利用小亚基核糖体RNA 技术分析温室黄瓜近根土壤古菌和真菌多样性

土壤古菌和真菌在温室生态系统是仅次于细菌的微生物,具有类似于细菌的重要生态功能。通过构建古菌16S rRNA和真菌18S rRNA基因克隆文库,分析温室黄瓜近根土壤古菌和真菌群落结构组成,为开发利用温室这一特殊的生态环境中丰富的微生物资源以及理解微生物与植物间的互作提供参考依据。采用研磨-冻融-溶菌酶-蛋白酶K-SDS热处理以及CTAB处理等理化方法,提取和纯化微生物总DNA,构建古菌16S rRNA和真菌18S rRNA基因克隆文库。利用DOTUR软件将古菌和真菌序列按照相似性97％的标准分成若干个可操作分类单元 (OTUs)。土壤古菌克隆文库主要包括泉古菌门和未分类的古菌两大类,并有少部分广域古菌类群,所有泉古菌均属于热变形菌纲,共45个OTUs;真菌克隆文库包括真菌门的大多数亚门真菌,共24个OTUs,未发现担子菌亚门真菌。古菌多样性比较丰富,且发现少量的广域古菌 (甲烷菌),这一情况可能与温室长期高温高湿,高有机质含量,土壤处于缺氧环境有关;土壤真菌的优势种群为子囊菌,占到土壤真菌的80％以上,这可能与绝大多数植物真菌性病害属于土传病害,通过菌丝体、菌核或子囊壳在土壤病残体中越冬有一定的关系。     

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.     

Metagenomic and small-subunit rRNA analyses reveal the genetic diversity of bacteria, archaea, fungi, and viruses in soil

Recent studies have highlighted the surprising richness of soil bacterial communities; however, bacteria are not the only microorganisms found in soil. To our knowledge, no study has compared the diversities of the four major microbial taxa, i.e., bacteria, archaea, fungi, and viruses, from an individual soil sample. We used metagenomic and small-subunit RNA-based sequence analysis techniques to compare the estimated richness and evenness of these groups in prairie, desert, and rainforest soils. By grouping sequences at the 97% sequence similarity level (an operational taxonomic unit [OTU]), we found that the archaeal and fungal communities were consistently less even than the bacterial communities. Although total richness levels are difficult to estimate with a high degree of certainty, the estimated number of unique archaeal or fungal OTUs appears to rival or exceed the number of unique bacterial OTUs in each of the collected soils. In this first study to comprehensively survey viral communities using a metagenomic approach, we found that soil viruses are taxonomically diverse and distinct from the communities of viruses found in other environments that have been surveyed using a similar approach. Within each of the four microbial groups, we observed minimal taxonomic overlap between sites, suggesting that soil archaea, bacteria, fungi, and viruses are globally as well as locally diverse.     

Metagenomic and Small-Subunit rRNA Analyses Reveal the Genetic Diversity of Bacteria, Archaea, Fungi, and Viruses in Soil

Recent studies have highlighted the surprising richness of soil bacterial communities; however, bacteria are not the only microorganisms found in soil. To our knowledge, no study has compared the diversities of the four major microbial taxa, i.e., bacteria, archaea, fungi, and viruses, from an individual soil sample. We used metagenomic and small-subunit RNA-based sequence analysis techniques to compare the estimated richness and evenness of these groups in prairie, desert, and rainforest soils. By grouping sequences at the 97% sequence similarity level (an operational taxonomic unit [OTU]), we found that the archaeal and fungal communities were consistently less even than the bacterial communities. Although total richness levels are difficult to estimate with a high degree of certainty, the estimated number of unique archaeal or fungal OTUs appears to rival or exceed the number of unique bacterial OTUs in each of the collected soils. In this first study to comprehensively survey viral communities using a metagenomic approach, we found that soil viruses are taxonomically diverse and distinct from the communities of viruses found in other environments that have been surveyed using a similar approach. Within each of the four microbial groups, we observed minimal taxonomic overlap between sites, suggesting that soil archaea, bacteria, fungi, and viruses are globally as well as locally diverse.     

云南热带户用沼气池的原核生物群落结构研究

【目的】揭示云南热带农村户用沼气池中的原核生物(细菌和古菌)的群落结构特征。【方法】采用16S r RNA基因克隆文库技术对云南(北)热带代表性气候区的户用沼气池中的原核生物(细菌和古菌)多样性进行研究。【结果】得到细菌330条有效序列,划分为108个OTUs,文库覆盖度为81.5%;古菌有效序列185条,划分为17个OTUs,文库覆盖度为97.8%。通过Gen Bank数据库进行相似性比对与系统发育分析,结果表明:大部分细菌为未知细菌(Unclassified bacteria,占24.19%),优势细菌类群归属拟杆菌门(Bacteroidetes,占23.58%)、绿弯菌门(Chloroflexi,占21.46%)、厚壁菌门(Firmicutes,占13.91%)和变形菌门(Proteobacteria,占8.74%);古菌主要的优势类群为乙酸盐营养型的甲烷八叠球菌目(Methanosarcinales)的鬃毛甲烷菌属(Methanosaeta,占76.75%);此外还检测到少量未培养的泉古菌门细菌(Crenarchaeota,占9.19%)。【结论】云南(北)热带代表性气候区的农村户用沼气池中的微生物种类十分丰富,不同微生物种类的丰度存在明显差异,并存在明显优势种群,且细菌比古菌具有更丰富的多样性。     

Selective phylogenetic analysis targeting 16S rRNA genes of hyperthermophilic archaea in the deep-subsurface hot biosphere

International drilling projects for the study of microbial communities in the deep-subsurface hot biosphere have been expanded. Core samples obtained by deep drilling are commonly contaminated with mesophilic microorganisms in the drilling fluid, making it difficult to examine the microbial community by 16S rRNA gene clone library analysis. To eliminate mesophilic organism contamination, we previously developed a new method (selective phylogenetic analysis [SePA]) based on the strong correlation between the guanine-plus-cytosine (G+C) contents of the 16S rRNA genes and the optimal growth temperatures of prokaryotes, and we verified the method's effectiveness (H. Kimura, M. Sugihara, K. Kato, and S. Hanada, Appl. Environ. Microbiol. 72:21-27, 2006). In the present study we ascertained SePA's ability to eliminate contamination by archaeal rRNA genes, using deep-sea hydrothermal fluid (117 degrees C) and surface seawater (29.9 degrees C) as substitutes for deep-subsurface geothermal samples and drilling fluid, respectively. Archaeal 16S rRNA gene fragments, PCR amplified from the surface seawater, were denatured at 82 degrees C and completely digested with exonuclease I (Exo I), while gene fragments from the deep-sea hydrothermal fluid remained intact after denaturation at 84 degrees C because of their high G+C contents. An examination using mixtures of DNAs from the two environmental samples showed that denaturation at 84 degrees C and digestion with Exo I completely eliminated archaeal 16S rRNA genes from the surface seawater. Our method was quite useful for culture-independent community analysis of hyperthermophilic archaea in core samples recovered from deep-subsurface geothermal environments.     

九龙江河口区nirS型反硝化细菌多样性及系统发育学分析

【目的】结合16S rRNA基因克隆文库和nirS基因克隆文库的分析,揭示九龙江河口区nirS型反硝化细菌多样性。【方法】选取九龙江河口区一富营养化采样点,分别采集水样及沉积物样品,进行理化因子的测定并提取细菌总DNA。以水样DNA构建16S rRNA基因克隆文库,以沉积物DNA构建nirS基因克隆文库,分析微生物群落结构的多样性并构建系统发育树。【结果】从16S rRNA基因克隆文库中获得86条有效序列,按97%的序列相似性划分为53个OTU,分别属于Proteobacteria门、Planctomycetes门、Bacteroidetes门、Actinobacteria门、Firmicutes门和Chloroflexi门。其中属于Proteobacteria门OTU的克隆子占克隆数的62.9%,是最优势的类群,分属于Alphaproteobacteria、Betaproteobacteria、Gammaproteobacteria和Deltaproteobacteria纲等。从nirS基因克隆文库中获得190条有效序列,翻译为氨基酸序列后,按82%的序列相似性划分为60个OTU,并定位到属的水平。其中Proteobacteria门是最优势的类群,占文库克隆子总数的71.6%,包括Alphaproteobacteria纲(5.8%)、Betaproteobacteria纲(49.0%)和Gammaproteobacteria纲(16.9%)。nirS基因克隆文库中丰度最高的OTU与GenBank中的一株可培养反硝化菌Thauera sp. R-26906具有100%的序列相似性。【结论】九龙江河口区的微生物以及亚硝酸盐还原酶基因(nirS)具有丰富的多样性。大部分NirS序列在GenBank中的最相似序列来源于河口、海湾等相似的环境。     

Microbial community analysis of a coastal hot spring in Kagoshima, Japan, using molecular- and culture-based approaches

Ibusuki hot spring is located on the coastline of Kagoshima Bay, Japan. The hot spring water is characterized by high salinity, high temperature, and neutral pH. The hot spring is covered by the sea during high tide, which leads to severe fluctuations in several environmental variables. A combination of molecular- and culture-based techniques was used to determine the bacterial and archaeal diversity of the hot spring. A total of 48 thermophilic bacterial strains were isolated from two sites (Site 1: 55.6°C; Site 2: 83.1°C) and they were categorized into six groups based on their 16S rRNA gene sequence similarity. Two groups (including 32 isolates) demonstrated low sequence similarity with published species, suggesting that they might represent novel taxa. The 148 clones from the Site 1 bacterial library included 76 operational taxonomy units (OTUs; 97% threshold), while 132 clones from the Site 2 bacterial library included 31 OTUs. Proteobacteria, Bacteroidetes, and Firmicutes were frequently detected in both clone libraries. The clones were related to thermophilic, mesophilic and psychrophilic bacteria. Approximately half of the sequences in bacterial clone libraries shared <92% sequence similarity with their closest sequences in a public database, suggesting that the Ibusuki hot spring may harbor a unique and novel bacterial community. By contrast, 77 clones from the Site 2 archaeal library contained only three OTUs, most of which were affiliated with Thaumarchaeota.     

Selective Phylogenetic Analysis Targeting 16S rRNA Genes of Hyperthermophilic Archaea in the Deep-Subsurface Hot Biosphere

International drilling projects for the study of microbial communities in the deep-subsurface hot biosphere have been expanded. Core samples obtained by deep drilling are commonly contaminated with mesophilic microorganisms in the drilling fluid, making it difficult to examine the microbial community by 16S rRNA gene clone library analysis. To eliminate mesophilic organism contamination, we previously developed a new method (selective phylogenetic analysis [SePA]) based on the strong correlation between the guanine-plus-cytosine (G+C) contents of the 16S rRNA genes and the optimal growth temperatures of prokaryotes, and we verified the method's effectiveness (H. Kimura, M. Sugihara, K. Kato, and S. Hanada, Appl. Environ. Microbiol. 72:21-27, 2006). In the present study we ascertained SePA's ability to eliminate contamination by archaeal rRNA genes, using deep-sea hydrothermal fluid (117°C) and surface seawater (29.9°C) as substitutes for deep-subsurface geothermal samples and drilling fluid, respectively. Archaeal 16S rRNA gene fragments, PCR amplified from the surface seawater, were denatured at 82°C and completely digested with exonuclease I (Exo I), while gene fragments from the deep-sea hydrothermal fluid remained intact after denaturation at 84°C because of their high G+C contents. An examination using mixtures of DNAs from the two environmental samples showed that denaturation at 84°C and digestion with Exo I completely eliminated archaeal 16S rRNA genes from the surface seawater. Our method was quite useful for culture-independent community analysis of hyperthermophilic archaea in core samples recovered from deep-subsurface geothermal environments.     

Communities of archaea and bacteria in a subsurface radioactive thermal spring in the Austrian Central Alps, and evidence of ammonia-oxidizing Crenarchaeota

Scanning electron microscopy revealed great morphological diversity in biofilms from several largely unexplored subterranean thermal Alpine springs, which contain radium 226 and radon 222. A culture-independent molecular analysis of microbial communities on rocks and in the water of one spring, the "Franz-Josef-Quelle" in Bad Gastein, Austria, was performed. Four hundred fifteen clones were analyzed. One hundred thirty-two sequences were affiliated with 14 bacterial operational taxonomic units (OTUs) and 283 with four archaeal OTUs. Rarefaction analysis indicated a high diversity of bacterial sequences, while archaeal sequences were less diverse. The majority of the cloned archaeal 16S rRNA gene sequences belonged to the soil-freshwater-subsurface (1.1b) crenarchaeotic group; other representatives belonged to the freshwater-wastewater-soil (1.3b) group, except one clone, which was related to a group of uncultivated Euryarchaeota. These findings support recent reports that Crenarchaeota are not restricted to high-temperature environments. Most of the bacterial sequences were related to the Proteobacteria (alpha, beta, gamma, and delta), Bacteroidetes, and Planctomycetes. One OTU was allied with Nitrospina sp. (delta-Proteobacteria) and three others grouped with Nitrospira. Statistical analyses suggested high diversity based on 16S rRNA gene analyses; the rarefaction plot of archaeal clones showed a plateau. Since Crenarchaeota have been implicated recently in the nitrogen cycle, the spring environment was probed for the presence of the ammonia monooxygenase subunit A (amoA) gene. Sequences were obtained which were related to crenarchaeotic amoA genes from marine and soil habitats. The data suggested that nitrification processes are occurring in the subterranean environment and that ammonia may possibly be an energy source for the resident communities.     

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。     

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.

     

西藏米拉山土壤古菌16S rRNA及amoA基因多样性?分析

摘要：【目的】硝化作用在全球土壤氮循环中具有重要的作用,虽然细菌一度被认为单独负责催化这个过程的限速步骤,但是最近一些研究结果表明泉古菌具有氨氧化的能力。本文通过构建古菌16S rRNA 基因克隆文库和氨氧化古菌amoA基因文库,分析西藏米拉山高寒草甸土壤中古菌及氨氧化古菌群落结构组成情况,为揭示青藏高原高寒草甸土壤古菌的多样性提供理论基础。【方法】采用未培养技术直接从土壤中提取微生物总DNA,分别利用通用引物构建古菌16S rRNA 基因和氨氧化古菌amoA基因克隆文库。【结果】通过构建系统发育树,表明古菌16S rRNA 基因克隆文库包括泉古菌门和未分类的古菌两大类,并且所有泉古菌均属于热变形菌纲。氨氧化古菌amoA基因克隆文库中序列均为泉古菌。通过DOTUR软件分析,古菌16S rRNA基因和古菌amoA基因克隆文库分别包括64个OTUs和 75个OTUs。【结论】西藏米拉山高寒草甸土壤中古菌多样性比较丰富,表明古菌在高寒草甸土壤的氮循环中可能具有重要的作用。所获得的一些序列与已知环境中土壤、淡水及海洋沉积物中获得的一些序列具有很高的相似性,其古菌及氨氧化古菌来自不同环境的可能性比较大,可能与青藏高原的地质历史变迁过程有关。米拉山古菌及氨氧化古菌与陆地设施土壤中相似性最高,说明与西藏米拉山高寒草甸土壤的退化有关。     

Life in extreme environments: microbial diversity in Great Salt Lake,Utah

Great Salt Lake (GSL) represents one of the world’s most hypersaline environments. In this study, the archaeal and bacterial communities at the North and South arms of the lake were surveyed by cloning and sequencing the 16S rRNA gene. The sampling locations were chosen for high salt concentration and the presence of unique environmental gradients, such as petroleum seeps and high sulfur content. Molecular techniques have not been systematically applied to this extreme environment, and thus the composition and the genetic diversity of microbial communities at GSL remain mostly unknown. This study led to the identification of 58 archaeal and 42 bacterial operational taxonomic units. Our phylogenetic and statistical analyses displayed a high biodiversity of the microbial communities in this environment. In this survey, we also showed that the majority of the 16S rRNA gene sequences within the clone library were distantly related to previously described environmental halophilic archaeal and bacterial taxa and represent novel phylotypes.     

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.     

A meta-analysis of the microbial diversity observed in anaerobic digesters

In this study, the collective microbial diversity in anaerobic digesters was examined using a meta-analysis approach. All 16S rRNA gene sequences recovered from anaerobic digesters available in public databases were retrieved and subjected to phylogenetic and statistical analyses. As of May 2010, 16,519 bacterial and 2869 archaeal sequences were found in GenBank. The bacterial sequences were assigned to 5926 operational taxonomic units (OTUs, based on ?97% sequence identity) representing 28 known bacterial phyla, with Proteobacteria (1590 OTUs), Firmicutes (1352 OTUs), Bacteroidetes (705 OTUs), and Chloroflexi (693 OTUs) being predominant. Archaeal sequences were assigned to 296 OTUs, primarily Methanosaeta and the uncharacterized WSA2 group. Nearly 60% of all sequences could not be classified to any established genus. Rarefaction analysis indicates that approximately 60% of bacterial and 90% of archaeal diversity in anaerobic digesters has been sampled. This analysis of the global bacterial and archaeal diversity in AD systems can guide future studies to further examine the microbial diversity involved in AD and development of comprehensive analytical tools.     

Molecular Diversity of the Rumen Microbiome of Norwegian Reindeer on Natural Summer Pasture

The molecular diversity of the rumen microbiome was investigated in five semi-domesticated adult female Norwegian reindeer (Rangifer tarandus tarandus) grazing on natural summer pastures on the coast of northern Norway (71.00° N, 25.30° E). Mean population densities (numbers per gram wet weight) of methanogenic archaea, rumen bacteria and ciliate protozoa, estimated using quantitative real-time polymerase chain reaction (PCR), were 3.17 × 10⁹, 5.17 × 10¹¹ and 4.02 × 10⁷, respectively. Molecular diversity of rumen methanogens was revealed using a 16S rRNA gene library (54 clones) constructed using pooled PCR products from the whole rumen contents of the five individual reindeer. Based upon a similarity criterion of <97%, a total of 19 distinct operational taxonomic units (OTUs) were identified, nine of which are potential new species. The 16S rRNA sequences generated from the reindeer rumen exhibited a high degree of sequence similarity to methanogens affiliated with the families Methanobacteriaceae (14 OTUs) and Methanosarcinaceae (one OTU). Four of the OTUs detected belonged to a group of uncultivated archaea previously found in domestic ruminants and thought to be dominant in the rumen together with Methanobrevibacter spp. Denaturing gradient gel electrophoresis profiling of the rumen bacterial 16S rRNA gene and the protozoal 18S rRNA gene indicated a high degree of animal variation, although some bands were common to all individuals. Automated ribosomal intergenic spacer analysis (ARISA) profiling of the ruminal Neocallimastigales population indicated that the reindeer are likely to contain more than one type of anaerobic fungus. The ARISA profile from one animal was distinct from the other four. This is the first molecular investigation of the ruminal methanogenic archaea in reindeer, revealing higher numbers than expected based on methane emission data available. Also, many of the reindeer archaeal 16S rRNA gene sequences were similar to those reported in domesticated ruminants in Australia, Canada, China, New Zealand and Venezuela, supporting previous findings that there seems to be no host type or geographical effect on the methanogenic archaea community structure in ruminants.     

Status of the phylogenetic diversity census of ruminal microbiomes

In this study, the collective microbial diversity in the rumen was examined by performing a meta-analysis of all the curated 16S rRNA gene (rrn) sequences deposited in the RDP database. As of November 2010, 13,478 bacterial and 3516 archaeal rrn sequences were found. The bacterial sequences were assigned to 5271 operation taxonomic units (OTUs) at species level (0.03 phylogenetic distance) representing 19 existing phyla, of which the Firmicutes (2958 OTUs), Bacteroidetes (1610 OTUs) and Proteobacteria (226 OTUs) were the most predominant. These bacterial sequences were grouped into more than 3500 OTUs at genus level (0.05 distance), but only 180 existing genera were represented. Nearly all the archaeal sequences were assigned to 943 species-level OTUs in phylum Euryarchaeota. Although clustered into 670 genus-level OTUs, only 12 existing archaeal genera were represented. Based on rarefaction analysis, the current percent coverage at species level reached 71% for bacteria and 65% for archaea. At least 78,218 bacterial and 24,480 archaeal sequences would be needed to reach 99.9% coverage. The results of this study may serve as a framework to assess the significance of individual populations to rumen functions and to guide future studies to identify the alpha and global diversity of ruminal microbiomes.     

Deep Sequencing and Ecological Characterization of Gut Microbial Communities of Diverse Bumble Bee Species

Gut bacterial communities of bumble bees are correlated with defense against pathogens. Further understanding this host-microbe association is vitally important as bumble bees are currently experiencing global population declines, potentially due in part to emergent diseases. In this study, we used pyrosequencing and community fingerprinting (ARISA) to characterize the gut microbial communities of nine bumble species from across the Bombus phylogeny. Overall, we delimited 74 bacterial taxa (operational taxonomic units or OTUs) belonging to Betaproteobacteria, Gammaproteobacteria, Bacilli, Actinobacteria, Flavobacteria and Alphaproteobacteria. Each bacterial community was taxonomically simple, containing an average of 1.9 common (relative abundance per sample > 5%) bacterial OTUs. The most abundant and prevalent (occurring in 92% of the samples) bacterial OTU, based on 16S rRNA sequences, closely matched that of the previously described Betaproteobacteria species Snodgrassella alvi. Bacteria that were first described in bee-related external environments dominated a number of gut bacterial communities, suggesting that they are not strictly dependent on the internal gut environment. The ARISA data showed a correlation between bacterial community structures and the geographic locations where the bees were sampled, suggesting that at least a subset of the bacterial species may be transmitted environmentally. Using light and fluorescent microscopy, we demonstrated that the gut bacteria form a biofilm on the internal epithelial surface of the ileum, corroborating results obtained from Apis mellifera.     

Comparison of microbial community compositions of injection and production well samples in a long-term water-flooded petroleum reservoir

Water flooding plays an important role in recovering oil from depleted petroleum reservoirs. Exactly how the microbial communities of production wells are affected by microorganisms introduced with injected water has previously not been adequately studied. Using denaturing gradient gel electrophoresis (DGGE) approach and 16S rRNA gene clone library analysis, the comparison of microbial communities is carried out between one injection water and two production waters collected from a working block of the water-flooded Gudao petroleum reservoir located in the Yellow River Delta. DGGE fingerprints showed that the similarities of the bacterial communities between the injection water and production waters were lower than between the two production waters. It was also observed that the archaeal composition among these three samples showed no significant difference. Analysis of the 16S rRNA gene clone libraries showed that the dominant groups within the injection water were Betaproteobacteria, Gammaproteobacteria and Methanomicrobia, while the dominant groups in the production waters were Gammaproteobacteria and Methanobacteria. Only 2 out of 54 bacterial operational taxonomic units (OTUs) and 5 out of 17 archaeal OTUs in the injection water were detected in the production waters, indicating that most of the microorganisms introduced by the injection water may not survive to be detected in the production waters. Additionally, there were 55.6% and 82.6% unique OTUs in the two production waters respectively, suggesting that each production well has its specific microbial composition, despite both wells being flooded with the same injection water.