Oceanogr., 39(4), 1994, 762-771
Q 1994, by the American Society of Limnology and Oceanography, Inc
Limnol.
Dissolved humic substances of vascular plant origin in a
coastal marine environment
Mary Ann Moran and Robert E. Hodson
Department
of Marine
Sciences, University
of Georgia, Athens 30602-2206
Abstract
Bacterial decomposition
of vascular plant detritus in coastal wetlands results in the conversion of
particulate organic matter to dissolved form and causes the release of humic substances into the bulk
dissolved organic carbon (DOC) pool. WC found that 34% of the DOC accumulating during degradation
of salt marsh grass (Spartina alternif7oru) from coastal wetlands of the southeastern U.S. fits the definition
of humic substances and that lignin is the primary source of the dissolved humic substances (66% of the
total). Although marine bacterioplankton
used the lignin-rich humic substances more slowly and less
efficiently than other components of the DOC pool, a significant fraction (24%) of these substances was
mineralized within 7 weeks. Concentrations
of vascular plant biomarkers (lignin phenols) indicate that
1 l-75% of the dissolved humic substances on the southeastern U.S. continental shelf is from vascular
plant-dominated
environments.
Calculations indicate that about half this material is contributed by the
coastal salt marshes and half by river export. Vascular plant influence was lower in the bulk DOC pool
(2-38%), indicating that terrestrially derived material is harbored preferentially
in the humic substances
subcomponent of marine DOC.
Solubilization
of particulate organic matter
by bacteria has recently been recognized as an
important feature of carbon cycling in marine
ecosystems and a quantitatively
important
route by which carbon is transferred from the
particulate to the dissolved phase (Moran and
Hodson 1989; Smith et al. 1992). This process
appears to be especially significant in coastal
salt marshes of the southeastern U.S., where
as much as 40% of the carbon in Spartina alterniflora detritus is converted to stable dissolved intermediates during bacterial decomposition (Moran and Hodson 1989). The
conversion of vascular plant detritus to dissolved organic matter (DOM) is significant
from an ecological standpoint because it provides plant-derived
carbon to populations of
salt marsh bacteria other than those attached
to the particulate detritus, including free-living
bacterioplankton
(Moran and Hodson 1989).
Furthermore, plant-derived
DOM is susceptible to export from the coastal salt marshes
to adjacent marine environments,
whereas
Acknowledgments
Edward Sheppard provided technical assistance in the
lab and field. WC appreciate the detailed reviews of this
manuscript by John Ertel, Rick Keil, and John Hedges.
This research was funded by grants NA80AA-DO0091
from the NOAA Office of Sea Grant and OCE 9 1- 16450
from the National Science Foundation.
plant material in particulate form generally is
not (Chalmers et al. 1985; Williams et al. 1992).
The ecological role of vascular plant-derived
DOM, both within coastal marshes and in marine ecosystems to which it has been exported,
will depend on its relative biological lability
among other factors. We know from previous
studies that rates of utilization
of DOM derived from Spartina detritus are highly variable: some components are rapidly assimilated
by bacteria over time scales of hours to days,
whereas other components have much longer
turnover times (Moran and Hodson 1989). The
refractory components are of particular interest because they appear to be enriched in lignin
byproducts and are potential precursors of humic substances (Moran and Hodson 1990b).
The refractory components are also the components most likely to persist long enough to
be available for export. Indeed, the export of
humified, lignin-rich
DOM to marine environments has been demonstrated by the identification of lignin biomarkers in the humic
substances of several coastal and oceanic systems (Meyers-Schulte and Hedges 1986; Moran et al. 199 la,b).
Although the rates of turnover and the ecological roles of chemically identifiable,
low
molecular weight components of marine DOM
have been studied for nearly three decades
(Wright and Hobbie 1966; Azam and Hodson
1977), little is known about the fate of refrac762
Coastal humic substances
tory, higher molecular weight dissolved organits, especially marine humic substances. Radiocarbon
dating
indicates
that humic
substances constitute the oldest component of
marine dissolved organic carbon (DOC) (Bauer
et al. 1992), which suggests a largely geochemical, rather than ecological, role for these compounds in oceanic environments.
Yet recent
studies of humic substances in freshwater environments demonstrate that some humic material is turning over on relatively short time
scales and that the turnover is driven by bacterial activity (Moran and Hodson 1990a;
Tranvik 1990; Tulonen et al. 1992). Thus, further study of the formation and turnover of
dissolved humic substances in marine ecosystems is warranted.
In this study, we begin to assemble a more
complete picture of the ecological role of vascular plant-derived humic substances in coastal marine environments.
We document the
microbially
mediated formation of dissolved
humic substances from S. alternijlora detritus
in coastal salt marshes of the southeastern U.S.,
measure rates of turnover of this material by
natural marine bacterioplankton,
and estimate
the contribution
of these coastal marshes to
the dissolved humic substances pool on the
continental shelf.
Methods
Radiolabeling S. alterniflora
lignocellulose-S. alterniflora plants were collected intact from a salt marsh on Sapelo Island, Georgia, and maintained in pots under natural light.
Plants were uniformly
radiolabeled by periodically, over a period of several months, placing Mylar bags over the plants and allowing
14C02 inside the bags to be incorporated into
plant material. Two weeks after the final addition of r4C02, aboveground portions of plants
were harvested by cutting culms at sediment
level. Additional S. alterniflora plants were labeled specifically in the lignin fraction of the
lignocellulose by incubating live cuttings in
sterile aqueous solutions
containing
[U14C]cinnamic acid, which had been synthesized enzymatically from commercially available [U-14C]phenylalanine.
The cinnamic acid,
an intermediate in the pathway of lignin biosynthesis, was incorporated primarily into the
lignin fraction of the plant material. After la-
763
beling, both types of plant material were dried
at 50°C and ground into small particles (<425
pm in size) in a Wiley mill. Unincorporated
radiolabel and labeled nonstructural
components were removed from the plant material
by serial extraction in boiling ethanol, ethanolbenzene [ 1 : 2 (vol : vol)], and water (Benner et
al. 1984) resulting in either uniformly labeled
lignocellulose
([U-14C]lignocellulosc;
lignin,
cellulose, and hemiccllulose all labeled at the
same specific activity) or lignin-labeled
lignocellulosc ([ 14C-lignin]lignocellulosc;
lignin
fraction labeled, cellulose and hemicellulosc
unlabclcd). These lignoccllulose preparations
are good models for naturally occurring Spartina detritus because lignoccllulose accounts
for the bulk (> 95%) of the remaining material
after natural plant senescence in the salt marsh.
Chemical fractionation and radioanalysis of
the 14C-labeled lignocellulose
(according to
methods of Benner et al. 1984) indicated that
the label in the [U-14C]lignocellulose
was distributed proportionately
among the major
chemical fractions
and that the [14C-ligninllignocellulose
was substantially free of radiolabeled contaminants. Specific activities of
the radiolabeled
lignocellulose
were determined by cornbusting subsamples at 900°C in
a Biological Oxidizer (Harvey Instruments) and
radioassaying evolved r4C02 after trapping in
ethanolamine-amended
scintillation
cocktail.
Specific activity of the lignin fraction of [14Cligninllignocellulose
was calculated assuming
that lignin accounts for 10% of the carbon in
S. alterniflora lignocellulose (Hodson et al.
1984).
Humic substance formation-Spartina
lignocellulose (2-2.5 g) was placed in acid-washed
l-liter flasks with 700 ml of half-strength artificial seawater (Sigma Sea Salts, 20 g liter-l)
prepared with autoclaved,
distilled
water.
Three milliliters
of a marine microbial inoculum were added to each flask. The inoculum
was obtained by concentrating
microorganisms from 1 liter of salt marsh water into a 20ml volume over 0.2~pm pore-size Nuclepore
filters. Inorganic nutrients were added at concentrations typical of marsh water: 10 PM N
(as ammonia and nitrate) and 1 PM P. Three
preparations of Spartina lignocellulose
were
incubated (two replicates each): [U-14C]lignocellulose, [ 14C-lignin]lignocellulose,
and unlabeled lignoccllulosc.
764
Moran and flodson
Flasks were incubated with constant stirring
and aeration to prevent accumulation of plant
detritus at the bottom of the flasks and to ensure aerobic conditions throughout the experiment. Elevation of flasks 1.5 cm above the
surface of the stir plates minimized effects of
the heat generated by continuous stirring. Aeration was provided by individual
aquarium
pumps equipped with in-line disposable filter
units (Gelman Acrodisc, 0.2~pm pore-size)
which were replaced weekly.
After 6 weeks, contents of the flasks were
harvested by sequential filtration
through
Whatman GFK glass-fiber filters and 0.2-pm
pore-size Nuclepore filters. For humic substance isolation and quantification,
filtrate was
acidified to pH 2 and pumped through 2.2- x
40-cm columns containing Amberlite XAD-8
resin. The nonhumic compounds (those passing through the XAD-8 resin without adhering) were collected in the column effluent. The
humic compounds (those concentrated on the
resin) were eluted in 0.1 N NaOH and then
passed through an Amberlite AGMP-50 cation exchange resin to remove sodium ions.
DOC concentrations in the humic and nonhumic fractions were determined by direct injection into a high-temperature
combustion
carbon analyzer (Shimadzu TOC-500). Radiolabel in these fractions was determined by assaying 1-ml subsamples added to 10 ml of scintillation fluid after the pH of each was adjusted
to neutral with concentrated NaOH or HCl.
Recovery of radiolabel after the humic substance isolation procedure averaged 8 1%; about
half of the unrecovered label (9% of the total)
was found to remain on the XAD-8 resin. The
humic and nonhumic fractions were stored
frozen until further use.
Humic substance utilization -Turnover
of
dissolved humic substances derived from
Spartina lignocellulose was measured in microcosms containing DOC, consisting of one
labeled fraction-either
humic or nonhumic
(formed during the decomposition
of [U14C]lignocellulose)-and
one unlabeled fraction (formed during the decomposition of unlabeled lignocellulose). This approach avoided
potential artifacts associated with the use of
physically isolated DOC components during
measurements of utilization
and allowed for
the possibility of bacterial cometabolism of the
more refractory compounds. The final DOC
mixture contained humic and nonhumic sub-
stances at their original concentrations. Six 125ml bottles were established with the humic
fraction containing the radiolabel, and six bottles were established with the nonhumic fraction containing the label. Final volume per
microcosm was 25 ml, and final DOC concentration (labeled plus unlabeled) was 6 mg C
liter-l. Three bottles of each set were inoculated with 1 ml of a salt marsh microbial inoculum prepared as described above; the other
three served as sterile controls. Bottles were
shaken gently (to minimize
aggregation of
[ 14C]DOC) at 100 rpm in the dark at room
temperature.
Experiments were terminated after 7 weeks.
A 5-ml aliquot was removed from each microcosm and stored at 4°C in 2% buffered
formaldehyde for later bacterial counts. The
remainder was passed through a 0.22~pm poresize Millipore filter. The filter was washed with
filter-sterilized
seawater to remove unincorporated [14C]DOC, placed in 10 ml of scintillation cocktail, and assayed to determine radioactivity
incorporated
into particulate
material (representing incorporation
into microbial biomass after correction for sterile controls). A l-ml aliquot of the filtrate was acidified to pH 2.5 to remove inorganic C and then
radioassayed to determine unincorporated
[ 14C]DOC. The remaining filtrate (N 20 ml) was
acidified to pH 2.0 and pumped through
XAD-8 columns (0.8 by 12 cm). Column effluent (nonhumic compounds) and column eluant from an NaOH wash (humic compounds)
were collected, concentrated by vortex evaporation to 1-ml volumes, dried onto glass-fiber
filters, and radioassayed by combustion and
trapping of 14C02 as described above. 14Cbudgets were determined and microbial mineralization was calculated by difference.
In these utilization
experiments we were
concerned about the possibility that the biological availability
of the DOC might be altered during the humic isolation procedure, for
example due to pH manipulations
or the loss
of a fraction of the DOC on the XAD-8 resin.
However, we have demonstrated previously
that the sum of the bacterial growth on humic
and nonhumic
fractions obtained by this
method equals the total growth in unfractionated DOC (Moran and Hodson 1990a), suggesting that alterations, if any, were minor.
Bacterial counts were performed on formaldehyde-preserved
samples filtered onto pre-
Coastal humic substances
blackened 0.2~pm pore-size Nuclepore filters
and stained with 0.0 1% acridinc orange for 3
min. Numbers of bacterial cells in inoculated
bottles averaged 1 X 1O7 ml-l; numbers of
cells in uninoculated
controls were indistinguishable from those on background filters
prepared with filter-sterilized
distilled water
instead of sample.
Lignin phenol analysis - Seawater samples
(38 liters) were collected at l-2-m depths on
the continental shelf of the southeastern U.S.
(between 30-32”N and 80-81OW) during a
cruise on the RV Blue Fin in June 1990. Water
was immediately passed through ashed, 293mm-diameter
Gelman A/E glass-fiber filters
( 1.6-pm approximate pore size) and acidified
to pH 2 with 6 N HCl. Subsamples of water
were removed and frozen for later determination of DOC concentration with a Shimadzu
TOC-500 carbon analyzer.
Humic substances were isolated by pumping
seawater (50 ml min-l) through a 2.2- x 40cm column of Ambcrlite XAD-8 resin, eluting
with 0.1 N NaOH, and passing the eluant
through a BioRad AG-MP50 cation exchange
resin (Moran et al. 199 la). Humic materials
were concentrated
by rotary evaporation,
freeze-dried, and stored frozen until oxidation.
Carbon content of humic substances was determined on a Perkin-Elmer
240C CHN analyzer.
Lignin phenols were quantified by gas chromatographic analysis of cupric oxide oxidation
products of humic substances (Ertel et al. 1984).
The oxidation procedure involved reacting 530 mg of freeze-dried humic substances with
alkaline cupric oxide for 3 h at 170°C. Three
lignin phenols (vanillic acid, vanillin, and acetovanillon) were quantified on an HP 5890 gas
chromatograph with an Alltech SE-30 and a
J&W DB- 170 1 silica capillary column, and lignin phenol concentrations
were normalized
based on vanillic acid concentrations (Moran
et al. 199 la). Ratios of the concentrations of
the three phenols to one another (acid : aldehyde; ketone : aldehyde; ketone : acid) were
consistent among the samples. Reproducibility of oxidation and GC analysis was 11+4%
(Moran et al. 199 la).
Results
Formation of humic DOC- Measurement
of radiolabel in dissolved organic compounds
after 6 weeks of S. alternijlora decomposition
765
indicated that 1.2 & 0.1% (n = 2) of the carbon
from particulate plant detritus and 3.6 + 0.6%
(n = 2) of the carbon specifically from the lignin fraction of the detritus had accumulated
in soluble form. Based on our previous measurements of the ratio of DOC accumulation
to particulate plant detritus loss (Moran and
Hodson 1990b), we estimate that 25% of the
bulk lignocellulose was degraded during this
time period, while 18% of the lignin component was degraded.
Water from the microcosms containing the
newly formed radiolabeled DOC was passed
through an XAD-8 resin to determine the percent of labeled compounds fitting the operational definition of humic substances. An average of 34.4% of the solubilized radiolabel
was associated with the humic fraction (Fig.
1). Percent humic C was also determined based
on analysis of DOC concentrations in the eluants from the XAD-8 resin; 37.4+4.8% (n =
4) of the DOC was humic, in good agreement
with the radioactivity-based
measurement.
From measurements of the radioactivity in the
accumulated DOC and the specific activity of
the uniformly
labeled plant lignocellulose
(68,889 dpm mg-l), we calculated that 11.7
mg of DOC accumulated per gram of initial
Spartina detritus during 6 weeks of decomposition, of which 4.0 mg g- 1were humic substances.
The importance of lignin as a source material for humic DOC was suggested by the
high percentage of lignin-derived
DOC (73%)
which fit the operational definition of humic
substances (Fig. 1). Based on the specific activity of the lignin fraction of the lignin-labeled
lignocellulose (55,420 dpm mg-l), we calculated that lignin was the source of 2.6 mg humic substances g-l Spartina detritus, or 65%
of the accumulated humic material (Fig. 1).
Lignin was the source of only 12% of the nonhumic DOC.
Utilization of humic DOC-Microbial
uptake of Spartina-derived DOC during a 7-week
period was calculated as percent label incorporated into microbial biomass (particulate
material >0.2 pm in diameter, corrected for
sterile controls) plus percent label mineralized
(Table 1). A total of 24.4% of the humic substances fraction was used, with 0.3% incorporated into microbial biomass and 24.1%
mineralized (1.2 + 0.3% overall assimilation efficiency). A total of 39.2% of the nonhumic
766
Moran and IIodson
Nonhumic
lignin-derived
polysaccharide-
#derived
Nonhu mic
POlYS#acchat *ide-de
Fig. 1. Classification
and source material for dissolved organic matter accumulating during decomposition
of
Spartina alterniftora from a coastal salt marsh. Values indicate the percentage of total DOC represented by each fraction.
DOC was used, with 1.6% incorporated into
biomass and 37.3% mineralized (4.3&0.7%
assimilation
efficiency). These percentages
should be considered minimum
estimates,
however, since some lignocellulose degradation products would also have been taken up
during the initial 6-week formation period,
prior to the start of the utilization experiments.
[14C]DOC remaining after the 7 weeks of
microbial processing was analyzed to determine the distribution of radiolabel between the
humic and nonhumic fractions. While - 1520% of the label in either fraction was associated with the alternate fraction after incubation, this same percent interconversion
was
also found in sterile controls (Table 2). For
DOC refractionated
on an XAD-8 column
without an incubation, the percent interconversion was also similar, suggesting that the
shifting of radiolabel between fractions may
be the result of slight differences in the conditions of chromatography
with the XAD-8
resin or a consequence of storage. However,
the assignment of vascular plant-derived DOC
into humic or nonhumic categories is apparcntly not subject to change, due to rapid biologically mediated interconversions.
Humic substances on the continental shelfConcentrations
of humic carbon in surface
seawater of the southeastern U.S. continental
shelf ranged from 0.07 to 0.45 mg C liter-l
(Table 3). In the nearshore region of the shelf,
humic substances were present in higher concentrations than on the outer shelf (Fig. 2A)
and accounted for a greater percentage of the
total DOC (19 vs. 12%; Fig. 2B). A region of
seawater with humic C concentrations lower
than typically found (shaded portion of Fig.
2A) was present on the midshelf. Temperature
and salinity profiles in the region suggested an
area of upwelled water of oceanic origin (L. R.
Pomeroy pers. comm.). Lignin phenols ranged
in concentrations from 0.23 to 4.2 pg liter-’
in surface water of the shelf (Fig. 2C) and accounted for O. l-0.9% of the humic C.
The lignin phenols on the shelf likely orig-
Table 1. 14Cbudgets for microbial utilization of DOC formed during decomposition of Spartina alterniflora detritus.
Percent mineralization
is calculated by difference (100 - percent recovered) for inoculated incubations. y1= 3 + 1 SD.
After 7-weeks incubation
Labeled
DOC fraction
Humic
Nonhumic
Treatment
Sterile
Inoculated
Sterile
Inoculated
dpm added as
[ 14C]DOC
8,225
8,225
13,925
13,925
Filter (dpm)
213+12
236-t5*
252k 18
476*66-f
Filtrale (dpm)
8,172+283
6,007 k690-f
13,044+ 129
8,263+ 1,212t
Percent recovered
101.9k3.3
75.9k8.3
95.5kl.O
62.7k8.3
* Measured counts in inoculated microcosms were significantly different from counts in sterile microcosms at P < 0.05 (Mann-Whitney test).
t Measured counts in inoculated microcosms were significantly different from counts in sterile microcosms at P < 0.0 1 (Mann-Whitney test).
Percent
mineralized
24.1
37.3
Coastal humic substances
Table 2. Distribution
of radiolabel between humic and
nonhumic fractions of Spartina-derived
DOC following a
7-week incubation with marine microorganisms.
Labeled
DOC fraction
Humic
Nonhumic
Final distribution of label
Treatment
No incubation
Sterile
Inoculated
No incubation
Sterile
Inoculated
% humic
% nonhumic
82.6k3.0
83.6-t-0.8
82.2k0.9
13.9t- 1.6
18.2+ 1.8
22.5k3.9
17.4k3.0
16.4kO.8
17.8kO.9
86.2+ 1.6
81.8k1.8
77.5k3.9
inate in the salt marshes along the Georgia and
South Carolina coastlines (280,000 ha in area;
Hopkinson and Hoffman 1984) and in the river systems that drain the Piedmont and coastal
plain (19,500,OOO ha of drainage area; Alberts
et al. 1990). The relative contributions of these
sources to the total exported DOC is not presently known, but a preliminary
estimate can
be made based on measured DOC flux from
the major rivers in the southeastern U.S. (Hopkinson and Hoffman 1984; Alberts et al. 1990)
and area-based DOC export from South Carolina and Georgia marshes (Hopkinson and
Hoffman 1984; Chalmers et al. 1985; Williams
et al. 1992). About 1,196 x lo3 t of DOC is
estimated to bc delivcrcd to the southeastern
767
U.S. shelf annually (Table 4); - 55% originates
in the coastal salt marshes and 45% is derived
from river export. If we assume that humic C
makes up 50% of the riverine DOC and 25%
of the salt marsh DOC, 433 x lo3 t of humic
C is estimated to be delivered to the shelf (Table 4); -38% originates in the coastal salt
marshes and 62% is derived from river export.
Lignin phenol data were used in a simple
mixing model to estimate terrestrial-intertidal
contribution to bulk DOC and humic C in shelf
seawater. The terrestrial-intertidal
end member (source material) used in the model was
estimated as a 1.O : 0.8 1 mixture of marsh and
river DOC, based on relative DOC contributions calculated in Table 4 and lignin phenol
concentrations shown in Table 3. Lignin phenols accounted for 0.55% of the DOC and
1.23% of the humic C in this composite terrestrial-intertidal
end member. The marine end
member was assumed to have no measurable
lignin phenol signal. For bulk DOC, the percent terrestrial-intertidal
contribution was calculated as
%DOC,,, = (%LIGsh,lfDoc X 1OO)/“~LIGterr,oc.
%DOC,,, is the percentage of bulk DOC in
shelf samples that is terrestrially-intertidally
Table 3. Concentrations
of DOC (mg liter-‘),
humic C (mg liter-l),
and vanillyl lignin phenols (pg liter-‘)
and
ratio of vanillic acid to vanillin in humic substances isolated from continental shelf water samples. The terrestrialintertidal component of the DOC and the humic substances subfraction are estimated from river (n = 2) and marsh
(n = 4) end members as described in the text. (Not determined-ND.)
Terrestrially-intertidally
W)
Sta.
1
la
2
4
6
7
8
9
10
14
15
17
18
19
21
24
River*
Marsh*
* From Moran et al. 1991~.
DOC
2.46
2.32
2.25
1.63
1.78
1.44
1.28
1.46
1.33
2.06
3.55
1.41
1.76
1.28
1.16
1.93
9.9kO.l
5.9rto.5
Humic C
Lignin
0.31
0.38
0.29
0.19
0.18
0.14
0.18
ND
0.07
0.45
0.42
0.19
0.28
0.29
0.14
0.39
5.3kO.l
1.5kO.2
2.40
2.93
1.84
1.09
0.39
0.71
0.80
0.53
0.23
3.96
2.69
1.07
3.11
4.22
0.67
2.16
157+ 13
17&3
Ad:Al V
1.05
0.88
0.74
1.51
0.65
0.67
0.74
1.38
1.42
0.75
0.80
0.99
0.71
1.29
0.72
1.49
0.77-to.03
0.88kO.13
derived
Total DOC
Humic DOC
11
14
9
8
3
6
40
39
32
29
11
26
23
4
2
22
9
9
20
38
13
17
45
33
29
57
75
25
28
Moran and Hodson
768
A
Savanilall
- ‘.Vo
_ 0.3
Bruns
Savannah
Fig. 2.
A-Humic
C concentrations
(mg liter-l);
B-humic
C expressed as a percent of DOC, C-lignin
phenol
concentrations(pg liter-‘); D-percent terrestrially derived DOC in watersof the southeasternU.S. continental shelf.
Stations consideredto be nearshore(*) and outer shelf(t) are indicated. Area of upwelled ocean water is shaded.
derived, %LIGshelfDOCis the percent lignin phenols in DOC from continental shelf samples,
and ohLIG,,,Doc is the percent lignin phenols
in DOC from terrestrial-intertidal
sources. For
humic C, the terrestrial-intertidal
contribution
was calculated as
OhHUMIC,,,
= (%LIGshelM x 1OO)/%LTG,,,H.
%HUMIC,,,
is the percentage of the humic
substances subcomponent of shelf DOC that
is terrestrially-intertidally
derived, %LIGshelm
is the percent lignin phenols in humic sub-
stances isolated from continental shelf samples, and %LIGterH is the percent lignin phenols
in humic substances isolated from terrestrialintertidal sources. Calculations based on these
models indicate that 2-38% of the total DOC
in continental shelf seawater is of marsh or
riverine origin (Table 3 and Fig. 2D). By contrast, 1 l-75% of the humic C in shelf seawater
is marsh or river derived (Table 3). In every
sample, terrestrial-intertidal
influence was calculated to be higher in the humic C fraction
than in the bulk DOC by a factor of 2-8.
769
Coastal humic substances
Discussion
The quantitative
importance
of bacterial
solubilization
in the formation of DOM in
aquatic environments has been demonstrated
(Moran and Hodson 1989, 1990b; Smith et al.
1992). However, little is known about the
chemical composition of this DOM or the ecological role it plays in aquatic ecosystems. In
previous studies of coastal marshes of the
southeastern U.S., we found evidence that the
DOM formed as a result of Spartina detritus
decomposition
was rich in lignin byproducts
and humic substances (Moran and Hodson
1989, 1990b). Uncertainty about the trophodynamic role of humic substances in marine
environments prompted us to concentrate our
attention on this chemical subcomponent of
vascular plant-derived DOM.
Formation of plant-derived dissolved humic
substances-Dissolved
humic substances are
defined in this study as those compounds which
adhere to an XAD-8 resin under acidic (pH 2)
conditions and back elute in base. This operational definition, based on the hydrophobicity of humic compounds, is the most widely
accepted method for identifying and isolating
aquatic humic substances (Thurman 1985). A
significant fraction (34%) of the dissolved material released from degrading salt marsh grass
fits this definition, whether measurements are
based on radioactivity
isolated by XAD-8
methodology or on DOC concentrations
in
resin eluants. Thus, vascular plant detritus degrading in coastal marshes can be an important
source of humic substances. Furthermore, these
operationally defined humic substances form
quite rapidly (within a few weeks of the initiation of decomposition) and therefore do not
necessarily require extended biological and
physical processing for their formation. In previous studies, Filip and Alberts (1988) documented the biologically mediated accumulation of humic substances from S. alterniflora
detritus after 10 months of decomposition, and
Fustec et al. (1989) demonstrated the formation of humic substances from the lignin fraction of plant detritus in alluvial soils after 7
weeks. Humic substances also have been extracted from partially degraded tissues of several other vascular plant species (Ertel and
Hedges 1985), and they have been found in
the culture medium of lignin-degrading
bacterial cultures growing on lignocellulose (McCarthy et al. 1986).
Table 4. Inputs of terrestrial-intertidal
DOC and humic C from Georgia and South Carolina rivers and salt
marshes to the southeastern U.S. continental shelf.
Humic C input
DOC input
(10’ t yr-‘)
Source
Rivers
Marshes
536* (45%)
660$ (55%)
268t (62%)
165§ (38%)
* Average of values calculated by Hopkinson and Hoffman 1984 (626 x 1O3
t yr-r) and Alberts et al. 1990 (445 X lo3 t yr-I).
t Assuming riverine DOC to be 50% humic C (Thurman 1985; Alberts et al.
1990; Moran et al. 199la).
$ Estimated from Hopkinson and Hoffman 1984, Chalmers et al. 1985, and
Williams et al. 1992, assuming a 7,000-m-wide zone of salt marsh along a
400,000-m coastline (Hopkinson and Hoffman 1984). Range = 36 l-9 18 x
10’ t.
4 Assuming salt marsh DOC to be 25% humic C (Moran et al. 199 1a).
Turnover of vascular plant-derived humic
substances-The ecological role of vascular
plant-derived humic substances in marine ecosystems will depend in large part on how rapidly and efficiently the humic substances are
assimilated by marine bacterioplankton.
Natural humic substances generally are considered
to be among the most refractory components
of the bulk DOC pool, although recent evidence from freshwater environments suggests
that humic substances can support significant
levels of bacterial secondary production (Moran and Hodson 1990a; Tranvik 1990; Tulonen et al. 1992). In our experiments, the nonhumic components of DOC were assimilated
more readily and more efficiently by natural
marine bacteria than were the humic components, although 24% of the humic substance
fraction was nonetheless used in a 7-week period. These plant-derived humic materials of
recent age (3 weeks average age) are apparently
more labile than dissolved humic substances
which have accumulated in natural environments, as only a few percent (< 4) of the humic
substances from a South Carolina reservoir or
a Georgia backwater swamp were readily assimilable by bacteria over similar time intervals (Moran and Hodson 1990a). Thus, significant heterogeneity of compounds included
in the humic substances definition is evident.
The compounds accumulating in natural environments are likely to be those that are most
resistant to biological attack (i.e. those having
the longest turnover times), yet relatively labile
compounds with ecologically relevant turnover times are also encompassed by this broad
chemical classification.
The differentially
radiolabeled plant materials used in these studies allowed us to deter-
_
770
Moran and Hodson
mine the relative importance of lignin and
nonlignin
components
of lignocellulose
as
source materials for humic substances. Lignin
is a highly aromatic, chemically heterogeneous
polymer that accounts for 10% or less of the
lignocellulose
in S. alterniflora (Moran and
Hodson 1990a) and other species of grasses,
yet it was the source material for 30% of the
DOC and 65% of the dissolved humic substances accumulating
from degrading Spartina. The vascular plant polysaccharides (cellulose and hemicellulose)
were presumably
used more rapidly by bacteria; although readily lost from the plant detritus during microbial
degradation, a smaller proportion of polysaccharides accumulated
in dissolved
form.
Moreover, the polysaccharide-derived
compounds that did accumulate were much less
likely to be humic matter. Lignin therefore
makes a disproportionate
contribution
to the
stable DOC accumulating from vascular plant
detritus in coastal marshes.
Terrestrial-intertidal contribution to humic
substances in a marine environment-The accumulation of vascular plant-derived DOC in
coastal marshes suggests a potential role, either
ecological or geochemical, for this material in
adjacent marine environments.
In this study
and a previous one (Moran et al. 199 1a), we
found that a measurable fraction of the DOC
in seawater on the southeastern U.S. shelf was
of terrestrial-intertidal
origin. On the basis of
calculations of export from southeastern U.S.
rivers and marshes (Table 4), about half the
terrestrial DOC present on the shelf originates
in the coastal salt marshes and half comes from
river export.
Terrestrial influence is greater in the humic
substances subcomponent
of shelf seawater
than in the bulk DOC (Table 3). This greater
terrestrial component in humic substances results from the mixing of humic-rich DOC in
river or coastal marsh water (-50 and 25%
humic substances, respectively) with the humic-poor DOC in ocean water (- 5-l 0% humic material). That is, bulk DOC in terrestrialintertidal source waters is diluted to a greater
extent by bulk marine DOC than humic substances in source waters are diluted by marine
humic substances. Biological considerations
also argue for greater terrestrial influence on
marine humic substances than on bulk DOC
because natural bacterial communities apparently assimilate the nonhumic components of
vascular plant-derived DOC more rapidly than
the humic component (Table 1) and because
there is no rapid biological interconversion between these two pools (Table 2). Biological
processing can therefore be expected to deplete
nonhumic components of terrestrial-intertidal
DOC relative to the humic component. The
simple mixing models we used to calculate terrestrial influence on shelf DOC do not include
considerations of biologically mediated changes
in DOC composition (i.e. conservative behavior of terrestrial material is assumed). If significant biological processing does occur and
if it involves discrimination
against the humic
substances fraction, the disparity in terrestrial
influence between bulk DOC and the humic
subcomponent would be greater than that estimated by the model.
Quantification
of the terrestrial component
of marine DOC and measurement of the turnover time of terrestrial material in the marine
environment are topics of special interest for
reconciling existing discrepancies in the global
carbon budget (Williams and Druffel 198 7;
Hedges 1992). Meyers-Schulte
and Hedges
(1986) used lignin phenol concentrations
in
marine humic substances to estimate that DOC
in open-ocean water is - 1% terrestrially derived. Because our results indicate greater terrestrial influence on humic substances than on
bulk DOC in marine environments,
there is
unlikely to be a significant but previously undiscovered pool of terrestrially derived material in the nonhumic component of marine
DOC which would greatly alter this estimate.
As expected, terrestrial-intertidal
contributions to the marine DOC pool are higher in
coastal ocean environments than in the open
ocean; we estimate that 2-38% (this study) and
3-36% (Moran et al. 199 la) of DOC in seawater on the southeastern U.S. continental shelf
is of terrestrial-intertidal
origin. The present
study also suggests that intertidal
environments can be an important source of DOC,
and of humic C in particular, to the ocean, yet
many current global carbon budgets consider
only the riverine export of terrestrial DOC
(Meybeck 1982; Mantoura and Woodward
1983). DOC entering marine environments
from intertidal sources may be dissimilar in
Coastal humic substances
composition, 14C age, or biological lability to
riverine DOC, and these factors may influence
its ecological and geochemical role in the ocean.
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Submitted: 22 March 1993
Accepted: 27 October 1993
Amended: 18 November 1993