| Abstract |
Cold seeps and gas hydrates sediments have an intrinsic role in the carbon cycle and therefore have become environments of
intensive study in recent years. They function as energy source for chemosynthetically based life,
can be used as potential energy resource and play an important role in slope stabilization.
Sulfide and methane are available in high concentrations and support a variety of highly adapted microorganisms and symbiont-bearing
invertebrates. The anaerobic oxidation of methane (AOM) is a key biogeochemical process at cold seeps and is assumed to be coupled to
sulfate reduction and mediated by a consortium of anaerobic methanotrophic Archaea (ANME)
and sulfate-reducing Bacteria (SRB). AOM acts as a scavenger to significant amounts of methane which could otherwise be released in
the atmosphere, contributing, to climate change. Another sink for methane in the ocean is the aerobic oxidation of methane (MOx)
whilst methane can be produced by microbially mediated Methanogenesis. Active methane seeps and gas hydrates have been identified on
a number of mud volcanoes (MV) in the Anaximander Mountains, East Mediterranean Sea.
The structure of highly complex
prokaryotic communities harboured in marine sediments is dictated by rapid changes of physical and geochemical factors along
sediment depth in hot-spot sites, like MVs. In this thesis, sediment from two mud volcanoes,
Amsterdam and Kazan were analysed and it was investigated whether (a) the community structure of Bacteria and Archaea vary concomitantly, (b) the prokaryotic assemblages
are dominated by one or several phylotypes with different relative abundances, implying a
specific or an opportunistic community, respectively, and also (c) which of these specific communities are involved in biogeochemical
processes related to mud volcanism.
Sediment samples were sampled every 5 cm of the top 30 cm sediment and the distribution of specific biomarkers derived from
ANME groups and SRB were investigated. High abundances of sn-2,3-di-O-isoprenoidal glycerol
ethers (mainly archaeol and sn-2-
hydroxyarchaeol) were retrieved. The
concentrations measured were much higher (3 to 12 times) in Amsterdam sediments related to
Kazan. In Amsterdam MV, maximum in archaeal biomarker abundances at 15-20 cmbsf (cm below sea floor) horizon correlated with the
methane-sulfate transition zone and the measured concentrations are the highest reported so far for mud volcano’s sediments. In
Kazan MV two local maxima were present at horizons 5-10 and 20-25, whereas the primer transition zone seemed to be placed from 7 to
13 cmbsf and a minor one at 20 to 25 cmbsf. Specific bacterial markers such us C16:1ω7c,
C18ω:17c showed dominance of sulphide oxidizing Bacteria (SOx) in the upper layers for both volcanoes. Characteristic fatty acids
C16:1ω5c, cyC17:0ω5,6 for AOM related SRB were also present in high concentrations.
The vertical distribution of the archaeal
and bacterial community composition was studied further with the construction of 16SS
RNA gene libraries from the same sites of Amsterdam and Kazan MVs. The vast majority of
all archaeal phylotypes retrieved in both mud volcanoes belong to Euryarchaeota. In
Amsterdam MV, ANME-1 and ANME-2 seem to co-dominate all archaeal libraries constructed
except the one from 30 cmbsf horizon, where ANME-3 was the dominant group. In bacterial
clone libraries Proteobacteria (mainly γ-, δ- and
ε- groups) dominated 0 and 5 cmbsf. Choloflexi- affiliated phylotypes were present in all sediment layers and dominated 10 cmbsf
horizon. Phylotypes belonging to candidate division JS1 were also present in all sediment horizons but exhibited their highest relative
abundance at 15 cmbsf. Phylotypes affiliated with δ-Proteobacteria had significant representation in all bacterial libraries and
showed substantial percentage at 25 cmbsf. The vast majority of all bacterial phylotypes retrieved was closely related with methane
seeps and gas hydrate environments. They were affiliated with putative sulfate-reducers, sulfide
oxidizers, sulfur reducers and methylotrophs
In Kazan MV, the majority of the
archaeal phylotypes were related to
uncultivated members of ANME-2, from habitats where AOM occurs. ANME-1 and ANME-3 groups were also present. In bacterial
clone libraries from Kazan MV, Proteobacteria were the most abundant and diverse bacterial
group, with the γ-Proteobacteria dominating in most sediment layers and were related to members from marine sediments involved in
methane cycling. The δ-Proteobacteria included several of the sulfate-reducers known to co-
occur with anaerobic methane oxidizers. The rest of the bacterial phylotypes were shared among 15 known phyla, with cultured and
uncultured representatives, and three
unaffiliated groups. In most cases, these phylotypes represented microorganisms from similar, marine and non-marine, habitats.
Diversity was calculated by Shannon-
Wiener diversity index (H) and Pielou evenness index (J). The Shannon diversity index for
Archaea was lower than for Bacteria in the same sediment horizon but indices did not co-vary for the two groups with sediment depth. For the
archaeal communities H was calculated in Amsterdam MV between 0.99 - 1.72 and in
Kazan MV 0.56 - 1.73. For the bacterial communities, H varied between 1.92 - 4.03 and
1.47 - 3.82 for Amsterdam and Kazan MV,
respectively. Pielou index showed similar distribution pattern with H in most horizons.
Cluster analysis using Morita similarity index revealed that more similarities are shared within different layers from the same MV rather
than between the two volcanoes. The level of similarity of bacterial communities, with the exception of the communities at 15 and 20
cmbsf for both volcanoes, was low when phylotypes were used, indicating little overlap across the rest sediment layers. So apart from
the clustered 15/20 horizons, each of the other horizons appeared to be a different habitat containing its own unique community of bacteria.
The presence and the functional
diversity of Archaea involved in methane metabolism (methanotrophs and methanogens) were investigated by detection and phylogenetic analysis of mcrA gene. This gene
encodes for methyl-coenzyme M reductase, an essential enzyme in both anaerobic methane oxidation and methane production. The mcrA
gene was present in all sediment horizons at Amsterdam MV and in all, but the surface horizon at Kazan MV. Putative methane
produces related to Methanogenium
organophilum were detected only in the deepest analyzed sediment layer for both MVs.
The rest of the phylotypes were affiliated with functional group mcr a,b, mcr c, mcr e and mcr
f. Generally, the results of mcrA analysis are in agreement with the phylogenetic analysis of 16S
rRNA gene for Kazan MV. In the case of Amsterdam MV there seems to be a failure in 146 detecting mcr a,b group (corresponding to
ANME-1) at 15 and 20 cmbsf.
AOM is a significant biogeochemical
process in many cold-seep sediments, evident in everything from pore water and sedimentary
geochemical profiles to authigenic carbonates to (chemosynthetic) macrofaunal abundance
and diversity. In sediments from both Amsterdam and Kazan mud volcano, measured pore water profiles of the concentrations of
sulfate, sulfide, detected high values of archaeol and hydroxyarchaeol and bacterial biomarkers, retrieved phylotypes in both 16s rRNA gene and
mcrA gene libraries, all indicate that AOM is presently occurring in the sediments of these two mud volcano.
Differences in the size, methane
concentrations and fluxes, sedimentological and geochemical characteristics and eruptive history
between Amsterdam and Kazan MV (two geographically close mud volcanoes) have
probably led to the formation on different prokaryotic communities in each of them.
Moreover, for each mud volcano, it seems that mud volcanism shapes mixed prokaryotic communities at the domain level, with Archaea
following different diversity patterns with sediment depth than Bacteria. Archaeal ribosomal RNA gene sequences are consistent
with dominance of AOM-like organisms, while the majority of recovered bacterial sequences
affiliate with sulfate-reducers, sulfide oxidizers, sulfur reducers and methylotrophs.
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Μοριακός και βιογεωχημικός χαρακτηρισμός των μικροοργανισμών που εμπλέκονται στην αναερόβια οξείδωση του μεθανίου και τη μεθανιογένεση σε ιζήματα μεθανιοϋδριτών της Ανατολικής Μεσογείου
