Characterization of microbial community structure in Gulf of Mexico gas hydrates: comparative analysis of DNA- and RNA-derived clone libraries

The characterization of microbial assemblages within solid gas hydrate, especially those that may be physiologically active under in situ hydrate conditions, is essential to gain a better understanding of the effects and contributions of microbial activities in Gulf of Mexico (GoM) hydrate ecosystems. In this study, the composition of the Bacteria and Archaea communities was determined by 16S rRNA phylogenetic analyses of clone libraries derived from RNA and DNA extracted from sediment-entrained hydrate (SEH) and interior hydrate (IH). The hydrate was recovered from an exposed mound located in the northern GoM continental slope with a hydrate chipper designed for use on the manned-submersible Johnson Sea Link (water depth, 550 m). Previous geochemical analyses indicated that there was increased metabolic activity in the SEH compared to the IH layer (B. N. Orcutt, A. Boetius, S. K. Lugo, I. R. Macdonald, V. A. Samarkin, and S. Joye, Chem. Geol. 205:239-251). Phylogenetic analysis of RNA- and DNA-derived clones indicated that there was greater diversity in the SEH libraries than in the IH libraries. A majority of the clones obtained from the metabolically active fraction of the microbial community were most closely related to putative sulfate-reducing bacteria and anaerobic methane-oxidizing archaea. Several novel bacterial and archaeal phylotypes for which there were no previously identified closely related cultured isolates were detected in the RNA- and DNA-derived clone libraries. This study was the first phylogenetic analysis of the metabolically active fraction of the microbial community extant in the distinct SEH and IH layers of GoM gas hydrate.     

Characterization of Microbial Community Structure in Gulf of Mexico Gas Hydrates: Comparative Analysis of DNA- and RNA-Derived Clone Libraries

The characterization of microbial assemblages within solid gas hydrate, especially those that may be physiologically active under in situ hydrate conditions, is essential to gain a better understanding of the effects and contributions of microbial activities in Gulf of Mexico (GoM) hydrate ecosystems. In this study, the composition of the Bacteria and Archaea communities was determined by 16S rRNA phylogenetic analyses of clone libraries derived from RNA and DNA extracted from sediment-entrained hydrate (SEH) and interior hydrate (IH). The hydrate was recovered from an exposed mound located in the northern GoM continental slope with a hydrate chipper designed for use on the manned-submersible Johnson Sea Link (water depth, 550 m). Previous geochemical analyses indicated that there was increased metabolic activity in the SEH compared to the IH layer (B. N. Orcutt, A. Boetius, S. K. Lugo, I. R. Macdonald, V. A. Samarkin, and S. Joye, Chem. Geol. 205:239-251). Phylogenetic analysis of RNA- and DNA-derived clones indicated that there was greater diversity in the SEH libraries than in the IH libraries. A majority of the clones obtained from the metabolically active fraction of the microbial community were most closely related to putative sulfate-reducing bacteria and anaerobic methane-oxidizing archaea. Several novel bacterial and archaeal phylotypes for which there were no previously identified closely related cultured isolates were detected in the RNA- and DNA-derived clone libraries. This study was the first phylogenetic analysis of the metabolically active fraction of the microbial community extant in the distinct SEH and IH layers of GoM gas hydrate.     

Prokaryotic diversity,composition structure,and phylogenetic analysis of microbial communities in leachate sediment ecosystems

In order to obtain insight into the prokaryotic diversity and community in leachate sediment, a culture-independent DNA-based molecular phylogenetic approach was performed with archaeal and bacterial 16S rRNA gene clone libraries derived from leachate sediment of an aged landfill. A total of 59 archaeal and 283 bacterial rDNA phylotypes were identified in 425 archaeal and 375 bacterial analyzed clones. All archaeal clones distributed within two archaeal phyla of the Euryarchaeota and Crenarchaeota, and well-defined methanogen lineages, especially Methanosaeta spp., are the most numerically dominant species of the archaeal community. Phylogenetic analysis of the bacterial library revealed a variety of pollutant-degrading and biotransforming microorganisms, including 18 distinct phyla. A substantial fraction of bacterial clones showed low levels of similarity with any previously documented sequences and thus might be taxonomically new. Chemical characteristics and phylogenetic inferences indicated that (1) ammonium-utilizing bacteria might form consortia to alleviate or avoid the negative influence of high ammonium concentration on other microorganisms, and (2) members of the Crenarchaeota found in the sediment might be involved in ammonium oxidation. This study is the first to report the composition of the microbial assemblages and phylogenetic characteristics of prokaryotic populations extant in leachate sediment. Additional work on microbial activity and contaminant biodegradation remains to be explored.     

Molecular analysis of the bacterial community in a continental high-temperature and water-flooded petroleum reservoir

Water from a continental high-temperature, long-term water-flooded petroleum reservoir in Huabei Oilfield in China was analysed for its bacterial community and diversity. The bacteria were characterized by their 16S rRNA genes. A 16S rRNA gene clone library was constructed from the community DNA, and using restriction fragment length polymorphism analysis, 337 randomly selected clones were clustered with 74 operational taxonomic units. Sequencing and phylogenetic analyses showed that the screened clones were affiliated with Gammaproteobacteria (85.7%), Thermotogales (6.8%), Epsilonproteobacteria (2.4%), low-G+C Gram-positive (2.1%), high-G+C Gram-positive, Betaproteobacteria and Nitrospira (each <1.0%). Thermopilic bacteria were found in the high-temperature water from the flooded petroleum reservoir, as well as mesophilic bacteria such as Pseudomonas-like clones. The mesophilic bacteria were probably introduced into the reservoir as it was being exploited. This work provides significant information on the structure of bacterial communities in high-temperature, long-term water-flooded petroleum reservoirs.     

Denaturing gradient gel electrophoresis (DGGE) analysis of bacterial community composition in deep-sea sediments of the South China Sea

The South China Sea, which is one of the largest marginal seas in the world, is predicted to have suitable accumulation conditions and exporting prospects for natural gas hydrate. The aim of this study was to explore the bacterial community composition of deep-sea sediments from such an ecosystem. DNA was extracted by five different methods and used as templates for PCR amplification of the V3 regions of the 16S rRNA gene. Denaturing gradient gel electrophoresis (DGGE) was used to separate the amplified products and analyse the 16S rRNA gene diversity of sediment samples. The results of DGGE indicated that the bacterial community composition is influenced by DNA extraction methods. Sequencing dominant bands demonstrated that the major phylogenetic groups identified by DGGE belong to Proteobacteria, Bacteroidetes, gram-positive bacteria and Archaea. Integrating different DNA extraction procedures are needed to analyse the actual bacterial diversity from environment when the amplification of 16S rRNA gene and construction of representative clone library were adopted.     

A culture-dependent bacterial community structure analysis based on liquid cultivation and its application to a marine environment

A method for analyzing culture-dependent bacterial community structure by liquid cultivation was established using 96-well microplates. Using 96-well microplates, this method can easily provide accurate enumeration of viable microorganisms and simultaneous separation of bacteria, which allowed us to analyze the bacterial community. Bacteria in diluted surface seawater were separated using 96-well microplates and cultivated with 1/5 ZoBell 2216E liquid medium. The 98 cultures obtained were subsequently applied to phylogenetic analysis based on 16S rRNA gene sequences. The bacterial diversity, evaluated by the Shannon–Weaver index, was relatively small but comparable to previously reported bacterial communities of several environments. The most abundant group was the family Rhodobacteraceae , which has been frequently detected in marine environments. Most bacteria were phylogenetically related to bacteria or uncultured clones detected in marine environments, but distant from published species. The analysis of bacterial community structure by liquid cultivation would be useful as an alternative culture-dependent approach.     

The molecular diversity of the methanogenic community in a hypereutrophic freshwater lake determined by PCR-RFLP

AIMS: To combine database-held sequence information with a programme of experimental molecular ecology to define the methanogenic community of a hypereutrophic lake by a PCR-restriction fragment length polymorphism (RFLP) analysis. METHODS AND RESULTS: Methanogen diversity in a hypereutrophic freshwater lake was analysed using 16S rDNA PCR-RFLP. Database-held 16S rRNA gene sequences for 76 diverse methanogens were analysed for specific restriction sites that permitted unequivocal differentiation of methanogens. Restriction digestion and agarose gel electrophoresis of the 16S rDNA from selected methanogen pure cultures generated observed restriction profiles that corroborated the expected patterns. This method was then tested by analysing methanogen diversity in samples obtained over 1 year from sediment and water samples taken from the same sampling site. CONCLUSIONS: Restriction analysis of the 16S rRNA gene sequences from 157 methanogen clones generated from lakewater and sediment samples showed that over 50% were similar to Methanoculleus spp. Furthermore, a total of 16 RFLP types (1-16) were identified, eight of which contained no cultured representative archaeal 16S rRNA gene sequences. SIGNIFICANCE AND IMPACT OF THE STUDY: This RFLP strategy provides a robust and reliable means to rapidly identify methanogens in the environment.     

Bacterial diversity in deep Mediterranean sediments: relationship with the active bacterial fraction and substrate availability

We investigated vertical distribution and depth-related patterns (from 670 to 2,570 metres) of bacterial diversity in sediment samples collected along a transect in the warm deep Mediterranean sea. Analyses of bacterial diversity were compared with the abundance of benthic bacteria, their metabolically active fraction and the substrates potentially available for their growth. The number of active bacteria was dependent upon the availability of organic substrate in the sediment deriving from phytopigment inputs from the photic layer. The T-RFLP analysis revealed that the surface layers of all sediments analysed were dominated by the same ribotypes, but clear shifts in bacterial community structure were observed in deeper sediment layers. High values of bacterial diversity (expressed as D, H') and evenness (as J) were observed at all stations (a total of 61 ribotypes was identified), and as a result of the large fraction of rare ribotypes (c. 35%), the overall bacterial diversity in the deep sea region investigated was among the highest reported so far in literature. Biodiversity parameters did not display any relationship with water depth, but ribotype richness was related with the number and percentage of active bacteria, suggesting a coupling between organic inputs stimulating bacterial growth and deep-sea bacterial diversity.     

Effect of sample handling on estimation of bacterial diversity in marine sediments by 16S rRNA gene sequence analysis

Abstract The diversity of bacterial communities in deep marine sediments, up to 503 metres below the sea floor of the Japan Sea, was investigated by sequence analysis of amplified 16S rRNA genes. The use of different sample handling procedures greatly affected the types and diversity of sequences obtained. DNA from sediment samples stored aerobically for up to 24 h before freezing was dominated by sequences belonging to the β- and γ-proteobacteria, many of which appeared to originate from aerobic bacteria. Sub-samples equilibrated anaerobically at 16°C, were then injected with a radiotracer and immediately frozen, to simulate the conditions of a typical control sample from a radiotracer based activity assay, contained mostly α-proteobacterial sequences. Pristine sediment samples taken anaerobically and frozen within 2 h contained the widest diversity of sequences from α-, γ-, δ-proteobacteria and Gram-positive bacteria, which appeared to have originated from predominantly anaerobic or facultative bacteria. It was clear that both samples that were not frozen immediately (within 2 h) showed signs of enrichment of specific bacterial groups. Our results strongly suggest that immediate freezing should always be employed when sediment samples are to be used to assess bacterial diversity by molecular methods.     

基于16S rRNA基因序列分析受砷和硫酸盐污染的土壤细菌多样性（英文稿）

为了了解普通耕地土壤(Nor-1)和受砷及硫酸盐污染土壤(Sul-1)中的细菌组成和多样性差异,对2个不同土壤样品直接提取总DNA,通过PCR扩增16S rRNA基因并建立文库,对文库克隆进行核糖体DNA扩增片段酶切分析(ARDRA)和测序,构建系统进化树。从Nor-1土壤样品中测序获得23个16S rRNA基因序列,分析序列系统发育关系表明,共包含Acidobacteria(12.3%,8/65)、Actinobacteria(3.1%,2/65)、Firmicutes(21.5%,14/65)、Nitrospira(3.1%,2/65)和Proteobacteria(60%,39/65)等5个不同细菌门。而从Sul-1土壤样品中测序获得19个16S rRNA基因序列,分析序列系统发育关系表明,共包含Firmicutes(29.5%,13/44)和Proteobacteria(70.5%,31/44)等2个不同细菌门。结果表明,受高浓度的砷和硫酸盐的影响,Sul-1土壤中细菌群落结构相较于普通耕地土壤(Nor-1)发生了明显的改变,多样性明显下降,但有大量具有较强的污染物降解能力的不动杆菌(Acinetobacter)相关序列在Sul-1土壤细菌群落中被发现。