Next Generation Sequencing to Define Prokaryotic and Fungal Diversity in the Bovine Rumen

A combination of Sanger and 454 sequences of small subunit rRNA loci were used to interrogate microbial diversity in the bovine rumen of 12 cows consuming a forage diet. Observed bacterial species richness, based on the V1–V3 region of the 16S rRNA gene, was between 1,903 to 2,432 species-level operational taxonomic units (OTUs) when 5,520 reads were sampled per animal. Eighty percent of species-level OTUs were dominated by members of the order Clostridiales, Bacteroidales, Erysipelotrichales and unclassified TM7. Abundance of Prevotella species varied widely among the 12 animals. Archaeal species richness, also based on 16S rRNA, was between 8 and 13 OTUs, representing 5 genera. The majority of archaeal OTUs (84%) found in this study were previously observed in public databases with only two new OTUs discovered. Observed rumen fungal species richness, based on the 18S rRNA gene, was between 21 and 40 OTUs with 98.4–99.9% of OTUs represented by more than one read, using Good’s coverage. Examination of the fungal community identified numerous novel groups. Prevotella and Tannerella were overrepresented in the liquid fraction of the rumen while Butyrivibrio and Blautia were significantly overrepresented in the solid fraction of the rumen. No statistical difference was observed between the liquid and solid fractions in biodiversity of archaea and fungi. The survey of microbial communities and analysis of cross-domain correlations suggested there is a far greater extent of microbial diversity in the bovine rumen than previously appreciated, and that next generation sequencing technologies promise to reveal novel species, interactions and pathways that can be studied further in order to better understand how rumen microbial community structure and function affects ruminant feed efficiency, biofuel production, and environmental impact.     

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.     

A meta-analysis of the publicly available bacterial and archaeal sequence diversity in saline soils

An integrated view of bacterial and archaeal diversity in saline soil habitats is essential for understanding the biological and ecological processes and exploiting potential of microbial resources from such environments. This study examined the collective bacterial and archaeal diversity in saline soils using a meta-analysis approach. All available 16S rDNA sequences recovered from saline soils were retrieved from publicly available databases and subjected to phylogenetic and statistical analyses. A total of 9,043 bacterial and 1,039 archaeal sequences, each longer than 250 bp, were examined. The bacterial sequences were assigned into 5,784 operational taxonomic units (OTUs, based on ≥97 % sequence identity), representing 24 known bacterial phyla, with Proteobacteria (44.9 %), Actinobacteria (12.3 %), Firmicutes (10.4 %), Acidobacteria (9.0 %), Bacteroidetes (6.8 %), and Chloroflexi (5.9 %) being predominant. Lysobacter (12.8 %) was the dominant bacterial genus in saline soils, followed by Sphingomonas (4.5 %), Halomonas (2.5 %), and Gemmatimonas (2.5 %). Archaeal sequences were assigned to 602 OTUs, primarily from the phyla Euryarchaeota (88.7 %) and Crenarchaeota (11.3 %). Halorubrum and Thermofilum were the dominant archaeal genera in saline soils. Rarefaction analysis indicated that less than 25 % of bacterial diversity, and approximately 50 % of archaeal diversity, in saline soil habitats has been sampled. This analysis of the global bacterial and archaeal diversity in saline soil habitats can guide future studies to further examine the microbial diversity of saline soils.     

Investigation of the FeFe-hydrogenase gene diversity combined with phylogenetic microbial community analysis of an anaerobic domestic sewage sludge

Biological hydrogen production through the anaerobic digestion is an environmental friendly alternative for satisfying future hydrogen demands. Microorganisms residing into waste water treatment plants are far from being exhaustively characterized and surveys on hydrogen production through FeFe-hydrogenase in such ecosystems are scarce. This study combined the analysis of 16S rRNA and [FeFe]-hydrogenase (hydA) genes with statistical tools to estimate richness and diversity of the microbial community of a domestic sewage treatment plant at the phylogenetic and functional levels. Archaeal groups were represented by 69 % of sequences assigned to Methanosarcinales and the remaining belonged to Methanomicrobiales. Within the bacterial library, 136 operational taxonomic units (OTUs) were distributed into 9 phyla, being 86 OTUs related to uncultivated bacteria. From these, 25 OTUs represented potential novel taxa within Synergistetes. Proteobacteria was the most predominant (36 % of the OTUs) and diversified phylogenetic group in the bacterial library, most of them assigned to the class Betaproteobacteria. Twenty-two putative hydA sequences were recovered into four distinct clusters and most of them were more closely related to each other than with sequences retrieved from databases, indicating they are hitherto undetected [Fe–Fe]-hydrogenase gene sequences. The richness estimates revealed that the number of sampled sequences was enough for full coverage of the archaeal diversity but not sufficient to cover both bacterial and hydA gene diversities. The results confirmed a great richness and diversity of bacterial and hydA sequences retrieved from the sewage sludge sample, suggesting such environment as a potential reservoir of new hydrogenase genes for biotechnological exploration.     

Bacterial diversity in the rumen of Indian Surti buffalo (Bubalus bubalis), assessed by 16S rDNA analysis

Bacterial communities in buffalo rumen were characterized using a culture-independent approach for a pooled sample of rumen fluid from 3 adult Surti buffaloes. Buffalo rumen is likely to include species of various bacterial phyla, so 16S rDNA sequences were amplified and cloned from the sample. A total of 191 clones were sequenced and similarities to known 16S rDNA sequences were examined. About 62.82% sequences (120 clones) had >90% similarity to the 16S rDNA database sequences. Furthermore, about 34.03% of the sequences (65 clones) were 85–89% similar to 16S rDNA database sequences. For the remaining 3.14%, the similarity was lower than 85%. Phylogenetic analyses were also used to infer the makeup of bacterial communities in the rumen of Surti buffalo. As a result, we distinguished 42 operational taxonomic units (OTUs) based on unique 16S r DNA sequences: 19 OTUs affiliated to an unidentified group (45.23% of total OTUs), 11 OTUs of the phylum Firmicutes, also known as the low G+C group (26.19%), 7 OTUs of theCytophaga-Flexibacter-Bacteroides phylum (16.66%), 4 OTUs of Spirochaetes (9.52%), and 1 OTU of Actinobacteria (2.38%). These include 10 single-clone OTUs, so Good’s coverage (94.76%) of 16S rRNA libraries indicated that sequences identified in the libraries represent the majority of bacterial diversity present in rumen.     

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。     

Phylogenetic diversity of bacterial communities in bovine rumen as affected by diets and microenvironments

Phylogenetic analysis was conducted to examine ruminal bacteria in two ruminal fractions (adherent fraction vs. liquid fraction) collected from cattle fed with two different diets: forage alone vs. forage plus concentrate. One hundred forty-four 16S rRNA gene (rrs) sequences were obtained from clone libraries constructed from the four samples. These rrs sequences were assigned to 116 different operational taxonomic units (OTUs) defined at 0.03 phylogenetic distance. Most of these OTUs could not be assigned to any known genus. The phylum Firmicutes was represented by approximately 70% of all the sequences. By comparing to the OTUs already documented in the rumen, 52 new OTUs were identified. UniFrac, SONS, and denaturing gradient gel electrophoresis analyses revealed difference in diversity between the two fractions and between the two diets. This study showed that rrs sequences recovered from small clone libraries can still help identify novel species-level OTUs.     

Bacterial and archaeal communities in the acid pit lake sediments of a chalcopyrite mine

Bacterial and archaeal community structures and diversity of three different sedimentary environments (BH1A, BH2A and BH3A) in the acid pit lake of a chalcopyrite mine at Touro (Spain) were determined by 16S rRNA gene PCR-DGGE and sequencing of clone libraries. DGGE of bacterial and archaeal amplicons showed that the sediments harbor different communities. Bacterial 16S rRNA gene sequences were assigned to Acidobacteria, Actinobacteria, Cyanobacteria, Planctomycetes, Proteobacteria, Chloroflexi and uncultured bacteria, after clustering into 42 operational taxonomic units (OTUs). OTU 2 represented approximately 37, 42 and 37 % of all sequences from sediments BH1A, BH2A and BH3A, respectively, and was phylogenetically related to uncultured Chloroflexi. Remaining OTUs were phylogenetically related to heterotrophic bacteria, including representatives of Ferrithrix and Acidobacterium genera. Archaeal 16S rRNA gene sequences were clustered into 54 OTUs. Most of the sequences from the BH1A sediment were assigned to Euryarchaeota, whereas those from BH2A sediment were assigned to Crenarchaeota. The majority of the sequences from BH3A sediment were assigned to unclassified Archaea, and showed similarities to uncultured and unclassified environmental clones. No sequences related to Acidithiobacillus and Leptospirillum, commonly associated with acid mine drainage, were detected in this study.     

共存于厌氧真菌分离培养液中瘤胃甲烷菌的检测及其多样性分析

对分离自山羊瘤胃的真菌分离培养液中甲烷菌进行16SrDNA扩增、DGGE分析、RFLP及测序分析,研究共存于真菌分离培养液中甲烷菌的种类及其多样性。DGGE结果显示:从厌氧真菌分离至第45代,甲烷菌多样性指数由1·32降至0·99,相似性最低为34·7%;第45代至62代,多样性指数由0·99升至1·15,相似性最低为89·2%。RFLP多态性分析69个克隆共得到5个操作分类单元,选择其中6个具有代表性的序列进行测序。序列及系统进化分析表明,属于其中3个操作分类单元的克隆最相似菌都是UnculturedarchaealsymbiontPA202,相似性均为95%,没有与这些克隆相似性较高的已培养甲烷菌;属于另外2个操作分类单元的克隆最相似菌都是Unculturedrumenmethanogen956,相似性均为97%,最相似已知菌为Methanobrevibactersp.NT7,相似性为97%。结果表明,真菌培养液中存在目前尚未分离培养的瘤胃甲烷菌。     

Comparative metagenomic analysis of bacterial populations in three full-scale mesophilic anaerobic manure digesters

While the use of anaerobic digestion to generate methane as a source of bioenergy is increasing worldwide, our knowledge of the microbial communities that perform biomethanation is very limited. Using next-generation sequencing, bacterial population profiles were determined in three full-scale mesophilic anaerobic digesters operated on dairy farms in the state of Vermont (USA). To our knowledge, this is the first report of a metagenomic analysis on the bacterial population of anaerobic digesters using dairy manure as their main substrate. A total of 20,366 non-chimeric sequence reads, covering the V1-V2 hypervariable regions of the bacterial 16S rRNA gene, were assigned to 2,176 operational taxonomic units (OTUs) at a genetic distance cutoff value of 5 %. Based on their limited sequence identity to validly characterized species, the majority of OTUs identified in our study likely represented novel bacterial species. Using a naïve Bayesian classifier, 1,624 anaerobic digester OTUs could be assigned to 16 bacterial phyla, while 552 OTUs could not be classified and may belong to novel bacterial taxonomic groups that have yet to be described. Firmicutes, Bacteroidetes, and Chloroflexi were the most highly represented bacteria overall, with Bacteroidetes and Chloroflexi showing the least and the most variation in abundance between digesters, respectively. All digesters shared 132 OTUs, which as a “core” group represented 65.4 to 70.6 % of sequences in individual digesters. Our results show that bacterial populations from microbial communities of anaerobic manure digesters can display high levels of diversity despite sharing a common core substrate.     

Composition, spatial distribution, and diversity of the bacterial communities in the rumen of cows fed different forages

The species composition, distribution, and biodiversity of the bacterial communities in the rumen of cows fed alfalfa or triticale were investigated using 16S rRNA gene clone library analyses. The rumen bacterial community was fractionated and analyzed as three separate fractions: populations in the planktonic, loosely attached to rumen digesta particles, and tightly attached to rumen digesta particles. Six hundred and thirteen operational taxonomic units (OTUs) belonging to 32 genera, 19 families, and nine phyla of the domain Bacteria were identified from 1014 sequenced clones. Four hundred and fifty one of the 613 OTUs were identified as new species. These bacterial sequences were distributed differently among the three fractions in the rumen digesta of cows fed alfalfa or triticale. Chao 1 estimation revealed that, in both communities, the populations tightly attached to particulates were more diverse than the planktonic and those loosely attached to particulates. S-Libshuff detected significant differences in the composition between any two fractions in the rumen of cows with the same diet and between the communities fed alfalfa and triticale diets. The species richness estimated for the communities fed alfalfa and triticale is 1027 and 662, respectively. The diversity of the rumen bacterial community examined in this study is greater than previous studies have demonstrated and the differences in the community composition between two high-fiber diets have implications for sample selection for downstream metagenomics applications.     

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.     

Bacteria and Archaea community structure in the rumen microbiome of goats (Capra hircus) from the semiarid region of Brazil

Most studies present in the literature about the rumen microbiome have focused on cattle and sheep. This is the first report of the characterization of the bacterial and archaeal communities present in the liquid and solid-associated fractions of the rumen from free ranging Moxotó breed goats using 16S rRNA gene libraries. PCR was used to amplify the 16S rRNA gene with bacterial and archaeal universal primers and sequences from each library constructed were obtained. Sequences of Bacteria from the phyla Bacteroidetes and Firmicutes were predominant. The overall dominant classes in the rumen were Clostridia and Bacteroidia, which are known to play a role in plant fiber degradation in other ruminants. Unclassified Bacteria accounted for 4.7% of the liquid fraction sequences and 16.4% of the solid fraction sequences. From the archaeal libraries only sequences from the phylum Euryarcheota were identified and were assigned to the class Methanobacteria of the genera Methanobrevibacter and Methanosphaera. A group of Archaea not previously known to be associated with the rumen was identified: uncultured methanogens belonging to the "uncultured marine bacteria" groups II and III. The local water contained high salt concentrations and this may explain the presence of these groups in the Moxotó goat rumen.     

Diversity and Community Composition of Methanogenic Archaea in the Rumen of Scottish Upland Sheep Assessed by Different Methods

Ruminal archaeomes of two mature sheep grazing in the Scottish uplands were analysed by different sequencing and analysis methods in order to compare the apparent archaeal communities. All methods revealed that the majority of methanogens belonged to the Methanobacteriales order containing the Methanobrevibacter, Methanosphaera and Methanobacteria genera. Sanger sequenced 1.3 kb 16S rRNA gene amplicons identified the main species of Methanobrevibacter present to be a SGMT Clade member Mbb. millerae (≥91% of OTUs); Methanosphaera comprised the remainder of the OTUs. The primers did not amplify ruminal Thermoplasmatales-related 16S rRNA genes. Illumina sequenced V6–V8 16S rRNA gene amplicons identified similar Methanobrevibacter spp. and Methanosphaera clades and also identified the Thermoplasmatales-related order as 13% of total archaea. Unusually, both methods concluded that Mbb. ruminantium and relatives from the same clade (RO) were almost absent. Sequences mapping to rumen 16S rRNA and mcrA gene references were extracted from Illumina metagenome data. Mapping of the metagenome data to16S rRNA gene references produced taxonomic identification to Order level including 2–3% Thermoplasmatales, but was unable to discriminate to species level. Mapping of the metagenome data to mcrA gene references resolved 69% to unclassified Methanobacteriales. Only 30% of sequences were assigned to species level clades: of the sequences assigned to Methanobrevibacter, most mapped to SGMT (16%) and RO (10%) clades. The Sanger 16S amplicon and Illumina metagenome mcrA analyses showed similar species richness (Chao1 Index 19–35), while Illumina metagenome and amplicon 16S rRNA analysis gave lower richness estimates (10–18). The values of the Shannon Index were low in all methods, indicating low richness and uneven species distribution. Thus, although much information may be extracted from the other methods, Illumina amplicon sequencing of the V6–V8 16S rRNA gene would be the method of choice for studying rumen archaeal communities.     

Diversity of culturable halophilic archaea isolated from Rambla Salada, Murcia (Spain)

We have studied the diversity of culturable halophilic Archaea at Rambla Salada, Murcia (south-eastern Spain). We made 8 samplings at different places in this habitat during the years 2006 and 2007 and isolated a total of 49 strains, which were identified by means of phenotypic tests and the hypervariable V1–V3 region of the 16S rRNA gene sequences (around 500 bp). The ribosomal data showed that the isolates belonged to 12 genera within the Halobacteriaceae family, with Haloferax and Natrinema being the most abundant. Five strains showed less than 97% sequence identity with validly described species and may well represent new taxa. All the strains grew best with around 25% w/v salts, required high concentrations of NaCl and magnesium and produced red to pink colonies. They were facultative anaerobes with both respiratory and fermentative metabolisms. The diversity of the archaeal community was analysed with the MOTHUR package. We identified 14 OTUs at the 3% genetic distance level and found quite high diversity. Rarefaction curves of richness estimators and diversity indices demonstrated that our collection of isolates represented the archaeal community at Rambla Salada that can be isolated under the conditions used in this work. This is the first report to be published on the culturable archaea at Rambla Salada, an area of considerable ecological interest.     

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.

     

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。     

Evaluation of different partial 16S rRNA gene sequence regions for phylogenetic analysis of microbiomes

Operational taxonomic units (OTUs) are conventionally defined at a phylogenetic distance (0.03—species, 0.05—genus, 0.10—family) based on full-length 16S rRNA gene sequences. However, partial sequences (700 bp or shorter) have been used in most studies. This discord may affect analysis of diversity and species richness because sequence divergence is not distributed evenly along the 16S rRNA gene. In this study, we compared a set each of bacterial and archaeal 16S rRNA gene sequences of nearly full length with multiple sets of different partial 16S rRNA gene sequences derived therefrom (approximately 440-700 bp), at conventional and alternative distance levels. Our objective was to identify partial sequence region(s) and distance level(s) that allow more accurate phylogenetic analysis of partial 16S rRNA genes. Our results showed that no partial sequence region could estimate OTU richness or define OTUs as reliably as nearly full-length genes. However, the V1-V4 regions can provide more accurate estimates than others. For analysis of archaea, we recommend the V1-V3 and the V4-V7 regions and clustering of species-level OTUs at 0.03 and 0.02 distances, respectively. For analysis of bacteria, the V1-V3 and the V1-V4 regions should be targeted, with species-level OTUs being clustered at 0.04 distance in both cases.     

Analysis of Rumen Methanogen Diversity in Water Buffaloes (Bubalus bubalis) Under Three Different Diets

The water buffalo (Bubalus bubalis) is a prominent livestock species for the production of milk and meat in many countries. We investigated the diversity of rumen methanogens in Mediterranean water buffaloes maintained in Brazil under different diets: corn silage, grazing pasture, or sugar cane. A total of 467 clones were isolated from three methanogen 16S rRNA gene clone libraries that each represented a distinct feed type. The 467 clones were assigned to 19 species-level operational taxonomic units (OTUs). Four OTUs were represented in all three libraries, eight OTUs were library-specific, six OTUs were found in only the corn silage and pasture grazing libraries, and one OTU was shared only between pasture grazing and sugar cane libraries. We found that Methanobrevibacter-related sequences were the most abundant in the water buffaloes sampled for our analysis, in contrast to previously reported studies showing that Methanomicrobium mobile-like methanogens were the most abundant methanogens in water buffaloes of Murrah and Surti breeds sampled in India. Considering the worldwide distribution of water buffaloes and the likely wide variety of diets provided, our results combined with studies from other groups support that larger scope analyses of microbiomes for this livestock species would provide great insight into the contribution of geographical location, breed, and diet in determining the population structure of rumen microorganisms.     

Species Composition of Cultivated and Noncultivated Bacteria from Short Filaments in an Icelandic Hot Spring at 88 degrees C

Samples of short pink-grayish filaments were collected from a hot spring in the Hengill area in southwestern Iceland at 85-88 degrees C, pH 6.9 and 1.7 mg/L sulfide. The species composition was studied by cloning and sequencing small subunit rRNA genes obtained by PCR amplifications from mat DNA. Using 98% sequence similarity as a cutoff value, a total of 5 bacterial operational taxonomic units (OTUs) and 6 archaeal OTUs were detected among 68 bacterial clones and 97 archaeal clones. Database matching showed that 80.5% of the archaeal sequences were 99% similar to Pyrobaculum islandicum and 14.5% were closest to the Korarchaeota clone sequence SRI306. About 87% of the bacterial sequences had the closest database match (99%) to the clone sequence SRI48 but were also found to be 99% identical with hydrogen-oxidizing strains previously isolated in this laboratory from hot springs in the same region. Out of 7 Thermus sequences, 4 were 100% identical to T. scotoductus NMX2 A.1 but 3 represented a new uncultivated Thermus species. Four different media, varying in organic nutrients and phosphate composition were used to isolate 81 aerobic thermophilic heterotrophs. Four isolates were Bacillus spp; but out of 77 Thermus isolates, 42 belonged to T. scotoductus and 35 to T. brockianus. T. scotoductus seemed to be preferably isolated on media low in nutrients and phosphate, whereas for T. brockianus it was the opposite. The T. scotoductus clones and isolates had 99-100% sequence similarity to each other. No T. brockianus sequences were found in the bacterial clone library.