washing. The pH of the 

supernatant after the last washing was between 3 and 4. The 

precipitate was re-suspended in water and lyophilized. This 

procedure yielded 0.1406 gram (g) of humic acid (84 percent 

of the DOC in the water).

Dialysis

Exhaustive dialysis of the black water was carried out by 

placing approximately 50 mL of the black water in a dialysis 

bag made of SpectraPor® 3 regenerated cellulose dialysis casing (Spectrum Laboratories, Rancho Dominguez, Cal.). Two 

different casings were used; one had a nominal 3,500 dalton 

(Da) molecular-weight cutoff and the other a cut off of 12,000 

Da (calibrated with globular proteins); both casings were 29 

millimeters (mm) in diameter. Each dialysis bag was suspended in a 1 liter (L) graduated cylinder filled with de-ionized water. The water in the cylinder was constantly stirred 

with a magnetic stirrer, and changed daily until no more color 

diffused out of the bag.

Spectral analyses

Solid-state 13C NMR spectra of the lyophilized fractions 

were measured on a Chemagnetics CMX 200-megahertz 

(MHz) proton frequency spectrometer (Wershaw and others, 

1998) equipped with a 7.5 mm Chemagnetics ceramic probe. 

The samples were packed in zirconia rotors and spun at 5000 

hertz (Hz). The acquisition parameters were contact time of 

l millisecond (ms) or 5 ms, pulse delay of 1 second (s), and 4.5 

microsecond (μs) 90o pulse.

Liquid-state 13C NMR spectra of the humic acid fraction dissolved in d6

 methyl sulfoxide (Aldrich Chemical Co., 

99.5 atom percent deuterium) were measured on a 500 MHz 

JEOL Eclipse spectrometer. The refocused insensitive nuclei 

enhanced by polarization transfer (INEPT) pulse sequence 

was used. The proton frequency was 500 MHz and the carbon 

frequency was 125 MHz. The measurements were made at 

constant temperature of 50o Centigrade (C); the following 

parameters were used: WALTZ proton decoupling with an offset of 5.0 parts per million (ppm), J constant of 140 Hz, carbon 

acquisition time of 0.521 s, 13C offset of 100 ppm. A total of 

16,384 data points were collected; the data were plotted with a 

line-broadening of 100 Hz.

Infrared (IR) spectra of the fractions were measured on 

a Perkin-Elmer 2000 Fourier transform spectrometer. Pellets 
Table 1. Elemental composition of the black water

[mg/L, milligrams per liter; μg/L, micrograms per liter]

Major Elements Minor Elements

Element Concentration 

(mg/L)

Element Concentration 

(μg/L)

Ca 210.00 Al 410.00

Fe 23.00 B 270.00

Mg 71.00 Be 0.96

Mn 5.30 Cd 2.20

Na 44.00 Co 40.00

Si (as SiO2) 120.00 Cr 10.00

Sr 1.50 Cu 81.00

Li 26.00

Mo 27.00

Ni 73.00

V 140.00

Zn 510.00

Table 2. Elemental analyses of black-water precipitate (BWP), 

wheat-straw alkali lignin (AL) and milled wood lignin (MWL)

Element BWP (percent) AL (percent)* MWL (percent)*

Carbon 51.67 64.9 58.6

Hydrogen 5.02 6.0 5.5

Oxygen 39.50 28.4 35.5

Nitrogen 1.68 0.6 0.4

Total 97.87 99.9 100.0

*Data from Pan and Sano (2000)

4 Biogeochemical Processes That Produce Dissolved Organic Matter From Wheat Straw 

for IR analysis were prepared by triturating with a mortar and 

pestle approximately 5 milligrams (mg) of sample with about 

250 mg of potassium bromide (KBr), and then pressing the 

mixture in a die. The NMR and IR spectra of the wheat straw 

were measured using the same methods as those used for the 

DOM fractions.

Ultraviolet-visible (UV-visible) spectra were obtained 

with a Spectral Instruments (Tucson, Ariz.) 400 Series 

spectrophotometer fitted with a 1 centimeter (cm) path length 

fiber-optic dip cell.

Molecular weight measurements

High-performance size-exclusion chromatography 

(HPSEC) with multi-angle laser-light scattering (MALLS) 

detection was performed at Chromaceutical Advanced Technologies, Inc., Hopkinton, Mass. A Waters Alliance 2695 

HPSEC system with a Waters 2996 photodiode array detector, 

Wyatt Optilab DSP refractive index detector, and Wyatt MiniDawn light scattering detector was used. The column was a 

Tosoh Biosep GMPWx1 column 7.8 mm in diameter and 300 

mm in length; the mobile phase was phosphate-buffered saline 

solution at pH 7.4 (0.138 molar sodium chloride, 10 millimolar phosphate, 200 ppm sodium azide). The flow rate was 

1 mL/min, and the injection volume was 100 μL. The column 

temperature was maintained at 35oC, the detector temperature 

at 40oC, and the sample temperature at 25oC. Two samples 

were subjected to HPSEC/MALLS analysis: untreated black 

water and the retentate from the exhaustive dialysis through 

the 12,000 Da membrane. The retentate was lyophilyzed after 

dialysis and then reconstituted in the phosphate-buffered saline 

solution for analysis.

From the MALLS data it is possible to calculate the number-average molecular weight (Mn

), weight-average molecular 

weight (Mw), the z-average molecular weight (Mz), and the 

root-mean-square (RMS) radius of the particles in solution 

(Wyatt, 1993). The ratio Mw/Mn provides a measure of the 

polydispersity of the particles in solution.

Biochemical Study Results

Elemental composition

Analysis of the black water (BW) yielded a DOC of 

2,900 mg/L. This DOC is very much higher than one normally finds in a natural water. The pH of the BW measured in 

the laboratory was 7.7. The elemental composition of the BW 

is given in Table 1.

Lowering the pH of the black water to pH 1 yielded 

a precipitate (humic acid) that contained 84 percent of the 

DOC in the black water. The elemental composition of the 

black-water precipitate (BWP) reported here is considerably 

different from that of alkali lignin (AL) or milled wood lignin 

(MWL) isolated from wheat straw (Table 2). The higher oxygen content and lower carbon content of the BWP compared to 

the wheat-straw AL and MWL indicate that the BWP contains 

significant carbohydrate material, as will be demonstrated 

in the following section. The higher nitrogen content of the 

BWP probably represents amino acids and proteins derived 

from the wheat straw. The concentrations of carbohydrates, 

proteins, and amino acids are apparently reduced during the 

lignin isolation procedures.

Results of carbohydrate analysis

Carbohydrate analyses of the BWP were performed. 

Phenol-sulfuric acid analysis of the water extract of BWP 

indicated that there are no free carbohydrates. When the 18 M 

sulfuric acid was added to the BWP suspension all of the 

suspended material went into solution. Colorimetric analysis 

indicated that 27 percent of BWP is insoluble carbohydrate. 

The results of specific carbohydrate analysis after hydrolysis 
0.0

230 250 310 330 350 370 390

WAVELENGTH, IN NANOMETERS (nm)

ADSORBANCE

270 290

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Figure 2. Ultraviolet-visible spectrum of black water.

Table 3. The 13C Chemical shifts of types of carbon atoms commonly found in DOM

[ppm, parts per million]

Chemical Shifts (ppm) Functional Groups

170-180 Carbonyl carbons in carboxylic acids and amides

100-160 Aromatic carbons

90-105 Anomeric carbons in carbohydrates

55-90 Aliphatic alcohols and ethers.

10-50 Aliphatic carbons

Biochemical Study Results 5

with trifluoroacetic acid and gas-liquid chromatographic analysis of the alditol acetates are as follows: rhamnose 6 percent, 

arabinose 34 percent, xylose 32 percent, mannose 8 percent, 

galactose 7 percent and glucose 14 percent. These results 

indicate that the major carbohydrate polymer present is an 

arabino-xylan, and indeed, Lawther and others (1995) pointed 

out that wheat straw hemicellulose is composed mainly of 

β-1-4 linked D-xylopyranose units with side chains composed 

of L-arabinose, D-glucuronic acid, D-glucuronic-4-O-methyl 

ether, D-galactose, and possibly D-glucose units. Calibration 

of the gas-liquid chromatographic results with standards indicated that the total carbohydrate content of the BWP sample 

is 35 percent. This result is probably more accurate than the 

27 percent result obtained colorimetrically.

Ultraviolet-visible, nuclear magnetic resonance 

and infrared spectral results

The UV-visible spectrum of the BW (figure 2) has a welldefined absorption band at 278 nanometers (nm) which has 

been shown to be characteristic of lignin (Sun and Tomkinson, 2002; Scalbert and others, 1986). Studies with model 

compounds show that a band in this region is indicative of 

nonconjugated phenolic groups (Goldschmid, 1971). Sun and 

Tomkinson (2002) and Scalbert and others (1986) also found a 

band near 320 nm in the spectra of alkali lignins isolated from 

wheat straw. They attributed this band to hydroxycinnamic 

acids conjugated to phenolic groups. Only a weak hump in 

this region is present in the spectrum of the BW indicating that 

these conjugated structures are in lower concentrations in the 

BW than in the wheat-straw alkali lignins. The reduced intensity of this band probably indicates that some of the lignin 
300 250 200 150 100 50 0 -50 -100

CHEMICAL SHIFT, IN PARTS PER MILLION (ppm)

Vertical

expansion

Wheat straw

A

Figure 3. (A) 13C NMR spectrum of wheat straw (one millisecond contact time), and (B) 13C NMR spectra of 

black-water fractions (one millisecond contact time).

6 Biogeochemical Processes That Produce Dissolved Organic Matter From Wheat Straw 

groups are attached to non-phenolic groups such as carbohydrates (see Sun and Tomkinson, 2002).

The NMR spectra of the BWP, the four major fractions of 

the wheat straw DOM, and of dry, undegraded wheat straw are 

shown in figure 3. A generalized list of the types of functional 

groups that are represented by the various regions of DOM 

spectra is given in Table 3.

The most prominent aromatic carbon bands in the wheat 

straw spectrum are at 153, 148, and 134 ppm. Hassi and others (1987) have shown that these bands are characteristic of 

guaiacylpropanoid and syringylpropanoid lignin monomeric 

units. The 153 ppm band represents the C-3 and C-5 carbon 

atoms of syringylpropanoid units in which there is an ether 

linkage at C-4. The 148 ppm band represents either the C-3 

and C-4 carbons of guaiacylpropanoid units or the C-3 and 

C-5 carbons of syringylpropanoid units in which C-4 is a phenol. The shoulder at 56 ppm probably represents methyl ether 

groups in lignin units.

Evidence of lignin structures is most apparent in the 

spectra of fractions 1, 2, 3, and BWP where the aromatic 

bands are much more prominent than they are in fraction 4. 

The 152 ppm band is the strongest aromatic band in these 

spectra. A shoulder at about 148 ppm is present in the spectra 

of fractions 2 and 3. Broad bands between about 129 and 134 

ppm also are present in the spectra of the first three fractions. 

Each of the spectra also has a broad band centered at about 

108 ppm. The 130-134 ppm bands probably represent the 

C-1 and C-4 carbons of syringylpropanoid units. These bands 

occur between 136-138 ppm if C-4 is an ether. However, if 

C-4 is a phenol then C-1 has a chemical shift of 132 ppm and 

C-4 a shift of 135 ppm, this might lead one to conclude that 

C-4 linkages in the fractions and BWP are phenolic. Unfortu-
300 250 200 150 100 50 0 -50 -100

CHEMICAL SHIFT, IN PARTS PER MILLION (ppm)

BWP

Fraction 1

Fraction 2

Fraction 3

Fraction 4

B

Figure 3. Continued.

Biochemical Study Results 7

nately, however, the strong 152 ppm band implies that C-4 is 

an ether linkage rather than a phenolic linkage. At the present 

time, this seeming contradiction cannot be explained. The 

bands centered at about 108 ppm most likely arise from C-2 

and C-6 carbons of syringylpropanoid units in which C-α is 

attached to an oxygen atom (Scalbert and others, 1986). The 

INEPT data discussed below also indicate that C-α is attached 

to an oxygen atom. The bands at about 73 ppm in the spectra 

of fractions 1, 2, 3 and BWP probably represent carbohydrate 

units conjugated to the lignin as discussed below. The 56 ppm 

band, which is the strongest band in each of the spectra, indicates that the lignin has not undergone demethylation.

Comparison of the ratios of the intensities of the 152 

ppm bands to the intensities of the 148 ppm bands (or shoulders) in the spectra of fractions 1, 2, and 3 and BWP to 

the 152 ppm:148 ppm ratio in the undegraded wheat straw 

spectrum indicates that syringyl units are in greater concentration than guaiacyl units in the lignin of the BW isolates than 

in the wheat-straw lignin. This is consistent with the findings of Almendros and others (1992) that the residual lignin 

in decaying wheat straw is enriched in guaiacyl units. They 

pointed out that syringyl lignin components are generally less 

condensed and less resistant to degradation than guaiacyl components. The syringyl components, therefore, will be more 

readily leached from the decomposing wheat straw than the 

guaiacyl components.

Carbonyl bands between 172 and 175 ppm are present in 

the NMR spectra of the wheat straw, the precipitate, and all of 

the fractions. These bands are relatively broad, and probably 

represent mixtures of aliphatic acids and esters. Fraction 1 

was isolated at the ambient pH of the black water (pH 7.7), 

and therefore it must also contain some ionized carboxylate 

groups. More definitive results are provided by the IR spectra. 

The IR spectra of the samples provide further evidence of 

the chemical structures of the samples (figure 4). The IR spectrum (not shown) of the undegraded wheat straw has a band at 

1,732 reciprocal centimeters (cm-1) that is consistent with the 

presence of aliphatic esters. A distinct carbonyl band is absent 

from the IR spectrum of fraction 1, however, a broad shoulder 

centered near 1,650 cm-1 is present. This shoulder and the 

band at 1,594 cm-1 probably represent the asymmetric stretching modes of different carboxylate groups. Fractions 2 and 3 

and BWP have well-resolved carbonyl bands at about 1,720 

cm-1 that are most likely representative of aliphatic carboxylic 
Table 4. The 13C Chemical shifts of protonated carbon atoms in hydroxyphenylpropanoid and carbohydrate units

Chemical Shifts (ppm) In Spectrum Carbon atom*

127.7 C-2, C-6 p-hydroxyphenyl

118.7 C-6 guaiasyl (with C-4 phenol)

114.5 C-5 guaiasyl (with C-4 phenol)

111.3 C-2 guaiasyl (with C-4 phenol)

106.1 C-2, C-6 syringyl (with αC-0)

104.0 C-2, C-6 syringyl (with C-4 phenol or ether) anomeric C carbohydrate

80-86.0 β-O-4; C-4 carbohydrate

71.5 HC-αOH (β-O-4 linkage)

62.3 C-5 carbohydrate (probably xylose)

59.9 HC-γOH (β-O-4 linkage)

55.7 Methyl ethers

*Data from Scalbert and other (1986) and Terashima and others (1997)

8 Biogeochemical Processes That Produce Dissolved Organic Matter From Wheat Straw 

acids. Lignin extracts of wheat straw generally have fewer 

carboxylic acids groups than the BW isolates. For example, 

the carbonyl band in the spectrum of the acid-insoluble fraction of the potassium-hydroxide extract of wheat straw (wheatstraw alkali lignin) measured by Sun and Tomkinson, (2002) is 

only present as a weak shoulder at 1,702 cm-1. Well-resolved 

bands characteristic of lignins (Faix, 1991) are in the fingerprint regions of the IR spectra of BWP and of fractions 1-3. 

The 1,127 cm-1 band indicates that the lignin contains both 

guaiacyl and syringyl units, and the shoulder at about 1,160 

cm-1 is indicative of p-hydroxyphenyl units; this shoulder was 

observed by Sun and Tomkinson (2002) in the IR spectrum 

of wheat-straw alkali lignin. The bands between 1,070 and 

1,030 cm-1 in the BWP and fractions 1-3 spectra most likely 

represent carbohydrates (Colthup and others, 1990). The relative intensities of these two bands are similar to those of the 

carbohydrate bands in wood.

The liquid-state refocused INEPT spectrum of BWP, 

which only shows protonated carbon atoms, provides further 

support for the presence of lignin and carbohydrate units (figure 5). The acquisition parameters of the INEPT experiment 

have been chosen such that all of the protonated carbon atoms 

will be seen in the spectrum; however, the response is not 

quantitative. All of the bands in the spectrum can be assigned 

to hydroxyphenylpropanoid lignin or carbohydrate units, as 

shown in table 4. The bands at 59.9, 71.5, and between 80-

86 ppm are all indicative of linkages between the β-carbon 

atoms of the propanoid side chains and the C-4 carbons of the 

aromatic rings (β-O-4) in the lignin units. No other types of 

linkages are detected in the INEPT spectrum. These results 

are consistent with the work of Terashima and others (1997) 

who found that between 65 and 74 percent of the linkages in 

wheat straw lignin are β-O-4, and that other types of linkages are in much lower concentrations. The sensitivity of the 

INEPT experiment in figure 5 apparently was inadequate to 

observe the other types of linkages. The 62.3 ppm band has a 

chemical shift very close to that attributed to the C-5 of xylose 

in hemicelluloses of barley straw lignin isolates (62.8 ppm) by 

Sun and others (2001).

Biogeochemical process implications of the 

spectral data

The IR and NMR spectra of all of the BW isolates except 

fraction 4, which comprises about 2 percent of the NOM in the 

BW, have spectral bands characteristic of lignin. The NMR 

spectrum (figure 4) and IR spectrum (not shown) of fraction 4 

indicate that it is most likely composed of a mixture of amino 

acids, sugar acids, and other aliphatic acids. This fraction 

probably represents a more complete degradation of the 

chemical components of wheat straw than the other fractions 

(Wershaw and others, 1996).

The carboxylic acid bands in the NMR and IR spectra 

of the BW isolates probably represent more than one type of 

carboxylate group. Lam and others (1992) have shown that 

cinnamic acids linked to carbohydrate and lignin groups are 

present in wheat straw. Additional carboxylate groups may 

arise from the microbial degradation of lignin. The microbial 

degradation of wheat straw lignin has been found to involve 

oxidative depolymerization, preferential removal of syringyl 

groups, and an increase in carbonyl and carboxylate groups 

(Almendros and others, 1992; Crestini and others, 1998; 

Chen and Chang, 1985; Gaines and others, 1996; Vane and 

others, 2001). Crestini and others (1998) have proposed that 

wheat-straw lignin also may undergo aromatic-ring cleavage 

during the early stages of biodegradation. Thus, the aliphatic 

carboxylic acid bands in fractions 2-4 and the BWP probably 

represent both cinnamic acids and carboxylates formed during 

aromatic-ring cleavage.

Aromatic-ring cleavage proceeds by the β–ketoadipate 

pathway. In this reaction sequence, ring cleavage takes place 

between two adjacent (ortho) hydroxyl groups on an aro-
2,000 cm-1 1,600

A - 1,594 cm-1 H - 1,225 cm-1

B - 1,506 cm-1 I - 1,127 cm-1

C - 1,464 cm-1 J - 1,085 cm-1

D - 1,463 cm-1 K - 1,035 cm-1

E - 1,419 cm-1 L - 1,034 cm-1

F - 1,328 cm-1 M - 835 cm-1

G - 1,267 cm-1

1,200 800 400

FREQUENCY IN RECIPROCAL CENTIMETERS (cm-1)

BWP

Fraction 1

Fraction 2

M

K L

J

I

G H

E

D

C

B F

A

Fraction 3

ABSORBANCE

Figure 4. Infrared spectra of black water (BW) fractions 1. 2, 3 and black water precipitate (BWP).

Biochemical Study Results 9

matic ring which are oxidized to carboxylates. The resulting muconic acid (or muconate) undergoes isomerization to 

muconolactone followed by hydrolysis and further oxidation 

to β–ketoadipate. All of the steps in this sequence are enzymatically mediated as described in detail by Gaines and others 

(1996). Further oxidation (β-oxidation) produces succinylCoA and acetyl-CoA.

Molecular weight of dissolved organic matter

The total organic carbon content (TOC) of the black 

water is 2.9 g/L which corresponds to about 5.8 g/L DOM 

assuming about 50 percent C in the DOM (see Table 2). This 

high concentration of the lignin-derived organic matter (OM) 

in the black water would initially appear to indicate this OM 

is made up of low molecular weight compounds. However, 

exhaustive dialysis through a 3,500 and 12,000 Da cutoff 

membrane indicated that 90 percent of the TOC would not 

penetrate the 3,500 Da membrane, and that 68 percent would 

not penetrate a 12,000 Da membrane.

Results of HPSEC/MALLS analysis of the DOM in the 

black water and 12,000 Da retentate are listed in table 5.

In principle, the MALLS technique, which measures scatteredlight intensity as a function of angle, provides an absolute 

measure of the sizes of the particles in solution. However, 

the calculations used to obtain the values listed in the table 

require the measurement of other optical parameters such as 
120

127.7 118.7

114.5

111.3 71.5 62.3

59.9

55.7

104.0

98.3 93.9

85.9

84.2

80.0

81.1

110 100 90

CHEMICAL SHIFT, IN PARTS PER MILLION (ppm)

130 80 70 60 50

Figure 5. 13C refocused INEPT spectrum of black water precipitate (BWP).

Table 5. HPSEC/MALLS results for BW and 12,000 Dalton samples

[Da, dalton, Mn, number average molecular weight; Mw, weight average 

molecular weight; Mz, z average molecular weight; RMS radius, root-meansquare radius; nm, nanometer]

Parameter Black Water 12 kDa Retentate

Mn (Da) 124,000.00 83,800.00

Mw (Da) 383,000.00 172,000.00

Mz (Da) 4,370,000.00 2,620,000.00

RMS radius (nm) 22.30 30.80

Polydispersity 3.10 2.05

10 Biogeochemical Processes That Produce Dissolved Organic Matter From Wheat Straw 

the refractive index increment in order to yield accurate results 

(see Wyatt, 1993, for a detailed discussion). The results in 

Table 5 indicate that both samples are polydisperse and composed mainly of relatively high molecular weight particles. 

The dialysis has reduced the polydispersity of the DOM; however, in both samples there is a substantial amount of very high 

molecular weight particles as indicated by the very high Mz 

values. The relatively low RMS radius compared to Mn and 

Mw values of the black-water DOM strongly suggest that the 

particles in solution are mainly compact spheroidal structures. 

The increase in size of the dialyzed sample probably indicates 

that the constituent molecules of the lyophilized material did 

not fold into their most compact configurations when re-dissolved. The lower Mn

, Mw, and Mz values of the 12,000 Da 

retentate compared to those of the black water probably are 

the result of differential solution with the highest molecular 

weight molecules not re-dissolving completely after freeze 

drying. Any undissolved material would be removed by the 

filtration prior to the chromatography.

Molecular weights from 324 to 17 x 106 Da have been 

reported for lignins extracted from wood (Goring, 1971). The 

results tabulated by Goring (1971) indicate that polydispersities (Mw/Mn

) as high as seven have been observed. There are 

at least two possible explanations for these results. One possibility is that the molecular weight measurements accurately 

reflect the actual molecular weight distribution of the lignin 
Biochemical Study Results 11

polymer in wood. The other possibility is that the lignin is 

present in the wood as a high-molecular weight polymer, and 

that the relatively harsh extraction procedures used to isolate 

lignin from woody tissue partially degrade the polymer, yielding fragments of widely varying size. More recently, Goring 

(1989) has proposed that most lignin molecules are relatively 

low-molecular weight, two-dimensional networks that can 

form molecular aggregates. Molecular weight measurements 

by Pan and Sano (2000) and Evtuguin and others (2001) provide support for this hypothesis. They found that the molecular weights (either Mw or Mn

) of lignin preparations isolated by 

such commonly used procedures as milling, alkaline, dioxane, 

or acetic acid extraction generally do not exceed 5,000 Da.

Pan and Sano (2000) measured the molecular weights 

of lignin isolated from wheat straw by three different methods—milling, acetic acid extraction, and alkaline extraction at 

160oC by size-exclusion chromatography (SEC). The isolates 

were acetylated prior to the chromatography; polystyrene 

molecular-weight standards were used. The Mw values of the 

acetylated lignin isolates ranged from 3,270 to 4,430 Da and 

the Mn values from 1,770 to 2,020 Da. Sun and Tompkinson (2002) measured similar molecular weights by SEC on 

alkaline-extracted wheat straw lignin.

Consideration of the way lignin forms in woody tissue provides a possible explanation for the large discrepancy 

between the measurements in this report and those of Pan and 

Sano (2000) and Sun and Tompkinson (2002). In woody terrestrial plants lignin is imbedded in the hemicellulose matrix 

of the cell walls. Lignin deposition is one of the final stages of 

xylem-cell differentiation (Donaldson, 2001). The deposition 

takes place in the interlamellar voids of the cell walls. During 

the deposition, chemical bonds form between the lignin and 

hemicellulose carbohydrates (Sun and others, 2000). Lam 

and others (1992) have shown that cinnamic acid groups in 

wheat lignin are bound to polysaccharides by ester linkages. 

Two different mechanisms have been proposed for the lignin 

polymerization. Lewis and his co-workers (see Burlat and 

others, 2001) have proposed that lignin deposition is initiated 

at specific dirigent binding sites in the cell wall. Monolignol-derived free radicals polymerize at these sites. Burlat 

and others (2001) pointed out that it has been proposed that 

“***lignification occurs through an iterative process of lignin 

assembly (following primary chain formation) in a template 

guided manner.” This process apparently favors β-O-4 linkages. Other workers, however, feel that lignin formation is 

a more “loosely ordered” process in which phenolic phenylpropanoid monomers other than p-hydroxyphenylpropanoid, 

guaiacylpropanoid, and syringylpropanoid monolignols can 

be incorporated into a lignin oligomer/polymer via undirected 

free-radical coupling (see Ralph and others, 2001). In either 

scenario the lignin oligomers ultimately link to the hemicellulose chains of the matrix.

Biogeochemical process implications of the 

molecular-weight data

The results described in this report indicate that differences in molecular weights between the lignin isolates and the 

black-water DOM samples are due to the fact that the lignin 

in the BW DOM is attached to hemicellulose chains, whereas 

the lignin isolates have been freed from the hemicellulose. 

Therefore, the low molecular weights measured by Pan and 

Sano (2000) and Sun and Tompkinson (2002) indicate that 

lignin monomeric units polymerize to form relatively small 

lignin oligomers. As the oligomers form, they bind to one or 

more hemicellulose polymeric chains to form a lignin-hemicellulose complex. Other lignin oligomers can attach to the 

same complex forming a network of interlinked lignin and 

hemicellulose units. The lignin units are bound to hemicellulose mainly by ester linkages. Wheat straw hemicellulose 

is composed mainly of β-1-4 linked D-xylopyranose units 

with side chains composed of L-arabinose, D-glucuronic acid, 

D-glucuronic-4-O-methyl ether, D-galactose, and possibly 

D-glucose units (Lawther and others, 1995).

The extraction procedures that have been developed 

to isolate lignin from woody tissue have been designed to 

produce lignin free of any associated carbohydrate material. 

For example, Guadalix and others (1997) showed that alkaline 

extraction of lignin from wheat straw causes ester cleavage. One would expect a similar type of cleavage during acid 

extraction. Thus, the 13C NMR spectra of BWP and fractions 

1, 2, and 3 all have carbohydrate strong bands in the region 

between 60 and 80 ppm (figure 2), whereas, carbohydrate 

bands are absent in this region of the spectra of milled wood 

lignin and alkali lignin.

Lawther and others (1995) measured Mw values ranging 

from 9,000 to 30,000 Da for hemicellulose fractions isolated 

by different procedures from wheat straw. The fractions with 

the higher molecular weights apparently are less degraded that 

those with lower molecular weights. These values are still 

much lower than those measured on the BW DOM samples 

reported here. The high molecular weights of the BW samples 

most likely are the result of substantial cross-linking of hemicellulose chains by lignin oligomers. In addition, some of the 

large particles detected may actually be aggregates of several 

lignin-hemicellulose complexes.

The relatively small RMS radius of the black water DOM 

indicates that the lignin-hemicellulose polymers fold into 

compact globular particles in which the nonpolar parts of the 

polymer form the interiors of the particles and the polar groups 

are on the exterior surfaces of the particles. This is similar to 

the folding of biopolymers such as proteins (Gellman, 1998). 

Cubberley and Iverson (2001) have shown that it is possible to 

synthesize flexible aromatic oligomers in which the aromatic 

rings will stack one on top of another to form compact pleated 

structures in solution. The oligomers that they synthesized 

were composed of electron-rich and electron-deficient monomeric units. In polar solvents they found that the stacking was 
12 Biogeochemical Processes That Produce Dissolved Organic Matter From Wheat Straw 

caused mainly by hydrophobic interactions, however, donoracceptor interactions also played a part.

Summary and Conclusions

Natural organic matter (NOM) has been studied for 

over 200 years. Most workers have attempted to elucidate 

the structures of chemical components of NOM by using the 

same methods that have been used to elucidate the chemical 

structures of pure compounds. These methods, however, are 

intrinsically unsuitable for this task because NOM isolates are 

complex mixtures of compounds that form molecular aggregates in solution. A more fruitful approach is to study the 

biochemical reactions that the chemical components of plant 

tissue undergo as they degrade into NOM. This type of study 

is generally unfeasible in natural systems because the NOM in 

these systems is normally derived from many different plant 

species. The results reported here are unique because the dissolved organic matter (DOM) component of NOM studied was 

derived mainly from a single species. Bales of wheat straw 

had been left in a field for several years; during this period of 

time, black water (BW) from the decomposing wheat straw 

accumulated in pools in the field.

The dissolved organic carbon content (DOC) and elemental composition of the BW were determined. Lowering the pH 

of the BW to pH 1 yielded a precipitate (BWP) that contained 

84 percent of the total DOC in the BW. The ultraviolet (UV)-

visible spectrum of the BW has a well-defined absorption 

band at 278 nanometers (nm) which has been shown to be 

characteristic of lignin.

The DOM from the BW was fractionated by polarity into 

four fractions on XAD-8 resin. The relative concentrations 

of the fractions were: fraction 1, 31 percent; fraction 2, 53 

percent; fraction 3, 14 percent; and fraction 4, 2 percent where 

fraction 1 was the least polar and fraction 4 the most polar. 

The BW DOM was also fractionated by dialysis using 3,500 

dalton (Da) and 12,000 Da membranes. The 3,500 Da membrane retained 90 percent of the total organic carbon (TOC) in 

the BW, and the 12,000 Da membrane retained 68 percent of 

the TOC.

The unfractionated DOM, the BWP, and the different 

fractions were characterized by infrared and nuclear magnetic 

resonance spectroscopy. The composition of the carbohydrates in the BWP was determined. The carbohydrate composition is typical of the composition of wheat straw hemicellulose.

The unfractionated BW and 12,000 Da retentate were 

analyzed by high-performance size-exclusion chromatography 

(HPSEC) with multi-angle laser-light scattering (MALLS) 

detection. The HPSEC/MALLS measurements indicate the 

majority of the DOM particles in the unfractionated BW have 

molecular weights on the order of 100,000 Da and root-meansquare (RMS) radii of about 22 nanometers (nm).

The results presented here indicate that the BW DOM 

is composed of hemicellulose chains cross-linked to lignin 

oligomers. These types of structures have been shown to exist 

in the hemicellulose matrix of plant cell walls. These crosslinked complexes apparently were released from partially 

degraded wheat-straw cell walls with little alteration. These 

lignin-hemicellulose polymers fold into compact globular 

particles in which the nonpolar parts of the polymer form the 

interiors of the particles and the polar groups are on the exterior surfaces of the particles. The tightly folded, compact conformation of these particles probably renders them relatively 

resistant to microbial degradation. This should be especially 

the case for the aromatic lignin structures that will be buried in 

the interiors of the particles.

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