Bacterial enzymes for dissimilatory sulfate reduction in a marine microbial mat (Black Sea) mediating anaerobic oxidation of methane

Anaerobic oxidation of methane (AOM) with sulfate is catalysed by microbial consortia of archaea and bacteria affiliating with methanogens and sulfate-reducing Deltaproteobacteria respectively. There is evidence that methane oxidation is catalysed by enzymes related to those in methanogenesis, but the enzymes for sulfate reduction coupled to AOM have not been examined. We collected microbial mats with high AOM activity from a methane seep in the Black Sea. The mats consisted mainly of archaea of the ANME-2 group and bacteria of the Desulfosarcina-Desulfococcus group. Cell-free mat extract contained activities of enzymes involved in sulfate reduction to sulfide: ATP sulfurylase (adenylyl : sulfate transferase; Sat), APS reductase (Apr) and dissimilatory sulfite reductase (Dsr). We partially purified the enzymes by anion-exchange chromatography. The amounts obtained indicated that the enzymes are abundant in the mat, with Sat accounting for 2% of the soluble mat protein. N-terminal amino acid sequences of purified proteins suggested similarities to the corresponding enzymes of known species of sulfate-reducing bacteria. The deduced amino acid sequence of PCR-amplified genes of the Apr subunits is similar to that of Apr of the Desulfosarcina/Desulfococcus group. These results indicate that the major enzymes involved in sulfate reduction in the Back Sea microbial mats are of bacterial origin, most likely originating from the bacterial partner in the consortium.     

南海Formosa冷泉区沉积物微生物多样性与分布规律研究

【目的】当前对全球冷泉生态系统微生物生态学研究显示，冷泉生态系统中主要微生物类群为参与甲烷代谢的微生物，它们的分布差异与所处冷泉区生物地球化学环境密切相关。但在冷泉区内也存在环境因子截然不同的生境，尚缺乏比较冷泉区内小尺度生境间微生物多样性和分布规律的研究。本研究旨在分析南海Formosa冷泉区内不同生境间微生物多样性差异，完善和理解不同环境因子对冷泉内微生物群落结构的影响。【方法】对采集自南海Formosa冷泉区不同生境(黑色菌席区、白色菌席区和碳酸盐岩区)沉积物样本中古菌和细菌16S rRNA基因进行测序，结合环境因子，比较微生物多样性差异，分析环境因子对微生物分布的影响。【结果】发现在Formosa冷泉内的不同生境中，甲烷厌氧氧化古菌(anaerobic methanotrophic archaea,ANME)是主要古菌类群，占古菌总体相对丰度超过70%；在菌席区ANME-1b和ANME-2a/b是主要ANME亚群，碳酸盐岩区则是ANME-1b。硫酸盐还原菌(sulfate-reducing bacteria,SRB)和硫氧化菌(sulfur-oxidizing bacteria...     

Diversity and abundance of aerobic and anaerobic methane oxidizers at the Haakon Mosby Mud Volcano, Barents Sea

Submarine mud volcanoes are formed by expulsions of mud, fluids, and gases from deeply buried subsurface sources. They are highly reduced benthic habitats and often associated with intensive methane seepage. In this study, the microbial diversity and community structure in methane-rich sediments of the Haakon Mosby Mud Volcano (HMMV) were investigated by comparative sequence analysis of 16S rRNA genes and fluorescence in situ hybridization. In the active volcano center, which has a diameter of about 500 m, the main methane-consuming process was bacterial aerobic oxidation. In this zone, aerobic methanotrophs belonging to three bacterial clades closely affiliated with Methylobacter and Methylophaga species accounted for 56%+/-8% of total cells. In sediments below Beggiatoa mats encircling the center of the HMMV, methanotrophic archaea of the ANME-3 clade dominated the zone of anaerobic methane oxidation. ANME-3 archaea form cell aggregates mostly associated with sulfate-reducing bacteria of the Desulfobulbus (DBB) branch. These ANME-3/DBB aggregates were highly abundant and accounted for up to 94%+/-2% of total microbial biomass at 2 to 3 cm below the surface. ANME-3/DBB aggregates could be further enriched by flow cytometry to identify their phylogenetic relationships. At the outer rim of the mud volcano, the seafloor was colonized by tubeworms (Siboglinidae, formerly known as Pogonophora). Here, both aerobic and anaerobic methane oxidizers were found, however, in lower abundances. The level of microbial diversity at this site was higher than that at the central and Beggiatoa species-covered part of the HMMV. Analysis of methyl-coenzyme M-reductase alpha subunit (mcrA) genes showed a strong dominance of a novel lineage, mcrA group f, which could be assigned to ANME-3 archaea. Our results further support the hypothesis of Niemann et al. (54), that high methane availability and different fluid flow regimens at the HMMV provide distinct niches for aerobic and anaerobic methanotrophs.     

Diversity, activity, and abundance of sulfate-reducing bacteria in saline and hypersaline soda lakes

Soda lakes are naturally occurring highly alkaline and saline environments. Although the sulfur cycle is one of the most active element cycles in these lakes, little is known about the sulfate-reducing bacteria (SRB). In this study we investigated the diversity, activity, and abundance of SRB in sediment samples and enrichment cultures from a range of (hyper)saline soda lakes of the Kulunda Steppe in southeastern Siberia in Russia. For this purpose, a polyphasic approach was used, including denaturing gradient gel electrophoresis of dsr gene fragments, sulfate reduction rate measurements, serial dilutions, and quantitative real-time PCR (qPCR). Comparative sequence analysis revealed the presence of several novel clusters of SRB, mostly affiliated with members of the order Desulfovibrionales and family Desulfobacteraceae. We detected sulfate reducers and observed substantial sulfate reducing rates (between 12 and 423 micromol/dm(3) day(-1)) for most lakes, even at a salinity of 475 g/liter. Enrichments were obtained at salt saturating conditions (4 M Na(+)), using H(2) or volatile fatty acids as electron donors, and an extremely halophilic SRB, strain ASO3-1, was isolated. Furthermore, a high dsr gene copy number of 10(8) cells per ml was detected in a hypersaline lake by qPCR. Our results indicate the presence of diverse and active SRB communities in these extreme ecosystems.     

Microbial stratification in deeply buried marine sediment reflects changes in sulfate/methane profiles

We examined sediments collected at Ocean Drilling Program (ODP) Leg 201 Site 1229 on the Peru Margin for microbial populations throughout the sediment column. Heterotrophic cultivation from these sediments yielded numerous colonies from various depths, including 49 bacterial isolates. At ODP Site 1229, there are significant interfaces of sulfate and methane, across which microbial cell numbers increase substantially. At these sulfate/methane transition zones (SMTZs), however, we observed a decrease in the success rate for the cultivation of bacterial colonies. Utilizing both direct plating and enrichment in different media, we cultivated isolates from the upper SMTZ around 30 m below seafloor (mbsf); however, similar attempts yielded no colonies from within the lower zone at 85 mbsf. The phylogenetic relationships of the 16S rRNA gene sequences for the isolates were determined and most were related to other organisms and sequences previously found in the subsurface belonging to the γ‐Proteobacteria, cytophaga–flavobacterium–bacteroides, high G + C Gram‐positives, and Firmicutes groups. The most diverse group of isolates from Site 1229 was found between the SMTZs at 50 mbsf. ODP Leg 201 Site 1228 was examined for comparison and yielded an additional 18 isolates from 16 to 179 mbsf that were similar to those found at Site 1229. Direct plating at Site 1228 also showed decreased colony formation in the area of sulfate/methane transition. Our results suggest that heterotrophic bacterial populations are affected by SMTZs in deeply buried sediment.     

Anaerobic methane oxidation and sulfate reduction in bacterial mats of coral-like carbonate structures in the Black Sea

A detailed study of the processes of anaerobic methane oxidation and sulfate reduction in the bacterial mats occurring on coral-like carbonate structures in the region of methane seeps in the Black Sea, as well as of the phenotypic diversity of sulfate-reducing bacteria developing in this zone, has been performed. The use of the radioisotopic method shows the microbial mat structure to be heterogeneous. The peak activity of the two processes was revealed when a mixture of the upper (dark) and underlying (intensely pink) layers was introduced into an incubation flask, which confirms the suggestion that methanotrophic archaea and sulfate-reducing bacteria closely interact in the process of anaerobic methane oxidation. Direct correlation between the rate of anaerobic methane oxidation and the methane and electron acceptor concentrations in the medium has been experimentally demonstrated. Several enrichment and two pure cultures of sulfate-reducing bacteria have been obtained from the near-bottom water and bacterial mats. Both strains were found to completely oxidize the substrates to CO2 and H2S. The bacteria grow at temperatures ranging from -1 to 18 (24) degrees C, with an optimum in the 10-18 degrees C range, and require the presence of 1.5-2.5% NaCl and 0.07-0.2% MgCl2 x 6H2O. Regarding the aggregate of their phenotypic characteristics (cell morphology, spectrum of growth substrates, the capacity for complete oxidation), the microorganisms isolated have no analogues among the psychrophilic sulfate-reducing bacteria already described. The results obtained demonstrate the wide distribution of psychrophilic sulfate-reducing bacteria in the near-bottom water and bacterial mats covering the coral-like carbonate structures occurring in the region of methane seeps in the Black Sea, as well as the considerable catabolic potential of this physiological group of psychrophilic anaerobes in deep-sea habitats.     

中国南海沉积环境可培养细菌多样性研究

【目的】探索海洋沉积环境中可培养细菌的多样性。【方法】采用纯培养分离及16S rRNA基因序列鉴定的方法,对我国南海海域20个沉积物样品进行细菌多样性分析。【结果】共获得200株细菌,分属于47个属,99个种。经系统进化分析,可培养菌株主要分布于4个类群:厚壁菌门(Firmicutes)、变形菌门(Proteobacteria)、放线菌门(Actinobacteria)和拟杆菌门(Bacteroidetes),优势类群为Firmicutes,其中芽孢杆菌属(Bacillus)所占比例为55.6%;而Actinobacteria和Bacteroidetes两个类群获得菌株较少;在Firmicutes和Actinobacteria两个类群中发现8个潜在新种和3个潜在新属级类群。【结论】初步研究结果表明,南海海洋沉积环境可培养微生物资源丰富,新物种资源多样;其中,芽孢杆菌为海洋沉积环境中的优势类群,随着样品深度的增加,细菌多样性呈现递减的趋势,深度可能是影响细菌多样性的一个重要因素;其次,分离培养基和分离方法直接关系到样品中可培养微生物多样性的发现,有待深入研究。     

Activity and community structures of sulfate-reducing microorganisms in polar,temperate and tropical marine sediments

Temperature has a fundamental impact on the metabolic rates of microorganisms and strongly influences microbial ecology and biogeochemical cycling in the environment. In this study, we examined the catabolic temperature response of natural communities of sulfate-reducing microorganisms (SRM) in polar, temperate and tropical marine sediments. In short-term sediment incubation experiments with ³⁵S-sulfate, we demonstrated how the cardinal temperatures for sulfate reduction correlate with mean annual sediment temperatures, indicating specific thermal adaptations of the dominant SRM in each of the investigated ecosystems. The community structure of putative SRM in the sediments, as revealed by pyrosequencing of bacterial 16S rRNA gene amplicons and phylogenetic assignment to known SRM taxa, consistently correlated with in situ temperatures, but not with sediment organic carbon concentrations or C:N ratios of organic matter. Additionally, several species-level SRM phylotypes of the class Deltaproteobacteria tended to co-occur at sites with similar mean annual temperatures, regardless of geographic distance. The observed temperature adaptations of SRM imply that environmental temperature is a major controlling variable for physiological selection and ecological and evolutionary differentiation of microbial communities.     

Response of sulfate-reducing bacteria to an artificial oil-spill in a coastal marine sediment

In situ mesocosm experiments using a calcareous sand flat from a coastal area of the island of Mallorca in the Mediterranean Sea were performed in order to study the response of sulfate-reducing bacteria (SRB) to controlled crude oil contamination, or heavy contamination with naphthalene. Changes in the microbial community caused by the contamination were monitored by a combination of comparative sequence analysis of 16S rRNA genes, fluorescence in situ hybridization, cultivation approaches and metabolic activity rates. Our results showed that crude oil and naphthalene negatively influenced the total microbial community as the natural increase in cell numbers due to the seasonal dynamics was attenuated. However, both contaminants enhanced the sulfate reduction rates, as well as the culturability of SRB. Our results suggested the presence of autochthonous deltaproteobacterial SRBs that were able to degrade crude oil or polycyclic aromatic hydrocarbons such as naphthalene in anaerobic sediment layers.     

Na+-dependent accumulation of sulfate and thiosulfate in marine sulfate-reducing bacteria

Uptake of ³⁵S-labelled sulfate and thiosulfate was studied in twenty sulfate-reducing bacteria. Micromolar additions of these substrates were highly accumulated by washed cells of freshwater and marine strains. In marine strains accumulation required Na⁺. Generally, the uptake capacity was increased after sulfate limitation during growth. With two marine species, Desulfovibrio salexigens and Desulfobacterium autotrophicum, the effects of various ionophores and inhibitors affecting the transmembrane pH or Na⁺ gradient or the membrane potential were studied. In both strains transport was reversible. There was no discrimination between sulfate and thiosulfate. With increasing additions the amount taken up increased, while the accumulation factor (C_in/C_out) decreased. Uptake was not directly correlated with the ATP level inside the cells. From these results and the action patterns of the inhibitors tested it is concluded that marine sulfate-reducing bacteria accumulate sulfate and thiosulfate electrogenically in symport with Na⁺ ions, while in freshwater strains protons are symported. The high-accumulating systems are induced only at low sulfate concentration, while low-accumulating systems are active at sulfate-sufficient conditions.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DCCD dicyclohexylcarbodiimide - ETH 157 N, N-dibenzyl-N,N-diphenyl-1,2-diphenylendioxydiacetamide - TCS 3,3,4,5-tetrachlorosalicylanilide     

Biogeochemical and molecular signatures of anaerobic methane oxidation in a marine sediment

Anaerobic methane oxidation was investigated in 6-m-long cores of marine sediment from Aarhus Bay, Denmark. Measured concentration profiles for methane and sulfate, as well as in situ rates determined with isotope tracers, indicated that there was a narrow zone of anaerobic methane oxidation about 150 cm below the sediment surface. Methane could account for 52% of the electron donor requirement for the peak sulfate reduction rate detected in the sulfate-methane transition zone. Molecular signatures of organisms present in the transition zone were detected by using selective PCR primers for sulfate-reducing bacteria and for Archaea. One primer pair amplified the dissimilatory sulfite reductase (DSR) gene of sulfate-reducing bacteria, whereas another primer (ANME) was designed to amplify archaeal sequences found in a recent study of sediments from the Eel River Basin, as these bacteria have been suggested to be anaerobic methane oxidizers (K. U. Hinrichs, J. M. Hayes, S. P. Sylva, P. G. Brewer, and E. F. DeLong, Nature 398:802-805, 1999). Amplification with the primer pairs produced more amplificate of both target genes with samples from the sulfate-methane transition zone than with samples from the surrounding sediment. Phylogenetic analysis of the DSR gene sequences retrieved from the transition zone revealed that they all belonged to a novel deeply branching lineage of diverse DSR gene sequences not related to any previously described DSR gene sequence. In contrast, DSR gene sequences found in the top sediment were related to environmental sequences from other estuarine sediments and to sequences of members of the genera Desulfonema, Desulfococcus, and Desulfosarcina. Phylogenetic analysis of 16S rRNA sequences obtained with the primers targeting the archaeal group of possible anaerobic methane oxidizers revealed two clusters of ANME sequences, both of which were affiliated with sequences from the Eel River Basin.     

The effect of sulfate and nitrate on methane formation in a freshwater sediment

A freshwater sediment from a ditch of a peat grassland near Zegveld (Province of Utrecht, The Netherlands) was investigated for its potential methanogenic and syntrophic activity and the influence of sulfate and nitrate on these potential activities. Methanogenesis started after a 10 days lagphase. After 35–40 days aceticlastic methanogens were sufficiently enriched to cause a net decrease of acetate. In the presence of sulfate methane formation was only slightly affected. The addition of nitrate led to an outcompetion of aceticlastic methanogens by nitrate reducers. When inorganic electron acceptors were absent, substrates like propionate and butyrate were converted by syntrophic methanogenic consortia. Addition of inorganic electron acceptors resulted in an outcompetition of the syntrophic propionate and butyrate degrading consortia by the sulfate and nitrate reducers.     

Inorganic carbon fixation by sulfate-reducing bacteria in the Black Sea water column

The Black Sea is the largest anoxic water basin on Earth and its stratified water column comprises an upper oxic, middle suboxic and a lower permanently anoxic, sulfidic zone. The abundance of sulfate-reducing bacteria (SRB) in water samples was determined by quantifying the copy number of the dsrA gene coding for the alpha subunit of the dissimilatory (bi)sulfite reductase using real-time polymerase chain reaction. The dsrA gene was detected throughout the whole suboxic and anoxic zones. The maximum dsrA copy numbers were 5 x 10(2) and 6.3 x 10(2) copies ml(-1) at 95 m in the suboxic and at 150 m in the upper anoxic zone, respectively. The proportion of SRB to total Bacteria was 0.1% in the oxic, 0.8-1.9% in the suboxic and 1.2-4.7% in the anoxic zone. A phylogenetic analysis of 16S rDNA clones showed that most clones from the anoxic zone formed a coherent cluster within the Desulfonema-Desulfosarcina group. A similar depth profile as for dsrA copy numbers was obtained for the concentration of non-isoprenoidal dialkyl glycerol diethers (DGDs), which are most likely SRB-specific lipid biomarkers. Three different DGDs were found to be major components of the total lipid fractions from the anoxic zone. The DGDs were depleted in (13)C relative to the delta(13)C values of dissolved CO(2) (delta(13)C(CO2)) by 14-19 per thousand. Their delta(13)C values [delta(13)C(DGD(II-III))] co-varied with depth showing the least (13)C-depleted values in the top of the sulfidic, anoxic zone and the most (13)C-depleted values in the deep anoxic waters at 1500 m. This co-variation provides evidence for CO(2) incorporation by the DGD(II-III)-producing SRB, while the 1:2 relationship between delta(13)C(CO2) and delta(13)C(DGD(II-III)) indicates the use of an additional organic carbon source.     

Multi-scale factors affecting composition,diversity, and abundance of sediment denitrifying microorganisms in Yangtze lakes

Applied Microbiology and Biotechnology - Sediment denitrification is the dominant nitrogen removal pathway in many aquatic habitats and can be regulated by local-, landscape-, and regional-scale...     

Diversity of piezophilic microorganisms in the closed ocean Japan Sea

Deep-sea sediment samples were collected at a depth of 3,064 m in the Japan Sea. Microorganisms in the sediment sample were cultivated under several pressure conditions, and the high-pressure adapted microbes were isolated. Two of the isolates exhibited piezophilic growth profiles. This is the first report to show the presence of piezophiles in the Japan Sea.     

The impact of the marine sewage outfalls on the sediment quality: The Black Sea and the Marmara case

Marine sewage outfalls are the cheapest method of disposal for municipalities with restricted funds, despite their negative effects on marine ecosystems. This study was conducted on the marine sewage outfalls of the Turkish cities Rize (Black Sea) and Yalova (Sea of Marmara). Heavy metals, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and organochlorine pesticides (OCPs) were investigated in the sediments for determine pollution degree. Two of Yalova stations were classified as having high contamination levels (1034.436 and 1476.483 ng/g, respectively) and all the other stations suffered moderate pollution in terms of ∑₁₆PAHs. ∑DTTs in sediments above probable effect levels (PELs), were observed both stations Yalova and Rize. The highest metal concentrations in the sediments from the eight stations ranged from 4.97 to 29.65 mg/kg for Ni; 7.57–44.14 mg/kg for Cr; 9.84–42.76 mg/kg for Cu; and 48.08–103.77 mg/kg for Zn. In sum, examination of pollutant sources provides clear evidence of an anthropogenic effect on the discharge environments.