Molecular phylogenetic diversity of the microbial community associated with a high-temperature petroleum reservoir at an offshore oilfield

The microbial community and its diversity in production water from a high-temperature, water-flooded petroleum reservoir of an offshore oilfield in China were characterized by 16S rRNA gene sequence analysis. The bacterial and archaeal 16S rRNA gene clone libraries were constructed from the community DNA and, using sequence analysis, 388 bacterial and 220 archaeal randomly selected clones were clustered with 60 and 28 phylotypes, respectively. The results showed that the 16S rRNA genes of bacterial clones belonged to the divisions Firmicutes, Thermotogae, Nitrospirae and Proteobacteria, whereas the archaeal library was dominated by methanogen-like rRNA genes (Methanothermobacter, Methanobacter, Methanobrevibacter and Methanococcus), with a lower percentage of clones belonging to Thermoprotei. Thermophilic microorganisms were found in the production water, as well as mesophilic microorganisms such as Pseudomonas and Acinetobacter-like clones. The thermophilic microorganisms may be common inhabitants of geothermally heated specialized subsurface environments, which have been isolated previously from a number of high-temperature petroleum reservoirs worldwide. The mesophilic microorganisms were probably introduced into the reservoir as it was being exploited. The results of this work provide further insight into the composition of microbial communities of high-temperature petroleum reservoirs at offshore oilfields.     

Phylogenetic Diversity of the Archaeal Community in a Continental High-Temperature,Water-Flooded Petroleum Reservoir

The diversity of an archaeal community was analyzed in the water from a continental high-temperature, long-term water-flooded petroleum reservoir in Huabei Oilfield in China. The archaea were characterized by their 16S rRNA genes. An archaeal 16S rDNA clone library was constructed from the DNA isolated from the formation water, and 237 randomly selected positive clones were clustered in 28 phylotypes by sequencing analyses. Phylogenetic analysis of these sequences indicated that the dominant members of the archaeal phylotypes were affiliated with the order Methanomicrobiales. Totally, the archaeal community was composed of methanogens belonging to four orders: Methanobacteriales, Methanococcales, Methanomicrobiales, and Methanosarcinales. Most of the clones clustered with sequences previously described for methanogens, but there was a difference in the relative distribution of sequences detected here as compared to that of previous studies. Some thermophilic methanogens detected had been previously isolated from a number of high-temperature petroleum reservoirs worldwide; thus, they might exhibit adaptations to the environments and be the common habitants of geothermally heated subsurface environments.     

Phylogenetic diversity of bacteria in an earth-cave in Guizhou province, southwest of China

The objective of this study was to analyze the phylogenetic composition of bacterial community in the soil of an earth-cave (Niu Cave) using a culture-independent molecular approach. 16S rRNA genes were amplified directly from soil DNA with universally conserved and Bacteria-specific rRNA gene primers and cloned. The clone library was screened by restriction fragment length polymorphism (RFLP), and representative rRNA gene sequences were determined. A total of 115 bacterial sequence types were found in 190 analyzed clones. Phylogenetic sequence analyses revealed novel 16S rRNA gene sequence types and a high diversity of putative bacterial community. Members of these bacteria included Proteobacteria (42.6%), Acidobacteria (18.6%), Planctomycetes (9.0%), Chloroflexi (Green nonsulfur bacteria, 7.5%), Bacteroidetes (2.1%), Gemmatimonadetes (2.7%), Nitrospirae (8.0%), Actinobacteria (High G+C Gram-positive bacteria, 6.4%) and candidate divisions (including the OP3, GN08, and SBR1093, 3.2%). Thirty-five clones were affiliated with bacteria that were related to nitrogen, sulfur, iron or manganese cycles. The comparison of the present data with the data obtained previously from caves based on 16S rRNA gene analysis revealed similarities in the bacterial community components, especially in the high abundance of Proteobacteria and Acidobacteria. Furthermore, this study provided the novel evidence for presence of Gemmatimonadetes, Nitrosomonadales, Oceanospirillales, and Rubrobacterales in a karstic hypogean environment.     

New evidence for bacterial diversity in the ascoma of the ectomycorrhizal fungus Tuber borchii Vittad

The microbial community associated with ascocarps of the ectomycorrhizal fungus Tuber borchii Vittad. was studied by both cultivation and direct extraction of bacterial 16S rRNA gene (rDNA) sequence approaches. The inner part of six T. borchii ascoma collected in North-Central Italy was used to establish a bacterial culture collection and to extract the total genomic DNA to obtain a library of 16S rDNAs representative of the truffle bacterial community. Most of the isolates were affiliated to the gamma-Proteobacteria, mainly Fluorescent pseudomonads; some isolates were members of the Bacteroidetes group and Gram-positive bacteria, mostly Bacillaceae. The majority of the clones from the library were alpha-Proteobacteria showing significant similarity values, of greater than 97%, with members of the Sinorhizobium/Ensifer Group, Rhizobium and Bradyrhizobium spp. not previously identified as Tuber-associated bacteria. Only a few bacterial strains belonging to this bacterial subclass were found in the culture collection and isolated on a medium specific for Rhizobium-like organisms. A few clones were members of the beta- and gamma-Proteobacteria; as well as low and high G+C Gram-positive bacteria. Our findings clearly indicate that a dual approach increases the information obtained on the structural composition of a truffle bacterial community as compared to that derived via cultivation or direct recovery of 16S rDNA sequences alone.     

Phylogenetic diversity and activity of anaerobic microorganisms of high-temperature horizons of the Dagang Oilfield (China)

The number of microorganisms of major metabolic groups and the rates of sulfate-reducing and methanogenic processes in the formation waters of the high-temperature horizons of Dagang oilfield have been determined. Using cultural methods, it was shown that the microbial community contained aerobic bacteria oxidizing crude oil, anaerobic fermentative bacteria, sulfate-reducing bacteria, and methanogenic bacteria. Using cultural methods, the possibility of methane production from a mixture of hydrogen and carbon dioxide (H2 + CO2) and from acetate was established, and this result was confirmed by radioassays involving NaH14CO3 and 14CH3COONa. Analysis of 16S rDNA of enrichment cultures of methanogens demonstrated that these microorganisms belong to Methanothermobacter sp. (M. thermoautotrophicus), which consumes hydrogen and carbon dioxide as basic substrates. The genes of acetate-utilizing bacteria were not identified. Phylotypes of the representatives of Thermococcus spp. were found among 16S rDNAs of archaea. 16S rRNA genes of bacterial clones belong to the orders Thermoanaerobacteriales (Thermoanaerobacter, Thermovenabulum, Thermacetogenium, and Coprothermobacter spp.), Thermotogales, Nitrospirales (Thermodesulfovibrio sp.) and Planctomycetales. 16S rDNA of a bacterium capable of oxidizing acetate in the course of syntrophic growth with H2-utilizing methanogens was found at high-temperature petroleum reservoirs for the first time. These results provide further insight into the composition of microbial communities of high-temperature petroleum reservoirs, indicating that syntrophic processes play an important part in acetate degradation accompanied by methane production.     

Turkey fecal microbial community structure and functional gene diversity revealed by 16S rRNA gene and metagenomic sequences

The primary goal of this study was to better understand the microbial composition and functional genetic diversity associated with turkey fecal communities. To achieve this, 16S rRNA gene and metagenomic clone libraries were sequenced from turkey fecal samples. The analysis of 382 16S rRNA gene sequences showed that the most abundant bacteria were closely related to Lactobacillales (47%), Bacillales (31%), and Clostridiales (11%). Actinomycetales, Enterobacteriales, and Bacteroidales sequences were also identified, but represented a smaller part of the community. The analysis of 379 metagenomic sequences showed that most clones were similar to bacterial protein sequences (58%). Bacteriophage (10%) and avian viruses (3%) sequences were also represented. Of all metagenomic clones potentially encoding for bacterial proteins, most were similar to low G+C Gram-positive bacterial proteins, particularly from Lactobacillales (50%), Bacillales (11%), and Clostridiales (8%). Bioinformatic analyses suggested the presence of genes encoding for membrane proteins, lipoproteins, hydrolases, and functional genes associated with the metabolism of nitrogen and sulfur containing compounds. The results from this study further confirmed the predominance of Firmicutes in the avian gut and highlight the value of coupling 16S rRNA gene and metagenomic sequencing data analysis to study the microbial composition of avian fecal microbial communities.     

Phylogenetic Diversity of Bacteria Associated with the Marine Sponge <Emphasis Type="Italic">Gelliodes carnosa</Emphasis> Collected from the Hainan Island Coastal Waters of the South China Sea

Several molecular techniques were employed to document the bacterial diversity associated with the marine sponge Gelliodes carnosa. Cultivation-dependent and cultivation-independent methods were used to obtain the 16S rRNA gene sequences of the bacteria. Phylogenetic analysis based on the 16S rRNA gene sequences showed that the bacterial community structure was highly diverse with representatives of the high G + C Gram-positive bacteria, cyanobacteria, low G + C Gram-positive bacteria, and proteobacteria (α-, β-, and γ-), most of which were also found in other marine environments, including in association with other sponges. Overall, 300 bacterial isolates were cultivated, and a total of 62 operational taxonomic units (OTUs) were identified from these isolates by restriction fragment length polymorphism (RFLP) analysis and DNA sequencing of the 16S rRNA genes. Approximately 1,000 16S rRNA gene clones were obtained by the cultivation-independent method. A total of 310 clones were randomly selected for RFLP analysis, from which 33 OTUs were acquired by further DNA sequencing and chimera checking. A total of 12 cultured OTUs (19.4% of the total cultured OTUs) and 13 uncultured OTUs (39.4% of the total uncultured OTUs) had low sequence identity (≤97%) with their closest matches in GenBank and were probably new species. Our data provide strong evidence for the presence of a diverse variety of unidentified bacteria in the marine sponge G. carnosa. A relatively high proportion of the isolates exhibited antimicrobial activity, and the deferred antagonism assay showed that over half of the active isolates exhibited a much stronger bioactivity when grown on medium containing seawater. In addition to demonstrating that the sponge-associated bacteria could be a rich source of new biologically active natural products, the results may have ecological implications. This study expands our knowledge of the diversity of sponge-associated bacteria and contributes to the growing database of the bacterial communities within sponges.     

Quantifying bacterial population dynamics in compost using 16S rRNA gene probes

Composting provides a dynamic setting for studying ecological topics such as succession, competition, and community stability in a relatively short period of time. This study used hierarchical small sub-unit-based rRNA gene probes to quantify the change in the relative abundance of phylogenetic groups common to compost in laboratory scale reactors. Bacterial 16S rRNA gene targets accounted for only 37% of all small subunit (SSU) rRNA genes initially, but increased to a maximum of 83% of the total at 84 h. The sum of rRNA genes detected using probes specific to Pseudomonas and low-G+C Gram-positive rRNA genes represented between 16% and 87% of the total. The lack of hybridization to the taxon-specific probes was most pronounced between 36 h and 60 h, when the pH was between 4.6 and 4.8. During this period the relative abundance of taxon-specific gene targets accounted for only 17–33% of the total bacterial rRNA gene targets. Pseudomonas-type 16S rRNA genes were the most abundant of the groups measured until 72 h. Those genes had their highest relative abundance at 12 h (78% of bacterial rRNA genes; 30% of all rRNA genes), after which time their relative abundance began to decline as the temperature increased. Prior to 72 h, 16S rRNA genes from low-G+C Gram-positive bacteria (LGC-GPB) represented less than 7% of the bacterial rRNA genes. However, by 84 h the relative abundance of LGC-GPB and Bacillus rRNA genes had increased to 60% and 18% of the bacterial rRNA gene targets, respectively (50% and 15% of all rRNA genes, respectively).     

Gram-positive bacteria with a high DNA G+C content are characterized by a common insertion within their 23S rRNA genes.

An insertion of about 100 bases within the central part of the 23S rRNA genes was found to be a phylogenetic marker for the bacterial line of descent of Gram-positive bacteria with a high DNA G + C content. The insertion was present in 23S rRNA genes of 64 strains representing the major phylogenetic groups of Gram-positive bacteria with a high DNA G+C content, whereas it was not found in 23S rRNA genes of 55 (eu)bacteria representing Gram-positive bacteria with a low DNA G + C content and all other known (eu)bacterial phyla. The presence of the insertion could be easily demonstrated by comparative gel electrophoretic analysis of in vitro-amplified 23S rDNA fragments, which contained the insertion. The nucleotide sequences of the amplified fragments were determined and sequence similarities of at least 44% were found. The overall similarity values are lower than those of 16S and 23S rRNA sequences of the particular organism. Northern hybridization experiments indicated the presence of the insertion within the mature 23S rRNA of Corynebacterium glutamicum.     

Bacterial succession in a petroleum land treatment unit

Bacterial community dynamics were investigated in a land treatment unit (LTU) established at a site contaminated with highly weathered petroleum hydrocarbons in the C(10) to C(32) range. The treatment plot, 3,000 cubic yards of soil, was supplemented with nutrients and monitored weekly for total petroleum hydrocarbons (TPH), soil water content, nutrient levels, and aerobic heterotrophic bacterial counts. Weekly soil samples were analyzed with 16S rRNA gene terminal restriction fragment (TRF) analysis to monitor bacterial community structure and dynamics during bioremediation. TPH degradation was rapid during the first 3 weeks and slowed for the remainder of the 24-week project. A sharp increase in plate counts was reported during the first 3 weeks, indicating an increase in biomass associated with petroleum degradation. Principal components analysis of TRF patterns revealed a series of sample clusters describing bacterial succession during the study. The largest shifts in bacterial community structure began as the TPH degradation rate slowed and the bacterial cell counts decreased. For the purpose of analyzing bacterial dynamics, phylotypes were generated by associating TRFs from three enzyme digests with 16S rRNA gene clones. Two phylotypes associated with Flavobacterium and Pseudomonas were dominant in TRF patterns from samples during rapid TPH degradation. After the TPH degradation rate slowed, four other phylotypes gained dominance in the community while Flavobacterium and Pseudomonas phylotypes decreased in abundance. These data suggest that specific phylotypes of bacteria were associated with the different phases of petroleum degradation in the LTU.     

Comparison of bacterial community in aqueous and oil phases of water-flooded petroleum reservoirs using pyrosequencing and clone library approaches

Bacterial communities in both aqueous and oil phases of water-flooded petroleum reservoirs were characterized by molecular analysis of bacterial 16S rRNA genes obtained from Shengli Oil Field using DNA pyrosequencing and gene clone library approaches. Metagenomic DNA was extracted from the aqueous and oil phases and subjected to polymerase chain reaction amplification with primers targeting the bacterial 16S rRNA genes. The analysis by these two methods showed that there was a large difference in bacterial diversity between the aqueous and oil phases of the reservoir fluids, especially in the reservoirs with lower water cut. At a high phylogenetic level, the predominant bacteria detected by these two approaches were identical. However, pyrosequencing allowed the detection of more rare bacterial species than the clone library method. Statistical analysis showed that the diversity of the bacterial community of the aqueous phase was lower than that of the oil phase. Phylogenetic analysis indicated that the vast majority of sequences detected in the water phase were from members of the genus Arcobacter within the Epsilonproteobacteria, which is capable of degrading the intermediates of hydrocarbon degradation such as acetate. The oil phase of reservoir fluid samples was dominated by members of the genus Pseudomonas within the Gammaproteobacteria and the genus Sphingomonas within the Alphaproteobacteria, which have the ability to degrade crude oil through adherence to hydrocarbons under aerobic conditions. In addition, many anaerobes that could degrade the component of crude oil were also found in the oil phase of reservoir fluids, mainly in the reservoir with lower water cut. These were represented by Desulfovibrio spp., Thermodesulfovibrio spp., Thermodesulforhabdus spp., Thermotoga spp., and Thermoanaerobacterium spp. This research suggested that simultaneous analysis of DNA extracted from both aqueous and oil phases can facilitate a better understanding of the bacterial communities in water-flooded petroleum reservoirs.     

Chilling and cultivar type affect the diversity of bacterial endophytes colonizing sweet pepper (Capsicum anuum L.)

A climate chamber experiment was conducted to assay the effect of low temperatures (chilling) on the diversity of bacteria colonizing the endospheres of two thermophilic sweet pepper (Capsicum anuum L.) cultivars, Milder Spiral and Ziegenhorn Bello. Structural diversity was analyzed by 16S rRNA-based terminal restriction fragment length polymorphism (T-RFLP) analysis and by the generation of 16S rRNA gene libraries to determine dominant community members in T-RFLP profiles. Cultivable community members colonizing lines Milder Spiral and Ziegenhorn Bello were identified by 16S rRNA gene analysis. T-RFLP profiles and 16S rRNA gene libraries revealed a high heterogeneity of community composition due to chilling and suggested further the existence of cultivar-specific communities. The majority of isolates obtained from the cultivar Milder Spiral were assigned as high-G+C Gram-positive bacteria (Microbacterium sp., Micrococcus sp., Rhodococcus sp.) and Firmicutes (Staphylococcus sp.). Of the isolated endophytes obtained from cultivar Zeigenhorn Bello, 93% were affiliated with Staphylococcus aureus and Bacillus sp. (Firmicutes). The experimental set-up was suited to demonstrate that chilling and cultivar type can influence the diversity of bacterial endophytes colonizing sweet pepper. We propose additional chilling experiments to investigate the effect of chilling on functional, plant-beneficial abilities of bacterial endophytes associated with low-temperature-sensitive crops, such as sweet pepper.     

Monitoring of activity dynamics of an anaerobic digester bacterial community using 16S rRNA polymerase chain reaction--single-strand conformation polymorphism analysis

The influence of parameter changes on the bacterial community of a laboratory-scale anaerobic digester fed with glucose was investigated using a culture-independent approach based on single-strand conformation polymorphism (SSCP) analysis of total 16S rDNA and 16S rRNA amplification products. With the digester operating at steady state, the 16S rDNA SSCP patterns of the bacterial community showed eight peaks, whereas the 16S rRNA patterns showed six peaks with a very prominent one corresponding to a Spirochaetes-related bacterium. An acidic shock at pH 6 caused an increase in the 16S rRNA level of two Clostridium-related bacteria. After a 1 week starvation period, the major bacteria present reverted to a basal 16S rRNA level proportional to their 16S rDNA level. Starvation revealed the presence of a previously undetected peak whose corresponding sequence was deeply branched into the low G+C Gram-positive bacteria phylum. Twenty-four hours after a spiked addition to the starved digester community of starch, glucose, lactate or sulphate, an upsurge in several new 16S rRNA-derived peaks was observed. Thus, the perturbation approach combined with 16S rRNA analysis revealed bacteria that had not been detected through 16S rDNA analysis.     

Diversity of bacterial community in freshwater of Woopo wetland

Diversity of bacterial community in water layer of Woopo wetland was investigated. Cultivable bacterial strains were isolated by the standard dilution plating technique and culture-independent 16S rRNA gene clones were obtained directly from DNA extracts of a water sample. Amplified rDNA restriction analysis (ARDRA) was applied onto both of the isolates and 16S rRNA gene clones. Rarefaction curves, coverage rate and diversity indices of ARDRA patterns were calculated. Representative isolates and clones of all the single isolate/clone phylotype were partially sequenced and analyzed phylogenetically. Sixty-four and 125 phylotypes were obtained from 203 bacterial isolates and 235 culture-independent 16S rRNA gene clones, respectively. Bacterial isolates were composed of 4 phyla, of which Firmicutes (49.8%) and Actinobacteria (32.0%) were predominant. Isolates were affiliated with 58 species. Culture-independent 16S rRNA gene clones were composed of 8 phyla, of which Proteobacteria (62.2%), Actinobacteria (15.5%), and Bacteroidetes (13.7%) were predominant. Diversity of 16S rRNA gene clones originated from cultivation-independent DNA extracts was higher than that of isolated bacteria.     

Phylogenetic diversity and activity of anaerobic microorganisms of high-temperature horizons of the Dagang oil field (P. R. China)

The number of microorganisms of major metabolic groups and the rates of sulfate reduction and methanogenesis processes in the formation waters of the high-temperature horizons of Dagang oil field have been determined. Using cultural methods, it was shown that the microbial community contained aerobic bacteria oxidizing crude oil, anaerobic fermentative bacteria, sulfate-reducing bacteria, and methanogens. Using cultural methods, the possibility of methane production from a mixture of hydrogen and carbon dioxide (H₂ + CO₂) and from acetate was established, and this result was confirmed by radioisotope methods involving NaH¹⁴CO₃ and ¹⁴CH₃COONa. Analysis of enrichment cultures 16S rDNA of methanogens demonstrated that these microorganisms belong to Methanothermobacter sp. (M. thermautotrophicus), which consumes hydrogen and carbon dioxide as basic substrates. The genes of acetate-utilizing bacteria were not revealed. Phylotypes of the representatives of Thermococcus spp. were found among archaeal 16S rDNA. 16S rRNA genes of bacterial clones belong to the orders Thermoanaerobacteriales (Thermoanaerobacter, Thermovenabulum, Thermacetogenium, and Coprothermobacter spp.), Thermotogales, Nitrospirales (Thermodesulfovibrio sp.) and Planctomycetales. 16S rDNA of a bacterium capable of oxidizing acetate in the course of syntrophic growth with H₂-utilizing methanogens was found in high-temperature petroleum reservoirs for the first time. These results provide further insight into the composition of microbial communities of high-temperature petroleum reservoirs, indicating that syntrophic processes play an important part in acetate degradation accompanied by methane production.     

Phylogenetic analysis of bacterial communities in mesophilic and thermophilic bioreactors treating pharmaceutical wastewater

The phylogenetic diversity of the bacterial communities supported by a seven-stage, full-scale biological wastewater treatment plant was studied. These reactors were operated at both mesophilic (28 to 32 degrees C) and thermophilic (50 to 58 degrees C) temperatures. Community fingerprint analysis by denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified V3 region of the 16S rRNA gene from the domain Bacteria revealed that these seven reactors supported three distinct microbial communities. A band-counting analysis of the PCR-DGGE results suggested that elevated reactor temperatures corresponded with reduced species richness. Cloning of nearly complete 16S rRNA genes also suggested a reduced species richness in the thermophilic reactors by comparing the number of clones with different nucleotide inserts versus the total number of clones screened. While these results imply that elevated temperature can reduce species richness, other factors also could have impacted the number of populations that were detected. Nearly complete 16S rDNA sequence analysis showed that the thermophilic reactors were dominated by members from the beta subdivision of the division Proteobacteria (beta-proteobacteria) in addition to anaerobic phylotypes from the low-G+C gram-positive and Synergistes divisions. The mesophilic reactors, however, included at least six bacterial divisions, including Cytophaga-Flavobacterium-Bacteroides, Synergistes, Planctomycetes, low-G+C gram-positives, Holophaga-Acidobacterium, and Proteobacteria (alpha-proteobacteria, beta-proteobacteria, gamma-proteobacteria and delta-proteobacteria subdivisions). The two PCR-based techniques detected the presence of similar bacterial populations but failed to coincide on the relative distribution of these phylotypes. This suggested that at least one of these methods is insufficiently quantitative to determine total community biodiversity-a function of both the total number of species present (richness) and their relative distribution (evenness).     

Bacterial diversity in water injection systems of Brazilian offshore oil platforms

Biogenic souring and microbial-influenced corrosion is a common scenario in water-flooded petroleum reservoirs. Water injection systems are continuously treated to control bacterial contamination, but some bacteria that cause souring and corrosion can persist even after different treatments have been applied. Our aim was to increase our knowledge of the bacterial communities that persist in the water injection systems of three offshore oil platforms in Brazil. To achieve this goal, we used a culture-independent molecular approach (16S ribosomal RNA gene clone libraries) to analyze seawater samples that had been subjected to different treatments. Phylogenetic analyses revealed that the bacterial communities from the different platforms were taxonomically different. A predominance of bacterial clones affiliated with Gammaproteobacteria, mostly belonging to the genus Marinobacter (60.7%), were observed in the platform A samples. Clones from platform B were mainly related to the genera Colwellia (37.9%) and Achromobacter (24.6%), whereas clones obtained from platform C were all related to unclassified bacteria. Canonical correspondence analyses showed that different treatments such as chlorination, deoxygenation, and biocide addition did not significantly influence the bacterial diversity in the platforms studied. Our results demonstrated that the injection water used in secondary oil recovery procedures contained potentially hazardous bacteria, which may ultimately cause souring and corrosion.     

Bacterial diversity in the bacterioneuston (sea surface microlayer): the bacterioneuston through the looking glass

The bacterioneuston is defined as the community of bacteria present within the neuston or sea surface microlayer. Bacteria within this layer were sampled using a membrane filter technique and bacterial diversity was compared with that in the underlying pelagic coastal seawater using molecular ecological techniques. 16S rRNA gene libraries of approximately 500 clones were constructed from both bacterioneuston and the pelagic water samples and representative clones from each library were sequenced for comparison of bacterial diversity. The bacterioneuston was found to have a significantly lower bacterial diversity than the pelagic seawater, with only nine clone types (ecotaxa) as opposed to 46 ecotaxa in the pelagic seawater library. Surprisingly, the bacterioneuston clone library was dominated by 16S rRNA gene sequences affiliated to two groups of organisms, Vibrio spp. which accounted for over 68% of clones and Pseudoalteromonas spp. accounting for 21% of the library. The dominance of these two 16S rRNA gene sequence types within the bacterioneuston clone library was confirmed in a subsequent gene probing experiment. 16S rRNA gene probes specific for these groups of bacteria were designed and used to probe new libraries of 1000 clones from both the bacterioneuston and pelagic seawater DNA samples. This revealed that 57% of clones from the bacterioneuston library hybridized to a Vibrio sp.-specific 16S rRNA gene probe and 32% hybridized to a Pseudoalteromonas sp.-specific 16S rRNA gene probe. In contrast, the pelagic seawater library resulted in only 13% and 8% of 16S rRNA gene clones hybridizing to the Vibrio sp. and Pseudoalteromonas sp. probes respectively. Results from this study suggest that the bacterioneuston contains a distinct population of bacteria and warrants further detailed study at the molecular level.     

Molecular phylogenetic diversity of bacteria associated with the leachate of a closed municipal solid waste landfill

A 16S rDNA-based molecular study was performed to determine the nature of the bacterial constituents of the leachate from a closed municipal solid waste landfill. Total community DNA was extracted and bacterial 16S rRNA genes were subsequently amplified and cloned. Recombinant rDNA clones in the library were randomly selected, and they were sequenced for a single run and then grouped. A total of 76 sequence types representing 138 randomly selected nonchimeric clones were identified. Full-length sequencing and phylogenetic analysis of the sequence types revealed that more than 90% of the screened clones were affiliated with low-G+C gram-positive bacteria (38.4%), Proteobacteria (35.5%), the Cytophaga Flexibacter Bacteroides group (11.6%), and Spirochaetes (5.1%). Minor portions were affiliated with Verrucomicrobia (2.9%), candidate division OP11 (2.2%), and the green nonsulfur bacteria, Cyanobacteria and the Deinococcus Thermus group (each <1.0%). Although some rDNA sequences clustered with genera or taxa that were classically identified within anaerobic treatment systems and expected with known functions, a substantial fraction of the clone sequences showed relatively low levels of similarity with any other reported rDNA sequences and thus were derived from unknown taxa. These results suggest that bacterial communities in landfill environment are far more complex than previously expected and remain largely unexplored.     

胜利油田单12高温油藏非培养和富集培养样品的细菌组成特性

[目的]通过比较分析油藏样品的微生物群落结构特点,认识油藏微生物的生态功能.[方法]利用3种油藏微生物研究中常用的富集培养方法,对胜利油田单12区块S12-4油井产出水样品进行了选择性富集培养,运用构建16S rRNA基因文库的方法分析了富集样品和非培养样品的细菌多样性.[结果]通过16S rRNA基因序列比对发现,非培养样品、异养菌富集样品、烃降解菌富集样品和硫酸盐还原菌富集样品中的优势菌分别为Pseudomonas属,Thermotoga属,Thermaerobacter属和Thermotoga属的成员.多样性分析结果表明,非培养样品的微生物多样性最丰富,同时非培养样品和富集样品的微生物群落结构存在很大的差异,富集样品中的微生物包括优势菌在油藏原位环境中含量很低.[结论]细菌组成差异的比较结果,对油藏微生物的生态功能研究和微生物驱油潜力评估具有重要意义.