Diversity of Microbial Communities in Production and Injection Waters of Algerian Oilfields Revealed by 16S rRNA Gene Amplicon 454 Pyrosequencing

The microorganisms inhabiting many petroleum reservoirs are multi-extremophiles capable of surviving in environments with high temperature, pressure and salinity. Their activity influences oil quality and they are an important reservoir of enzymes of industrial interest. To study these microbial assemblages and to assess any modifications that may be caused by industrial practices, the bacterial and archaeal communities in waters from four Algerian oilfields were described and compared. Three different types of samples were analyzed: production waters from flooded wells, production waters from non-flooded wells and injection waters used for flooding (water-bearing formations). Microbial communities of production and injection waters appeared to be significantly different. From a quantitative point of view, injection waters harbored roughly ten times more microbial cells than production waters. Bacteria dominated in injection waters, while Archaea dominated in production waters. Statistical analysis based on the relative abundance and bacterial community composition (BCC) revealed significant differences between production and injection waters at both OTUs_0.03 and phylum level. However, no significant difference was found between production waters from flooded and non-flooded wells, suggesting that most of the microorganisms introduced by the injection waters were unable to survive in the production waters. Furthermore, a Venn diagram generated to compare the BCC of production and injection waters of one flooded well revealed only 4% of shared bacterial OTUs. Phylogenetic analysis of bacterial sequences indicated that Alpha-, Beta- and Gammaproteobacteria were the main classes in most of the water samples. Archaeal sequences were only obtained from production wells and each well had a unique archaeal community composition, mainly belonging to Methanobacteria, Methanomicrobia, Thermoprotei and Halobacteria classes. Many of the bacterial genera retrieved had already been reported as degraders of complex organic molecules and pollutants. Nevertheless, a large number of unclassified bacterial and archaeal sequences were found in the analyzed samples, indicating that subsurface waters in oilfields could harbor new and still-non-described microbial species.     

胜利油藏不同时间细菌群落结构的比较

利用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)和构建16S rRNA基因克隆文库2种方法,对孤岛油田两口井(注水井G和采油井L)在相距9个月的2个时间点(A和B)所采集样品的细菌群落结构进行了比较。DGGE图谱聚类分析表明注水井在2个时间点的微生物群落结构相似性为48.1%,而采油井的相似性只有28.7%。16S rRNA基因克隆文库结果表明,A时间点样品G中的优势菌群为Betaproteobacteria、Gammaproteobacteria,还有Deferribacteres、Firmicutes、Bacteroidetes等;而样品L中,Gammaproteobacteria中的Moraxellaceae含量达到97%。B时间点G中除了优势菌Betaproteobacteria之外,Deferribacteres的数量显著增加,成为优势菌;而L在B时间点优势菌除Gammaproteobacteria外,还有Betaproteobacteria和Firmicutes。采油井中的微生物群落结构随时间发生了显著改变,而注水井变化不显著。这一结果部分揭示了微生物采油过程中地层微生物群落的变化规律,有助于进一步阐明微生物驱油的机理。     

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.     

Microbial diversity in production waters of a low-temperature biodegraded oil reservoir

Microbial communities in two production waters of a low-temperature and low-salinity petroleum reservoir in Canada were examined using cultural and molecular approaches. The predominant cultivated microorganisms were homoacetogens but sulfate-reducers, acetoclastic methanogens and denitrifiers also gave significant counts. The dominant members of the culturable population were affiliated with the Firmicutes, the "Deltaproteobacteria", the "Epsilonproteobacteria", the Spirochaetes and the Euryarchaeota. 16S rRNA gene clone libraries were also constructed from the total DNA collected from production waters. The bacterial library was entirely composed by a single phylotype related to Arcobacter. The archaeal phylotypes were generally very closely related to members of the orders Methanosarcinales and Methanomicrobiales. Consistent with earlier observations, our data suggest that methanogenesis is a dominant terminal process in the reservoir. Moreover, the cross-evaluation of culture-dependent and -independent techniques also indicates that, contrary to most studies, both acetoclastic and lithotrophic methanogens may be involved in this process. This first investigation of the microbial diversity in a non water-flooded low-temperature and low-salinity petroleum reservoir expands substantially our knowledge of the extent of microbial diversity and highlights the complexity of microbial communities involved in the oil field food chain.     

Microbial diversity in degraded and non-degraded petroleum samples and comparison across oil reservoirs at local and global scales

Microorganisms have shown their ability to colonize extreme environments including deep subsurface petroleum reservoirs. Physicochemical parameters may vary greatly among petroleum reservoirs worldwide and so do the microbial communities inhabiting these different environments. The present work aimed at the characterization of the microbiota in biodegraded and non-degraded petroleum samples from three Brazilian reservoirs and the comparison of microbial community diversity across oil reservoirs at local and global scales using 16S rRNA clone libraries. The analysis of 620 16S rRNA bacterial and archaeal sequences obtained from Brazilian oil samples revealed 42 bacterial OTUs and 21 archaeal OTUs. The bacterial community from the degraded oil was more diverse than the non-degraded samples. Non-degraded oil samples were overwhelmingly dominated by gammaproteobacterial sequences with a predominance of the genera Marinobacter and Marinobacterium. Comparisons of microbial diversity among oil reservoirs worldwide suggested an apparent correlation of prokaryotic communities with reservoir temperature and depth and no influence of geographic distance among reservoirs. The detailed analysis of the phylogenetic diversity across reservoirs allowed us to define a core microbiome encompassing three bacterial classes (Gammaproteobacteria, Clostridia, and Bacteroidia) and one archaeal class (Methanomicrobia) ubiquitous in petroleum reservoirs and presumably owning the abilities to sustain life in these environments.

     

Phylogenetic diversity, composition and distribution of bacterioplankton community in the Dongjiang River, China

Bacterioplankton community compositions in the Dongjiang River were characterized using denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone library construction. Water samples in nine different sites were taken along the mainstem and three tributaries. In total, 24 bands from DGGE gels and 406 clones from the libraries were selected and sequenced, subsequently analyzed for the bacterial diversity and composition of those microbial communities. Bacterial 16S rRNA gene sequences from freshwater bacteria exhibited board phylogenetic diversity, including sequences representing the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Actinobacteria, Bacteriodetes, Verrucomicrobia, and candidate division TM7. Members of Betaproteobacteria group were the most dominant in all sampling sites, followed by Gammaproteobacteria, Alphaproteobacteria, and Actinobacteria. DGGE profiles and the ∫-LIBSHUFF analysis revealed similar patterns of bacterial diversity among most sampling sites, while spatial distribution variances existed in all sites along the river basin. Statistical analysis showed that bacterial species distribution strongly correlated with environmental variables, such as nitrate and ammonia, suggesting that nitrogen nutrients may shape the microbial community structure and composition in the Dongjiang River. This study had important implications for the comparison with other rivers elsewhere and contributed to the growing data set on the factors that structure bacterial communities in freshwater ecosystems.     

Microbial communities in long-term, water-flooded petroleum reservoirs with different in situ temperatures in the Huabei Oilfield, China

The distribution of microbial communities in the Menggulin (MGL) and Ba19 blocks in the Huabei Oilfield, China, were studied based on 16S rRNA gene analysis. The dominant microbes showed obvious block-specific characteristics, and the two blocks had substantially different bacterial and archaeal communities. In the moderate-temperature MGL block, the bacteria were mainly Epsilonproteobacteria and Alphaproteobacteria, and the archaea were methanogens belonging to Methanolinea, Methanothermobacter, Methanosaeta, and Methanocella. However, in the high-temperature Ba19 block, the predominant bacteria were Gammaproteobacteria, and the predominant archaea were Methanothermobacter and Methanosaeta. In spite of shared taxa in the blocks, differences among wells in the same block were obvious, especially for bacterial communities in the MGL block. Compared to the bacterial communities, the archaeal communities were much more conserved within blocks and were not affected by the variation in the bacterial communities.     

Archaeal and Bacterial Communities Associated with the Surface Mucus of Caribbean Corals Differ in Their Degree of Host Specificity and Community Turnover Over Reefs

Comparative studies on the distribution of archaeal versus bacterial communities associated with the surface mucus layer of corals have rarely taken place. It has therefore remained enigmatic whether mucus-associated archaeal and bacterial communities exhibit a similar specificity towards coral hosts and whether they vary in the same fashion over spatial gradients and between reef locations. We used microbial community profiling (terminal-restriction fragment length polymorphism, T-RFLP) and clone library sequencing of the 16S rRNA gene to compare the diversity and community structure of dominant archaeal and bacterial communities associating with the mucus of three common reef-building coral species (Porites astreoides, Siderastrea siderea and Orbicella annularis) over different spatial scales on a Caribbean fringing reef. Sampling locations included three reef sites, three reef patches within each site and two depths. Reference sediment samples and ambient water were also taken for each of the 18 sampling locations resulting in a total of 239 samples. While only 41% of the bacterial operational taxonomic units (OTUs) characterized by T-RFLP were shared between mucus and the ambient water or sediment, for archaeal OTUs this percentage was 2-fold higher (78%). About half of the mucus-associated OTUs (44% and 58% of bacterial and archaeal OTUs, respectively) were shared between the three coral species. Our multivariate statistical analysis (ANOSIM, PERMANOVA and CCA) showed that while the bacterial community composition was determined by habitat (mucus, sediment or seawater), host coral species, location and spatial distance, the archaeal community composition was solely determined by the habitat. This study highlights that mucus-associated archaeal and bacterial communities differ in their degree of community turnover over reefs and in their host-specificity.     

昆明盐矿古老岩盐沉积中的原核生物多样性

应用PCR-DGGE和rRNA分析法研究了昆明盐矿古老岩盐沉积中的原核生物多样性。样品的细菌DGGE分析得到27条带,古菌得到18条带。样品与纯培养得到的19个属菌株的DGGE图谱对比分析发现,细菌18个属菌株,只有1个属菌株与样品中的1条带迁移位置都不一致;古菌1个属的菌株不与样品中任何条带迁移位置一致。表明纯培养所得菌株并非该环境中的优势类群。同时,建立了样品细菌和古菌的16S rDNA克隆文库,从中分别挑取36个细菌克隆和20个古菌克隆进行ARDRA分析。细菌可分为10个OTUs,其中3个OTUs是优势类群,分别占38.9%,25.0%,16.7%,其余7个OTUs各含有1个克隆。古菌分为8个OTUs,没有明显的优势类群。每个OTU的代表克隆16S rDNA序列分析表明,细菌分属3大类群:α-Proteobacteria,γ-Proteobacteria和Actinobacteria,以Pseudomonas属菌为优势,含有其它岩盐沉积中没有发现的Actinobacteria。古菌主要是Halorubrum属、Haloterrigena属菌和未培养古菌。本研究表明,昆明盐矿古老岩盐沉积具有较丰富的原核生物多样性,含有大量未知的、未培养或不可培养的原核生物,但在原核生物物种组成和丰度上,免培养与此前的纯培养研究结果存在一定差异。因此,结合使用两类方法才能较全面地认识高盐极端环境微生物的多样性。     

Molecular community analysis of magnesium-rich bittern brine recovered from a Tunisian solar saltern

The microbial community of a magnesium-rich bittern brine saturated with NaCl (380-400 g/L) from a Tunisian solar saltern was investigated using a molecular approach based on 16S rRNA gene analysis and viability tests. The results revealed the existence of microbial flora. Viability test assessment showed that 46.4% of this flora was viable but not detectable by culturability tests. 16S rRNA genes from 49 bacterial clones and 38 archaeal clones were sequenced and phylogenetically analyzed. Eleven operational taxonomic units (OTUs) determined by the DOTUR program with 97% sequence similarity were generated for Bacteria. These OTUs were affiliated with Bacteroidetes and Gammaproteobacteria. The archaeal community composition exhibited more diversity with 38 clones, resulting in 13 OTUs affiliated with the Euryarchaeota phylum. Diversity measurement showed a more diverse archaeal than bacterial community at the saturated pond.     

Archaea Appear to Dominate the Microbiome of Inflatella pellicula Deep Sea Sponges

Microbes associated with marine sponges play significant roles in host physiology. Remarkable levels of microbial diversity have been observed in sponges worldwide through both culture-dependent and culture-independent studies. Most studies have focused on the structure of the bacterial communities in sponges and have involved sponges sampled from shallow waters. Here, we used pyrosequencing of 16S rRNA genes to compare the bacterial and archaeal communities associated with two individuals of the marine sponge Inflatella pellicula from the deep-sea, sampled from a depth of 2,900 m, a depth which far exceeds any previous sequence-based report of sponge-associated microbial communities. Sponge-microbial communities were also compared to the microbial community in the surrounding seawater. Sponge-associated microbial communities were dominated by archaeal sequencing reads with a single archaeal OTU, comprising ∼60% and ∼72% of sequences, being observed from Inflatella pellicula. Archaeal sequencing reads were less abundant in seawater (∼11% of sequences). Sponge-associated microbial communities were less diverse and less even than any other sponge-microbial community investigated to date with just 210 and 273 OTUs (97% sequence identity) identified in sponges, with 4 and 6 dominant OTUs comprising ∼88% and ∼89% of sequences, respectively. Members of the candidate phyla, SAR406, NC10 and ZB3 are reported here from sponges for the first time, increasing the number of bacterial phyla or candidate divisions associated with sponges to 43. A minor cohort from both sponge samples (∼0.2% and ∼0.3% of sequences) were not classified to phylum level. A single OTU, common to both sponge individuals, dominates these unclassified reads and shares sequence homology with a sponge associated clone which itself has no known close relative and may represent a novel taxon.     

Monitoring exogenous and indigenous bacteria by PCR-DGGE technology during the process of microbial enhanced oil recovery

A field experiment was performed to monitor changes in exogenous bacteria and to investigate the diversity of indigenous bacteria during a field trial of microbial enhanced oil recovery (MEOR). Two wells (26-195 and 27-221) were injected with three exogenous strains and then closed to allow for microbial growth and metabolism. After a waiting period, the pumps were restarted and the samples were collected. The bacterial populations of these samples were analyzed by denaturing gradient gel electrophoresis (DGGE) with PCR-amplified 16S rRNA fragments. DGGE profiles indicated that the exogenous strains were retrieved in the production water samples and indigenous strains could also be detected. After the pumps were restarted, average oil yield increased to 1.58 and 4.52 tons per day in wells 26-195 and 27-221, respectively, compared with almost no oil output before the injection of exogenous bacteria. Exogenous bacteria and indigenous bacteria contributed together to the increased oil output. Sequence analysis of the DGGE bands revealed that Proteobacteria were a major component of the predominant bacteria in both wells. Changes in the bacteria population in the reservoirs during MEOR process were monitored by molecular analysis of the 16S rRNA gene sequence. DGGE analysis was a successful approach to investigate the changes in microorganisms used for enhancing oil recovery. The feasibility of MEOR technology in the petroleum industry was also demonstrated.     

Characterization of the prokaryotic diversity in cold saline perennial springs of the Canadian high Arctic

The springs at Gypsum Hill and Colour Peak on Axel Heiberg Island in the Canadian Arctic originate from deep salt aquifers and are among the few known examples of cold springs in thick permafrost on Earth. The springs discharge cold anoxic brines (7.5 to 15.8% salts), with a mean oxidoreduction potential of -325 mV, and contain high concentrations of sulfate and sulfide. We surveyed the microbial diversity in the sediments of seven springs by denaturing gradient gel electrophoresis (DGGE) and analyzing clone libraries of 16S rRNA genes amplified with Bacteria and Archaea-specific primers. Dendrogram analysis of the DGGE banding patterns divided the springs into two clusters based on their geographic origin. Bacterial 16S rRNA clone sequences from the Gypsum Hill library (spring GH-4) were classified into seven phyla (Actinobacteria, Bacteroidetes, Firmicutes, Gemmatimonadetes, Proteobacteria, Spirochaetes, and Verrucomicrobia); Deltaproteobacteria and Gammaproteobacteria sequences represented half of the clone library. Sequences related to Proteobacteria (82%), Firmicutes (9%), and Bacteroidetes (6%) constituted 97% of the bacterial clone library from Colour Peak (spring CP-1). Most GH-4 archaeal clone sequences (79%) were related to the Crenarchaeota while half of the CP-1 sequences were related to orders Halobacteriales and Methanosarcinales of the Euryarchaeota. Sequences related to the sulfur-oxidizing bacterium Thiomicrospira psychrophila dominated both the GH-4 (19%) and CP-1 (45%) bacterial libraries, and 56 to 76% of the bacterial sequences were from potential sulfur-metabolizing bacteria. These results suggest that the utilization and cycling of sulfur compounds may play a major role in the energy production and maintenance of microbial communities in these unique, cold environments.     

珠三角养殖水体中参与氮循环的微生物群落结构

【目的】为了研究珠三角地区养殖水体中各种含氮化合物循环转化的特征及其相关功能微生物的群落结构。【方法】构建人工模拟养殖系统,采用15N-稳定性同位素探测技术(stable isotope probing,15N-SIP)标记参与氮素迁移的微生物,通过氯化铯-溴化乙锭密度梯度超高速离心法分离15N标记的DNA,并以此构建含15N-DNA的细菌和古菌16S rRNA基因克隆文库。【结果】15N标记的基因组DNA经过超高速密度梯度离心后成功与14N-DNA分离;对克隆文库序列的分析表明:细菌文库得到的19个可操作分类单元(Operational TaxaUnits,OTUs)分别归为变形菌门(Proteobacteria)和浮霉菌门(Planctomycetes)。变形菌门为绝对优势类群,占整个细菌克隆文库的99.2%,其中优势菌群为丛毛胞菌属(Comamonas)(15.7%)、亚硝化单胞菌属(Nitrosomonas)(12.4%)、肠杆菌科(Enterobacteriaceae)(11.5%)和硝化杆菌属(Nitrobacter)(11.5%);古菌文库得到的9个OTUs归为奇古菌门(Thaumarchaeota)、泉古菌门(Crenarchaeota)和广古菌门(Euryarchaeota)。【结论】将15N-SIP技术应用于珠三角养殖水体氮素循环微生物群落结构的研究中,得到了丰富的氮素循环微生物群落的组成,这些信息为进一步分离纯化氮素降解微生物提供了重要的参考,为营造健康的水产养殖环境提供科学依据。     

Methanogen community structure in the rumens of farmed sheep, cattle and red deer fed different diets

Development of inhibitors and vaccines that mitigate rumen-derived methane by targeting methanogens relies on knowledge of the methanogens present. We investigated the composition of archaeal communities in the rumens of farmed sheep (Ovis aries), cattle (Bos taurus) and red deer (Cervus elaphus) using denaturing gradient gel electrophoresis (DGGE) to generate fingerprints of archaeal 16S rRNA genes. The total archaeal communities were relatively constant across species and diets, and were less variable and less diverse than bacterial communities. There were diet- and ruminant-species-based differences in archaeal community structure, but the same dominant archaea were present in all rumens. These were members of three coherent clades: species related to Methanobrevibacter ruminantium and Methanobrevibacter olleyae; species related to Methanobrevibacter gottschalkii, Methanobrevibacter thaueri and Methanobrevibacter millerae; and species of the genus Methanosphaera. Members of an archaeal group of unknown physiology, designated rumen cluster C (RCC), were also present. RCC-specific DGGE, clone library analysis and quantitative real-time PCR showed that their 16S rRNA gene sequences were very diverse and made up an average of 26.5% of the total archaea. RCC sequences were not readily detected in the DGGE patterns of total archaeal 16S rRNA genes because no single sequence type was abundant enough to form dominant bands.     

Bacterial diversity in five Icelandic geothermal waters: temperature and sinter growth rate effects

The microbial ecology associated with siliceous sinters was studied in five geochemically diverse Icelandic geothermal systems. Bacterial 16S rRNA clone libraries were constructed from water-saturated precipitates from each site resulting in a total of 342 bacterial clone sequences and 43 species level phylotypes. In near-neutral, saline (2.6–4.7% salinity) geothermal waters where sinter growth varied between 10 and ~300 kg year⁻¹ m⁻², 16S rRNA gene analyses revealed very low (no OTUs could be detected) to medium (9 OTUs) microbial activity. The most dominant phylotypes found in these waters belong to marine genera of the Proteobacteria. In contrast, in alkaline (pH = 9–10), meteoric geothermal waters with temperature = 66–96°C and <1–20 kg year⁻¹m⁻² sinter growth, extensive biofilms (a total of 34 OTUs) were observed within the waters and these were dominated by members of the class Aquificae (mostly related to Thermocrinis), Deinococci (Thermus species) as well as Proteobacteria. The observed phylogenetic diversity (i.e., number and composition of detected OTUs) is argued to be related to the physico-chemical regime prevalent in the studied geothermal waters; alkaliphilic thermophilic microbial communities with phylotypes related to heterotrophic and autotrophic microorganisms developed in alkaline high temperature waters, whereas halophilic mesophilic communities dominated coastal geothermal waters.     

Diverse microbial communities inhabit Antarctic sponges

Genetic techniques were employed to investigate the archaeal, bacterial and eukaryotic communities associated with the Antarctic sponges Kirkpatrickia varialosa, Latrunculia apicalis, Homaxinella balfourensis, Mycale acerata and Sphaerotylus antarcticus. The phylogenetic affiliation of sponge-derived bacteria was assessed by 16S rRNA sequencing of cloned DNA fragments. Denaturing gradient gel electrophoresis (DGGE) was used to determine the stability of bacterial associations within each sponge species and across spatial scales. Of the 150 archaeal clones from L. apicalis, K. varialosa and M. acerata screened by restriction fragment length polymorphism (RFLP) analysis, four unique operational taxonomic units (OTUs) were observed and all clustered closely together within the Crenarchaeota. Of the 250 sponge-derived bacterial clones screened by RFLP analysis, 61 were unique OTUs that were not detected during examination of 160 seawater-derived clones. Rarefaction analysis indicated that the clone libraries represented between 44 and 83% of the total estimated diversity. Phylogenetic analysis of sequence data revealed that the bacterial communities present in Antarctic sponges primarily clustered within the Gamma and Alpha proteobacteria and the Cytophaga/Flavobacterium of Bacteroidetes group. Bacterial DGGE analysis for replicate sponge and seawater samples at each Antarctic site revealed that bacterial communities were consistently detected within a particular species regardless of the collection site, with six bacterial bands exclusively associated with a single sponge species. Phylogenetic analysis of sequence data from eukaryotic DGGE analysis revealed that the communities present in Antarctic sponges fell into diatom and dinoflagellate clusters with many sequences having no known close relatives. In addition, seven eukaryotic sequences that were not detected in seawater samples or other sponge species were observed in K. varialosa.     

Microbial diversity with dominance of 16S rRNA gene sequences with high GC contents at 74 and 98 °C subsurface crude oil deposits in Japan

We prepared DNA from the production waters of oil deposits and wellheads of the high- and hypertemperature Japanese oil wells #AR39 (depth, 1230 m; temperature, 74 °C; pressure, 2.92 MPa) and #SR123 (depth, 1687 m; temperature, 98 °C; pressure, 11.3 MPa) to detect indigenous bacterial and archaeal microorganisms. We used PCR to amplify the 16S rRNA genes of microbial communities and characterized them based on their sequences. A few species of microorganisms with high GC contents were detected in samples from oil deposits, whereas the microbial constituents and their GC contents were diverse in wellhead samples. A comparison of the composition of the microbial communities found that the predominant indigenous populations in the #SR123 oil deposit were Thermotoga hypogea-, Thermotoga petrophila- and Thermodesulfobacterium commune-like bacteria with a 61-63% GC content in their 16S rRNA gene sequences, and Archaeoglobus fulgidus-like archaea with a 65% GC content, whereas the major population in #AR39 comprised Thermacetogenium phaeum- and Fervidobacterium pennavorans-like bacteria and Methanothermobacter thermautotrophicus-like archaea with a 60%, 60% and 61% GC content, respectively.     

Massive dominance of Epsilonproteobacteria in formation waters from a Canadian oil sands reservoir containing severely biodegraded oil

The subsurface microbiology of an Athabasca oil sands reservoir in western Canada containing severely biodegraded oil was investigated by combining 16S rRNA gene- and polar lipid-based analyses of reservoir formation water with geochemical analyses of the crude oil and formation water. Biomass was filtered from formation water, DNA was extracted using two different methods, and 16S rRNA gene fragments were amplified with several different primer pairs prior to cloning and sequencing or community fingerprinting by denaturing gradient gel electrophoresis (DGGE). Similar results were obtained irrespective of the DNA extraction method or primers used. Archaeal libraries were dominated by Methanomicrobiales (410 of 414 total sequences formed a dominant phylotype affiliated with a Methanoregula sp.), consistent with the proposed dominant role of CO(2) -reducing methanogens in crude oil biodegradation. In two bacterial 16S rRNA clone libraries generated with different primer pairs, >?99% and 100% of the sequences were affiliated with Epsilonproteobacteria (n?=?382 and 72 total clones respectively). This massive dominance of Epsilonproteobacteria sequences was again obtained in a third library (99% of sequences; n?=?96 clones) using a third universal bacterial primer pair (inosine-341f and 1492r). Sequencing of bands from DGGE profiles and intact polar lipid analyses were in accordance with the bacterial clone library results. Epsilonproteobacterial OTUs were affiliated with Sulfuricurvum, Arcobacter and Sulfurospirillum spp. detected in other oil field habitats. The dominant organism revealed by the bacterial libraries (87% of all sequences) is a close relative of Sulfuricurvum kujiense - an organism capable of oxidizing reduced sulfur compounds in crude oil. Geochemical analysis of organic extracts from bitumen at different reservoir depths down to the oil water transition zone of these oil sands indicated active biodegradation of dibenzothiophenes, and stable sulfur isotope ratios for elemental sulfur and sulfate in formation waters were indicative of anaerobic oxidation of sulfur compounds. Microbial desulfurization of crude oil may be an important metabolism for Epsilonproteobacteria indigenous to oil reservoirs with elevated sulfur content and may explain their prevalence in formation waters from highly biodegraded petroleum systems.     

Study of microbial community structures in UASB sludge treating municipal wastewater by denaturing gradient gel electrophoresis of 16S rDNA

The structures of microbial communities in lab-scale upflow anaerobic sludge blanket (UASB) reactors for treating municipal wastewater with different ratios of COD soluble/COD total were studied using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes.The microbial structure of the inoculum sludge obtained from a full-scale UASB reactor of treating potato processing wastewater was compared with the structures of sludges collected from three lab-scale UASB reactors after eight months feeding with raw municipal wastewater, with CEPS (chemically enhanced primary sedimentation) pretreated municipal wastewater, and with a synthetic municipal sewage, respectively. Computer-aided numerical analysis of the DGGE fingerprints showed that the bacterial community underwent major changes. The sludges for treating raw and CEPS pretreated wastewater had very similar bacterial and archaeal communities (82%and 96% similarity) but were different from that for treating the synthetic sewage. Hence, despite similar % COD in the particulate form in the synthetic and the real wastewater, the two wastewaters were selected for different microbial communities. Prominent DGGE bands of Bacteria and Archaea were purified and sequenced. The 16S rRNA gene sequences of the dominant archaeal bands found in the inoculum, and UASB sludge fed with raw sewage, CEPS pretreated wastewater, and synthetic sewage were closely associated with Methanosaeta concilii. In the UASB sludge fed with synthetic sewage, another dominant band associated with an uncultured archaeon 39-2 was found together with M. concilii.