FEMS Microbiology Ecology 54 (2005) 47–56
www.fems-microbiology.org
Characterization of a deep-sea microbial mat from an active cold
seep at the Milano mud volcano in the Eastern Mediterranean Sea
Sander K. Heijs
a,*
, Jaap S. Sinninghe Damsté b, Larry J. Forney
a,1
a
b
Department of Microbiology, Center for Ecological and Evolutionary Studies, University of Groningen, 9750 AA Haren, The Netherlands
Royal Netherlands Institute for Sea Research (NIOZ), Department of Marine Biogeochemistry and Toxicology, Den Burg, The Netherlands
Received 14 October 2004; received in revised form 6 February 2005; accepted 17 February 2005
First published online 10 March 2005
Abstract
A white, filamentous microbial mat at the Milano mud volcano in the Eastern Mediterranean Sea was sampled during the Medinaut cruise of the R/V Nadir in 1998. The composition of the mat community was characterized using a combination of phylogenetic and lipid biomarker methods. The mat sample was filtered through 0.2 and 5-lm filters to coarsely separate unicellular and
filamentous bacteria. Analyses of 16S rRNA gene sequences amplified from the total community DNA from these fractions showed
that similar archaeal populations were present in both fractions. However, the bacterial populations in the fractions differed from
one another, and were more diverse than the archaeal ones. Lipid analysis showed that bacteria were the dominant members of the
mat microbial community and the relatively low d13C carbon isotope values of bulk bacterial lipids suggested the occurrence of
methane- and sulfide-based chemo(auto)trophy. Consistent with this, the bacterial populations in the fractions were related to
Alpha-, Gamma- and Epsilonproteobacteria, most of which were chemoautotrophic bacteria that utilize hydrogen sulfide (or reduced
sulfur compounds) and/or methane. The most common archaeal 16S rRNA gene sequences were related to those of previously identified Archaea capable of anaerobic methane oxidation. Although the filamentous organisms observed in the mat were not conclusively identified, our results indicated that the Eastern Mediterranean deep-sea microbial mat community might be sustained on a
combination of methane- and sulfide-driven chemotrophy.
2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Deep-sea microbial mat; Chemotrophy; Sulfide; Methane; Diversity
1. Introduction
Previous studies have shown the presence of dense filamentous microbial mats in various marine environments [1–5]. The populations in these microbial mat
communities are sustained through chemotrophy, in
which energy is derived from the oxidation of chemical
compounds by microorganisms, as light is not available
*
Corresponding author. Fax: +31 50 363 2154.
E-mail address: [email protected] (S.K. Heijs).
1
Current address: Department of Biological Sciences, University of
Idaho, Moscow, ID 83844-3051, Idaho, USA.
as an energy source. The microorganisms present in
these chemotrophy-based ecosystems are usually sulfideor sulfur-oxidizing, methane-oxidizing and heterotrophic
prokaryotes [6–9]. Some members of these communities
have previously been characterized using culturedependent and light- and electron-microscopy methods,
and the presence of the filamentous sulfur-oxidizing bacteria Beggiatoa sp., Thioploca sp., Leucothrix, Thiotrix
and Desmanthos has been confirmed [2,10–12]. Other
studies performed, using culture-independent techniques, have shown that microbial mat communities in
vent or cold seep environments can be quite diverse
and include prokaryotes such as Epsilonproteobacteria
0168-6496/$22.00 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsec.2005.02.007
48
S.K. Heijs et al. / FEMS Microbiology Ecology 54 (2005) 47–56
[13,14] or even Archaea [15], that are responsible for the
filamentous structure of the mats. These organisms are
thought to participate in either sulfide- or methanebased chemoautotrophy. Most recently, Mills and coworkers investigated the microbial communities in
mat-like structures in cold-seep environments in the
Gulf of Mexico [16]. This investigation showed a microbial mat community, including bacteria that could play
a role in sulfide-based aerobic chemotrophy as well as
Archaea, which might play a role in anaerobic methane
oxidation.
In the present study, seven mud volcanoes (at 1600–
2000 m depth) in the eastern Mediterranean Sea were
explored with the Nautile submersible during the
‘‘MEDINAUT’’ cruise of the R/V Nadir in 1998. Their
central parts actively seeped methane to the bottom
waters and were covered by thick carbonate crust pavements. Microbial aggregates were visually discernible
from the Nautile. White, filamentous material, tentatively assumed to contain Beggiatoa aggregates, was collected at the Milano mud volcano [5].
The aim of the current study was to identify the dominant members of the microbial community in these
mats, using 16S ribosomal RNA (rRNA) gene-based
methods and lipid biomarker analysis in combination
with compound-specific carbon isotope measurements.
The integrated use of these techniques enabled us to
determine which microorganisms form the dominant
members of the mat community, and indicate whether
the community was based on sulfide- and/or methanedriven chemoautotrophy.
2. Materials and methods
2.1. Site description and sampling
Mat filaments were collected using a titanium vacuum bottle (Fig. 1: photograph showing the filaments
being sampled with the titanium bottle), operated from
the submersible Nautile and support vessel R/V Nadir.
Samples were taken from the summit of the Milano
mud volcano at 3344.0 0 N, 2446.7 0 E at a water depth
of 1958 m in the Olympi field. Upon return of the submersible to the support vessel, filamentous mat material
was transferred to two sterile 10-ml Greiner tubes,
sealed, immediately frozen at 80 C and kept frozen
until analysis.
2.2. DNA isolation
For the isolation of DNA, the mat samples were
thawed on ice and mixed well, after which 10 ml was
transferred from each tube to a 50-ml sterile Greiner
tube. The tube containing the mat material was subjected to mild sonication for 3 min at 100 W in a sonication bath (Branson B1540) filled with water and melting
ice. This sonication was repeated twice. Subsequently,
the samples were separated into two fractions, by subsequent filtration through 5 and 0.2-lm pore size filters.
We assumed that the 5-lm fraction consisted of larger
microorganisms (possibly filaments), whereas the 0.2lm fraction contained smaller prokaryotes, including
free-living cells and filament-associated microorganisms
Fig. 1. Sampling of the deep-sea microbial mat with a vacuum titanium bottle from the Nautile submersible at the Milano mud volcano in the
Eastern Mediterranean Sea. Insert shows a light microscopy image of the microbial community at 400· magnification. The scale bar indicates a
length of 5 lm.
S.K. Heijs et al. / FEMS Microbiology Ecology 54 (2005) 47–56
that were dislodged during sonication. The two fractions
obtained by filtration were designated Milano WF1 5l
and Milano WF2 0.2l.
Total DNA was isolated using the Wizard Genomic
DNA Isolation Kit (Promega Benelux B.V., Leiden,
The Netherlands), following a procedure that was
slightly modified from that recommended by the manufacturer. The filter for each of the two filter fractions was
cut into small pieces using a sterile scalpel and placed in
2-ml Eppendorf tubes. To each tube, 355 ll of 0.1 M
Tris/50 mM EDTA (pH 9.0), 40 ll of lysozyme solution
(20 mg ml1) and 15 ll of 20% sodium dodecyl sulfate
(SDS) was added. The tubes were incubated for 1 h at
37 C, shaken at 5 min intervals, and subsequently centrifuged at 16,000g for two min, after which the supernatant was collected for each filter fraction and placed on
ice. To the remaining pellet, 600 ll of sterile Milli-Q
water was added, followed by incubation at 80 C for
5 min. After centrifugation at 16,000g for 2 min, the
supernatants were recovered, pooled with the previous
supernatant for each filter fraction and placed on ice.
After this, DNA was extracted from the pellets and
supernatants from each filter fraction, using the manufacturerÕs protocol. The extracted DNA was precipitated
with 0.8 volume of iso-propanol, collected by centrifugation, and washed with ice-cold 70% ethanol. After centrifugation, the pellet was air-dried, suspended in
100 ll of 10 mM Tris (pH 8.0), and rehydrated overnight at 4 C. As a final step, the DNA solution was
cleaned with a WIZARD DNA cleanup kit (Promega
Benelux B.V., Leiden, The Netherlands) and concentrated to a volume of 50 ll. The presence of of high
molecular weight DNA was confirmed by gel electrophoresis and the concentration of DNA was measured
spectrophotometrically.
2.3. Amplification of 16S rRNA genes, cloning and
sequencing
The 16S rRNA genes were amplified using primers
specific for bacterial and archaeal 16S ribosomal RNA
genes. Eubacterial 16S ribosomal RNA genes were
amplified using the B8F (5 0 -AGAGTTTGATCMTGGCTCAG-3 0 ) forward primer [17] and the
universal U1406R (5 0 -ACGGGCGGTGTGTRC-3 0 ) reverse primer [18]. Archaeal 16S ribosomal RNA genes
were amplified with the A2F (5 0 -TTCCGGTTGATCCYGCCGGA-3 0 ) forward primer [19] in combination
with the universal U1406R primer. For PCR, 1 ll of
DNA template was used in 25-ll reactions that contained 10.2 mM Tris, 2.3 mM MgCl2, 50 mM KCl, 2%
DMSO, 5 lg BSA, 0.2 mM of each dNTP, 0.2 lM of
each primer and 0.5 U Taq DNA polymerase. Samples
were amplified in a Perkin–Elmer GeneAmp PCR System 9700 (Perkin–Elmer Applied Biosystems Netherlands, Nieuwerkerk a/d IJsel, The Netherlands) using
49
the following program: 95 C for 5 min; 35 cycles of
94 C for 1 min, 57.5 C for 30 s, 72 C for 4 min, with
a final elongation step of 72 C for 7 min.
PCR products were purified using QIA quick spin columns (Invitrogen BV, Groningen, The Netherlands) and
were cloned in the pGEM-T easy vector system (Promega)
using Escherichia coli JM109. Cloned inserts were
amplified by PCR using the pGEM-T specific primers
T7 (5 0 -TAATACGACTCACTATAGGG-3 0 ) and SP6
(5 0 -GATTTAGGTGACACTATAG-3 0 ). PCR mixtures
were as described above, with the following PCR conditions: 94 C for 5 min; 30 cycles of 94 C for 1 min, 48 C
for 30 s, 72 C for 4 min, with a final elongation step of 72
C for 7 min. From each library, clones with inserts were
selected and partial 16S rRNA gene sequences were
determined using an ABI PRISM 3100 Genetic Analyzer
(Perkin–Elmer Applied Biosystems, USA). Either the
forward PCR primer or T7 vector primer was used in
addition to a U515 (5 0 -GTGCCAGCMGCCGCGG-3 0 )
forward primer with a Taq DyeDeoxy terminator
sequencing kit (Applied Biosystems).
2.4. Sequence analysis
Partial sequences were manually edited in Chromas
1.45 (www.technelysium.com.au) and contig assemblies
were done in Bioedit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html), using the built-in algorithm according to Huang (1992) [20]. This approach resulted in
bacterial and archaeal 16S rRNA gene sequences ranging from 800 base pairs (bp) to 1300 bp.
Chimeric sequences were detected by using the chimera detection utility at the Ribosomal Database
Project homepage (http://rdp.cme.msu.edu/html/analyses.html). Nearest relatives in Genbank were identified
using the basic local alignment search tool (BLAST)
from the NCBI website (http://www.ncbi.nlm.nih.gov/
blast). Selected sequences and their close relatives were
aligned using the fast aligner utility of the ARB software
package [21]. Alignments were checked manually using
the secondary structure of the 16S rRNA gene. 16S
rRNA gene sequences showing more than 97% similarity with each other were considered to belong to the
same phylotype. In addition, sequences were divided
into phylogenetic groups that were consistent with taxonomically related phyla and orders. These groups were
assigned by determining the taxonomic class of the nearest GenBank relative. Sequences that could not be
linked to previously identified bacterial or archaeal taxonomic classes were listed as unclassified.
Evolutionary distances were calculated according to
the Kimura two-parameter correction method [22], after
which neighbor joining trees were constructed with 1000
bootstrap samplings using Treecon-W [23]. The Shannon–Weaver indices of diversity were calculated for all
samples on the basis of the phylotype distribution using
50
S.K. Heijs et al. / FEMS Microbiology Ecology 54 (2005) 47–56
the PAST program (http://folk.uio.no/ohammer/past/).
Similarities between the two filter fractions on the basis
of phylotypes and phylogenetic groups were determined
using the Morisita–Horn index of similarity [24].
16S rRNA gene sequences determined in this study
were deposited in Genbank under Accession Nos.
AY592799–AY592933.
2.5. Lipid analysis
Filamentous mat material (not sonicated or filtered)
was used for the analysis of lipids. A pellet of the filamentous mat material was prepared by centrifugation
at 16,000g for 30 min. Lipids were ultrasonically extracted from the pellet using methanol, methanol/dichloromethane (1:1, v/v) and dichloromethane. An aliquot
of the total lipid extract was methylated with diazomethane in diethyl ether, and filtered over a small SiO2 column with ethyl acetate as the eluent. Alcohols in the
eluate were converted to their trimethylsilyl ether derivatives with bis(trimethylsilyl)trifluoracetamide (BSTFA)
in pyridine. Individual compounds were analyzed by gas
chromatography (GC) and GC–mass spectrometry
(MS). Another aliquot of the extract was used for analysis of glycerol dialkyl glycerol tetraethers (GDGTs) by
high performance liquid chromatography/atmospheric
pressure chemical ionization mass spectrometry
(HPLC–APCI-MS) as described elsewhere [25].
GC–MS was performed with a HP 5890 gas chromatograph (Hewlett–Packard, Palo Alto, CA, USA),
equipped with a CP-Sil5 capillary column (25 m,
0.32 mm i.d., 0.12 lm film thickness) (Chrompack, Middelburg, The Netherlands) coupled to a VG Autospec
Ultima Q mass spectrometer (VG, Manchester, UK).
The column temperature was programmed from 70 C
to 130 C at a rate of 20 C/min and then to 320 C
(15 min isothermal) at 4 C/min. Isotope-ratio-monitoring gas chromatography–mass spectrometry (irmGC–MS) was performed using a Finnigan Delta-plus
XL-irm-GC–MS system (Finnigan, Bremen, Germany),
equipped with an on-column injector and fitted with a
25-m · 0.32 mm fused silica capillary column coated
with a 0.12-lm film of CP-Sil5 (Chrompack, Middelburg, The Netherlands). Helium was used as the carrier
gas and the oven was programmed as described for the
GC analyses. Isotopic values were calculated by integrating the m/z 44, 45 and 46 ion currents of the peaks
produced by combustion of the chromatographically
separated compounds and those of CO2-spikes produced by admitting CO2 with a known 13C content at
regular intervals into the mass spectrometer. Duplicate
analyses were carried out and the results were averaged
to obtain a mean value. Reported d13C values are expressed against the Vienna Pee Dee Belemnite standard
(VPDB), after correction for the addition of carbon during derivatization, and have an error of less than ±1.0&.
3. Results and discussion
To investigate the microbial community that constituted the white filamentous mat at the Milano mud volcano, the dominant morphotypes were studied with light
microscopy, whereas the composition of the microbial
community present was characterized using phylogenetic methods. Light microscopy revealed a microbial
community that consisted of large filaments, as well as
various coccoid- and rod-shaped organisms (see Fig. 1,
insert). These results indicated that the microbial community in the mat was more diverse than expected when
the mat was sampled. Although the majority of the cells
appeared intact, freeze-thawing of the mat material
diminished the quality of the sample in such a way that
it was not suitable for scanning or transmission electron
microscopy. As a result, the details of bacterial cell morphology could not be determined.
3.1. Diversity and similarity
The diversity of the Milano mud volcano microbial
mat community was studied by cloning and sequencing
of 16S rRNA genes. Two filter fractions were obtained
by filtration through 5 lm and a 0.2-lm pore size filters
to distinguish between larger (filament- or particle-associated) and smaller prokaryotes (free-living microorganisms or prokaryotes liberated by sonication). Taking
into account that freeze-thawing of the mat material
may have introduced a bias in the relative abundances
of the sequences obtained, the results of this study do
not necessarily represent the in situ microbial community structure. However, given the amount of intact (filamentous) cells visible during microscopic examination,
we assume that our approach identified the most abundant prokaryotes and the filaments, and distinguished
filaments from smaller prokaryotes. From each filter
fraction, about 45 bacterial 16S rRNA gene sequences
were determined. In total, these 90 clones represented
52 distinct bacterial phylotypes and comprised nine bacterial phylogenetic clades (Table 1). A total of 31 and 14
archaeal 16S rRNA sequences were obtained for Milano
WF1 5l and Milano WF2 0.2l, respectively. These represented eight archaeal phylotypes within three phylogenetic clades (Table 1). Rarefaction analysis of the
distribution of phylotypes yielded asymptotic accumulation curves (data not shown), which indicated that the
clone libraries represented the most abundant microbial
populations in the microbial mat.
The 5-lm and 0.2-lm filter fractions contained distinctive bacterial phylotypes. Only four out of the 52
bacterial phylotypes were common to Milano WF1 5l
and Milano WF2 0.2l. In contrast, most archaeal phylotypes were found in both samples (Table 1). This
apparent difference in the distribution of the microbial
populations of Milano WF1 5l and Milano WF2 0.2l
S.K. Heijs et al. / FEMS Microbiology Ecology 54 (2005) 47–56
51
Table 1
Phylogeny and distribution of populations found in a deep-sea microbial mat at the Milano mud volcano (Eastern Mediterranean)
Phylotypes#,+
Domain Phylogenetic Group
Archaea
,,
,,
Bacteria
,,
,,
,,
,,
,,
,,
,,
,,
#
+
a,b,c,d,e,f
1
Milano WF1 5 µm
Milano WF2 0.2 µm
Novel Group
N.R.
2
A-14
2A-12(3) a, 2A-15(9)b
Novel Methanosarcinales
1A-01(14)a, 1A-14(12 )b, 1A-15(2), 1A-26, 1A-28, 1A-07
Thermoplasmales related
N.R.
2A-10
Total number of sequences
31
14
Shannon-Weaver diversity index
1.24
1.03
1
Morisity-Horn similarity value
0.85 (0.98)
Acidobacteria
1B-06
N.R.
N.R.
Actinobacteria
1B-23
c
2B-15, 2B-23c
Alphaproteobacteria
1B-22, 1B-24(2), 1B-28, 1B-33, 1B-41 , 1B-38
N.R.
CFB (Cytophaga-Flavobacterium)
1B-07, 1B-10, 1B-15, 1B-19
N.R.
Chloroflexi
1B-16, 1B-17, 1B-48
2B-11(3), 2B-40(2), 2B-48(5)d
Deltaproteobacteria
1B-09d 1B-18, 1B-39, 1B-40, 1B-43
2B-10(6)e 2B-14, 2B-16, 2B-24(2), 2B-30(6),
Epsilonproteobacteria
1B-36(3)e
2B-41, 2B-46(2), 2B-47
2B-04, 2B-08, 2B-13(2)f, 2B-20, 2B-25, 2B-29(4), 2B-42
Gammaproteobacteria
1B-03, 1B-05, 1B-20, 1B-21, 1B-25, 1B-27, 1B-32,
f
1B-42, 1B-44(5) , 1B-45(3), 1B-46, 1B-47(3)
OP-11
N.R.
2B-09, 2B-32, 2B-37
Total number of sequences
44
46
Shannon-Weaver diversity index
3.45
2.83
Morisity-Horn similarity value1
0.29 (0.63)
= Novel phylotypes with <97% sequence similarity to known 16S rDNA sequences are shown.
= Numbers in bold font within parentheses are the number of clones belonging to each phylotype.
= Clones in the same phylotype, but in different size fractions of the sample are designated by similar underlining and superscript symbol.
= Number shows Morisity-Horn similarity value at phylotype-level. Similarity values at phylogenetic group level are shown between brackets.
was quantified by Morisita–Horn indices of similarity.
The values showed that there was low to moderate similarity at the phylotype and phylogenetic group levels for
the bacterial populations in the two fractions. For the
archaeal populations these similarity values were relatively high (Table 1). The Shannon–Weaver diversity
values were calculated to determine the level of diversity
in the microbial mat community; these values were highest for bacterial phylotypes and relatively low for archaeal phylotypes (Table 1). Comparing both filter
fractions, the overall diversity was highest in the 5-lm
filter fraction (WF1, Table 1). These data show that
the bacterial community of the Milano filamentous
mat was more diverse than the archaeal one, with clear
differences in the composition of the bacterial populations between filter fractions Milano WF1 5l and Milano WF2 0.2l (Table 1).
3.2. Microbial mat community analysis and lipid
composition
The relatedness of the most abundant sequences in
the microbial mat from the Milano mud volcano and
previously reported sequences is presented in Figs. 2
and 3. Neither the archaeal nor the bacterial 16S rRNA
gene sequences showed high resemblance to sequences
from cultivated species, which indicated that the main
fraction of the prokaryotic community consisted of novel organisms.
The archaeal 16S rRNA gene sequences were all related to those of members of the Euryarchaeota (Fig.
2) and no sequences related to members of the
Crenarchaeota were detected. Most archaeal 16S rRNA
sequences from Milano WF2 0.2l and all sequences
from Milano WF1 5l were distantly related (i.e.,
<90% sequence similarity) to those of cultured members
of the Methanosarcinales, a phylum known to encompass methanogens. The majority of these Methanosarcinales-related sequences formed two distinct clades that
were closely (>96% sequence similarity) affiliated with
the ANME-2AB and ANME2-C sequences. These sequences have been linked to the anaerobic oxidation
of methane (AOM) in previous studies [26–28]. Therefore, our results suggest that the Archaea in the microbial mat could play a role in the oxidation of methane.
However, this tentative conclusion could not be fully
substantiated by the results of the lipid analyses. The
only archaeal lipid identified was GDGT without cyclopentane rings (i.e., GDGT-0). This lipid was found in
relatively low amounts compared to bacterial lipids,
indicating that Archaea were not abundant members
of the microbial community. GDGT-0 is a membrane lipid that is only known to occur in quite disparate groups
of Archaea; hyperthermophilic and mesophilic members
of the Crenarchaeota, methanogens and euryarchaeota
capable of AOM [29–34]. In view of the high numbers
of archaeal 16S rRNA gene sequences from this study
that were phylogenetically related to (euryarchaeal)
ANME-2 sequences (Fig. 2), it would be plausible to
attribute GDGT-0 to members of the Euryarchaeota
capable of AOM. However, in other studies, the distribution of GDGTs in samples from sites with AOM is always characterized by an approximately equal
abundance of three GDGTs: GDGT-0 and GDGTs
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S.K. Heijs et al. / FEMS Microbiology Ecology 54 (2005) 47–56
0.05 substitutions/site
M59138 Methanosarcina mazei
AF419638 Unc. archaeon C1R019
AF354133 Unc. archaeon Eel 36a2A5
Methanosarcinales
Sulfolobus acidocaldarius
ANME-1
100
Thermoplasmales
related
Novel
D14876
100
ANME-2AB
98
AF419650 Unc. archaeon AT_R007 Guaymas Basin (AOM sediments)
Milano WF2 0.2µ A 12 (3 sequences)
99100 Milano WF1 5µ A 01 (14 sequences)
91
Milano WF1 5µ A 26
Milano WF1 5µ A 28
91
AF354128 Unc. archaeon Eel 36a2A4
Milano
WF1 5µ A 15 (2 sequences)
61
AF354134 Unc. archaeon SB 24a1F10
99 62
100 Milano WF2 0.2µ A 15 (9 sequences)
61
Milano WF1 5µ A 14 (12 sequences)
Milano WF1 5µ A 07
72
100
AF356643 Unc. archaeon G72_C12 H2S related
M59141 Methanosaeta thermoacetophila
71
AF412943 Unc. euryarchaeote CH4 seep Black Sea
100
AF134386 Unc. archaeon TA1a
X03407 Halobacterium halobium
AF339746 Thermoplasma volcanium
62
Milano WF2 0.2µ A 10
99
100
AY053471 Unc. archaeon AT425 ArD10 gashydrate
AF095265 Methanobacterium uliginosum
AF419629 Unc. archaeon a2b037 Guaymas Basin (AOM sediments)
93
99
97
AY341269 Unc. archaeon ZAR100 anoxic sulfur spring
100
Milano WF2 0.2µ A 14
AF083072 Cenarchaeum symbiosum (marine group I)
AF419653 Unc. archaeon AT_R021 Guaymas Basin (AOM sediments)
X99559 Pyrodictium abyssi
AF191225 Acidolobus aceticus
74
ANME-2C
52
Fig. 2. Phylogenetic tree of archaeal 16S rRNA sequences retrieved from a deep-sea microbial mat at the summit of the Milano mud volcano in the
Eastern Mediterranean. The tree was constructed using sequences longer than 800 bp and neighbor-joining analysis using 1000 bootstrap replicates
was used to infer the topology. Phylogenetic groups detected are indicated in brackets. The bar represents 5% sequence divergence.
containing one or two cyclopentane rings [30–34]. Recently, Blumenberg and co-authors have linked GDGTs
to the ANME-1 group, whereas sn-2-hydroxyarchaeol
and crocetane were most abundant in ANME-2 dominated carbonate reefs [35]. Thus, the GDGT distribution
in the microbial mat from the Milano mud volcano differs substantially from the pattern expected for Archaea
involved in AOM. One way to determine if the GDGT-0
in the mat originated from Archaea capable of AOM
would be to determine the carbon isotopic composition
of the biphytane skeletons of GDGT-0. If the GDGT-0
originated from Archaea involved in AOM, a low d13C
value would be expected (cf. [30,31,34]). Unfortunately,
the amount of material available was not sufficient to
perform this assay. Therefore, direct evidence for the
involvement of the ANME-2 related sequences in
AOM is lacking.
The majority of the bacterial 16S rRNA gene sequences from Milano WF1 5l and Milano WF2 0.2l
were affiliated with Alpha-, Gamma-, Delta- and Epsilonproteobacteria (Fig. 3). The most abundant deltaproteobacterial phylotypes in the mat were found in
Milano WF2 0.2l. These deltaproteobacterial sequences were related (>94% similarity) to sequences
of known sulfate reducing bacteria (SRB), such as Desulfocapsa sulfexigens, Desulfovibrio sp. and Desulfosarcina sp. In both filter fractions, there were alpha- and
epsilon-proteobacterial 16S rRNA gene sequences related to the sulfide-oxidizing bacteria Sulfitobacter sp.
(>96% sequence similarity), Thiomicrospira sp. and Sulfurimonas autotrophica (93–95% sequence similarity),
hydrocarbon seep bacteria (>96% sequence similarity),
the aerobic methylotroph Methyloarcula terricola
(<92% sequence similarity) and deep sea sulfide-based
chemotrophic Alphaproteobacteria (>95% sequence
similarity) [36–38]. In addition, sequences of Gammaproteobacteria related to sulfur oxidizing bacteria
(>93–98% sequence similarity), aerobic methylotrophs
S.K. Heijs et al. / FEMS Microbiology Ecology 54 (2005) 47–56
0.05 substitutions/site
53
DSVRR168 Desulfosarcina variabilis
68
81
100
77
Milano WF2 0.2µ B 40 (2 sequences)
UDE535247 Unc. delta-proteobacterium clone Hyd8
DMU45989 Desulfonema magnum
100
95
72
Milano WF1 5µ B 18
U13928 Geobacter sulfurreducens
AF354663 Desulfovibrio mediterraneus
95
Epsilon-proteobacteria
Alpha-proteobacteria
Gamma-proteobacteria
Milano WF2 0.2µ B 11 (3 sequences)
Milano WF1 5µ B 39
Y13672 Desulfocapsa sulfexigens
56
100
Milano WF2 0.2µ B 48 (5 sequences)
53
100 Milano WF1 5µ B 09
75
M59297 Bdellovibrio bacteriovorus
X99237 Desulfovibrio halophilus
Milano
WF1 5µ B 06
56
Milano WF1 5µ B 43
50
100
AJ241020 Delta-proteobacterium Sva0679
93
AJ404370.1 Nautilia lithotrophica
100
AJ309655.2 Caminibacter hydrogeniphilus
AY211663.1 Unc. epsilon-proteobacterium clone GoM GC185 538E
100 AB013263 Epsilon-proteobacterium deep sea NKB11
100
Milano WF2 0.2µ B 30 (6 sequences)
99
L40808 Thiomicrospira denitrificans
73
Milano WF2 0.2µ B 46 (2 sequences)
99
58
AB088431.1 Sulfurimonas autotrophica strain:OK10
100
Milano WF2 0.2µ B 47
Milano WF2 0.2µ B 24 (2 sequences)
84
AF029044 Benzene mineralizing consortium clone SB-17
98
AB091292.1 Sulfurovum lithotrophicum
8654
Milano WF2 0.2µ B 16
80
61 AF449250.1 Unc. epsilon-proteobacterim clone R76-B76 (Riftia tube)
68 AF154091 Hydrocarbon seep bacterium BPC056
Milano WF2 0.2µ B 10 (6 sequences)
66 Milano WF1 5µ B 36 (3 sequences)
Milano WF2 0.2µ B 15
100
AB015579 Alpha-proteobacterium BD 7-3
Milano WF1 5µ B 22
AF030437 Methyloarcula terricola
98
AF254105 Slope strain EI1*
74
Milano WF1 5µ B 38
96 62
81
AF026462 Roseobacter sp.
100
SSP534230
Sulfitobacter sp. HEL-78
100
89
99
Milano WF1 5µ B 28
M63810 Alpha-proteobacterium marine plankton clone 83
Milano WF1 5µ B 33
74
Milano WF2 0.2µ B 23
59 100 Milano WF1 5µ B 41
94
Milano WF1 5µ B 24 (2 sequences)
82
AB013259 1 Unidentified alpha-proteobacterium NKB7
AY034139.1 Thiobacillus prosperus
AY496953.1 White Point filamentous Sulfur-oxidizing bacterium
100 Milano WF2 0.2µ B 29 (4 sequences)
100
AF035956 Beggiatoa sp.(large bacterium from Bay of Concepcion, Chile)
UGA240986 Unc. gamma-proteobacterium Sva0071
99 Milano WF1 5µ B 20
100
Milano WF2 0.2µ B 42
Milano WF1 5µ B 27
8378
100 AB015252 Gamma-proteobacterium ITB 148
M90415 Symbiont of Solemya velum
Milano WF2 0.2µ B 13 (2 sequences)
70
100 Milano WF1 5µ B 44 (5 sequences)
AY354128.1 Unc. bacterium clone pIR3BG09
100 Milano WF1 5µ B 32
87
Milano WF2 0.2µ B 08
79
Milano WF1 5µ B 05
92
AF069959 Thiomicrospira crunogena
Milano WF2 0.2µ B 25
AF154089 Hydrocarbon seep bacterium BPC036
Milano WF2 0.2µ B 20
Milano WF2 0.2µ B 04
AF304197 Methylobacter marinus
67
Milano WF1 5µ B 03
63
AF150796 Methylomonas sp
100
Delta-proteobacteria
Milano WF1 5µ B 40
Y17712 Malonomonas rubra
62
Milano WF1 5µ B 42 O 44
U29164 Methanotrophic gill symbiont of mussel
Milano WF1 5µ B 47 (2 sequences)
57 7195 Milano WF1 5µ B 35
88 U05595 Methanotrophic gill symbiont of mussel
99
A14565
Milano WF1 5µ B 45 (3 sequences)
Milano WF1 5µ B 21
Escherichia coli
Fig. 3. Phylogenetic tree of bacterial 16S rRNA sequences related to Proteobacteria in a deep-sea microbial mat collected from the Milano mud
volcano in the Eastern Mediterranean. The tree was constructed using sequences longer than 800 bp and neighbor-joining analysis using 1000
bootstrap replicates was used to infer the topology. Phylogenetic groups detected are indicated in brackets. The bar represents 5% sequence
divergence.
(>93% sequence similarity) and methanotrophs (>95%
sequence similarity) [37,39,40] were present in both filter fractions.
These analyses support the differences between the
communities found in the two filter fractions, and suggest that the most abundant bacteria in the microbial
54
S.K. Heijs et al. / FEMS Microbiology Ecology 54 (2005) 47–56
communities consisted mainly of chemoautotrophs that
use reduced sulfur compounds and/or methane, as well
as sulfate-reducing prokaryotes. Despite the pitfalls
associated with linking function and phylogeny, we
can tentatively assume that functional properties are
conserved among these phylogenetically related populations as shown previously [41–47]. Taking this assumption into account, we tentatively conclude that
methane oxidation and the production and consumption
of reduced sulfur compounds occurred in the Milano
microbial mat. This conclusion was, to some extent, corroborated by the lipid analysis, which identified the
C16:1, C16:0 and C18:1 fatty acids as the most abundant
low molecular-weight lipids. Since these fatty acids are
widespread in both the bacterial and eukaryotic domains of life, it was not possible to determine a link to
specific bacteria. However, their predominance in the total low molecular-weight lipids provided supporting evidence that bacteria were the dominant members of the
microbial communities in the microbial mat. Their stable carbon isotopic compositions showed low d13C values of 51&, 43& and 52&, respectively, which
do not directly suggest the existence of microbial communities involved in AOM, as more negative values
would be expected [26]. The d13C values are too light
for Rubisco autotrophy [48], but can be explained by
assuming the assimilation of a carbon compound partially formed by oxidation of isotopically light (13C-depleted) methane. In addition, these d13C values
support chemo(auto)trophic processes such as aerobic
methane oxidation and chemoautotrophic dissolved
inorganic carbon (DIC) fixation (perhaps by sulfideoxidizing bacteria) [49]. The data from the lipid analysis
therefore supported the occurrence of chemotrophic
communities involved in methane and sulfide oxidation,
as was deduced from the prokaryotic sequence data.
3.3. Identification of the filamentous bacteria
While the data obtained demonstrate the existence of
a diverse microbial community in the microbial mat, the
identity of the filamentous organisms observed remains
obscure. Individuals viewing the mat from the Nautile
submersible in 1998 suggested that the filamentous
organisms might be Beggiatoa sp. [5]. Consistent with
this presumption, our data indicate that organisms closely related to those previously shown to be present in
a cold seep-associated mat [16] were also present in the
Milano microbial mat. These sequences were affiliated
with those of a large marine, sulfur-oxidizing Beggiatoa
spp. isolated in the Bay of Concepcion, Chile (Teske
et al., unpublished results), but constituted only about
5% of the total phylotypes found, as judged from their
prevalence in the clone libraries. This could be misleading, because of possible biases in lysis efficiency, 16S
rRNA copy number, PCR amplification and other fac-
tors [50–52]. Unfortunately, there was insufficient material available to identify the filamentous prokaryotes
using fluorescent in situ hybridization techniques [53].
Therefore, we can only speculate on the possible identity
of other filamentous organisms, besides Beggiatoa-related sequences. We hypothesize that the relatively
abundant 16S rRNA gene sequences in the Milano
WF1 5l filter fraction, which were absent in the Milano
WF2 0.2l sample, represented filamentous organisms.
Likely candidates are the Gammaproteobacteria related
to sulfide- or methane-oxidizing endosymbionts. Unfortunately, these endosymbionts have no cultured representatives and therefore, the morphology and identity
of these ‘‘potential’’ filaments remains unresolved.
Our results show the existence of a diverse deep-sea
microbial mat community in which filamentous organisms were not conclusively identified. However, Beggiatoa-related prokaryotic organisms could contribute to
the filamentous structure and appearance of the deepsea mat. Assuming that functional properties are conserved among phylogenetically related populations,
we provide evidence for the notion that the deep-sea
microbial mat community is based on a combination
of methane- and sulfide-driven chemotrophy, both aerobic and anaerobic. This could provide valuable insights for future deep-sea investigations and
cultivation attempts.
Acknowledgements
Samples of microbial filaments were obtained during
the French–Dutch ‘‘MEDINAUT’’ expedition, an integrated geological, geochemical and biological study of
mud volcanism and fluid seepage in the eastern Mediterranean Sea. We thank the officers and crew of the Nadir
R/V and the Nautile submersible and the Medinaut and
Medineth Scientific Party for their helpful co-operation
during seagoing activities. We thank Maria Schneider
and Mayee Wong of the University of Idaho (Moscow,
USA) for their help and support during DNA sequencing, and Stephen Bent (University of Idaho, Moscow,
USA) for help with Morisita–Horn similarity calculations. We thank two anonymous reviewers and Dick
van Elsas (University of Groningen) for their useful
comments. Financial support was provided by the
Dutch funding organization, NWO-ALW (ProjectGrant 809.63.013).
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