南美白对虾肠道微生物群落的分子分析

采用分子生物学手段16S rDNA克隆文库方法对实验室养殖条件下的南美白对虾肠道细菌进行了多样性研究。用限制性片段长度多态性(RFLP)方法从文库中筛选出可能不同细菌来源的克隆子12个,测定其16S rDNA片段核甘酸序列,将所获得的序列与GenBank数据库进行BLAST比对,结果表明:南美白对虾肠道的16S rDNA克隆文库中126个克隆子分属2个不同的细菌类群:变形细菌(Proteobacteria)和厚壁细菌(Firmicutes),其中厚壁细菌为优势菌群占到75.4%,且与最相似序列同源性均低于94%;变形细菌占到24.6%,与最相似序列同源性均高于98%,分别为希瓦氏菌属(Shewanella),泛菌属(Pantoea),Aranicola属,假单胞菌属(Pseudomonas)和弧菌属(Vibrio)。     

Microbial diversity in Maras salterns, a hypersaline environment in the Peruvian Andes

Maras salterns are located 3,380 m above sea level in the Peruvian Andes. These salterns consist of more than 3,000 little ponds which are not interconnected and act as crystallizers where salt precipitates. These ponds are fed by hypersaline spring water rich in sodium and chloride. The microbiota inhabiting these salterns was examined by fluorescence in situ hybridization (FISH), 16S rRNA gene clone library analysis, and cultivation techniques. The total counts per milliliter in the ponds were around 2 x 10(6) to 3 x 10(6) cells/ml, while the spring water contained less than 100 cells/ml and did not yield any detectable FISH signal. The microbiota inhabiting the ponds was dominated (80 to 86% of the total counts) by Archaea, while Bacteria accounted for 10 to 13% of the 4',6'-diamidino-2-phenylindole (DAPI) counts. A total of 239 16S rRNA gene clones were analyzed (132 Archaea clones and 107 Bacteria clones). According to the clone libraries, the archaeal assemblage was dominated by microorganisms related to the cosmopolitan square archaeon "Haloquadra walsbyi," although a substantial number of the sequences in the libraries (31% of the 16S rRNA gene archaeal clones) were related to Halobacterium sp., which is not normally found in clone libraries from solar salterns. All the bacterial clones were closely related to each other and to the gamma-proteobacterium "Pseudomonas halophila" DSM 3050. FISH analysis with a probe specific for this bacterial assemblage revealed that it accounted for 69 to 76% of the total bacterial counts detected with a Bacteria-specific probe. When pond water was used to inoculate solid media containing 25% total salts, both extremely halophilic Archaea and Bacteria were isolated. Archaeal isolates were not related to the isolates in clone libraries, although several bacterial isolates were very closely related to the "P. halophila" cluster found in the libraries. As observed for other hypersaline environments, extremely halophilic bacteria that had ecological relevance seemed to be easier to culture than their archaeal counterparts.     

Bacterial diversity in biofilms formed on condenser tube surfaces in a nuclear power plant

To elucidate the bacterial diversity in biofilms formed on a condenser tube from a nuclear power plant, 16S rRNA gene sequences were examined using a PCR-cloning-sequencing approach. Twelve operational taxonomic units were retrieved in the clone library, and the estimated species richness was low (13.2). Most of the clones (94.7%) were affiliated with α-Proteobacteria; Planctomycetes and γ-Proteobacteria were much rarer. Interestingly, except for one clone belonging to Pseudoalteromonas, most of the sequences displayed sequence similarities <97% of those of the closest type strains. Based on 16S rRNA phylogenetic analysis, most bacteria were assigned to novel taxa above the species level. The low species richness and unusual bacterial composition may be attributable to selective pressure from chlorine in the cooling water. To prevent or control bacterial biofilms in cooling circuits, additional studies of the physiology and ecology of these species will be essential.     

Composition and variability of biofouling organisms in seawater reverse osmosis desalination plants

Seawater reverse osmosis (SWRO) membrane biofouling remains a common challenge in the desalination industry, but the marine bacterial community that causes membrane fouling is poorly understood. Microbial communities at different stages of treatment processes (intake, cartridge filtration, and SWRO) of a desalination pilot plant were examined by both culture-based and culture-independent approaches. Bacterial isolates were identified to match the genera Shewanella, Alteromonas, Vibrio, and Cellulophaga based on 16S rRNA gene sequencing analysis. The 16S rRNA gene clone library of the SWRO membrane biofilm showed that a filamentous bacterium, Leucothrix mucor, which belongs to the gammaproteobacteria, accounted for nearly 30% of the clone library, while the rest of the microorganisms (61.2% of the total clones) were related to the alphaproteobacteria. 16S rRNA gene terminal restriction fragment length polymorphism (T-RFLP) analysis indicated that bacteria colonizing the SWRO membrane represented a subportion of microbes in the source seawater; however, they were quite different from those colonizing the cartridge filter. The examination of five SWRO membranes from desalination plants located in different parts of the world showed that although the bacterial communities from the membranes were not identical to each other, some dominant bacteria were commonly observed. In contrast, bacterial communities in source seawater were significantly different based on location and season. Microbial profiles from 14 cartridge filters collected from different plants also revealed spatial trends.     

Phylogenetic diversity of winter bacterioplankton of eutrophic siberian reservoirs as revealed by 16S rRNA gene sequence

Using 16S rRNA gene sequence analyses we investigated the bacterial diversity of winter bacterioplankton of two eutrophic Siberian reservoirs. These reservoirs show similarity in phytoplankton community composition in spring and autumn but tend to differ in summer in exhibiting cyanobacterial bloom. Forty-eight unique partial 16S RNA gene sequences retrieved from two libraries were mostly affiliated with the class Actinobacteria, b subdivision of the class Proteobacteria, and the phylum Cytophaga-Flavobacterium-Bacteroides. The clone library of the pond exhibiting summer cyanobacterial bloom showed more diversity in sequence composition. A significant number of bacterial 16S rRNA gene clones were closely related to freshwater bacteria previously found in different aquatic ecosystems. This finding confirms the assumption that some bacterial clades are globally distributed.     

Diversity and abundance of uncultured cytophaga-like bacteria in the Delaware estuary

The Cytophaga-Flavobacterium group is known to be abundant in aquatic ecosystems and to have a potentially unique role in the utilization of organic material. However, relatively little is known about the diversity and abundance of uncultured members of this bacterial group, in part because they are underrepresented in clone libraries of 16S rRNA genes. To circumvent a suspected bias in PCR, a primer set was designed to amplify 16S rRNA genes from the Cytophaga-Flavobacterium group and was used to construct a library of these genes from the Delaware Estuary. This library had several novel Cytophaga-like 16S rRNA genes, of which about 40% could be grouped together into two clusters (DE clusters 1 and 2) defined by sequences initially observed only in the Delaware library; the other 16S rRNA genes were classified into an additional four clades containing sequences from other environments. An oligonucleotide probe was designed for the cluster with the most clones (DE cluster 2) and was used in fluorescence in situ hybridization assays. Bacteria in DE cluster 2 accounted for about 10% of the total prokaryotic abundance in the Delaware Estuary and in a depth profile of the Chukchi Sea (Arctic Ocean). The presence of DE cluster 2 in the Arctic Ocean was confirmed by results from 16S rRNA clone libraries. The contribution of this cluster to the total bacterial biomass is probably larger than is indicated by the abundance of its members, because the average cell volume of bacteria in DE cluster 2 was larger than those of other bacteria and prokaryotes in the Delaware Estuary and Chukchi Sea. DE cluster 2 may be one of the more abundant bacterial groups in the Delaware Estuary and possibly other marine environments.     

Bacteria associated with mucus and tissues of the coral Oculina patagonica in summer and winter

The relative abundance of bacteria in the mucus and crushed tissue of the Mediterranean coral Oculina patagonica was determined by analyses of the 16S rRNA genes of isolated colonies and from a 16S rRNA clone library of extracted DNA. By SYBR gold staining, the numbers of bacteria in mucus and tissue samples were 6.2 x 10(7) and 8.3 x 10(8)/cm2 of coral surface, respectively, 99.8% of which failed to produce colonies on Marine Agar. From analysis of mucus DNA, the most-abundant bacterium was Vibrio splendidus, representing 68% and 50% of the clones from the winter and summer, respectively. After removal of mucus from coral by centrifugation, analyses of DNA from the crushed tissue revealed a large diversity of bacteria, with Vibrio species representing less than 5% of the clones. The most-abundant culturable bacteria were a Pseudomonas sp. (8 to 14%) and two different alpha-proteobacteria (6 to 18%). Out of a total 1,088 16S rRNA genes sequenced, 400 different operational taxonomic units were identified (> 99.5% identity). Of these, 295 were novel (< 99% identical to any sequences in the GenBank database). This study provides a comprehensive database for future examinations of changes in the bacterial community during bleaching events.     

Bacterial biodiversity from Roopkund Glacier, Himalayan mountain ranges, India

The bacterial diversity of two soil samples collected from the periphery of the Roopkund glacial lake and one soil sample from the surface of the Roopkund Glacier in the Himalayan ranges was determined by constructing three 16S rRNA gene clone libraries. The three clone libraries yielded a total of 798 clones belonging to 25 classes. Actinobacteria was the most predominant class (>10% of the clones) in the three libraries. In the library from the glacial soil, class Betaproteobacteria (24.2%) was the most predominant. The rarefaction analysis indicated coverage of 43.4 and 41.2% in the samples collected from the periphery of the lake thus indicating a limited bacterial diversity covered; at the same time, the coverage of 98.4% in the glacier sample indicated most of the diversity was covered. Further, the bacterial diversity in the Roopkund glacier soil was low, but was comparable with the bacterial diversity of a few other glaciers. The results of principal component analysis based on the 16S rRNA gene clone library data, percentages of OTUs and biogeochemical data revealed that the lake soil samples were different from the glacier soil sample and the biogeochemical properties affected the diversity of microbial communities in the soil samples.     

Microbial Diversity in Maras Salterns, a Hypersaline Environment in the Peruvian Andes

Maras salterns are located 3,380 m above sea level in the Peruvian Andes. These salterns consist of more than 3,000 little ponds which are not interconnected and act as crystallizers where salt precipitates. These ponds are fed by hypersaline spring water rich in sodium and chloride. The microbiota inhabiting these salterns was examined by fluorescence in situ hybridization (FISH), 16S rRNA gene clone library analysis, and cultivation techniques. The total counts per milliliter in the ponds were around 2 × 10⁶ to 3 × 10⁶ cells/ml, while the spring water contained less than 100 cells/ml and did not yield any detectable FISH signal. The microbiota inhabiting the ponds was dominated (80 to 86% of the total counts) by Archaea, while Bacteria accounted for 10 to 13% of the 4′,6′-diamidino-2-phenylindole (DAPI) counts. A total of 239 16S rRNA gene clones were analyzed (132 Archaea clones and 107 Bacteria clones). According to the clone libraries, the archaeal assemblage was dominated by microorganisms related to the cosmopolitan square archaeon “Haloquadra walsbyi,” although a substantial number of the sequences in the libraries (31% of the 16S rRNA gene archaeal clones) were related to Halobacterium sp., which is not normally found in clone libraries from solar salterns. All the bacterial clones were closely related to each other and to the γ-proteobacterium “Pseudomonas halophila” DSM 3050. FISH analysis with a probe specific for this bacterial assemblage revealed that it accounted for 69 to 76% of the total bacterial counts detected with a Bacteria-specific probe. When pond water was used to inoculate solid media containing 25% total salts, both extremely halophilic Archaea and Bacteria were isolated. Archaeal isolates were not related to the isolates in clone libraries, although several bacterial isolates were very closely related to the “P. halophila” cluster found in the libraries. As observed for other hypersaline environments, extremely halophilic bacteria that had ecological relevance seemed to be easier to culture than their archaeal counterparts.     

转几丁质酶和葡聚糖酶双价基因棉花对土壤细菌种群多样性的影响

以转几丁质酶和葡聚糖酶双价基因棉花为研究对象,非转基因受体棉花为对照,通过比较可培养细菌数量和基于16S rRNA克隆文库细菌种群分析,评价外源双价基因的导入在苗期、蕾期、花铃期和吐絮期对棉花根际细菌群落多样性的影响。结果表明,可培养细菌的数量不受外源双价基因的影响,随着棉花生育期的交替而变化,以代谢旺盛的花铃期最多。构建的转基因和非转基因不同生育期根际土壤细菌16S rRNA文库容量为2400个克隆,涵盖了细菌的283个属。其中,Acidobacterium是最大优势类群,共包括624个克隆,其次为未知细菌种群和Flavisolibacter。比较转基因和非转基因棉花根际土壤细菌的种群结构,结果显示,同一生育期内前者种群的多样性显著低于后者,二者的共有类群随着生长发育的进行而增多。研究结果说明几丁质酶基因和葡聚糖酶基因对棉花根际细菌种群多样性有着不同程度的削减作用,但是随着种植时间的延长,该差异呈现逐渐缩小的趋势。     

Diversity of bacteria and polyketide synthase associated with marine sponge Haliclona sp.

The microbial community associated with a marine sponge (Haliclona sp.) collected from Tateyama city, Japan was studied using 16S rRNA gene clone libraries. Two DNA templates were prepared using methods recommended for Gram-positive and Gram-negative bacteria in the Qiagen kit manual. From each DNA template, two 16S rRNA genes were PCR amplified, using the combination of universal bacterial primer 27f and primers 1385r and 1492r, respectively. A total of 347 clones were sequenced and compared with those available in DNA data banks. These sequences were members of ten bacterial phyla. Interestingly, more than 30 % of the clones represent novel sequences. A comparison of these sequences with sequences in a library prepared from DNA extracted from the surrounding water shows minimum DNA contamination. Taxonomically, the highest diversity was detected in the clone library prepared using a combination of primers 27f and 1492r and DNA isolated using the Gram-positive bacteria protocol. The potential of Haliclona sp.-associated bacteria to produce secondary metabolites was studied by cloning and sequencing the polyketide synthase (PKS, type 1) gene using the same DNA samples. Analysis of partial sequences derived from the sponge metagenome revealed 27 unique ketosynthase domains of PKS type I. This study suggests strongly that this Haliclona sp. plays host to diverse novel bacteria with a potential to produce novel polyketides.     

Vibrio sp. as a potentially important member of the Black Band Disease (BBD) consortium in Favia sp. corals

Black Band Disease (BBD) is a well-described disease plaguing corals worldwide. It has been established that ecological and environmental stress factors contribute to the appearance and progression of the disease, believed to be caused by a diverse microbial consortium. We have identified and characterized Vibrio sp. associated with BBD in Eilat reef corals using both culture-dependent and -independent methods. Direct sampling using 16S rRNA gene clone libraries showed seasonal dynamics in the diversity of BBD-associated Vibrios . In the two sampling periods, BBD-associated Vibrio clones showed similarities to different groups: October samples were similar to known pathogens, while December samples were similar to general aquatic Vibrio sp. Cultured bacterial isolates of Vibrio sp. were highly homologous (≥99%) to previously documented BBD-associated bacteria from the Caribbean, Bahamas and Red Seas, and were similar to several known coral pathogens, such as Vibrio coralliilyticus . The proteolytic activity of Vibrio sp., as measured using casein- and azocasein-based assays, directly correlated with temperature elevation and peaked at 26–28 °C, with the microorganisms producing more proteases per bacterial cell or increasing the rate of proteolytic activity of the same proteases (potentially metalloproteases). This activity may promote coral tissue necrosis and aid in ensuing progression of the coral BBD.     

High similarity between bacterioneuston and airborne bacterial community compositions in a high mountain lake area

The bacterioneuston (bacteria inhabiting the air–water interface) is poorly characterized and possibly forms a unique community in the aquatic environment. In high mountain lakes, the surface film is subjected to extreme conditions of life, suggesting the development of a specific and adapted bacterioneuston community. We have studied the surface film of a remote high mountain lake in the Pyrenees by cloning the PCR-amplified 16S rRNA gene and comparing with bacteria present in underlying waters (UW), and airborne bacteria from the dust deposited on the top of the snow pack. We did not detect unusual taxa in the neuston but rather very common and widespread bacterial groups. Betaproteobacteria and Actinobacteria accounted for >75% of the community composition. Other minor groups were Gammaproteobacteria (between 8% and 12%), Alphaproteobacteria (between 1% and 5%), and Firmicutes (1%). However, we observed segregated populations in neuston and UW for the different clades within each of the main phylogenetic groups. The soil bacterium Acinetobacter sp. was only detected in the snow–dust sample. Overall, higher similarities were found between bacterioneuston and airborne bacteria than between the former and bacterioplankton. The surface film in high mountain lakes appears as a direct interceptor of airborne bacteria useful for monitoring long-range bacterial dispersion.     

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.     

Phylogenetic analysis of the succession of bacterial communities in the Great South Bay (Long Island)

Bacterial community composition and succession were examined over the course of the summer season in the Great South Bay, Long Island, NY, USA, using a 16S rDNA clone library approach. There was a progression of changes in dominant species in the libraries during the summer of 1997. The July library had several groups dominant, the SAR407 relatives of the alpha-Proteobacteria (24%) and the SAR86 (18%), sulfur-oxidizing symbiont relatives (8%) of the gamma-Proteobacteria, and unidentified Cytophaga-Flexibacter representatives (22%). In August, the Cytophaga-Flexibacter (Gelidibacter sp. and unidentified Cytophaga-Flexibacter representative) and Cyanobacteria (Synechococcus sp.) increased to 28% and 14%, respectively. High GC Gram-positives appeared at 18%, and beta-Proteobacteria (Ralstonia sp.) at 10%. By September these groups had either declined or were absent, while the SAR86 cluster, Pseudoalteromonas and Alteromonas of the gamma-Proteobacteria were dominant in the community (61%). The dominance of open ocean bacteria along with the presence of Aureococcus anophagefferens (Pelagophyceae) in July suggests possible open ocean coupling to bloom events. Many clones in this study were related to previously described clones from a wide distribution of marine environments, substantiating the cosmopolitan nature of pelagic bacteria. Only one isolated bacterium was closely related to 16S rDNA found in the August library.     

Diversity of microbes associated with the marine sponge, Haliclona simulans, isolated from Irish waters and identification of polyketide synthase genes from the sponge metagenome

Samples of the sponge Haliclona simulans were collected from Irish waters and subjected to a culture-independent analysis to determine the microbial, polyketide synthase (PKS) and non-ribosomal peptide synthase (NRPS) diversity. 16S rRNA gene libraries were prepared from total sponge, bacterial enriched sponge and seawater samples. Eight phyla from the Bacteria were detected in the sponge by phylogenetic analyses of the 16S rRNA gene libraries. The most abundant phylum in the total sponge library was the Proteobacteria (86%), with the majority of these clones being from the γ- Proteobacteria (77%); two groups of clones were dominant and together made up 69% of the total. Both of these groups were related to other sponge-derived microbes and comprised novel genera. Within the other bacterial phyla groups of clones representing novel candidate genera within the phyla Verrucomicrobia and Lentisphaerae were also found. Selective enrichment of the bacterial component of the sponge prior to 16S rRNA gene analysis resulted in a 16S rRNA gene library dominated by a novel genus of δ- Proteobacteria , most closely related to the Bdellovibrio . The potential for the sponge microbiota to produce secondary metabolites was also analysed by polymerase chain reaction amplification of PKS and NRPS genes. While no NRPS sequences were isolated seven ketosynthase (KS) sequences were obtained from the sponge metagenome. Analyses of these clones revealed a diverse collection of PKS sequences which were most closely affiliated with PKS from members of the Cyanobacteria , Myxobacteria and Dinoflagellata .     

免培养法研究野生川金丝猴肠道内生细菌多样性

【目的】了解野生川金丝猴(Rhinopithecus roxellana)肠道内生细菌的组成及其多样性。【方法】提取川金丝猴肠道内生细菌总DNA,选用细菌通用引物799F和1492R对总DNA进行16S rRNA基因特异性扩增,构建川金丝猴肠道内生细菌16S rRNA基因克隆文库,对阳性克隆进行限制性内切酶片段长度多态性(PCR-RFLP)分析,并对HaeⅢ酶切带谱菌株进行测序,构建系统发育树。【结果】根据酶切带谱分析和测序结果,将随机挑取的157个阳性克隆归为27个不同的可操作分类单元(OTUs)。系统发育分析表明这些克隆序列有62.10%属于厚壁菌门(Firmicutes),其中包括梭菌属(Clostridium)、Cellulosilyticum属、Robinsoniella属、Anaerofustis属、Blautia属和Anaerovorax属,有37.90%属于未培养细菌。【结论】川金丝猴肠道内生细菌多样性丰富,并且可能存在新的分类单元。     

Identification of active bacterial communities in a model drinking water biofilm system using 16S rRNA-based clone libraries

Recent phylogenetic studies have used DNA as the target molecule for the development of environmental 16S rRNA gene clone libraries. As DNA may persist in the environment, DNA-based libraries cannot be used to identify metabolically active bacteria in water systems. In this study, an annular reactor was used to generate model drinking water biofilms grown on polycarbonate slides. High-quality RNA was extracted from 2-month-old biofilms and used to generate 16S rRNA-based clones. Sequencing analyses of 16S rRNA-based clones suggested that the active bacterial fraction consisted of a few dominant bacterial groups related to Nevskia ramosa and to uncultured bacteria. Several of these bacterial groups were closely related to clones characterized in a DNA-based clone library also generated in this study. Altogether, these results suggest that some of the predominant drinking water bacteria identified using DNA-based techniques are indeed active.     

Detection of stratified microbial populations related to Chlorobium and Fibrobacter species in the Atlantic and Pacific oceans.

A gene lineage (SAR406) related to Chlorobium and Fibrobacter species was found in 16S rRNA gene clone libraries prepared from samples from two oceans. The clone libraries were constructed from total picoplankton genomic DNA to assess bacterial diversity in the lower surface layer. The samples were collected by filtration from a depth of 80 m at a site in the western Sargasso Sea and from a depth of 120 m at a site in the Pacific Ocean, approximately 70 km from the Oregon coast. The PCR and primers which amplified nearly full-length 16S rRNA genes were used to prepare the clone libraries. Among the diverse gene clones in these libraries were two related clones (SAR406 and OCS307) which could not be assigned to any of the major bacterial phyla. Phylogenetic analyses demonstrated that these genes were distant relatives of the genus Fibrobacter and the green sulfur bacterial phylum, which includes the genus Chlorobium. The inclusion of SAR406 in phylogenetic trees inferred by several methods resulted in support from bootstrap replicates for the conclusion that Fibrobacter and Chlorobium species and SAR406 are a monophyletic group. An oligonucleotide probe that selectively hybridized to clone SAR406 was used to examine the distribution of this gene lineage in vertical profiles from the Atlantic and Pacific Oceans and in monthly time series at 0 and 200 m in the Atlantic Ocean. During stratified periods, the genes were most abundant slightly below the deep chlorophyll layer. Seasonal changes in the surface abundance of SAR406 rDNA were highly correlated with chlorophyll a levels (r = 0.75).     

Diversity of bovine rumen methanogens In vitro in the presence of condensed tannins, as determined by sequence analysis of 16S rRNA gene library

Molecular diversity of rumen archaeal populations from bovine rumen fluid incubated with or without condensed tannins was investigated using 16S rRNA gene libraries. The predominant order of rumen archaea in the 16S rRNA gene libraries of the control and condensed tannins treatment was found to belong to a novel group of rumen archaea that is distantly related to the order Thermoplasmatales, with 59.5% (15 phylotypes) and 81.43% (21 phylotypes) of the total clones from the control and treatment clone libraries, respectively. The 16S rRNA gene library of the control was found to have higher proportions of methanogens from the orders Methanomicrobiales (32%) and Methanobacteriales (8.5%) as compared to those found in the condensed tannins treatment clone library in both orders (16.88% and 1.68% respectively). The phylotype distributed in the order Methanosarcinales was only found in the control clone library. The study indicated that condensed tannins could alter the diversity of bovine rumen methanogens.