Petroleum degradation and associated microbial signatures at the Chapopote asphalt volcano, Southern Gulf of Mexico

At the Chapopote Knoll in the Southern Gulf of Mexico, deposits of asphalt provide the substrate for a prolific cold seep ecosystem extensively colonized by chemosynthetic communities. This study investigates microbial life and associated biological processes within the asphalts and surrounding oil-impregnated sediments by analysis of intact polar membrane lipids (IPLs), petroleum hydrocarbons and stable carbon isotopic compositions (δ¹³C) of hydrocarbon gases. Asphalt samples are lightly to heavily biodegraded suggesting that petroleum-derived hydrocarbons serve as substrates for the chemosynthetic communities. Accordingly, detection of bacterial diester and diether phospholipids in asphalt samples containing finely dispersed gas hydrate suggests the presence of hydrocarbon-degrading bacteria. Biological methanogenesis contributes a substantial fraction to the methane captured as hydrate in the shallow asphalt deposits evidenced by significant depletion in ¹³C relative to background thermogenic methane. In sediments, petroleum migrating from the subsurface stimulates both methanogenesis and methanotrophy at a sulfate-methane transition zone 6-7 m below the seafloor. In this zone, microbial IPLs are dominated by archaeal phosphohydroxyarchaeols and archaeal diglycosidic diethers and tetraethers. Bacterial IPLs dominate surface sediments that are impregnated by severely biodegraded oil. In the sulfate-reduction zone, diagnostic IPLs indicate that sulfate-reducing bacteria (SRB) play an important role in petroleum degradation. A diverse mixture of phosphohydroxyarchaeols and mixed phospho- and diglycosidic archaeal tetraethers in shallow oil-impregnated sediments point to the presence of anaerobic methane-oxidizing ANME-2 and ANME-1 archaea, respectively, or methanogens. Archaeal IPLs increase in relative abundance with increasing sediment depth and decreasing sulfate concentrations, accompanied by a shift of archaeol-based to tetraether-based archaeal IPLs. The latter shift is suggested to be indicative of a community shift from ANME-2 and/or methanogenic archaea in shallower sediments to ANME-1/methanogenic archaea and possibly benthic archaea in deeper sediments.     

Stable carbon isotopic compositions of intact polar lipids reveal complex carbon flow patterns among hydrocarbon degrading microbial communities at the Chapopote asphalt volcano

Seepage of asphalt forms the basis of a cold seep system at 3000 m water depth at the Chapopote Knoll in the southern Gulf of Mexico. Anaerobic microbial communities are stimulated in the oil-impregnated sediments as evidenced by the presence of intact polar membrane lipids (IPLs) derived from archaea and Bacteria at depths up to 7 m below the seafloor. Detailed investigation of stable carbon isotope composition (δ¹³C) of alkyl and acyl moieties derived from a range of IPL precursors with distinct polar head groups resolved the complexity of carbon metabolisms and utilization of diverse carbon sources by uncultured microbial communities. In surface sediments most of the polar lipid-derived fatty acids with phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG) head groups could be tentatively assigned to autotrophic sulfate-reducing bacteria, with a relatively small proportion involved in the anaerobic oxidation of methane. Derivatives of phosphatidyl-(N)-methylethanolamine (PME) were abundant and could be predominantly assigned to heterotrophic oil-degrading bacteria. Archaeal IPLs with phosphate-based hydroxyarchaeols and diglycosidic glyceroldibiphytanylglyceroltetraethers (GDGTs) were assigned to methanotrophic archaea of the ANME-2 and ANME-1 cluster, respectively, whereas δ¹³C values of phosphate-based archaeols and mixed phosphate-based and diglycosidic GDGTs point to methanogenic archaea. At a 7 m deep sulfate-methane transition zone that is linked to the upward movement of gas-laden petroleum, a distinct increase in abundance of archaeal IPLs such as phosphate-based hydroxyarchaeols and diglycosidic archaeol and GDGTs is observed; their δ¹³C values are consistent with their origin from both methanotrophic and methanogenic archaea. This study reveals previously hidden, highly complex patterns in the carbon-flow of versatile microbial communities involved in the degradation of heavy oil including hydrocarbon gases that would not have been evident from classical compound-specific isotope analyses of either bulk IPL or apolar lipid derivatives.     

海洋沉积物中现存微生物化学标志物完整极性膜脂研究进展

完整极性膜脂作为活的微生物细胞的化学标志物,能够反映海洋沉积物中现存微生物群落结构和生物量等信息.与生物学方法相比,完整极性膜脂分析技术具有无需培养、快速和普适性等特点.综述了海洋沉积物中细菌和古菌的细胞膜完整极性膜脂的组成特点及其在生物地球化学和微生物生态学等研究中的应用,重点评述了在生物地球化学循环中有特殊作用的微生物,如厌氧氨氧化细菌、甲烷氧化古菌、氨氧化古菌、具有四醚膜脂结构的海洋泉古菌等,或者是一些特殊生态系统,如冷泉、海底深部生物圈等研究中完整极性膜脂应用的进展.还简要介绍了完整极性膜脂的分析方法,并对其应用前景进行了展望.     

Fossilization and degradation of intact polar lipids in deep subsurface sediments: A theoretical approach

Intact polar membrane lipids (IPLs) are frequently used as markers for living microbial cells in sedimentary environments. The assumption with these studies is that IPLs are rapidly degraded upon cell lysis and therefore IPLs present in sediments are derived from in situ microbial production. We used a theoretical approach to assess whether IPLs in surface sediments can potentially represent fossilized IPLs derived from the upper part of the water column and whether IPLs can be preserved during sediment burial. Previous studies which examined the degradation kinetics of IPLs show that phospholipids, i.e. ester-linked lipids with a phosphor-containing head group, degrade more rapidly than glycosidic ether lipids, i.e. ether-linked lipids with a glycosidically bound sugar moiety. Based on these studies, we calculate that only a minor fraction of phospholipids but a major fraction of glycosidic ether lipids biosynthesized in the upper part of the water column can potentially reach deep-sea surface sediments. Using a simple model and power law kinetic degradation parameters reported in the literature, we also evaluated the degradation of IPLs during sediment burial. Our model predicts a log-log relationship between IPL concentrations and depth, consistent with what has been observed in studies of IPLs in subsurface sediments. Although our results do not exclude production of IPLs in subsurface sediment, they do suggest that IPLs present in the deep biosphere may contain a substantial fossil component potentially masking in situ IPL production.     

Advection and diffusion determine vertical distribution of microbial communities in intertidal sediments as revealed by combined biogeochemical and molecular biological analysis

Porewater advection stimulates nutrient exchange and microbial activity in shallow marine sediments, whereas element cycling in deeper diffusion-dominated sediments is comparatively slow due to limited nutrient supply. We studied the vertical distribution of microbial communities and organic matter (OM) cycling in these contrasting porewater regimes down to 5 m depth at an intertidal flat of the southern North Sea. Archaea, Bacteria and Eukarya were targeted, combining intact polar lipid (IPL) analysis with qualitative and quantitative molecular biological techniques. The largely sandy section 1 of the core (<75 cm) is characterized by rapid burial of fresh marine OM and intense porewater advection. This supply fuels heterotrophic microbes, as evident from the ¹³C isotopic composition of total organic carbon and IPL derivatives. Major sources of OM are algae and cyanobacteria, as suggested by the elevated amount of eukaryotic 18S rRNA gene copies and phosphate-free IPLs. The relative abundance of most phospholipids remained largely constant over the entire core, except for diphosphatidylglycerol, which represented about half of total IPL abundance in the lower part of section 1 (>50 cm) and the diffusion-dominated section 2 (75–490 cm). This suggests bacteria adapting their membranes in response to increasing physicochemical stress and starvation in the nutrient limited, fine grained sediments of section 2 with less bioavailable, predominantly terrestrial, OM. Relative amounts of bacterial acyl ether and diether phospholipids increased in this lower section and were assigned to sulfate reducers and yet uncultured myxobacteria. Archaea were an order of magnitude less abundant than Bacteria, and were affiliated mainly with Methanosarcinales and Methanomicrobiales. Accordingly, the archaeal IPL composition was typical for a methanogenic community. IPLs not exclusively derived from in situ microbial production emphasize that these biomarkers have to be interpreted with caution in sediments with complex hydrogeology. Our results demonstrate that contrasting subsurface flow regimes significantly impact on the vertical zonation of biogeochemical properties and microorganisms in marine sediments.     

Assessing the ratio of archaeol to caldarchaeol as a salinity proxy in highland lakes on the northeastern Qinghai–Tibetan Plateau

The ratio of archaeol to caldarchaeol (the ACE index) has been proposed recently as an index for paleosalinity reconstruction and is based principally on archaeal core lipids (CLs) from coastal salt pans (Turich, C., Freeman, K.H., 2011. Archaeal lipids record paleosalinity in hypersaline systems. Organic Geochemistry 42, 1147–1157). We have examined possible relationships between salinity and ACE in both CLs and intact polar lipids (IPLs) from suspended particulate matter (SPM) and surface sediments of lakes and surrounding soils on the northeastern Qinghai–Tibetan Plateau. Our results showed that ACE values were positively correlated with salinity in all samples; however, CL ACE values were systematically higher than IPL ACE values, probably due to different degradation kinetics of intact polar (IP) archaeol and IP caldarchaeol. On the other hand, surface sediment ACE values from both CLs and IPLs were lower than SPM ACE values, probably due to enhanced production of caldarchaeol relative to archaeol in the sediment. Our results demonstrate that the ACE proxy reflects changes in salinity in diverse environments on the Qinghai–Tibetan Plateau, which is promising for paleosalinity reconstruction; however, caution should be used when applying the salinity proxy before we have a better understanding of degradation kinetics of archaeal IPLs and in situ production of caldarchaeol and archaeol in sediments.     

Distributions and assemblages of microbial communities along a sediment core retrieved from a potential hydrate-bearing region offshore southwestern Taiwan

Assessing the impacts of methane released from hydrate-bearing environments on global carbon cycling would require detailed insights into the distributions and capacities of microbial communities at different horizons of sediment column. In this study, we conducted geochemical, gene abundance and diversity analyses for a sediment core retrieved from a potential hydrate-bearing region off southwestern Taiwan. Geochemical profiles were characterized by a sulfate-to-methane transition with decreasing total organic carbon and nitrogen in sediments, and increasing dissolved inorganic carbon, ammonium and total sulfur in sediments. Bacterial and archaeal 16S rRNA and amoA gene abundances decreased with depth. In contrast, ANME-1 and -2 16S rRNA gene abundances increased significantly across the sulfate-to-methane transition and peaked at different horizons below this interface. A total of 124,379 bacterial and 130,351 archaeal reads were recovered through tag-pyrosequencing of 16S rRNA genes and categorized into 9014 bacterial and 6394 archaeal operational taxonomic units on the basis of 97% sequence similarity, respectively. Major bacterial phyla/divisions and archaeal groups (>5% of the total reads) detected included Chloroflexi, Planctomycetes, OP9, Deltaproteobacteria, BHI80-139, MBG-B, Halobacteria, MCG, Thermoplasmata, ANME-1 and MG-I. The abundance variations of most major OTUs (>0.5% of the total reads) were statistically correlated with those of geochemical parameters. These lines of evidence suggest that the populations represented by the major OTUs or detected by group-specific primers were compartmentalized into different horizons and involved directly or indirectly in the cycling of methane, sulfate, organic carbon and nitrogen. Overall, this study demonstrates that the deep sequencing coverage combined with the quantification of gene abundance and geochemical characterization would enable to uncover the detailed distributions and potential metabolic capabilities of specific groups from complexly structured microbial communities in methane-rich marine sediments.     

南海东沙深海冷泉区973-5重力柱沉积物古菌多样性

中国南海东沙一带冷泉发育,但目前国内外对深海冷泉区微生物研究甚少,特别是缺乏利用高通量测序的记录。对东沙深海冷泉区973-5站位(该站位水深约3 000 m)长约935 cm的重力岩心进行了高通量分析。结果显示:该站位微生物细胞丰度为5.3×10⁸~34.0×10⁸个/g,随深度变深而增加,其变化趋势与甲烷含量变化可对比,与粒度、有机碳的变化也具有相关性。测序结果显示,岩心中主要古菌类群是MBGB(39.9%)、C3(15.8%)以及ANME-1(12.0%),随着深度的变化群落组成有所改变。硫酸盐-甲烷界面(SMI,760 cm)上下出现了大量的MBGB和ANME-1类群,pH也不断增加,暗示了这一区域存在不断增强的甲烷厌氧氧化作用。岩心底部出现了一定量的ANME-1和ANME-2类群,暗示除了在SMI附近甲烷氧化和硫酸盐还原反应强烈,其下部可能还有水合物的分解与甲烷的上涌,为ANME类群生存提供了营养物质。与东沙海区其他站位相比,973-5站位的甲烷通量较高,但没有发现产甲烷菌,推测该区沉积物中高浓度的甲烷来源为周边浅部或深部断裂系统运移供给。     

Polar and neutral isopranyl glycerol ether lipids as biomarkers of archaea in near-surface sediments from the Nankai Trough

The molecular and carbon isotopic compositions of polar isopranyl glycerol ether lipids, which are direct indicators of viable archaea, and neutral isopranyl glycerol ether lipids, which are derived from polar lipids via hydrolysis, in near-surface sediments from a methane seep in the Nankai Trough (off central Japan) were investigated. Procedures for extracting, separating and derivatizing polar and neutral ether lipids for detection using gas chromatography were first examined with one sediment sample and a cultivated methanogen. For all sediment samples, archaeol and hydroxyarchaeol were detected in both the polar and neutral ether lipid fractions. Acyclic and cyclic biphytanes were also detected in both types of lipid fractions after treatment with HI/LiAlH₄ for ether cleavage and alkylation. The δ¹³C values of archaeol, sn-2-hydroxyarchaeol, and sn-3-hydroxyarchaeol in the sample from 0.82 m below the seafloor were lower than −100‰ relative to PDB, indicating that diverse living methanotrophic archaea are present in the seep sediments. Biphytanes released from polar ether lipids in the same sample were less depleted in δ¹³C (−71‰ to −36‰). The wide range of δ¹³C values suggests that the biphytanes were derived not only from methanotrophic but also from non-methanotrophic archaea, and that the relative contributions of the methanotrophic and non-methanotrophic archaea differed, depending on the biphytane compound. The vertical profiles and δ¹³C values of the neutral ether lipids were similar to those of the intact polar ether lipids, suggesting that neutral ether lipids derived from fossil archaea in the samples had mainly been lost by the time of sampling.     

Structural diversity and fate of intact polar lipids in marine sediments

Marine sediments harbor an enormous quantity of microorganisms, including a multitude of novel species. The habitable zone of the marine sediment column begins at the sediment-water interface and probably extends to depths of several thousands of meters. Studies of the microbial diversity in this ecosystem have mostly relied on molecular biological techniques. We used a complementary method - analysis of intact polar membrane lipids - to characterize the in-situ microbial community in sediments covering a wide range of environmental conditions from Peru Margin, Equatorial Pacific, Hydrate Ridge, and Juan de Fuca Ridge. Bacterial and eukaryotic phospholipids were only detected in surface sediments from the Peru Margin. In contrast, deeply buried sediments, independent of their geographic location, were dominated by archaeal diether and tetraether lipids with various polar head groups and core lipids. We compared ring distributions of archaeal tetraether lipids derived from polar glycosidic precursors with those that are present as core lipids. The distributions of these related compound pools were distinct, suggestive of different archaeal sources, i.e., the polar compounds derive from sedimentary communities and the core lipids are fossil remnants from planktonic communities with possible admixtures of decayed sedimentary archaea. This in-situ production of distinct archaeal lipid populations potentially affects applications of the TEX86 paleotemperature proxy as demonstrated by offsets in reconstructed temperatures between both pools. We evaluated how varying cell and lipid stabilities will influence the sedimentary pool by using a box-model. The results are consistent with (i) a requirement of continuous inputs of freshly synthesized lipids in subsurface sediments for explaining the observed distribution of intact polar lipids, and (ii) decreasing lipid inputs with increasing burial depth.     

南海北部海马冷泉区表层沉积物的AOM生物标志化合物特征及意义

选取采自南海天然气水合物赋存区海马冷泉,管状蠕虫区（ROV06站位）和贻贝区（HM101站位）的2个表层沉积物柱状样品,提取其中的生物标志化合物,对其种类和稳定碳同位素进行了测定,用以探讨海底表层沉积物中的有机质来源、微生物种群分布及其对冷泉渗漏活动的响应特征. 两个站位的沉积物中均发现了大量与甲烷厌氧氧化古菌（ANME）有关的生物标志物,如2,6,11,15?四甲基十六烷（crocetane）、2,6,10,15,19?五甲基二十烷（PMI）等类异戊二烯烃,古醇（archaeol）、sn2?羟基古醇（sn2?OH?Ar）等,以及来源于硫酸盐还原菌（SRB）的异构/反异构脂肪酸iso?C₁₅和ai?C₁₅等. 这些生物标志物均具有极低的碳同位素特征（古菌生标δ13C值低至-126‰,硫酸盐还原菌生标δ13C值低至?89‰）,表明沉积物中发生了甲烷厌氧氧化作用（AOM）. ROV06和HM101站位沉积物中均检测到了crocetane,大多数sn2?羟基古醇/古醇大于1,同时ai?C₁₅/iso?C₁₅脂肪酸比值小于2,这说明两个站位沉积物中的甲烷厌氧氧化古菌主要以ANME?2/DSS为主,指示甲烷渗漏强度较强. ROV06站位的表层沉积物含有crocetane,但sn2?羟基古醇/古醇小于1,且ai?C_15/iso?C₁₅脂肪酸比值大于2.1,指示了ANME?1/DSS和ANME?2/DSS混合存在的种群特征,说明ROV06站位顶部甲烷渗漏强度有减小的趋势. 根据古菌种群ANME?2化合物对甲烷的碳同位素分馏（Δ:-50‰）及古菌生物标志物（PMI、古醇、sn2?羟基古醇）的平均δ13C值,计算得到甲烷δ13C值（-58‰~-53‰）,显示甲烷为热成因和生物成因混合气. 虽然ROV06和HM101站位的甲烷具有相近的δ13C值,但ROV06站位的SRB生物标志物比HM101站位要更加亏损13C（Δδ13C:18‰）,这可能与管状蠕虫的共生菌（硫氧化菌）吸收硫化物并释放出硫酸盐有关,因为其不断释放出的硫酸盐很可能极大地增强了甲烷厌氧氧化作用,使沉积物中含有更多13C亏损的无机碳.      

Phosphate-free ornithine lipid contents in Desulfovibrio spp. respond to growth temperature

Eight sulfate-reducing Desulfovibrio strains isolated from intertidal sediments of the North Sea were investigated for their intact polar lipid (IPL) composition. They contained two types of IPLs, phospholipids and aminolipids. The dominating phospholipids were phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and, in lower concentration, diphosphatidylglycerol (DPG). Aminolipids formed a significant IPL fraction in all strains and high resolution tandem mass spectrometry assigned them as phosphate-free ornithine lipids (OLs). In intertidal flat sediments microorganisms may face strong temperature change on varying timescales and it is crucial for the microbes to maintain constant membrane fluidity, e.g. by modification of their membrane lipid composition. We therefore investigated whether or not these strains employ the same strategies for adjusting their membrane composition to growth temperature and, in particular, how OLs are modified. In all strains the relative OL content was found to be higher at higher growth temperature, in most cases at the expense of PE content and less often PG content. The fatty acid (FA) side chains of the main PE and PG species were similar, i.e. both were dominated by C₁₇ or C₁₈, whereas C₁₅ FAs were additionally found as major OL side chains. The temperature-related side chain variation was similar for all IPLs: unsaturated FA content was lower at higher temperature. The corresponding FA patterns after hydrolysis revealed elevated branched FA content and anteiso/iso ratio at higher growth temperature. As the temperature-related changes in the IPL side chains were similar for all strains, we conclude that side chain modification plays a major role in the maintenance of membrane fluidity at higher temperature and that alternative roles of OLs in the membrane adaptation of Desulfovibrio spp. other than melting point adjustment are possible.     

南海北部冷泉碳酸盐岩中系列藿烷酸的检出及意义

对南海东沙海域九龙甲烷礁冷泉碳酸盐岩的藿烷酸系列化合物进行了研究, 结果显示冷泉碳酸盐岩中含有17β(H), 21β(H)-30-藿烷酸至17β(H), 21β(H)-33-藿烷酸系列化合物. 其中site1样品TVG3-C2(ANME-2古菌种群为主)中的17β(H), 21β(H)-32-藿烷酸化合物亏损13C(-69.8‰), 且其同位素值较接近iso-/aiso-C_15:0脂肪酸(-75.2‰~-90.0‰), 说明其母质摄入了甲烷来源的碳.尽管site2(TVG13-C3和TVG14-C2)和site3(TVG8-C5)自生碳酸盐岩中藿烷酸化合物都表现出了非渗漏特征(-30.7‰~-40.3‰, ANME-1古菌种群为主), 但这些样品中藿烷酸的δ13C值同样较接近硫酸盐还原菌来源的iso-/anteiso-C_15:0的δ13C值(-32.5‰~-49.8‰).由此说明大多数存在于以ANME-1种群为主的渗漏环境中的硫酸盐还原菌并没有参与到甲烷厌氧氧化作用中, 而导致藿烷酸化合物和硫酸盐还原菌来源的生物标志物并不明显亏损13C, 并且δ13C值相近.      

Intact Polar and Core Tetraether Lipids in Sediments from the Haiyang 4 Cold-seep of the Northern South China Sea and their Implications

A number of cold seeps have been discovered in the northern South China Sea (SCS) including the Haiyang 4 cold-seep area where Core 973-5 was collected. Intact polar lipids (IPLs) and core lipids (CLs) were analyzed separately in sediments from Core 973-5. The most abundant lipid biomarkers were isoprenoidal GDGTs (isoGDGTs), with Crenarchaeol and GDGT-0 predominating. IPL-isoGDGTs and CL-isoGDGTs were mainly derived from Thaumarchaeota. IPL-isoGDGTs were mainly produced and retained in situ thus containing most of the in situ microbiological information. Branched GDGTs were predominantly derived from generated in marine production, and mixed with some terrestrial inputs. All IPLs groups presented a high value in the sulfate-methane transition zone (SMTZ). Furthermore, IPL and CL-MI, IPL-R0/4 showed the highest values within the SMTZ, while IPL and CL-R4/i had the lowest values at the SMTZ, suggesting that the contribution of Methanophila and methanogenic to GDGTs increased, while the contribution of ammonia-oxidizing Archaea to GDGTs decreased at the SMTZ.     

Differential degradation of intact polar and core glycerol dialkyl glycerol tetraether lipids upon post-depositional oxidation

Archaeal and bacterial glycerol dialkyl glycerol tetraether lipids (GDGTs) are used in various proxies, such as TEX₈₆ and the BIT index. In living organism, they contain polar head groups (intact polar lipids – IPLs). IPL GDGTs have also been detected in ancient marine sediments and it is unclear whether or not they are fossil entities or are part of living cells. In order to determine the extent of degradation of IPL GDGTs over geological timescales, we analyzed turbidite deposits, which had been partly reoxidized for several kyr after deposition on the Madeira Abyssal Plain. Analysis of core lipid (CL) and IPL-derived GDGTs showed a reduction in concentration by two orders of magnitude upon post-depositional oxidation, while IPL GDGTs with a mono- or dihexose head group decreased by 2–3 orders of magnitude. The BIT index for CL- and IPL-derived GDGTs increased substantially upon oxidation from 0.1 to up to 0.5. Together with changing MBT/CBT values, this indicates preferential preservation of soil-derived branched GDGTs over marine isoprenoid GDGTs, combined with in situ production of branched GDGTs in the sediment. The TEX₈₆ value for IPL-derived GDGTs decreased by 0.07 upon oxidation, while that of CL GDGTs showed no significant change. Isolation of IPLs revealed that the TEX₈₆ value for monohexose GDGTs was 0.55, while the that for dihexose GDGTs was substantially higher, 0.70. Thus, the decrease in TEX₈₆ for IPL-derived GDGTs was in agreement with the dominance of monohexose GDGTs in the oxidized turbidite, probably caused by a combination of in situ production as well as selective preservation of terrestrial isoprenoid GDGTs. Due to the low amount of IPL GDGTs vs. CL GDGTs, the impact of IPL degradation on CL-based TEX₈₆ paleotemperature estimates was negligible.     

Diagnostic lipid biomarker and stable carbon isotope signatures of microbial communities mediating the anaerobic oxidation of methane with sulphate

The anaerobic oxidation of methane (AOM) with sulphate is the most important sink for methane in marine environments. This process is mediated by a consortium of methanotrophic archaea and sulphate reducing bacteria. So far, three groups of anaerobic methane oxidisers (ANME-1, -2 and -3) related to the methanogenic Methanosarcinales and Methanomicrobiales were discovered. The sulphate reducing partner of ANME-1 and -2 are two different eco-types of SRB related to the Desulfosarcina/Desulfococcus cluster (Seep-SRB1), whereas ANME-3 is associated with Desulfobulbus spp. (DBB). In this article, we reviewed literature data to assign statistically significant lipid biomarker signatures for a chemotaxonomic identification of the three known AOM communities. The lipid signatures of ANME-2/Seep-SRB1 and ANME-3/DBB are intriguingly similar, whereas ANME-1/Seep-SRB1 shows substantial differences to these AOM communities. ANME-1 can be distinguished from ANME-2 and -3 by a low ratio of the isoprenoidal dialkyl glycerol diethers sn2-hydroxyarchaeol and archaeol combined with a comparably low stable carbon isotope difference of archaeol relative to the source methane. Furthermore, only ANME-1 contains substantial amounts of isoprenoidal glycerol dialkyl glycerol tetraethers (GDGTs), however, with the probable exception of the ANME-2c sub-cluster. In contrast to the ANME-1 archaea, the tail to tail linked hydrocarbon tetramethylhexadecane (crocetane) is unique to ANME-2, whereas pentamethylicosenes (PMIs) with 4 and 5 double bonds without any higher saturated homologues were only found in ANME-3. The sulphate reducing partner of ANME-1 can be discerned from those of ANME-2 and -3 by a low ratio of the fatty acids (FAs) C_16:1ω5 relative to i-C_15:0 and, although to a lesser degree, by a high abundance of ai-C_15:0 relative to i-C_15:0. Furthermore, substantial amounts of ¹³C depleted non-isoprenoidal monoalkyl glycerol ethers (MAGEs) were only found in the sulphate reducing partners of ANME-2 and -3. A differentiation of these SRB is possible based on the characteristic presence of the FAs cy-C_17:0ω5,6 and C_17:1ω6, respectively. Generally, the data analysed here show overlaps between the different AOM communities, which highlights the need to use multiple lipid signatures for a robust identification of the dominating microbes involved.     

Contrasting Bacterial Dynamics in Subtropical Estuarine and Coastal Waters

Seasonal succession and composition of both attached and free-living bacterial communities were studied in subtropical estuarine and coastal waters with contrasting hydrographic conditions. A higher abundance of attached bacteria was recovered in the estuarine waters containing high concentrations of dissolved organic carbon (DOC) resulting from the freshwater discharge in the adjacent Pearl River, and Proteobacteria, including ??-, ??-, and ??-groups, predominated the attached community at both stations. Free-living bacterial communities at both stations showed higher diversity and lower seasonality than their attached counterparts, and ??-Proteobacteria accounted for the highest proportion at both stations. Redundancy analysis (RDA) demonstrated that, in addition to the obvious temperature effects, DOC and microphytoplankton (>20???m Chl a) drive the temporal variation of attached bacteria at the estuarine and coastal stations, respectively. On the other hand, picophytoplankton (<2???m Chl a) and dissolved oxygen concentration explained most of the free-living bacterial community succession at the estuarine station, while those at the coastal station were associated with micro- and picoplankton (Chl a fractions of <2 and >20???m). These findings suggest that temperature and bottom?Cup effects play a more important role for the spatial?Ctemporal variations of both attached and free-living bacterial communities in the subtropical estuarine and coastal waters.     

南海北部冷泉区沉积物中微生物丰度与甲烷浓度变化关系的初步研究

对2006年“海洋四号”在南海北部陆坡和深水冷泉活动区获得的两个重力活塞岩心中微生物丰度变化和甲烷浓度变化的关系进行了初步研究。荧光显微镜观察和直接计数结果表明,研究区冷泉微生物细胞形态有多种类型,但常见和占优势的是杆状、球状和链状。微生物丰度随不同站位和深度变化于106-105细胞个体/g之间。甲烷浓度高异常站位的微生物丰度大于甲烷浓度低的站位。在同一岩心中,微生物的丰度变化也和甲烷浓度变化相吻合,甲烷浓度高,微生物丰度大,反之亦然。该结果表明了冷泉微生物对沉积物中甲烷浓度的增加或减少十分敏感。     

Separation of core and intact polar archaeal tetraether lipids using silica columns: Insights into living and fossil biomass contributions

Intact polar lipids (IPLs) are frequently used as biomarkers for living microbial cells and can be separated from core lipids (i.e. lipids without polar headgroups), which are mainly derived from fossil (i.e. dead) cell material, using column chromatography. We have compared the effect of various silica column conditions on the separation and recovery of archaeal glycerol dialkyl glycerol tetraether (GDGT) core lipids, glycolipids and phosphoglycolipids using authentic standards and direct analysis with various high performance liquid chromatography-mass spectrometry (HPLC-MS) techniques. The commonly used procedure to separate these compound classes using dichloromethane, acetone and methanol as eluents, respectively, did not separate core GDGTs from glyco- and phosphoglyco-GDGTs. In contrast, a recently described procedure using hexane:ethyl acetate (3:1, v:v), ethyl acetate and methanol achieved both high recovery and successful separation of core GDGTs from the other IPLs. Application of the method to a geothermally heated soil and suspended particulate matter from the North Sea showed that it separates most of the core GDGTs from the other IPLS and that considerable qualitative and quantitative differences can occur between core and IPL-GDGTs. We conclude that the method is therefore appropriate for the separation of intact archaeal IPLs and their fossil analogues.     

Comparison of extraction and work up techniques for analysis of core and intact polar tetraether lipids from sedimentary environments

Glycerol dibiphytanyl glycerol tetraether-based intact polar lipids (IPL GDGTs) are used as biomarkers for living Archaea and are analyzed utilizing a variety of extraction and quantification techniques. Most IPL GDGT studies have used a modified Bligh–Dyer extraction method, but it has been suggested that Soxhlet extraction may be more efficient for environmental samples and biomass. We investigated the impact of three different extractions (Soxhlet, Bligh–Dyer and accelerated solvent extraction, ASE), two IPL quantification methods and two work up techniques (Na₂SO₄ and SiO₂ column) on the amount and distribution of CL (core lipid)- and IPL-derived GDGTs and crenarchaeol-based IPLs in marine sediments from the Arabian Sea and Icelandic shelf, as well as a microbial mat from a Dutch beach. The different extraction procedures gave a similar yield of CL- and IPL-derived GDGTs. Direct analysis of crenarchaeol IPLs showed, however, that, while GDGTs with a monohexose head group were not affected by the extraction method, there was a large effect on IPL GDGTs containing dihexose or hexose, phosphohexose head groups. Quantification of IPL-derived GDGTs by way of either separation over a silica column or by subtraction of CL GDGTs in the total lipid extract before and after hydrolysis gave similar results, but the ‘subtraction-method’ had a relatively large quantification error. However, the silica column, as well as drying over a Na₂SO₄ column, resulted in a loss of the hexose, phosphohexose IPLs by up to 80%. Based on the results, a modified Bligh–Dyer extraction with as little further treatment as possible is recommended to allow measurement of the full range of IPL GDGTs in sediments.