Molecular analysis of fungal diversity associated with three bryophyte species in the Fildes Region, King George Island, maritime Antarctica

The fungal communities associated with three bryophytes species (the liverwort Barbilophozia hatcheri, the mosses Chorisodontium aciphyllum and Sanionia uncinata) in the Fildes Region, King George Island, maritime Antarctica, were studied using clone library analysis. Fungal communities showed low diversity; the 680 clones belonged to 93 OTUs. Of these, 78 belonged to the phylum Ascomycota, 13 to the phylum Basidiomycota, 1 to the phylum Zygomycota, and 1 to an unknown phylum. Among the OTUs, the most common orders in the Ascomycota were Helotiales (42 OTUs) and Chaetothyriales (14 OTUs) and the most common orders in the Basidiomycota were Sebacinales (3 OTUs) and Platygloeales (3 OTUs). Most OTUs clustered within clades that contained phylotypes identified from samples in Antarctic or Arctic ecosystems or from bryophytes in other ecosystems. In addition, we found that host-related factor may shape the fungal communities associated with bryophytes in this region. This is the first systematic study of the fungal community in Antarctic bryophytes to be performed using culture-independent method and the results may improve understanding of the endophytic fungal evolution and ecology in the Antarctic ecosystem.     

Intra- and Interspecific Comparisons of Bacterial Diversity and Community Structure Support Coevolution of Gut Microbiota and Termite Host

We investigated the bacterial gut microbiota from 32 colonies of wood-feeding termites, comprising four Microcerotermes species (Termitidae) and four Reticulitermes species (Rhinotermitidae), using terminal restriction fragment length polymorphism analysis and clonal analysis of 16S rRNA. The obtained molecular community profiles were compared statistically between individuals, colonies, locations, and species of termites. Both analyses revealed that the bacterial community structure was remarkably similar within each termite genus, with small but significant differences between sampling sites and/or termite species. In contrast, considerable differences were found between the two termite genera. Only one bacterial phylotype (defined with 97% sequence identity) was shared between the two termite genera, while 18% and 50% of the phylotypes were shared between two congeneric species in the genera Microcerotermes and Reticulitermes, respectively. Nevertheless, a phylogenetic analysis of 228 phylotypes from Microcerotermes spp. and 367 phylotypes from Reticulitermes spp. with other termite gut clones available in public databases demonstrated the monophyly of many phylotypes from distantly related termites. The monophyletic “termite clusters” comprised of phylotypes from more than one termite species were distributed among 15 bacterial phyla, including the novel candidate phyla TG2 and TG3. These termite clusters accounted for 95% of the 960 clones analyzed in this study. Moreover, the clusters in 12 phyla comprised phylotypes from more than one termite (sub)family, accounting for 75% of the analyzed clones. Our results suggest that the majority of gut bacteria are not allochthonous but are specific symbionts that have coevolved with termites and that their community structure is basically consistent within a genus of termites.     

高通量测序分析大兴安岭典型森林土壤细菌和真菌群落特征

【背景】大兴安岭地区是我国对气候变化响应最敏感的区域,而土壤微生物在维持寒区森林生态系统结构和功能中发挥重要作用。【目的】探究大兴安岭不同森林类型土壤微生物群落结构及与环境因子的关系。【方法】采用Illumina MiSeq高通量测序技术,分析3种典型森林(白桦林、落叶松林和樟子松林)土壤细菌和真菌群落组成和多样性。【结果】3种林型中共获得2 786个细菌操作分类单元(Operational Taxonomic Unit,OTU),隶属于38门531属,其中优势菌门为变形菌门(Proteobacteria, 31.45%-40.32%)、酸杆菌门(Acidobacteria, 14.24%-40.16%)和放线菌门(Actinobacteria,7.13%-22.15%);1803个真菌OTU隶属于8门263属,其中优势菌门为担子菌门(Basidiomycota,40.43%-62.75%)和子囊菌门(Ascomycota,35.81%-53.68%)。主坐标分析表明,3种林型中细菌和真菌群落组间差异远大于组内差异。Mantel检验结果显示:细菌群落结构与pH、总氮(Total Nitrogen,TN)、总磷(Total Phosphorus,TP)和含水量(Soil Water Content,SWC)具有显著相关性(P0.05),其中pH的相关性系数最大;真菌群落结构与SWC、TN和TP具有显著相关性(P0.05),其中TN的相关性系数最大。冗余分析结果发现,TP与变形菌门、担子菌门相对丰度呈显著正相关,TN和SWC与酸杆菌门、子囊菌门呈显著正相关,pH与放线菌门呈显著正相关(P0.05)。【结论】不同林型间土壤微生物群落结构存在显著差异,明晰其分布规律及主要环境驱动因子,是把握寒区森林生态系统过程的关键。     

Diversity of microorganisms in decaying maize stalks revealed by a molecular method

Microbial diversity in decaying maize stalk was characterized by constructing and analyzing rRNA gene clone library. Total 47 OTUs were obtained from 82 bacterial clones, including Proteobacteria (64.6%), Actinobacteria (30.5%), Bacteroidetes (2.4%) and Firmicutes (2.4%). Most proteobacterial clones were members of Rhizobium, Pseudomonas and Stenotrophomonas. Eighty-four percent of Actinobacteria was related to Microbacterium. Only 14 OTUs were identified from 124 fungal clones, including Ascomycota (88%) and Basidiomycota (12%). Sixty percent of Ascomycota were members of Eupenicillium and Paecilomyces but all Basidiomycota were close to Kurtzmanomyces nectairei.     

Microbial communities in contrasting freshwater marsh microhabitats

Heterotrophic microorganisms are widely recognized as crucial components of ecosystems; yet information on their community structure and dynamics in benthic freshwater habitats is notably scarce. Using denaturing gradient gel electrophoresis (DGGE), we determined the composition of bacterial and fungal communities in a freshwater marsh over four seasons. DGGE revealed diverse bacterial communities in four contrasting microhabitats. The greatest compositional differences emerged between water-column and surface-associated bacteria, although communities associated with sediment also differed from those on plant litter and epiphytic biofilms. Sequences of bacterial clones derived from DGGE bands belonged to the Alphaproteobacteria (31%), Actinobacteria (19%) and Bacteriodetes (19%). Betaproteobacteria were notably absent. Fungal clones obtained from leaf litter were mainly Ascomycota , but two members of the Basidiomycota were also identified. Overall, habitat type was the most important factor explaining variation in bacterial communities among samples, whereas temporal patterns in community composition were less pronounced in spite of large seasonal variation in environmental conditions such as temperature. The observed differences among bacterial communities in different microhabitats were not caused by random variation, but rather appeared to be determined by habitat characteristics, as evidenced by largely congruent community profiles of replicate samples taken at 10–100 m distances within the marsh.     

Assessment of soil fungal communities using pyrosequencing

Pyrosequencing, a non-electrophoretic method of DNA sequencing, was used to investigate the extensive fungal community in soils of three islands in the Yellow Sea of Korea, between Korea and China. Pyrosequencing was carried out on amplicons derived from the 5′ region of 18S rDNA. A total of 10,166 reads were obtained, with an average length of 103 bp. The maximum number of fungal phylotypes in soil predicted at 99% similarity was 3,334. The maximum numbers of phylotypes predicted at 97% and 95% similarities were 736 and 286, respectively. Through phylogenetic assignment using BLASTN, a total of 372 tentative taxa were identified. The majority of true fungal sequences recovered in this study belonged to the Ascomycota (182 tentative taxa in 2,708 reads) and Basidiomycota (172 tentative taxa in 6,837 reads). The predominant species of Ascomycota detected have been described as lichen-forming fungi, litter/wood decomposers, plant parasites, endophytes, and saprotrophs: Peltigera neopolydactyla (Lecanoromycetes), Paecilomyces sp. (Sordariomycetes), Phacopsis huuskonenii (Lecanoromycetes), and Raffaelea hennebertii (mitosporicAscomycota). The majority of sequences in the Basidiomycota matched ectomycorrhizal and wood rotting fungi, including species of the Agaricales and Aphyllophorales, respectively. A high number of sequences in the Thelephorales, Boletales, Stereales, Hymenochaetales, and Ceratobasidiomycetes were also detected. By applying high-throughput pyrosequencing, we observed a high diversity of soil fungi and found evidence that pyrosequencing is a reliable technique for investigating fungal communities in soils.     

Contrasting Diversity and Host Association of Ectomycorrhizal Basidiomycetes versus Root-Associated Ascomycetes in a Dipterocarp Rainforest

Root-associated fungi, including ectomycorrhizal and root-endophytic fungi, are among the most diverse and important belowground plant symbionts in dipterocarp rainforests. Our study aimed to reveal the biodiversity, host association, and community structure of ectomycorrhizal Basidiomycota and root-associated Ascomycota (including root-endophytic Ascomycota) in a lowland dipterocarp rainforest in Southeast Asia. The host plant chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) region and fungal internal transcribed spacer 2 (ITS2) region were sequenced using tag-encoded, massively parallel 454 pyrosequencing to identify host plant and root-associated fungal taxa in root samples. In total, 1245 ascomycetous and 127 putative ectomycorrhizal basidiomycetous taxa were detected from 442 root samples. The putative ectomycorrhizal Basidiomycota were likely to be associated with closely related dipterocarp taxa to greater or lesser extents, whereas host association patterns of the root-associated Ascomycota were much less distinct. The community structure of the putative ectomycorrhizal Basidiomycota was possibly more influenced by host genetic distances than was that of the root-associated Ascomycota. This study also indicated that in dipterocarp rainforests, root-associated Ascomycota were characterized by high biodiversity and indistinct host association patterns, whereas ectomycorrhizal Basidiomycota showed less biodiversity and a strong host phylogenetic preference for dipterocarp trees. Our findings lead to the working hypothesis that root-associated Ascomycota, which might be mainly represented by root-endophytic fungi, have biodiversity hotspots in the tropics, whereas biodiversity of ectomycorrhizal Basidiomycota increases with host genetic diversity.     

Diversity of prokaryotes associated with soils around coal-fire gas vents in MaNasi county of Xinjiang, China

Bacterial and archaeal diversity in surface soils of three coal-fire vents was investigated by T-RFLP analysis and clone libraries of 16S rRNA genes. Soil analysis showed that underground coal fires significantly influenced soil pH, moisture and NO₃ ^? content but had little effect on other elements, organic matter and available nutrients. Hierarchical cluster analysis showed that bacterial community patterns in the soils were very similar, but abundance varied with geographic distance. A clone library from one soil showed that the bacterial community was mainly composed of Firmicutes, Proteobacteria, Acidobacteria, Bacteroidetes, Planctomycetes, Actinobacteria, and unidentified groups. Of these, Firmicutes was the most abundant, accounting for 71.4 % of the clones, and was mainly represented by the genera Bacillus and Paenibacillus. Archaeal phylotypes were closely related to uncultivated species of the phyla Crenarchaeota (97.9 % of clones) and Thaumarchaeota (2.1 %). About 28 % of archaeal phylotypes were associated with ammonia oxidization, especially phylotypes that were highly related to a novel, ammonia-oxidizing isolate from the phylum Thaumarchaeota. These results suggested that microbial communities in the soils were diverse and might contain a large number of novel cultivable species with the potential to assimilate materials by heterotrophic metabolism at high temperature.     

Bacterial Succession in a Petroleum Land Treatment Unit

Bacterial community dynamics were investigated in a land treatment unit (LTU) established at a site contaminated with highly weathered petroleum hydrocarbons in the C₁₀ to C₃₂ range. The treatment plot, 3,000 cubic yards of soil, was supplemented with nutrients and monitored weekly for total petroleum hydrocarbons (TPH), soil water content, nutrient levels, and aerobic heterotrophic bacterial counts. Weekly soil samples were analyzed with 16S rRNA gene terminal restriction fragment (TRF) analysis to monitor bacterial community structure and dynamics during bioremediation. TPH degradation was rapid during the first 3 weeks and slowed for the remainder of the 24-week project. A sharp increase in plate counts was reported during the first 3 weeks, indicating an increase in biomass associated with petroleum degradation. Principal components analysis of TRF patterns revealed a series of sample clusters describing bacterial succession during the study. The largest shifts in bacterial community structure began as the TPH degradation rate slowed and the bacterial cell counts decreased. For the purpose of analyzing bacterial dynamics, phylotypes were generated by associating TRFs from three enzyme digests with 16S rRNA gene clones. Two phylotypes associated with Flavobacterium and Pseudomonas were dominant in TRF patterns from samples during rapid TPH degradation. After the TPH degradation rate slowed, four other phylotypes gained dominance in the community while Flavobacterium and Pseudomonas phylotypes decreased in abundance. These data suggest that specific phylotypes of bacteria were associated with the different phases of petroleum degradation in the LTU.     

The Native Bacterioplankton Community in the Central Baltic Sea Is Influenced by Freshwater Bacterial Species

The Baltic Sea is one of the largest brackish environments on Earth. Despite extensive knowledge about food web interactions and pelagic ecosystem functioning, information about the bacterial community composition in the Baltic Sea is scarce. We hypothesized that due to the eutrophic low-salinity environment and the long water residence time (>5 years), the bacterioplankton community from the Baltic proper shows a native “brackish” composition influenced by both freshwater and marine phylotypes. The bacterial community composition in surface water (3-m depth) was examined at a single station throughout a full year. Denaturing gradient gel electrophoresis (DGGE) showed that the community composition changed over the year. Further, it indicated that at the four extensive samplings (16S rRNA gene clone libraries and bacterial isolates from low- and high-nutrient agar plates and seawater cultures), different bacterial assemblages associated with different environmental conditions were present. Overall, the sequencing of 26 DGGE bands, 160 clones, 209 plate isolates, and 9 dilution culture isolates showed that the bacterial assemblage in surface waters of the central Baltic Sea was dominated by Bacteroidetes but exhibited a pronounced influence of typical freshwater phylogenetic groups within Actinobacteria, Verrucomicrobia, and Betaproteobacteria and a lack of typical marine taxa. This first comprehensive analysis of bacterial community composition in the central Baltic Sea points to the existence of an autochthonous estuarine community uniquely adapted to the environmental conditions prevailing in this brackish environment.     

The search for sulfate-reducing bacteria in mat samples from the lost city hydrothermal field by molecular cloning

The work is dedicated to searching for microorganisms of the domain Bacteria capable of dissimilatory sulfate reduction in the samples of microbial mats from a carbonate chimney in the Lost City hydro-thermal field. Cloning of 16S rRNA genes, the universal phylogenetic marker, and dsrAB, the functional marker for sulfate reduction, revealed phylotypes related to spore-forming Desulfotomaculum. No members of the Deltaproteobacteria, comprising the most numerous bacterial group with demonstrated capacity for dissimilatory sulfate reduction, were found. The phylogenetic position of 16S rRNA clones from the mats suggests that this microbial community is a unique consortium, where the energy flow is related to hydrogen of hydrothermal origin, while mass growth of primary produces results from utilization of sulfide formed by sulfate-and sulfur-reducing microorganisms.     

Niche heterogeneity determines bacterial community structure in the termite gut (Reticulitermes santonensis)

Differences in microenvironment and interactions of microorganisms within and across habitat boundaries should influence structure and diversity of the microbial communities within an ecosystem. We tested this hypothesis using the well characterized gut tract of the European subterranean termite Reticulitermes santonensis as a model. By cloning and sequencing analysis and molecular fingerprinting (terminal restriction fragment length polymorphism), we characterized the bacterial microbiota in the major intestinal habitats - the midgut, the wall of the hindgut paunch, the hindgut fluid and the intestinal protozoa. The bacterial community was very diverse (> 200 ribotypes) and comprised representatives of several phyla, including Firmicutes (mainly clostridia, streptococci and Mycoplasmatales-related clones), Bacteroidetes, Spirochaetes and a number of Proteobacteria, all of which were unevenly distributed among the four habitats. The largest group of clones fell into the so-called Termite group 1 (TG-1) phylum, which has no cultivated representatives. The majority of the TG-1 clones were associated with the protozoa and formed two phylogenetically distinct clusters, which consisted exclusively of clones previously retrieved from the gut of this and other Reticulitermes species. Also the other clones represented lineages of microorganisms that were exclusively recovered from the intestinal tract of termites. The termite specificity of these lineages was underscored by the finding that the closest relatives of the bacterial clones obtained from R. santonensis were usually derived also from the most closely related termites. Overall, differences in diversity between the different gut habitats and the uneven distribution of individual phylotypes support conclusively that niche heterogeneity is a strong determinant of the structure and spatial organization of the microbial community in the termite gut.     

天山一号冰川前沿生态系统真菌群落结构演替及分布格局

【目的】分析真菌群落结构和多样性随着一号冰川退缩前沿年代序列的变化,揭示真菌群落的演替轨迹及环境因子对群落组成的影响。【方法】采用宏基因组学研究方法,结合生物信息学和统计学分析技术,对取自一号冰川末端表面冰尘,底部和前沿14个样品进行总DNA的提取,ITS基因的扩增并使用Illumina Miseq平台测序,通过相关生物地理化学特性综合分析在不同年代序列下真菌群落结构及其演替规律。【结果】经测序,筛选和质控分析获得185103条rawreads,占78.3%的非单序列在97%的相似度聚类分析共得到300个操作分类单元(OTU),共划分为6个门:子囊菌门(Ascomycota,52.7%)、担子菌门(Basidiomycota,16.9%)、壶菌门(Chytridiomycota,15.1%)、接合菌门(Zygomycota,2.4%)和球囊菌门(Glomeromycota,1.2%)。从演替初期到后期阶段虽然子囊菌的序列数逐渐下降而担子菌出现缓慢上升趋势,但子囊菌随着土壤年代序列的增加始终为优势类群,壶菌在冰川底部和前沿基层普遍存在且丰度仅次于子囊菌和担子菌。我们在缺乏植被的最新退缩基层发现依靠自养型宿主存活的活体营养菌,如Taphrinomycetes、Urediniomycetes和Ustilaginomycetes。从冰川底部和前沿基层检测到丰度较高的酵母菌,而粪生真菌(coprophilous fungi)仅仅出现在冰川前沿基层,共23个操作分类单元。球囊菌仅在前沿部分样品中存在,有着十分狭小的生态位分布。【结论】一号冰川前沿随着年代序列的增加真菌群落存在明显的演替轨迹和多样性的显著变化,不同生态位真菌类群组成的相似性较低且都存在明显的指示性真菌类群。     

Microbial Biogeography along an Estuarine Salinity Gradient: Combined Influences of Bacterial Growth and Residence Time

Shifts in bacterioplankton community composition along the salinity gradient of the Parker River estuary and Plum Island Sound, in northeastern Massachusetts, were related to residence time and bacterial community doubling time in spring, summer, and fall seasons. Bacterial community composition was characterized with denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA. Average community doubling time was calculated from bacterial production ([¹⁴C]leucine incorporation) and bacterial abundance (direct counts). Freshwater and marine populations advected into the estuary represented a large fraction of the bacterioplankton community in all seasons. However, a unique estuarine community formed at intermediate salinities in summer and fall, when average doubling time was much shorter than water residence time, but not in spring, when doubling time was similar to residence time. Sequencing of DNA in DGGE bands demonstrated that most bands represented single phylotypes and that matching bands from different samples represented identical phylotypes. Most river and coastal ocean bacterioplankton were members of common freshwater and marine phylogenetic clusters within the phyla Proteobacteria, Bacteroidetes, and Actinobacteria. Estuarine bacterioplankton also belonged to these phyla but were related to clones and isolates from several different environments, including marine water columns, freshwater sediments, and soil.     

Diversity of bacteria in surface ice of Austre Lovénbreen glacier, Svalbard

Two 16S rRNA gene clone libraries Cores 1U and 2U were constructed using two ice core samples collected from Austre Lovénbreen glacier in Svalbard. The two libraries yielded a total of 262 clones belonging to 59 phylotypes. Sequences fell into 10 major lineages of the domain Bacteria, including Proteobacteria (alpha, beta, gamma and delta subdivisions), Bacteroidetes, Actinobacteria, Firmicutes, Acidobacteria, Deinococcus-Thermus, Chloroflexi, Planctomycetes, Cyanobacteria and candidate division TM7. Among them, Bacteroidetes, Actinobacteria, Alphaproteobacteria and Cyanobacteria were most abundant. UniFrac data showed no significant differences in community composition between the two ice cores. A total of nineteen bacterial strains from the genera Pseudoalteromonas and Psychrobacter were isolated from the ice cores. Phylogenetic and phenotypic analyses revealed a close relationship between the ice core isolates and bacteria in marine environments, indicating a wide distribution of some bacterial phylotypes in both terrestrial and marine ecosystems.     

Phylogenetic diversity of bacteria associated with the endemic freshwater sponge Lubomirskia baicalensis

In this study, for the first time the diversity of bacteria associated with the endemic freshwater sponge Lubomirskia baicalensis collected from the Sousern Basin of Lake Baikal was investigated employing cultivation-independent approaches. In total, 102 bacterial 16S rRNA clones were screened using restriction fragment length polymorphism (RFLP) and 30 were selected for sequencing. BLASTN and phylogenetic analysis based on near full length 16S rDNA sequences showed that 22 operational taxonomic units (OTUs) were clustered in six known phyla: Actinobacteria (8 OTUs), alpha-Proteobacteria (4 OTUs), beta-Proteobacteria (4 OTUs), Verrucomicrobia (4 OTUs), Nitrospiracea (1 OTU) and Bacteroidetes (1 OTU). Remarkably all phylotypes were affiliated to uncultured microorganisms, however, all alpha-Proteobacteria sequences were closely related to bacteria derived from the freshwater sponge Spongilla lacustris. Our results reveal a high diversity in the L. baicalensis bacterial community and provide an insight into microbial ecology and diversity within freshwater sponges inhabiting the ancient Lake Baikal ecosystem.     

Phylogenetic Profiling and Diversity of Bacterial Communities in the Death Valley,an Extreme Habitat in the Atacama Desert

The Atacama Desert, one of the driest deserts in the world, represents a unique extreme environmental ecosystem to explore the bacterial diversity as it is considered to be at the dry limit for life. A 16S rRNA gene (spanning the hyper variable V3 region) library was constructed from an alkaline sample of unvegetated soil at the hyperarid margin in the Atacama Desert. A total of 244 clone sequences were used for MOTHUR analysis, which revealed 20 unique phylotypes or operational taxonomic units (OTUs). V3 region amplicons of the 16S rRNA were suitable for distinguishing the bacterial community to the genus and specie level. We found that all OTUs were affiliated with taxa representative of the Firmicutes phylum. The extremely high abundance of Firmicutes indicated that most bacteria in the soil were spore-forming survivors. In this study we detected a narrower diversity as compared to other ecological studies performed in other areas of the Atacama Desert. The reported genera were Oceanobacillus (representing the 69.5 % of the clones sequenced), Bacillus, Thalassobacillus and Virgibacillus. The present work shows physical and chemical parameters have a prominent impact on the microbial community structure. It constitutes an example of the communities adapted to live in extreme conditions caused by dryness and metal concentrations .

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-015-0539-3) contains supplementary material, which is available to authorized users.     

Intra- and interspecific comparisons of bacterial diversity and community structure support coevolution of gut microbiota and termite host

We investigated the bacterial gut microbiota from 32 colonies of wood-feeding termites, comprising four Microcerotermes species (Termitidae) and four Reticulitermes species (Rhinotermitidae), using terminal restriction fragment length polymorphism analysis and clonal analysis of 16S rRNA. The obtained molecular community profiles were compared statistically between individuals, colonies, locations, and species of termites. Both analyses revealed that the bacterial community structure was remarkably similar within each termite genus, with small but significant differences between sampling sites and/or termite species. In contrast, considerable differences were found between the two termite genera. Only one bacterial phylotype (defined with 97% sequence identity) was shared between the two termite genera, while 18% and 50% of the phylotypes were shared between two congeneric species in the genera Microcerotermes and Reticulitermes, respectively. Nevertheless, a phylogenetic analysis of 228 phylotypes from Microcerotermes spp. and 367 phylotypes from Reticulitermes spp. with other termite gut clones available in public databases demonstrated the monophyly of many phylotypes from distantly related termites. The monophyletic "termite clusters" comprised of phylotypes from more than one termite species were distributed among 15 bacterial phyla, including the novel candidate phyla TG2 and TG3. These termite clusters accounted for 95% of the 960 clones analyzed in this study. Moreover, the clusters in 12 phyla comprised phylotypes from more than one termite (sub)family, accounting for 75% of the analyzed clones. Our results suggest that the majority of gut bacteria are not allochthonous but are specific symbionts that have coevolved with termites and that their community structure is basically consistent within a genus of termites.     

Characterisation of the microbial diversity in a pig manure storage pit using small subunit rDNA sequence analysis

The microbial community structure of pig manure slurry (PMS) was determined with comparative analysis of 202 bacterial, 44 archaeal and 33 eukaryotic small subunit (SSU) rDNA partial sequences. Based on a criterion of 97% of sequence similarity, the phylogenetic analyses revealed a total of 108, eight and five phylotypes for the Bacteria, Archaea and Eukarya lineages, respectively. Only 36% of the bacterial phylotypes were closely related (>or=97% similarity) to any previously known sequence in databases. The bacterial groups most often represented in terms of phylotype and clone abundance were the Eubacterium (22% of total sequences), the Clostridium (15% of sequences), the Bacillus-Lactobacillus-Streptococcus subdivision (20% of sequences), theMycoplasma and relatives (10% of sequences) and the Flexibacter-Cytophaga-Bacteroides (20% of sequences). The global microbial community structure and phylotype diversity show a close relationship to the pig gastrointestinal tract ecosystem whereas phylotypes from the Acholeplasma-Anaeroplasma and the Clostridium purinolyticum groups appear to be better represented in manure. Archaeal diversity was dominated by three phylotypes clustering with a group of uncultured microorganisms of unknown activity and only distantly related to the Thermoplasmales and relatives. Other Archaea were methanogenic H2/CO2 utilisers. No known acetoclastic Archaea methanogen was found. Eukaryotic diversity was represented by a pluricellular nematode, two Alveolata, a Blastocystis and an Entamoebidae. Manure slurry physico-chemical characteristics were analysed. Possible inhibitory effects of acetate, sulphide and ammonia concentrations on the microbial anaerobic ecosystem are discussed.     

Culture-Independent Analysis of Gut Bacteria: the Pig Gastrointestinal Tract Microbiota Revisited

The phylogenetic diversity of the intestinal bacterial community in pigs was studied by comparative 16S ribosomal DNA (rDNA) sequence analysis. Samples were collected from a total of 24 pigs representing a variety of diets, ages, and herd health status. A library comprising 4,270 cloned 16S rDNA sequences obtained directly by PCR from 52 samples of either the ileum, the cecum, or the colon was constructed. In total, 375 phylotypes were identified using a 97% similarity criterion. Three hundred nine of the phylotypes (83%) had a <97% sequence similarity to any sequences in the database and may represent yet-uncharacterized bacterial genera or species. The phylotypes were affiliated with 13 major phylogenetic lineages. Three hundred four phylotypes (81%) belonged to the low-G+C gram-positive division, and 42 phylotypes (11.2%) were affiliated with the Bacteroides and Prevotella group. Four clusters of phylotypes branching off deeply within the low-G+C gram-positive bacteria and one in the Mycoplasma without any cultured representatives were found. The coverage of all the samples was 97.2%. The relative abundance of the clones approximated a lognormal distribution; however, the phylotypes detected and their abundance varied between two libraries from the same sample. The results document that the intestinal microbial community is very complex and that the majority of the bacterial species colonizing the gastrointestinal tract in pigs have not been characterized.