Conclusions

This dissertation focused on the study of different microbial communities involved in the process of AOM. This work began with the identification of several intact polar lipids (IPLs) from few samples phylogenetically dominated by each of three anaerobic methanotrophic consortia (ANME-1, -2 and 3 and sulfate reducer bacterial partners). After the identification of several diagnostic IPLs characteristic of the each AOM-community, these lipids were analyzed in a variety of globally distributed cold seep systems. Among these hot spots of AOM, different habitats were analyzed such as anoxic water bodies, mud volcanoes, oil fields, gas hydrate environments and hydrothermal vents. In the course of this work, it was possible to address several open questions regarding AOM-research: (1) identification of communities involved in AOM based on few diagnostic of IPLs, (2) microbial-derived IPL diversity in AOM hot spots and (3) environmental factors influencing the dominance and distribution of AOM-communities.

This work is the first to demonstrate that IPLs, which are biomarkers associated to living biomass, enable not only the distinction of the three main groups of AOM-mediating microbes from a wide variety of methane-bearing habitats (Chapter II and III) but, more importantly, provides additional insights on the environmental factors influencing the distribution of these communities (Chapter III).

The three phylogenetically distinct clusters of Euryarchaeota called ANME-1, -2 and -3 (e.g., Hinrichs et al., 1999; Boetius et al., 2000; Lösekann et al., 2007) which have been observed in association with sulfate-reducing bacteria (SRB) of the Desulfosarcina/Desulfococcus group (Boetius et al., 2000; Orphan et al., 2001; Michaelis et al., 2002, ‘‘ANME-1/DSS and -2/DSS aggregates”) or Desulfobulbus spp (Lösekann et al., 2007, ‘‘ANME-3/DBB aggregates”) exhibit a characteristic IPL composition.

ANME-1, which is not directly affiliated with any of the major orders of methanogens (Hinrichs et al., 1999; Orphan et al., 2001; Knittel et al., 2005) is characterized by the production of glyceroldialkylglyceroltetraether (GDGTs) with glycosidic and phospho as well as mixed glycosidic and phospho headgroups. The main glycosidic-GDGT in

ANME-1 system, is diglycosyl-GDGT (2Gly-GDGT, Rossel et al., 2008; Chapter II and Chapter III), a lipid also frequently observed in deep subsurface (Biddle et al., 2006; Lipp et al., 2008), as well among several species within the order Methanomicrobiales (Koga et al., 1998). In addition to glycosidic-GDGTs, GDGTs with mixed glycosidic and phospho or only phospho headgroups were dominated by 2Gly-GDGT-PG and 2PG-GDGT (Chapter III), which have been also previously reported in Methanobacterium thermoautotrophicum (Koga et al., 1993). Interestingly, contribution of 2Gly-GDGT, 2Gly-GDGT-PG and 2PG-GDGT varied depending of the ANME-1 habitat. Beside the general dominance of 2Gly-GDGT, the contribution of 2Gly-GDGT-PG and 2PG-GDGT was much higher in sediment than in carbonate reefs dominated by ANME-1.

Different from ANME-1, diagnostic IPLs of ANME-2 were archaeols with both glycosidic and phospho headgroups, which also occur in Methanocaldococcus jannaschii, Methanococcus voltae and Methanothirx soehngenii (Koga et al., 1993; Sturt et al., 2004). Within the glycosidic archaeols the main IPLs were 2Gly-archaeol (2Gly-AR), 2Gly-MAR (2Gly-macrocyclic archaeol), 2Gly-hydroxyarchaeol (2Gly-OH-AR), whereas the major phospho-archaeols were PG-OH-AR, phosphatidylserine-OH-AR (PS-OH-AR) and phosphatidylinositol-OH-AR (PI-(PS-OH-AR) (Chapter III). Similar to ANME-1 systems, archaeal IPLs containing phospho headgroups were more abundant in sediments than in carbonate reefs.

ANME-3, contrary to ANME-2 and ANME-1 contained neither glycosidic-archaeols nor GDGT-based IPLs. However, the phospho-glycosidic-archaeols composition was very similar to ANME-2, although with a generally less contribution of PI-OH-AR (Chapter III). The phylogenetic affiliation of ANME-2 and ANME-3 with the order Methanosarcinales, was consistent with the dominance of archaeol and hydroxyarchaeol with both glycosidic and phospho headgroups (Kates, 1997; Koga et al., 1998).

Among the major bacterial IPLs, relative high abundance of phosphatidylethanolamine (PE), phosphatidyl-(N)-methylethanolamine (PME) and phosphatidyl-(N,N)-dimethylethanolamine (PDME) with diacylglycerol (DAG) bond type, were found in ANME-2/DSS and ANME-3/DBB dominated settings (Rossel et al., 2008; chapter II and chapter III). PE is the major phospholipid type of SRB such as Desulfosarcina variabilis (Rütters et al., 2001) and its occurrence together with PME and

PDME in anoxic waters and surface sediments from the Black Sea has been also suggested to derive from SRB (Schubotz et al., submitted). However PME and PDME have been also described in some methanotrophic bacteria (Makula, 1978; Fang et al., 2000) as well as sulfide oxidizers (Barridge and Shively, 1968). The presence of PME and PDME seems to be a general feature of ANME-3/DBB dominated systems, although it needs to be taken into account, that a fraction of these two IPLs may derived either from aerobic methanotrophic bacteria or from sulfide oxidizers, both which contain similar membrane lipids (Barridge and Shively, 1968; Makula, 1978; Fang et al., 2000).

Other bacterial IPLs, which contributed mainly to ANME-2/DSS dominated mats, were the non-phospho IPLs ornithine lipids (OL), surfactin and betaine lipids (BL), with the latter characterized by odd fatty acid chains (BL-odd) (Chapter III). OL have been reported in SRB, and sulfur and iron oxidizing bacteria (Knoche and Shively, 1972;

Makula and Finerty, 1975), whereas surfactin is a lipopeptide with surface active properties common of Bacillus sp. (Vater et al., 1986) that may also be produced by an unknown bacteria in the mats from the Black Sea. On the other hand, BL-odd, contrary to BL with even fatty acid chains, have been suggested to derive from bacteria, do to their occurrence in deep anoxic water of the Black Sea (Schubotz et al., submitted).

Based on IPL distribution, it was possible to observe a clear separation within the chimney-like structures and the sediment habitats. ANME-1 and ANME-2/DSS inhabiting carbonate reefs contained high abundance of glycosidic-IPLs and IPL with non-phospho headgroups. Both archaeal (2Gly-GDGT, 2Gly-AR, 2Gly-MAR, 2Gly-OH-AR) and bacterial IPL (OL, surfactin and BL odd) composition point to the low abundance of phospho-IPLs in carbonate mats compared to sediments. Dissolved phosphate in sediment pore water has been shown to be strongly adsorbed on calcium carbonate (Cole et al., 1953; de Kanel and Morse, 1978). Therefore, limitation of dissolved phosphate in AOM carbonate mats from the Black Sea is likely responsible for the generally low abundance of IPLs with phospho headgroups in both ANME-1/DSS and ANME-2a/DSS dominated mats (Chapter III).

Beside the general differences in IPL composition of ANME-1, -2 and -3 communities, additional variations in the IPL pattern in relation to several environmental

variables provided new insights into the ecological niches dominated by these communities (Chapter III). ANME-1/DSS, in which the diagnostic IPL was 2Gly-GDGT, dominates habitats with higher temperature and lower oxygen content in bottom waters compared to the systems in which ANME-2/DSS and ANME-3/DBB inhabit. This relationship between ANME-1/DSS and temperature is in agreement with the detected higher AOM-activity of ANME-1/DSS at higher temperatures (up to 24°C) compared to ANME-2/DSS (up to 15°C) based on in vitro experiments (Nauhaus et al., 2005).

Furthermore, the dominance of ANME-1/DSS in low oxygen bottom waters is in agreement with previous field observations, which suggest that ANME-1/DSS may be more sensitive to oxygen than ANME-2/DSS (Knittel et al., 2005). Based on IPL diversity, ANME-2/DSS systems were separated in two groups: the carbonate reefs and the sediments. ANME-2/DSS dominated sediments were characterized not only by lower temperature and higher oxygen content in bottom waters, but also by higher methane and sulfate concentrations. These environmental variables were accompanied by the presence of PG-OH-AR and PI-OH-AR. On the other hand, ANME-2/DSS dominated carbonate mats were associated with higher sulfate reduction rates (SRR) and to the occurrence of 2Gly-OH-AR and 2Gly-MAR. These differences between carbonate reefs and sediments dominated by ANME-2/DSS could be explained by the presence of sulfide oxidizing bacteria (SOB) in the sediments, which efficiently remove sulfide and produce sulfate.

The environmental characteristics, as well as the archaeal IPL composition of ANME-3 and ANME-2 from sediments, suggest that these two communities dominate in similar environments, although due to the fact that the lowest temperatures were observed at ANME-3/DBB dominated sediments from Håkon Mosby Mud Volcano, it is possible that temperature may also select for either ANME-2/DSS or ANME-3/DBB.

IPL data in general was in good agreement with the phylogenetic information based on FISH methods. Nevertheless, in a few cases both methods have some discrepancies due to several potential reasons. It was observed that in sediments dominated by ANME-2c/DSS according to FISH counting, the contribution of ANME-1 derived GDGT-based IPLs was higher than the ANME-2/DSS IPL signal. The high contribution of GDGT-based IPLs was probably due to the presence of extremely large ANME-1 cells in this setting. Additionally, FISH methods could also underestimate

archaeal abundance, especially ANME-1, due to the low permeability of their membranes compared to the bacterial phospholipid (Wagner et al., 2003).

The evaluation of apolar lipids distribution provided a poor taxonomic separation between the three AOM-communities (Chapter III). This was probably due to the lack of GDGTs in our data set, which is the main core lipid of ANME-1, but also to the presumed longer turnover times of apolar lipids than of IPLs. Apolar signals may integrate longer periods in the geologic evolution of the studied seep systems, in which community changes are likely to occur resulting in a mixed signal from current and past microbial communities.

Furthermore, IPL behavior on marine sediment systems was evaluated using an experimental approach (Chapter IV). Incubations were performed using slurries of sediments with (sterile condition) and without sterilization (active condition), in which membrane lipid of archaea (2Gly-GDGT) and bacteria (C16-PC) were spiked. Both sterile and active conditions were incubated at 5°C and 40°C. According to our results both archaeal and bacterial IPLs were degraded under sterile conditions. However, after 465 days of incubation under active conditions, an increase of both IPLs was observed (although the bacterial IPL only increased at 5°C). This suggests that the microbial communities were growing. Unfortunately, degradation of IPLs in the active conditions could not be proved because the IPLs produced and degraded were indistinguishable.

Therefore, an improved experimental approach is necessary.

We demonstrated that few IPLs enable the distinction of AOM-communities, although the diversity of IPLs identified in methane-bearing habitats is very high (chapter III and V). Among the archaeal IPLs identified, GDGT-based and archaeol-based IPLs with glycosidic, mixed phospho and glycosidic or pure phospho headgroups were observed. Bacterial IPLs were also diverse having not only different phospho headgroups but also containing non-phospho IPLs. Structural information and fragmentation patterns of diverse IPL classes are provided in this thesis (Chapter V) as base for further IPL identification in AOM systems.

The results obtained during this thesis provide a clear distinction between the major microbial communities involved in AOM in marine sediments (ANME-1, -2 and -3

and SRB partners) based on IPL distribution. Additionally these results demonstrate that IPLs varied not only according to the community type but also in relation to the habitat characteristics. Furthermore, IPL distribution was also related to several environmental factors selecting for one of the three major AOM-community types. Thus, allowing to define the ecological niches dominated by each of these groups.