0016.7037179/l lOI-180380?.

00/0

The characterization of plant tissues by their

lignin oxidation products

JOHN I. HEDGES and DALE C. MANN*

Department of Oceanography, WB-10 University of Washington, Seattle, WA 98195, U.S.A.

(Received I I December 1978; uccepted in revised form 12 July 1979)

Abstract-The cupric oxide oxidation products of 23 vascular and nonvascular plant tissues have been
measured. Compositional data for vanillyl, syringyl, and cinnamyl phenols are presented in the form of
five lignin parameters which are related to plant variety, lignin concentration, and tissue type. On the
basis of these parameters, the 23 plant tissue samples are resolved into distinct compositional regions
corresponding to: (a) nonvascular plants, (b) gymnosperm woods, (c) nonwoody gymnosperm tissues, (d)
angiosperm woods, and (e) nonwoody angiosperm tissues. The same five parameters also can be detet-
mined for organic materials in soils and sediments and used either to discriminate between compo-
sitionally different organic mixtures or to estimate the relative amounts of each of the above types of
plant materials in the deposits.

INTRODUCTION MENZEL, 1974; HEDGES and PARKER, 1976; HEDGES

PHOTOSYNTHETICplants are the ultimate source of
essentially all organic material and the predominant
direct contributors of biological substances to soils
and sediments. Accordingly, a major thrust of organic
geochemical research has been to relate specific com-
ponents of the organic mixtures in soils and sediments
to their plant sources (EGLINGTON and MURPHY,
1969). Although the particular compounds to be used
as source indicators must be selected on the basis of
the problem at hand, abundant, chemically stable
substances which are unambiguously characteristic
a particular plant class, tissue type, or geographic
region, generally serve best.
Lignin compounds and associated phenolic sub-
stances in vascular plants satisfy these criteria for use-
ful geochemical tracers. Lignin compounds
phenolic polymers of relatively high chemical stability
which occur as a major component of the conductive
tissues of vascular plants, but are essentially absent
from all other living organisms. Vascular plants occur
almost exclusively on land where they account for a
major portion of total biomass. In addition, lignins of
the two most abundant classes of vascular plants, the
gymnosperms (nonflowering plants including conifers)
and angiosperms (flowering plants including hard-
wood trees, herbs and grasses) are compositionally
different. As a result of these clear-cut biochemical
and geographical distribution patterns, lignin com-
pounds can be applied as indicators of specific vascu-
lar plant sources or as tracers of land-derived organic
matter in lacustrine and marine environments
(BURG= et al., 1964; LEO and BARGHOORN, 1970;
POCKLINGTONand MACGREGOR, 1973; GARDNER and
and MANN, 1979).
Lignin polymers are commonly characterized on
the basis of their nitrobenzene or cupric oxide oxi-
dation products. Cupric oxide is a more convenient
oxidizing agent for geochemical samples because
nitrobenzene can form interfering reaction by-pro-
ducts (HEDGES and PARKER, 1976). The oxidation of
lignin compounds with alkaline cupric oxide yields as
major reaction products six vanillyl and syringyl
phenols in the forms of aldehydes, ketones, and car-
boxylic acids (HEDGESand PARKER, 1976). Ferulic and
of
p-coumaric acids are also produced by the oxidation
of lignins in nonwoody vascular plant tissues (SMITH,
1955; HEDGES and PARKER, 1976). All eight of these
phenolic oxidation products are essentially unique to
vascular plants and are formed in abundance patterns
are
which reflect plant taxonomy, tissue type, and lignin
content (SARKANEN and LUDWIG, 1971). Although
these relationships are generally recognized, the study
reported here is the first presentation of these distinc-
tive compositional patterns as reflected by lignin par-
ameters which are of general geochemical application.
EXPERIMENTAL
Samples
Lignin parameters reported here are calculated from
compositional data for 12 different plant tissues published
by HEDGESand PARKER(1976) and from previously unpub-
lished results for an additional 11 plant samples analyzed
by the authors. Wood and leaf (or needle) samples were
collected in late summer from living trees growing in
Seattle, Washington. Wood samples are homogenized cross
sections of branches l-2 cm in diameter which were
stripped of their bark. All tissue samples were freeze-dried
and ground to a fine powder before analysis.

Cupric oxide oxidation and chromatography

* Present address: Ecosystems Department, Battelle
Pacific Northwest Laboratories, Richland, WA 99352, Plant tissue samples were oxidized with alkaline cupric
U.S.A. oxide (HEDGES and PARKER, 1976). Phenolic oxidation
G.C.A. 43/l,+ 1803
1804 J. I. HELIGESand D. C. MANN

products were extracted and analyzed as outlined in the lit acid analogs of these aldehydes are included here
previous reference except that high resolution gas chroma- to account for a larger percentage of the total oxi-
tographic separations were made with 30-m by 0.25-mm
dation product because plant tissues and sedimentary
i.d. glass capillary columns coated with either SE-30 or
SP-2100 liquid phases. Individual lignin parameters can be lignins sometimes yield relatively high levels of non-
determined with a precision of approximately 7t loo/, aldehgdic phenols (HEDGES and PARKER. 1976).
(HEDGES and MANN, 1979). Almost all gymnosperms produce only vanillyt
phenols when reacted with cupric oxide, whereas
RESULTS AND DISCUSSIONS angiosperms produce syringyl phenols in addition to
the vanillyl counterparts and, thus. have S/V ratios
Lignin parameters greater than zero.
A total of five lignin parameters are described here. The second lignin parameter, the ‘C over V ratio‘
Each bears an independent unit of geochemical infor- (C/V), has no known previous analog. C/V is defined
mation or has a specific interpretjve application. The as the total mg of ~coumaric and ferulic acids
first parameter, the ‘S over V ratio’ (S/V). is defined as divided by the total mg of the three vanillyl phenols
the total yield in milligrams of syringealdehyde, aceto- produced the described cupric oxide oxidation. C/V is
syringone, and syringic acid divided by the total mg useful for discrimination between woody and non-
of vanillin, acetovanillone, and vanillic acid produced woody vascular plant tissues, because only the latter
from the cupric oxide oxidation of a plant (or sedi- typically produce significant amounts of the two cin-
ment) sample. Similar ratios have been employed by namyl phenols. Cinnamyl phenols are relatively abun-
wood chemists for the characterization of plant type, dant oxidation products of nonwoody tissues.
but are usually determined from the relative yields of accounting for approximately 5 of the total pllenolic
syringealdehyde and vanilfin produced by nitroben- yield from these samples (Table 1). In general. p-cou-
zene oxidation (BRAUNS and BRAUNS, 1960; SAR- maric acid is the more abundant oxidation product
KANENand LUDWIG, 1971). The ketone and carboxy- (Table 2). However, leaves of the angiosperm A~icrrr-

Table 1. Lignin parameters for vascular and nonvascular plant tissues

Latin name Common name :~; oc s/v CY V S C

Nonvascular plants
Anacystis nidulans* (W) t&e-green alga ND u 0 0
Lactarius deliciousus* (W) mushroom ND u 0 0
Sargassum sp.* (W) brown alga ND U 0 0
mean values ND U 0 0
Gymnosperm woods
Pinus caribeae* slash pine 44 13 0 0
Pinus centora lodge pole pine 49 4.2 0 0
Pinus taeda* loblolly pine 47 9.2 0 0
Ps~udotsuga menziesii Douglas fir 47 6.1 0 0
Thuja pli~ata red cedar 4X X.5 0 0
mean values 47 X.2 0 0
Nonwoody gymnosperm tissues
Pinus centoru (L) lodge pole pine Sf 0 0.53 1.9 0 I .o
Pseudotsuga menziesii (L) Douglas fir 52 0.06 0.57 2.1 0.12 I.2
Thuja plicata (L) red cedar 52 0 0.3x 2.1 0 0.X0
mean values 52 0.02 0.49 2.0 0.04 1.0
Angiosperm woods
Acer saccharinurn* silver maple 42 2.X 0 6.S 1X 0
Afnus ruhra red alder 47 2.0 0 3.6 7.2 0
Castanea saiina Oregon oak 46 1.5 0 X.0 12 0
Ifex opaca* American holly 64 5.2 0 2.7 14 f)
Phoenix dactyiifrra* fir palm 38 1.2 0 6.X X.2 0
mean values 47 2.5 0 5.5 12 0
Nonwoody angiosperm tissues
Alnus rubra (L) red alder 50 0.44 0.54 I.6 0.7 0.X7
Avicennia germinans* (W) black mangrove 47 1.2 1.1 1.3 1.5 I .4
Canavalia maritima* (W) jackbean 44 2.0 I.1 0.7 1.4 0.74
Castanea satina (L) Oregon oak 46 1.2 0.36 1.6 2.0 0.58
Ifex opaca’ (L) American holly 60 2.6 0.44 1.1 2.9 0.4X
Spartina a~ter~i~ora* (W) cord grass 44 1.t 1.0 3.0 1.9 3. I
Zostera marina (L) eel grass ND U CJ 0 0 ND
mean values 49 1.4 0.76 1.6 1.7 1.2

* Data from HEDGES and PARKER (1976).

Abbreviations: %OC, weight percentage of organic carbon; U, mathematically undefined due to division by zero; W.
whole plant sample; L, leaf or needle sample; ND, not determined. Definitions of the five lignin parameters are given in
the text.
 The characterization
Table 2. Cinnamyl phenol compositions of nonwoody plant tissues
Latin name
Gymnosperms
Pinus centora
Ps~dotsuga menziesii
Thuja pticata
Angiosperms
Alnus rubra
Avicennia germinans*
Canavalia maritima*
Castanea satina
1 lex opaca*
Spartina alterni$ora*
Zostera Marie
Abbreviations and symbols as
nia germinans produce only p-coumaric acid whereas
the angiosperm Spartina produces slightly
alternijiora
greater amounts of feruhc acid. Ferulic acid was also
produced in small amounts from the bark of the holly
tree, llex opuca, (HEDGES and PARKER, 1976), but
further analyses are needed to determine whether tree
barks characteristically produce cinnamyl phenols.
C/V and S/V are intensive parameters which can be
obtained directly from the chromatographic data
without knowing sample size, injected amount, or the
percentage of organic carbon in the sample.
The three extensive parameters, V, S, and C, are
defined, respectively, as the total yields in mg of (a)
vanillin, acetovanillone and vanillic acid, (b) syringe-
aldehyde, acetosyringone and syringic acid and (c)
p-coumaric and ferulic acid produced from LOOmg of
organic carbon in the sample. These nonratio par-
ameters are not as easily determined as S/V and C/v,
but have the advantages that they indicate the abso-
lute phenolic yields from individual plant tissues and,
therefore, more closely reflect relative lignin concen-
trations.
Compositional patterns
The specific plants which were analyzed include a
limited number of taxonomic groups and individually
may not be important sources of sedimentary organic
material. However, the five broad categories of plant
tissues that are represented by these samples (nonvas-
cular plant tissues as well as woods and herbaceous
tissues of g~nospe~s and angiosperms) are abun-
dant, widely distributed, and likely present in many
soils and recent sediments. Lignin parameters for the
23 plant samples are given in Table 1. Lignin par-
ameter values of individual plant tissues are plotted in
four different compositional regions in Fig. 1.
The ratios, C/V and S/V, are used in a two-
dimensional compositional plot (a) in Fig. 1. The
gymnos~~ wood, nonw~dy gymnos~~ tissue,
angiosperm wood, and nonwoody angiosperm tissue
samples are resolved into nonoverlapping areas in the
plane defined by C/V and S/V. Both gymnosperm and
of plant tissues 1805
pCoumaric Ferulic
Tissue acid, acid,
type mgig sample mg/g sample
L 3.3 1.8
L 5.7 0.7
L 3.5 0.7
L 3.5 0.8
W 6.6 0.0
W 2.2 1.0
L 1.8 0.8
L 1.4 1.4
W 5.9 7.0
L ND ND
in Table 1. Sample weights are on a dry basis.
angiosperm woods have composition points along the
line C/v = 0, because p-coumaric and ferulic acids
are not produced by the cupric oxide oxidation of
these samples. All gymnosperm tissue samples except
Douglas fir needles (Sfl = 0.06) do not produce syr-
ingyl phenols and, therefore, have composition points
on the line S/V = 0. The gymnosperm woods produce
neither cinnamyl nor syringyl phenols and plot at the
origin. By comparison, the nonwoody angiosperm tis-
sues (except Z&era mnrina) produce both families of
phenols and are the only tissue category to have com-
position points separate from both axes.
Composition plots (b), (c), and (d) in Fig. 1 rep
resent the three possible combinations of V, S, and C
in two-dimensional plots. Each of the three latter
plots provides a unique compositional perspective.
Plot (b) is similar to (a) except characteristic distribu-
tion patterns of cinnamyl phenols are now reflected
by C instead of C/V, and patterns of syringyl phenols
by S rather than by S/V. With one exception, Zostera
marina, the four major categories of vascular plant
tissues are again resolved into nonoverlapping com-
positional ranges in patterns similar to (a). Using
absolute parameters, however, nonvascular plant
composition points can also be included and plot at
the origin along with compositional points for gym-
nosperm woods which also produce neither syringyl
nor cinnamyl phenols.
Plot (c), S vs V, reflects the relative abundances of
syringyl and vanillyl phenols produced from the tissue
samples. Gymnosperm tissue com~sitions fall along
the line S = 0, whereas most angiosperm composi-
tions plot in a wedge-shaped region which corre-
sponds to a S/V range of about 14. Within both
taxonomic regions, woody and nonwoody tissues are
clearly resolved owing to the higher V and S values of
the woods which correspond to their greater lignin
contents.
With the exception of the sea grass, Zosteru marina,
nonvascular and vascular plant tissues occupy separ-
ate compositional regions in plot (c). In general, the
production of measurable amounts of vanillyl phenols
1806 J. 1. HEDGESand D. C. MANN

20

15

IO
S
2 A a
A 5
A a a a
[

0
t
8
@I cl “99 0

i I I I I I I 1
0 0.4 0.8 1.2 0 I 2 3 4
=/v C

I I

WI’
a

.
G GG nn
0 G AAG GAA AGG G
O- 8 n’

I I 1
0 5 IO 15 0 5 IO
v V

Fig. I. Plots of lignin parameters for individual plant tissues in two dimensional com~sitionai regions.
Symbols: (G)--gymnosperm woods, @-nonwoody gymnosperm tissues. (A)-angiosperm woods,
(atnonwoody angiosperm tissues, (m-nonvascular plant tissues. All circled letters plot at the origin of
the corresponding region

(V > 0) is a positive test for vascular plant tissues. Eel
grass is an exception to this classification, possibly
having lost lignin in the process of adapting to a mar-
ine en~ronment (DEN HARTOG, 1970). Although this
result is an exception to taxonomic classifications
based upon phenohc distributions, it is in accordance
with the general observation that marine plants are
devoid of lignins (HEDGES and PARKER,1976).
Plot (d) in Fig. 1, C vs V, also results in separation
(excluding Zostera marina) of the vascular and non-
vascular plant samples. Woods and nonwoody tissue
samples occupy separate compositional regions, but
cannot be resolved on a taxonomic basis using only
these two parameters.
A major reason for presenting phenolic compo-
sitional patterns for individual plant tissue samples in
terms of both ratio and absolute parameters is to pro-
vide both qualitative and quantitative methods for the
analysis of lignin mixtures in soils and sediments. The
choice of the appropriate parameters for mixture
analysis depends both upon sample type and the in-
formation that is desired.
The ratio parameters, S/V and C/V, are defined
only in terms of lignin-derived phenols. Ratio par-
ameters, therefore, can be determined directly for mix-
tures of vascular plant remains in natural samples
even if large quantities of other types of organic
materials are present. The ratios can then be used
either to identify compositional relationships between
lignin mixtures in different samples or to qualitatively
determine the types of plant tissues which are present
in individual mixtures.
By comparison, the absolute parameters, V, S and
C, are defined in terms of the total organic carbon in
the sample and decrease in proportion to the extent of
dilution of vascular plant remains by nonlignified
organic matter. Consequently, under circumstances of
simple mixing of lignin-free organic material with a
 The character~ation
lignin mixture of constant composition, V, S, and C
values (or any sum thereof) for the total sample can
be used to determine the relative amount of vascular
plant material that is present.
Absolute parameters may also be used to estimate
the relative weights of specific types of vascular plant
tissues within a sample. To make these estimations it
is necessary either to isolate the vascular plant
remains physically (by sieving or flotation) or to
determine their concentration by independent means
so that phenolic yields can be determined only for
lignin-bearing materials. The resulting parameters for
a soil or sediment sample can then be plotted in
figures such as 1 (b), (c), or (d) and the relative weights
of the four major classes of vascular plant tissues esti-
mated by interpolation between mean values for cor-
responding types of plant tissue samples. Such estima-
tions involve a number of assumptions (HEDGESand
MANN, 1979), but are significantly simplified by the
preliminary result that three of the four classes of
vascular plant tissues have at least one absolute lignin
parameter which is near zero.
CONCLUSIONS
The five lignin parameters introduced here are use-
ful for the classification of plant tissues and for the
analysis of organic mixtures in soils and recent sedi-
ments. In such applications vascular plants are dis-
tinguished from nonvascular plants because only vas-
cular plants produce vanillyl phenols (V > 0) upon
oxidation with cupric oxide. Among the vascufar
plants themselves, angiosperms characteristically pro-
duce syringyl phenols (S/V and S > 0) whereas gym-
nosperms do not. Nonwoody tissues of both gymno-
sperms and angiosperms can be distinguished from
woods by their lower yields of vanillyl and syringyl
phenols and by the characteristic production of cinna-
my1 phenols (C/V and C > 0). In the following paper
(HEDGESand MANN, 1979) lignin compositional par-
ameters are used to identify compositionally dissimi-
of plant tissues 1807
lar lignin mixtures in recent marine sediments from
different depositional regions, to test for chemical
alteration of lignin compounds in sediment cores, and
to estimate the relative abundances of different classes
of plant tissues in coastal marine sediments.
Acknowled~eme~rs-Acknowledgement is made to the
donors of the Petroleum Research Fund, administered by
the ACS, for partial support of this research. This research
was also funded in part by DOE contract
AT(4S- I)-2225-T40. D. MANN received partial support
through a federally funded work study program at the Uni-
versity of Washington. We thank DR ROY CARPENTER, his
students, and research associates for tiumerous contribu-
tions. This is contribution ilO from the Department of
Oceanography, University of Washington.
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