BIODEEP (EVK3-2000-00042) First year Scientific Report
Work Package number 3: Analysis of the species diversity, community structures and
phylogeny of micro-organism and meiofauna in the Mediterranean deep hypersaline anoxic
basins (DHABs)
Lead contractor: RUG
Participants: Conisma, UESSEX, IMBC, CNR
Start date: month 0
3.1 Objectives
Analysis of the Species diversity, community structures and phylogeny of micro-organisms and
meiofauna in Mediterranean deep hypersaline anoxic basins (DHAB).
Specific goals:
1. Development of methods for the analysis of DHABs prokaryotic communities.
2. Characterisation and comparison of microbial diversity in the four sampled basins.
3. Characterisation and comparison of meiofaunal and macrofaunal diversity in the four
sampled basins.
Methodology and Scientific Achievements
Methodologies
Sampling: During the calibration period, methods for storage and analysis of DHABs samples have
been developed. Prokaryote Liquid samples (seawater, brines and seawater-brines interface) were
collected using Niskin bottles attached to a CTD rosette carried by MODUS and were divided in
fractions to be used for culturing dependent and those used for culture independent analysis.
Meiobenthic samples and macrofauna samples were collected using a Bowers/Conelly multiplecorer with 63.5 cm2 corers and a 0.25m2 USNEL box-corer respectively.
DNA extraction: For the culture independent analysis, samples were filtered directly through 0.1
µm or 0,22 µm pore size sterile filters, immediately after their recovery on board. The filters and
sediment samples were stored at –20°C on board for subsequent analysis. Different protocols were
used for cell lysis and extraction of total DNA from the cells retained on the filters.
Prokaryotic 16S rDNA amplification and analysis: To determine the prokaryotic species
composition and abundance, Bacterial an Archaeal specific 16S rDNA fragments were amplified by
polymerase chain reaction (PCR) using specific oligonucleotide primers. For terminal restriction
length polymorphism (t-RFLP) studies, fluorescent labelled (FAM, HEX) oligonucleotides were
used. PCR products were purified using commercial available PCR purification kits (Qiagen). To
determine the 16S rDNA nucleotide sequence from individual organisms the obtained PCR product
was cloned into the pGEM-T cloning vector and transformed to competent Escherichia coli cells.
Analysis of the amplified 16S rDNA fragments (nucleotide sequence and restriction fragment
lengths) was performed on ABI 310 and ABI377 automated DNA sequencer.
In situ hybridisation: Information on conserved physiological characteristics from phylogenetic
related bacteria in the DHABs will be obtained by fluorescent in situ hybridisation (FISH). For this
purpose 16S rRNA targeted taxon- (group-, subclass-, genus-, species-, clone-) specific fluorescent
labelled oligonucleotide probes were developed that specifically react with microorganisms that are
expected to be present in the DHABs. The whole-cell hybridisation was evaluated with the Zeiss
epifluorescence microscope equipped with 3FL CY3-specific filters set (BP 610/75; FT 545/30; LP
565). Counter staining with DAPI was used as reference.
Meio-and macrofauna analysis: Immediately after sampling, the sediment samples were placed for
15 minutes in a MgCl2 solution for relaxation and then fixed with a neutralised formaldehyde
solution to a final concentration of 4%. In the laboratory, samples are stained with Rose Bengal
solution (0.5 g l-1) and sieved trough 500 and 32µm mesh. The fauna from the fraction remained on
the 32 µm sieve was extracted by triplicate centrifugation in Ludox TM (density 1.15 g cm-3). All
stained meiobenthic animals (metazoa and foraminifera) in the supernatant and in the residual
sediment are counted and identified under a stereomicroscope.
BIODEEP (EVK3-2000-00042) First year Scientific Report
Scientific achievements during first year
Partner 1c (CoNISMa) and 12 (CNR) counted microbial abundance by DAPI fluorescence staining
and microscopy counting. Approximately 104 cells/ml were counted in the seawater column
overlaying the brines, 106 cells/ml at the seawater-brine interface and 104-105 cells/ml in the brine
bodies. For all brines measured, the interfaces are clearly enriched in microorganisms demonstrate
that the chemocline between seawater and the brine pools acts as an in situ particle trap where
microorganisms can flourish due to the increased concentration in available nutrients. Cell numbers
in the Discovery brine body was found to be almost three orders of magnitude lower than in the
other brines, which may be inherent to the divergent chemical composition in the Discovery basin.
Partner 1c also determined the diversity in microbial communities in the 4 DHABs using various
DNA-fingerprinting methods such as ITS-Homoduplex Heteroduplex Polymorphism (ITS-HHP),
Automated Ribosomal Intergenic spacer analysis (ARISA), Amplified Ribosomal DNA Restriction
Analysis (ARDRA) and Denaturing Gradient Gel Electrophoresis (DGGE). All the fingerprints
showed that in the upper seawater column the bacterial pool is similar at all the depths analysed
(1500, 2500 and 3300 m) and between the different areas (Discovery and Bannock). The brine
pools showed to host bacterial communities different from the seawater, with high variability
among the four basins sampled, suggesting that DHABs are environments with a relatively high
biodiversity. Archaea in the brines are under investigation. Libraries constructed on a 150 bp
fragment of the 16SrDNA gene, which include the V5 hypervariable region, have been screened for
insert sequences. The retrieved clones will be compared with the total community diversity
described by Single Strand Conformation Polymorphism (SSCP). At present, in Urania and
L’Atalante brines sequences with partial homology to Halobacterium sp., Thermococcus sp.,
uncultured Euryarchaeota, unidentified Methanogen and other Archaea have been found.
Partner 5 (RUG) focussed on the microbial community diversity by sequence analysis of the
complete 16S rDNA genes of Bacteria and Archaea and on the presence of sulphate reducing
bacteria (SRB) by screening for the conserved sulphite reductase gene. DNA extraction and PCR
amplification of both archaeal and bacterial 16S rDNA was successful for all samples,
demonstrating their presence in all samples. The sulphite reductase gene could only be amplified
from brine and brine interface samples and not from the overlying water column.
Sequence analysis was performed on a number of clones selected from 1500 bp long bacterial and
archaeal 16S rDNA clone libraries obtained from the brine-interface samples. Sequencing of initial
700 bases (corresponding to position 700-1400 of the 16S rDNA gene of E. coli) from 96 bacterial
16S rDNA clones from the Discovery interface have been aligned and compared with the GenBank
database using BLAST. 36 different bacterial species were identified (>97% similarity), indicating a
high bacterial biodiversity. Dominant species found are Dechloromarinus chlorophilus (~99%
similarity) and Thiomicrospira spp. (~97.5% similarity). 16 other sequences showed homology with
sequences from other clones present in the database of GenBank (>95% similarity). Surprisingly,
64 out of the 96 clones had low sequence similarity (<95%) with sequences present in the GenBank
database, indicating that the majority of the bacterial species present in the brine-interface of the
Discovery basin are not yet cultured or sequenced. In addition, 16 sequences, although having a
similarity below 97%, clustered within the groups of sulphate reducing bacteria showing that
bacterial species play an important role in the sulphur cycle in this ecosystem. A preliminary
comparison between the Discovery brine and the first 36 sequences obtained from the bacterial 16S
rDNA gene of the L’Atalante brine interface showed in general a low homology. Sequencing of
Archaeal 16S rDNA sequences are in progress. An initial survey revealed sequences homologous to
know halotolerant or halophilic archaea or to uncultured archaea obtained from hypersaline lakes or
deep-sea ecosystems but also sequences that have no match in the database.
BIODEEP (EVK3-2000-00042) First year Scientific Report
Partner 6 (UESSEX) was also successful in DNA extraction and 16S rDNA amplification from oxic
seawater samples, interfaces and brines of L’Atalante, Urania and Bannock basins. Only samples
obtained from Discovery basin yielded rather low molecular weight DNA or RNA only. The
analysis of the restriction digestion of the PCR products revealed a relatively high similarity
between the samples from oxic seawater overlaying the brines (Discovery at 2500 m and 3300 m
depth and Bannock at 3000 m depth). Relatively high similarity was also observed between the
deepest oxic seawater sample (3500 m depth near Discovery) and the Urania interface sample, and
between the samples from l’Atalante and Bannock basin brines and l’Atalante basin interface.
Urania basin brine has the most different microbial community on the basis of t-RFLP analysis.
Partner 12 used the FISH technique to detect specific groups of microorganisms in the samples
collected from the various brines. They observed a distribution of Archaea and Bacteria (FISH with
CY3-labelled 16S rRNA oligonucleotide probes) within the sampled layers that was coherent with
the DAPI counts since the ARCH + EUB bacterial counts were always corresponding to the 0.726.47% of the total prokaryotic density (TD). The experimental work pointed out a negative side
effect of CY3-counter staining on the DAPI counts. In fact, in all double stained filters (DAPI+
either ARCH or EUB), the TD was decreased ten-fold. According to the TD values, the EUB
vertical distribution showed a decrease from the upper to the lower interface of the basins followed
by a significant increase in the Body Brine layer, with the exception of the Discovery basin Body
Brine where no FISH signal was recorded. This suggests that the Body Brine environments of the
L’Atalante, Urania and Bannock basins are much more suitable for Archaeal development than the
Interface layers. Three 16S rRNA-targeted probes specific for halophilic bacterial taxa were
optimised for in situ hybridisation. One of these three, the THT1 probe, was specifically designed to
complement the 20A2530 clone sequence obtained from the L’Atalante Lower Interface which
shows similarity to the Thermotoga group of Eubacteria. The THT1 probes matched with
complementary sequences in all DHABs samples, with the exception of the Discovery Body Brine
where no FISH signals were visualised. The highest THT1 FISH counts were recorded in the
L’Atalante Upper Interface sample, where the THT1 signal covered about 15% of the total number
of EUB cells. Analyses with the other two probes ARC94 (specific to the gram negative,
aerotolerant bacterium Arcobacter ssp.) and HAB1 (specific to the strictly anaerobic and
moderately halophilic bacterium Haloanaerobium spp.) are in progress.
Partner 11, which is specialised in the meio- and macrofaunal diversity, found a rather low
meiofaunal density from the non-brine sites of the L’Atalante, Urania and Bannock basins which
ranged from 11.7 (Urania basin) to 26 ind. 10cm-2 (L’Atalante, MCAT03). At Discovery basin
meiofaunal density was considerably higher (43.1 ind. 10cm-2). The brine samples of the L’Atalante
and Discovery basins displayed higher densities compared to those from the non-brine sites (41.545.3 and 191.1 ind. 10cm-2 respectively). However, at Urania and Bannock basins the opposite
trend was observed.
In the normal non-brine sediment samples of the L’Atalante, Urania and Bannock basins
nematodes were the most abundant taxon accounting for 44.7 to 64.4% of the total, followed by
foraminifera (25 to 43.9 %). At the Discovery basin, foraminifera were the most abundant taxon
(50.4%) followed by nematodes (40.9%), while at all basins, all the remaining meiobenthic taxa
comprised less than 20 % of the total. Community structure in the brine sediments differed
considerably among the four basins. At the Discovery basin the meiobenthos was dominated by
copepods and foraminifera (mostly planktonic species) comprising 75% of the total, followed by
juvenile bivalves, nematodes, allogromiid foraminifera, other crustacea. The L’Atalante basin was
characterised by various meiofaunal taxa; nematodes, foraminifera, copepods, juvenile molluscs
(unid. bivalves and gastropods). At the Urania basin juvenile bivalves (unid. sp.) dominated the
community (61.5%) followed by copepods, foraminifera and other crustacean taxa.
At the Bannock basin juvenile gastropods (unid. sp.) were the most abundant taxon (45%) at station
MCBANN01, followed by the above-mentioned bivalves, nematodes, planktonic copepods and
BIODEEP (EVK3-2000-00042) First year Scientific Report
foraminifera. At station MCBANN02, the meiobenthos community was comprised of various taxa
such as nematodes, foraminifera, crustacea and juvenile molluscs.
Macrofaunal density values obtained from the non-brine sites of the Discovery and Urania basins
range from 60 to 88 ind m-2 and are usual for deep-sea environments. In contrast to this, the
corresponding value from the only brine site (Urania basin) available was much less (24 ind. m -2).
The same trend was observed for biomass, with values ranging from 0.003-0.005 g m-2 outside the
brines (Discovery, Urania), dropping to 0.001 inside the brine (Urania basin). Macrofaunal
community structure is differs dramatically between the non-brine and brine sites. Polychaeta are
dominant outside the brine sediments with values ranging from 62-73% and 59-90% of the total
abundance and biomass respectively, while Crustacea, the second most dominant group, ranged
from 20-38% and 6-39% of the total abundance and biomass respectively. In contrast, the brine site
(Urania basin) is dominated by Crustacea (67% and 95% of the total abundance and biomass
respectively).
3.3 Socio-economic Relevance and Policy Implication
The result obtained so far already implies a large biodiversity with the majority of microbial species
still being uncultured or unidentified. Because of this large novel biodiversity in this ecosystem, still
unaffected by human interference, there is a reasonable change of finding new genetic or
biochemical traits that might prove valuable for medical or biotechnological applications.
3.4 Discussion and Conclusions
The total amount of microorganisms obtained from the samples seems to be sufficient for DNA
isolation and PCR amplification to study the prokaryotic community structure by culture
independent techniques. However for the culture dependent approach, larger sample sizes are
essential and to be able to clone specific genes without amplification the amount of DNA obtained
is probably insufficient. Unfortunately none of the partners in WP3 have been able to isolate DNA
or amplify DNA fragments from the sediments samples even though several protocols have been
applied. The reason is still unknown, but may be due to either a low cell density or the presence of
factors inhibiting DNA isolation.
Initial results already indicate large biodiversity with the majority of microbial species still being
uncultured or unidentified. There seems to be divergence in the brine communities in which the
l’Atalante and Bannock basins are more similar to each other than to the community inhabiting the
Urania basin brine. This is reflected in the chemical composition of the brines, which is rather
similar for l’Atalante and Bannock basins, whereas the Urania brine body is characterised by unique
high sulphide and methane concentrations. The fingerprints from brine samples were less similar to
each other than the oxic seawater samples A, C, and D from different depths and locations. The
brines therefore have a microbial community unique to the respective location.
3.5 Plan and Objectives for the Next Period
All of the partners within WP3 are now fully involved in their proposed research. Calibration of the
essential techniques has been successful as well as the initial steps required for obtaining data
(DNA-isolation, optimising FISH probes and fingerprinting techniques, etc.). Each partner will now
focus on the analysis of the large amount of samples that were obtained during the cruise or were
generated by DNA amplification or other techniques. Different, optimised DNA extraction
protocols will have to be applied to the sediment samples to obtain microbial community profiles.
These community profiles will be compared with those from anoxic enrichment cultures of
sediment samples in order to determine whether enriched microorganisms are abundant in the
sediments.