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Applied and Environmental Microbiology, March 2000, p. 1126-1132, Vol. 66, No. 3
Department of Marine Biogeochemistry and
Toxicology, Netherlands Institute for Sea Research, 1790AB Den Burg
(Texel),1 and Department of
Microbiology, Centre for Ecological Evolutionary Studies, University of
Groningen, 9750 AA Haren,2 The Netherlands
Received 14 October 1999/Accepted 5 January 2000
Although abundant geochemical data indicate that anaerobic methane
oxidation occurs in marine sediments, the linkage to specific microorganisms remains unclear. In order to examine processes of
methane consumption and oxidation, sediment samples from mud volcanoes
at two distinct sites on the Mediterranean Ridge were collected via the
submersible Nautile. Geochemical data strongly indicate
that methane is oxidized under anaerobic conditions, and
compound-specific carbon isotope analyses indicate that this reaction
is facilitated by a consortium of archaea and bacteria. Specifically,
these methane-rich sediments contain high abundances of
methanogen-specific biomarkers that are significantly depleted in
13C ( Methane can have a stronger
greenhouse effect than CO2, and recent work has highlighted
its potential climatic impact on glacial timescales (24) and
in relation to major geologic events (8, 12). Consequently,
controls on methane production and consumption are important concerns
in the evaluation of past and future climate change. In marine
sediments, anaerobic methane oxidation could be the dominant pathway
for methane consumption (2, 4, 6, 15, 25, 26), but the
organisms involved have not been isolated and the mechanism remains
controversial. Methanogenic archaea operating in reverse (9, 11,
14) or novel, previously uncharacterized archaea (13)
have been proposed to play a vital role, but current evidence remains ambiguous.
Methane tends to be highly depleted in 13C, and organisms
that consume methane either directly or indirectly via heterotrophic consumption of methanotroph biomass will be similarly depleted in
13C. For this study, we determined distributions and carbon
isotope abundances of organic components in methane-rich mud volcano
sediments of the Eastern Mediterranean Ridge. In particular, we
examined the Samples.
Using a bordeaux core, samples were collected from
surface sediments (upper 30 cm) of several mud volcano flows (mud
breccias) in the Olimpi (Milano and Napoli mud volcanoes) and
Anaximander (Amsterdam mud volcano) fields (10, 21, 38)
using the submersible Nautile during the 1998 cruising of
the R/V Nadir. Samples were then frozen at
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Biomarker Evidence for Widespread Anaerobic Methane
Oxidation in Mediterranean Sediments by a Consortium of Methanogenic
Archaea and Bacteria
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
13C values are as low as 
95
).
Biomarkers inferred to derive from sulfate-reducing bacteria and other
heterotrophic bacteria are similarly depleted. Consistent with previous
work, such depletion can be explained by consumption of
13C-depleted methane by methanogens operating in reverse
and as part a consortium of organisms in which sulfate serves as the terminal electron acceptor. Moreover, our results indicate that this
process is widespread in Mediterranean mud volcanoes and in some
localized settings is the predominant microbiological process.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
13C values of compounds derived from
specific organisms (i.e., biomarkers) presumed to play a major role in
methane oxidation, including methanogens, aerobic methanotrophs,
sulfate-reducing bacteria, and anaerobic heterotrophic bacteria. From
these results, we evaluate the controls on and microorganisms
responsible for methane oxidation in these sediments and the conditions
under which methane oxidation occurs.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
20°C.
Sampling via submersible provided a unique opportunity to obtain
sediments from a variety of highly specific settings, including readily
identifiable methane seeps, microbial mats, and brine pools (Table
1). In addition to these apparently "active" sites, we analyzed mud breccia profiles from Milano and Napoli mud volcanoes. In contrast to previous work with similar goals,
this site-specific sampling allowed a detailed comparison of microbial
processes in diverse settings.
TABLE 1.
Evidence for methane incorporation by methanogenic
archaea in Mediterranean mud volcano sediments
Extraction and separation. Samples (8 to 25 g of sediment) were freeze-dried and extracted via sonication in a sequence of solvent mixtures with increasing dichloromethane/methanol ratios (0:1 three times, 1:1 three times, and 1:0 three times; solvent volume was ca. three times the sediment volume). For two samples (Amsterdam seep and Milano seep), 100 g of sediment was extracted for 24 h with a Soxhlet apparatus and a dichloromethane-methanol mixture (7.5:1 [vol/vol]). Elemental sulfur was removed from the total extracts by adding ca. 100 mg of activated copper and stirring the sample for 4 h.
Aliquots of total extracts (65%) were separated into acetone-soluble and insoluble fractions (16). The soluble component was further separated on an alumina column (activated for 2 h at 150°C; 3 to 30 g of alumina, depending on sample size) into apolar and polar fractions by using three times the column volume of hexane-dichloromethane (9:1 [vol/vol]) and methanol as the eluents, respectively. In the polar fraction, fatty acids were methylated by refluxing at 60°C for 5 min in BF3 (in methanol), and alcohols were converted to trimethylsilyl derivatives with 25 µl each of bis(trimethylsilyl)trifluoracetamide (BSTFA) and pyridine heated at 60°C for 30 min.Analysis of biomarkers. Apolar and polar fractions were analyzed by gas chromatography (GC) and GC-mass spectrometry (MS) for identification. GC-MS was conducted with a Hewlett-Packard 5890 gas chromatograph interfaced with a VG Autospec Ultima Q mass spectrometer operated at 70 eV with a mass range m/z of 50 to 800 and a cycle time of 1.8 s (resolution, 1,000). A fused-silica CP-Sil 5 capillary column (25 m by 0.32 mm, df = 0.12 µm) was used with helium as the carrier gas. Samples were injected at 70°C, and the temperature was programmed to increase at 20°C/min to 130°C and at 4°C/min to 320°C and held constant for 15 min. Compound identifications were based on mass spectra and retention times. The structures of sn-2- and sn-3-hydroxyarchaeol were confirmed by comparison to standards isolated from methanogens (K.-U. Hinrichs et al., unpublished data). Abundances of compounds were determined by GC with a flame ionization detector and the run conditions described above. Quantification was based on comparison of peak areas to standards [2,3-dimethyl-5-(1,1-d2-hexadecyl)thiophene] added to the sample after column chromatography.
Isotope ratio monitoring GC-MS.
Isotope ratio monitoring
GC-MS was performed on a Finnigan Delta C device and used to determine
compound-specific 13C values. The GC conditions are the
same as those used during GC-MS analyses. The 13C value
of bishomohopanoic acid was determined by measurement of a derivatized
total lipid extract. 13C values are expressed against
virtual Pee Dee Belemnite (VPDB), have been corrected for the addition
of carbon during derivatization, and have an error of less than
±1.0, unless otherwise noted (based on analytical accuracy and
precision of measurements of coinjected standards
C20 and
C24 perdeuterated n-alkanesand considering the
probable influence of coelution in some cases).
MPN counts. Most probable number (MPN) counts were performed as follows. Surface sediment samples collected via the Nautile were suspended (1:10 [vol/vol]) in basal media (i.e., growth media without substrates added) for the functional groups of interest aboard the Nadir. For anaerobic organisms, the headspace of the suspension was kept anoxic by flushing with nitrogen gas. Following suspension, the inoculations were sonicated three times at 10 s (frequency, 41.7 kHz, 100 W) to detach microorganisms from the mineral matrix. After precipitation of the sediment, the supernatant was used in dilution series (up to a dilution of 108, 3, 5, or 8 replicates [see below]). Selective growth media and specific incubation conditions were used for methanogenic archaea (medium prepared according to reference 22, final volume of 10 ml in 18-ml Hungate tubes with H2/CO2 headspace, five replicates), sulfate reducers (medium prepared according to reference 35, final volume of 10 ml in 18-ml Hungate tubes with N2 headspace, five replicates), colorless sulfur bacteria (medium prepared according to reference 35, with a final volume of 200 µl in microtiter plates with air headspace, eight replicates) and methane-oxidizing bacteria (medium prepared according to reference 37, with a final volume of 20 ml in 120-ml butyl-rubber-stoppered crimp-seal vials with air headspace, three replicates). The dilution series were incubated for at least 10 weeks at in situ temperature (14°C), after which growth was evaluated. The presence of sulfide-oxidizing (colorless sulfur) bacteria was ascertained by color changes in response to pH variations, that of sulfate-reducing bacteria was ascertained by a measured increase in sulfide concentration, that of methanogens was ascertained by an increase in the concentration of methane, and that of aerobic methanotrophs was ascertained by a decrease in the concentration of methane.
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Geochemical background. Mud volcanoes in the Mediterranean Sea appear to be caused by tectonic compression resulting in the extrusion of methane-rich sediments (mud breccias) (21, 38). The methane-rich character of extruded mud breccias has been established by high methane concentrations at depth (> ca. 1 m) in multiple cores from the Olimpi mud volcano area (10). It is unlikely that this methane is generated in the mud breccia after emplacement, because methane concentrations appear to decrease with increasing age of the mudflow.
There is considerable evidence that much of the extruded methane is oxidized by anaerobic processes. Indirect evidence for methane oxidation derives from the widespread occurrence of authigenic carbonate in mud breccia profiles and abundant 13C-depleted carbonate crusts at the sediment-water interface (many crusts have13C values below 20 [unpublished results of the
MEDINAUT investigation])presumably precipitated from inorganic
carbon-rich waters generated during methane oxidation. Second, in
multiple cores collected from Ocean Drilling Program sites 970 and 971 (10) (Milano and Napoli domes of the Olimpi field) and
during the 1999 Medineth cruise (unpublished data from the Milano,
Napoli, and Moscow domes of the Olimpi field and Amsterdam, Kula, and
Kazan domes of the Anaximander field), methane concentrations increase
with depth by as much as 4 orders of magnitude in the upper 1 m of
the mud breccias. In most cases, this appears to be associated with the
zone of sulfate reduction (7) and results in the generation
of inorganic carbon and hydrogen sulfide. Such geochemical profiles are
expected if methane is oxidized by an anaerobic consortium of
methanogens and sulfate-reducing bacteria (9, 11, 14). It is
highly unlikely that the methane is oxidized aerobically, because
oxygen did not penetrate more than 2 cm into mud volcano sediments
investigated during the MEDINAUT cruise.
Molecular biogeochemistry of active sites.
Mud volcano
sediments contain abundant biomarkers diagnostic of archaea and,
specifically, methanogens (Fig. 1). In a
sample collected directly from a seep on the Napoli mud volcano
(MN16CC1), the two most abundant compounds are diphytanylglycerol
diether (archaeol, compound I), a lipid diagnostic of archaea
(19), and sn-3-hydroxyarchaeol (compound IIa),
with lesser amounts of sn-2-hydroxyarchaeol (compound IIb).
Hydroxyarcheaol is found predominantly in methanogens (20),
particularly in the orders Methanosarcinales and
Methanococcales (3, 20, 32). Moreover, 2,6,10,15,19-pentamethylicosane (PMI; compound VI) (5) and PMI with one to five double bonds (denoted as a group by V), observed only in cultures of Methanosarcina mazei and
Methanolobus bombayensis (30), were identified
and are consistent with a predominance of methanogenic archaea in this
sample. Other abundant compounds include crocetane (compound VII)
(27) and crocetene (compound VIII), as well as
biphytanediols (31) with zero, one (compound III), or two
(compound IV) cyclopentane rings, all of which have been attributed to
the archaea. These results differ from previous investigations of
archaeal lipids in cold seeps (9, 13). This is the first
report of a co-occurrence of irregular isoprenoids such as PMI and
crocetane with archaeol and hydroxyarchaeol. It is also the first
reported occurrence of biphytanediols in such settings. In total,
unambiguous archaeal lipids comprise over 70% of the polar lipid
fraction and over 60% of the total lipid extract.
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13C values below 65, significantly lower than those
of other compounds in the same sample and profoundly depleted relative
to photoautotrophic and some bacterial biomarkers (Fig.
2). This suggests that these methanogenic
archaea assimilate rather than produce 13C-depleted
methane, a process that has been previously proposed as reverse
methanogenesis (14).
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13C values of iso- and anteiso-C15 and
-C17 fatty acids (compounds XIII and XIV), compounds
abundant in sulfate-reducing bacteria but present only in trace
quantities in most chemolithotrophic and methanotrophic bacteria
(18), are ca. 68, somewhat enriched relative to but
within the range of values represented by archaea-derived biomarkers
(Fig. 2). Low 13C values are also observed for
diplopterol (compound IX) and diploptene (compound X)bacterial
biomarkers that are not observed in sulfate-reducing bacteria, but
which are present in many chemoorganotrophs (29).
Similar carbon isotopic relationships are present in other samples
(Table 1). In a bacterial mat collected from beneath a seep-related
brine pool on Napoli dome, compounds derived from archaea and bacteria
dominate the extractable lipid fraction. Diplopterol and archaeol are
among the most abundant compounds in the polar fraction, and PMI and
crocetane are the most abundant compounds in the apolar fraction. The
13C values of methanogen (archaeol, 85; PMI,
77), inferred sulfate reducer (anteiso-C15 fatty acid,
82), and inferred chemoorganotroph (diplopterol, 53)
biomarkers are all low and comparable to those observed in the seep
sample. The same compounds are also present in significant quantities
and characterized by low 13C values in samples collected
from bulk brine pool sediments (MN16BB1), a carbonate crust on the
Napoli (MN16BT4) mud volcano, and seeps on both the Milano (MN5CC1) and
Amsterdam (MN13CC2) mud volcanoes.
MPN counts at active sites.
Table
2 shows the results of the enumeration of
functional groups of microorganisms involved in the production and
consumption of sulfide and methane in the sediments of cold seeps.
Unfortunately, due to poor core recovery, insufficient sediment was
collected to perform MPN counts on the Napoli seep sample. Also,
because surface sediment samples were the focus of these initial MPN
studies, the abundances of methanogenic archaea and sulfate-reducing
bacteria, anaerobic organisms, are probably underestimated.
Nonetheless, it appears that methanogenic archaea are present in
relatively high numbers (between 102 and 104
organisms/cm3 of sediment) at active sites on the Milano
(Olimpi region) and Amsterdam (Anaximander region) mud volcanoes.
Significantly lower numbers were obtained for one site on the Amsterdam
mud volcano and for sediments from beneath the brine pool on Napoli mud
volcano. The numbers of sulfate-reducing bacteria are in the same order of magnitude as methanogenic archaea. Of the aerobic organisms, sulfide-oxidizing bacteria were detected on the Milano and Amsterdam mud volcanoes, with maximum numbers up to 2.4 × 104
organisms/cm3 sediment, and methane-oxidizing bacteria were
not detected at any of the sites studied despite surface sediment
sampling explicitly designed to ascertain the presence of such
organisms.
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Mud breccia profiles.
We also examined a depth profile (Fig.
3) collected from a mud breccia (MN16CC2)
1 m from the Napoli seep described above, but where the methane
flux to surface sediments appears to be significantly lower. Because
mud breccias are deposited as episodic events, depth profiles of
compound distributions should not be interpreted as representing a
stratigraphic record. Rather, they likely record the response of
microorganisms to current geochemical conditions. Thus, these settings
are ideal for studying the distribution and carbon isotopic
compositions of compounds generated solely under anaerobic conditions.
In the Napoli mud breccia profile, the abundances of archaeol, PMI, and
hydroxyarchaeol increase with increasing depth. Similarly, in a mud
breccia profile developed for the Milano mud volcano, archaeol and
hydroxyarchaeol are absent in the upper 10 cm, but are present in a
sample from a depth of 24 to 27 cm. In the deepest samples at both
sites, the methanogen-derived biomarkers are depleted in
13C (Table 1), suggesting that these compounds are
generated during methane consumption. Thus, in two distinct sites,
methanotrophic activity is concentrated below the maximum depth of
oxygen penetration (<2 cm). Additionally, in the Napoli profile,
diplopterol and fatty acid concentrations also increase with depth,
providing further evidence that these compounds are related to a
consortium of prokaryotes that includes methanogens.
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13C values of archaeol and PMI decrease
significantly with depth in the Napoli mud breccia (Fig. 3). Assuming
that archaea in shallower sediments also assimilate methane, the
biomarker 13C values indicate that methane
13C values also decrease with depth. In other words,
methane 13C values increase as the distance from the
methane source (underlying sediments) increases. Because methane
oxidation results in 13C enrichment of the residual methane
pool, the observed trend is expected if extensive anaerobic methane
oxidation is occurring. The organisms responsible for anaerobic methane
oxidation have yet to be isolated and studied, and the isotope effect
of this process is unknown. Nonetheless, the isotope effect is almost certainly negative, and if we make the tenuous assumption that the
values for anaerobic and aerobic methane oxidation are similar (ca.
9) (1), approximately 95% of the methane must be
oxidized to explain the observed 30 shift. It is likely that carbon
isotope fractionation during methanotrophy is highly variable
(17), and the value of 95% should be considered only a
first-order estimate. Nonetheless, it is probably a minimum estimate,
because it ignores methane oxidation that occurred at depths greater
than 20 cm. In fact, methane oxidation at greater depths is a likely
explanation for the approximately 30 difference between archaeal
lipid 13C values in the lowermost mud breccia sample and
the nearby seep. To explain a 60 change in methane
13C values, at least 99% of the methane must be
oxidized. Although these estimates are tentative, they strongly agree
with methane profiles generated during the recent Medineth cruise. In
contrast to archaeal lipids, bishomohopanol 13C values
do not change (and its abundance decreases with depth), confirming that
this compound is derived neither directly nor indirectly from
methanotrophic activity.
Interpretation.
It is unlikely that the low 13C
values of methanogen-derived biomarkers arise during methanogenic
metabolism in these settings. Such 13C-depleted biomarker
values can arise during methylotrophic methanogenesis utilizing organic
substrates (34), but that would require extensive methane
oxidation by either bacteria or other archaea to provide substrate
carbon. (Exogenous organic carbon inputs appear to be small
[36] and are not a likely source of substrate carbon.) Multiple lines of evidence indicate that aerobic methanotrophy by
bacteria does not provide substrates for methanogenesis. Diagnostic fatty acids (23) and methylhopanoids (33, 39) of
methanotrophic bacteria were not observed, and MPN analyses of multiple
samples failed to produce any evidence for the presence of aerobic
methanotrophs. Bishomohopanol (compound XII), a nonspecific bacterial
biomarker, is present, but in a much lower concentration than archaeal
lipids, and this compound has a significantly higher 13C
value than expected for a methanotroph (Fig. 2). The 13C
value for the hopanoid diplopterol would be consistent with an aerobic
methanotroph source (Fig. 2), but in two mud breccia profiles, the
abundance of this compound is greatest below the maximum depth of
oxygen penetration, precluding an origin from aerobic bacterial
methanotrophs. Finally, the geochemical evidence strongly suggests that
methane is oxidized anaerobically. Thus, aerobic methanotrophic
bacteria are present in either low abundances or absent, are probably
not responsible for significant methane oxidation, and cannot supply
substrates for methanogenesis.
13C values, and methanogens
utilizing such carbon could likewise have low 13C
values. However, this explanation implicitly requires that some other
organism is responsible for methane oxidation to generate such depleted
inorganic carbon. Based on the above discussion, this organism is not
likely an aerobic bacterial methanotroph. However, it has been proposed
that a new order of obligately methanotrophic archaea distinct from
methanogens exists in California methane seeps (13).
Currently, there is no direct evidence that members of the newly
discovered order of archaea in the California seeps are indeed the
primary methane oxidizers at that site or that they are obligate
methanotrophs. Nonetheless, it is possible that obligately
methanotrophic archaea do exist; if such organisms are present in our
samples, they could oxidize methane and generate CO2
through an as-yet-undetermined pathway. This depleted CO2 could then be assimilated by the methanogens and result in the observed
13C depletion of methanogen biomarkers.
Alternatively, it is possible that nominally methanogenic archaea
operating in reverse (14) are responsible for the bulk of
methane oxidation in these settings. The low 13C values
of such highly diagnostic methanogenic archaeal biomarkers as
polyunsaturated PMI and hydroxyarchaeol are entirely consistent with
this explanation. They are also consistent with the MPN counts, which
clearly indicate that organisms capable of methanogenesis are present
in these settings. Moreover, these findings sustain several previous
investigations that strongly indicate that anaerobic methane oxidation
is accomplished by a consortium of prokaryotes (9, 11, 14).
Because of the abundance of methanogen biomarkers and the similarity of
methanogen and less-specific archaeal biomarker 13C
values, we believe that reverse methanogenesis is a dominant pathway
for methane oxidation in these settings. However, it is also possible
that both facultative and obligate methane-oxidizing archaea are
present in our samples, perhaps accounting for the variability in lipid
distributions and MPN counts among different sites, and this will be
discussed in future work.
Reverse methanogenesis as a reaction, whether employed by facultative
or obligate methanotrophs, probably occurs in tandem with sulfate
reduction (14). The latter reaction consumes H2 and allows methane oxidation to remain thermodynamically favorable under anoxic conditions (14). MPN analyses, very-low-sulfate pore water concentrations (7), geochemical profiles of both sulfate and HS
obtained during the Medineth
investigation, and the presence of elemental sulfur in cores from
throughout the sampling area (this work) clearly indicate that sulfate
reducers are abundant and active in these sediments. Although it is
unclear what would serve as the carbon substrate for such sulfate
reducers, carbon ultimately derived from 13C-depleted
methane is the most likely source in these settings. This provides an
explanation for the low 13C values observed for the
biomarkers for sulfate-reducing bacteria, iso- and
anteiso-C15 and -C17 fatty acids (Fig. 2).
Although some sulfate-reducing bacteria are facultatively autotrophic,
sulfate reduction would be facilitated in these sediments by a supply
of organic substrates. Chemoorganotrophic bacteria such as acetogens
could consume methanogen biomass anaerobically and play an important
role in the generation of organic substrates such as acetate or
propionate. The presence of such organisms could explain the low
13C values of diplopterol and diploptene. Hopanoid
compounds have not yet been observed in anaerobic bacteria;
nonetheless, the abundance profiles of these compounds in the mud
breccia (Fig. 3) clearly indicate that these compounds are generated at
depth and under anoxic conditions. The co-occurrence and predominance of 13C-depleted diplopterol, iso- and
anteiso-C15 and -C17 fatty acids, and
methanogen biomarkers in multiple seep samples, a brine sample, a
carbonate crust, and a mat less than 0.5 cm thick suggest a close
coupling of methanotrophy, sulfate reduction, and chemoorganotrophy. Ultimately, then, sulfate serves as the terminal electron acceptor for
methane oxidation by a complex consortium of archaea and bacteria.
In addition to the several settings investigated on the Napoli mud
volcano, similar patterns of 13C depletion in biomarkers
for methanogens and bacteria were observed in samples collected from
two sites on the Milano mud volcano, which like the Napoli volcano is
part of the Olimpi field, and one site on the Amsterdam mud volcano,
which is located nearly 500 km to the east in the Anaximander
mountains. At all three sites, depleted methanogen biomarkers are
present, and in two of the three sites, archaeal lipids are the most
abundant compounds (Table 1). Thus, anaerobic methane oxidation by a
consortium of prokaryotes is a widespread and important process in
Mediterranean mud volcano sediments, and, in sites of highly localized
methane release, methanogens appear to account for the vast majority of prokaryotic biomass.
The implications of these results are profound on both a local and
global basis. In mud volcano settings, methanogens appear to play a
significant but poorly understood role in regulating the methane flux
into Mediterranean bottom waters. It appears that a significant
portionpotentially the majorityof methane released from
Mediterranean mud volcano sediments is consumed by these organisms. If
so, a diffusive flux of methane released by increasing bottom water
temperatures (28) and subsequent clathrate dissolution could
be largely consumed anaerobically, mitigating its impact on bottom
water redox states and methane flux to the atmosphere.
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ACKNOWLEDGMENTS |
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We are indebted to the captain and crew of the R/V Nadir and the submersible Nautile. We are also grateful to the crew of the R/V Logachev and the Medineth scientific party. We are particularly grateful to S. K. Heijs, who assimilated and assisted in the interpretation of MPN counts. Two anonymous reviewers provided very useful comments on the manuscript.
We also thank The Research Council for Earth and Life Sciences (ALW) of the Netherlands Organization for Scientific Research (NWO) for their support of this work (grants NWO 750.199.01 and ALW 809.63.010).
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Marine Biogeochemistry and Toxicology, Netherlands Institute for Sea Research, P.O. Box 59, 1790AB Den Burg (Texel), The Netherlands. Phone: (31) 222 369550. Fax: (31) 222 319674. E-mail: damste{at}nioz.nl.
This is NIOZ publication number 3439.
G. Aloisi de Larderel, J. L. Charlou, G. de Lange,
J. P. Donval, A. Fiala-Medioni, J.-P. Foucher, R. Haese,
P. Henry, J. Mascle, G. Nobbe, H. Pelle, C. Pierre, M. Sibuet, and
J. M. Woodside.
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
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