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.     

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.     

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。     

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.     

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.     

Illuminating Microbial Dark Matter in Meromictic Sakinaw Lake

Despite recent advances in metagenomic and single-cell genomic sequencing to investigate uncultivated microbial diversity and metabolic potential, fundamental questions related to population structure, interactions, and biogeochemical roles of candidate divisions remain. Numerous molecular surveys suggest that stratified ecosystems manifesting anoxic, sulfidic, and/or methane-rich conditions are enriched in these enigmatic microbes. Here we describe diversity, abundance, and cooccurrence patterns of uncultivated microbial communities inhabiting the permanently stratified waters of meromictic Sakinaw Lake, British Columbia, Canada, using 454 sequencing of the small-subunit rRNA gene with three-domain resolution. Operational taxonomic units (OTUs) were affiliated with 64 phyla, including more than 25 candidate divisions. Pronounced trends in community structure were observed for all three domains with eukaryotic sequences vanishing almost completely below the mixolimnion, followed by a rapid and sustained increase in methanogen-affiliated (∼10%) and unassigned (∼60%) archaeal sequences as well as bacterial OTUs affiliated with Chloroflexi (∼22%) and candidate divisions (∼28%). Network analysis revealed highly correlated, depth-dependent cooccurrence patterns between Chloroflexi, candidate divisions WWE1, OP9/JS1, OP8, and OD1, methanogens, and unassigned archaeal OTUs indicating niche partitioning and putative syntrophic growth modes. Indeed, pathway reconstruction using recently published Sakinaw Lake single-cell genomes affiliated with OP9/JS1 and OP8 revealed complete coverage of the Wood-Ljungdahl pathway with potential to drive syntrophic acetate oxidation to hydrogen and carbon dioxide under methanogenic conditions. Taken together, these observations point to previously unrecognized syntrophic networks in meromictic lake ecosystems with the potential to inform design and operation of anaerobic methanogenic bioreactors.     

Pyrosequencing reveals highly diverse and species-specific microbial communities in sponges from the Red Sea

Marine sponges are associated with a remarkable array of microorganisms. Using a tag pyrosequencing technology, this study was the first to investigate in depth the microbial communities associated with three Red Sea sponges, Hyrtios erectus, Stylissa carteri and Xestospongia testudinaria. We revealed highly diverse sponge-associated bacterial communities with up to 1000 microbial operational taxonomic units (OTUs) and richness estimates of up to 2000 species. Altogether, 26 bacterial phyla were detected from the Red Sea sponges, 11 of which were absent from the surrounding sea water and 4 were recorded in sponges for the first time. Up to 100 OTUs with richness estimates of up to 300 archaeal species were revealed from a single sponge species. This is by far the highest archaeal diversity ever recorded for sponges. A non-negligible proportion of unclassified reads was observed in sponges. Our results demonstrated that the sponge-associated microbial communities remained highly consistent in the same sponge species from different locations, although they varied at different degrees among different sponge species. A significant proportion of the tag sequences from the sponges could be assigned to one of the sponge-specific clusters previously defined. In addition, the sponge-associated microbial communities were consistently divergent from those present in the surrounding sea water. Our results suggest that the Red Sea sponges possess highly sponge-specific or even sponge-species-specific microbial communities that are resistant to environmental disturbance, and much of their microbial diversity remains to be explored.     

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

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

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.     

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.     

The prokaryotic community of a historically mining-impacted tropical stream sediment is as diverse as that from a pristine stream sediment

Mining negatively affects the environment by producing large quantities of metallic tailings, such as those contaminated with arsenic, with harmful consequences for human and aquatic life. A culture-independent molecular analysis was performed to assess the prokaryotic diversity and community structural changes of the tropical historically metal-contaminated Mina stream (MS) and the relatively pristine Mutuca stream (MTS) sediments. A total of 234 bacterial operational taxonomic units (OTUs) were affiliated with 14 (MS) and 17 (MTS) phyla and 53 OTUs were associated with two archaeal phyla. Although the bacterial community compositions of these sediments were markedly distinct, no significant difference in the diversity indices between the bacterial communities was observed. Additionally, the rarefaction and diversity indices indicated a higher bacterial diversity than archaeal diversity. Most of the OTUs were affiliated with the Proteobacteria and Bacteroidetes phyla. Alphaproteobacteria, Gemmatimonadetes and Actinobacteria were only found in the MS clone library. Crenarchaeal 16S rDNA sequences constituted 75 % of the MS archaeal clones, whereas Euryarchaeota were dominant in the MTS clones. Despite the markedly different characteristics of these streams, their bacterial communities harbor high diversity, suggesting that historically mining-impacted sediments promote diversity. The findings also provide basis for further investigation of members of Alphaproteobacteria as potential biological indicators of arsenic-rich sediments.     

Different response of bacteria,archaea and fungi to process parameters in nine full-scale anaerobic digesters

Biogas production is a biotechnological process realized by complex bacterial, archaeal and likely fungal communities. Their composition was assessed in nine full-scale biogas plants with distinctly differing feedstock input and process parameters. This study investigated the actually active microbial community members by using a comprehensive sequencing approach based on ribosomal 16S and 28S rRNA fragments. The prevailing taxonomical units of each respective community were subsequently linked to process parameters. Ribosomal rRNA of bacteria, archaea and fungi, respectively, showed different compositions with respect to process parameters and supplied feedstocks: (i) bacterial communities were affected by the key factors temperature and ammonium concentration; (ii) composition of archaea was mainly related to process temperature; and (iii) relative abundance of fungi was linked to feedstocks supplied to the digesters. Anaerobic digesters with a high methane yield showed remarkably similar bacterial communities regarding identified taxonomic families. Although archaeal communities differed strongly on genus level from each other, the respective digesters still showed high methane yields. Functional redundancy of the archaeal communities may explain this effect. 28S rRNA sequences of fungi in all nine full-scale anaerobic digesters were primarily classified as facultative anaerobic Ascomycota and Basidiomycota. Since the presence of ribosomal 28S rRNA indicates that fungi may be active in the biogas digesters, further research should be carried out to examine to which extent they are important players in anaerobic digestion processes.     

Characterization of rhizosphere prokaryotic diversity in a horizontal subsurface flow constructed wetland using a PCR cloning-sequencing based approach

Performance of biological wastewater treatment systems may be related to the composition and activity of microbial populations they contain. However, little information is known regarding microbial community inhabiting these ecosystems. The purpose of this study was to investigate archaeal and bacterial diversity, using cultivation-independent molecular techniques, in a constructed wetland receiving domestic wastewater. Two 16S rRNA gene libraries were constructed using total genomic DNA and amplified by PCR using primers specific for archaeal and bacterial domains. A high microbial diversity was detected. The Proteobacteria phylum is the most abundant and diversified phylogenetic group representing 31.3 % of the OTUs, followed by the Bacteroidetes (14.8 %), Planctomycetales (13.8 %), Actinobacteria (12 %), and Chloroflexi (8.2 %). Sequences affiliated with minor phylogenetic divisions such as the TM7, Nitrospira, OP10, and BRC1 are represented by <6 % of total OTUs. The Archaea domain was represented by the Thaumarchaeota phylum dominated by the Candidatus Nitrososphaera genus.     

Bacterial and archaeal phylogenetic diversity associated with swine sludge from an anaerobic treatment lagoon

Over the last decades, the demand for pork products has increased significantly, along with concern about suitable waste management. Anaerobic-lagoon fermentation for swine-sludge stabilization is a good strategy, although little is known about the microbial communities in the lagoons. Here, we employed a cloning- and sequencing-based analysis of the 16S rRNA gene to characterize and quantify the prokaryotic community composition in a swine-waste-sludge anaerobic lagoon (SAL). DNA sequence analysis revealed that the SAL library harbored 15 bacterial phyla: Bacteroidetes, Cloroflexi, Proteobacteria, Firmicutes, Deinococcus-Thermus, Synergystetes, Gemmatimonadetes, Chlorobi, Fibrobacteres, Verrucomicrobia and candidates division OP5, OP8, WWE1, KSB1, WS6. The SAL library was generally dominated by carbohydrate-oxidizing bacteria. The archaeal sequences were related to the Crenarchaeota and Euryarchaeota phyla. Crenarchaeota predominated in the library, demonstrating that it is not restricted to high-temperature environments, being also responsible for ammonium oxidation in the anaerobic lagoon. Euryarchaeota sequences were associated with the hydrogenotrophic methanogens (Methanomicrobiales and Methanobacteriales). Quantitative PCR analysis revealed that the number of bacterial cells was at least three orders of magnitude higher than the number of archaeal cells in the SAL. The identified prokaryotic diversity was ecologically significant, particularly the archaeal community of hydrogenotrophic methanogens, which was responsible for methane production in the anaerobic lagoon. This study provided insight into the archaeal involvement in the overall oxidation of organic matter and the production of methane. Therefore, the treatment of swine waste in the sludge anaerobic lagoon could represent a potential inoculum for the start-up of municipal solid-waste digesters.     

Robustness of archaeal populations in anaerobic co-digestion of dairy and poultry wastes

The objective of this study is to investigate the responses of methanogen populations to poultry waste addition by comparing the archaeal microbial populations in continuous anaerobic digesters with or without the addition of poultry waste as a co-substrate. Poultry waste was characterized as an organic/nitrogen-rich substrate for anaerobic digestion. Supplementing dilute dairy waste with poultry waste for anaerobic co-digestion to increase organic loading rate by 50% resulted in improved biogas production. Elevated ammonia derived from poultry waste did not lead to process inhibition at the organic loadings tested, demonstrating the feasibility of the anaerobic co-digestion of dairy and poultry wastes for improved treatment efficiency. The stability of the anaerobic co-digestion process was linked to the robust archaeal microbial community, which remained mostly unchanged in community structure following increases in organic loading and ammonia levels. Surprisingly, Crenarchaeota archaeal populations, instead of the Euryarchaeota methanogens, dominated the archaeal communities in the anaerobic digesters. The ecological functions of these abundant non-methanogen archaeal populations in anaerobic digestion remain to be identified.     

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

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

     

Phylogenetic analysis of 16S rRNA gene sequences reveals distal gut bacterial diversity in wild wolves (Canis lupus)

The aim of this study was to describe the microbial communities in the distal gut of wild wolves (Canis lupus). Fecal samples were collected from three healthy unrelated adult wolves captured at the nearby of Dalai Lake Nature Reserve in Inner Mongolia of China. The diversity of fecal bacteria was investigated by constructing PCR-amplified 16S rRNA gene clone libraries using the universal bacterial primers 27 F and 1493 R. A total of 307 non-chimeric near-full-length 16S rRNA gene sequences were analyzed and 65 non-redundant bacteria phylotypes (operational taxonomical units, OTUs) were identified. Seventeen OTUs (26%) showed less than 98% sequence similarity to 16S rRNA gene sequences were reported previously. Five different bacterial phyla were identified, with the majority of OTUs being classified within the phylum Firmicutes (60%), followed by Bacteroidetes (16.9%), Proteobacteria (9.2%), Fusobacteria (9.2%) and Actinobacteria (4.6%). The majority of clones fell within the order Clostridiales (53.8% of OTUs). It was predominantly affiliated with five families: Lachnospiraceae was the most diverse bacterial family in this order, followed by Ruminococcaceae, Clostridiaceae, Peptococcaceae and Peptostreptococcaceae.     

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 (α, β, γ, and δ), Bacteroidetes, and Planctomycetes. One OTU was allied with Nitrospina sp. (δ-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.     

Predictive functional profiling using marker gene sequences and community diversity analyses of microbes in full-scale anaerobic sludge digesters

Anaerobic digestion (AD) is widely used in treating the sewage sludge, as it can reduce the amount of sludge, eliminate pathogens and produce biofuel. To enhance the operational performance and stability of anaerobic bioreactors, operational and conventional chemical data from full-scale sludge anaerobic digesters were collected over a 2-year period and summarized, and the microbial community diversity of the sludge sample was investigated at various stages of the AD process. For the purpose of distinguishing between the functional and community diversity of the microbes, Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) software was used to impute the prevalence of 16S rDNA marker gene sequences in the difference in various sludge samples. Meanwhile, a taxa analysis was also carried out to investigate the different sludge samples. The microbial community diversity analysis of one AD sludge sample showed that the most dominant bacterial genera were Saccharicrinis, Syntrophus, Anaerotruncus and Thermanaerothrix. Among archaea, acetoclastic Methanosaeta represented 56.0 %, and hydrogenotrophic Methanospirillum, Methanoculleus, Methanothermus and Methanolinea accounted for 41.3 % of all methanogens. The taxa, genetic and functional prediction analyses of the feedstock and AD sludge samples suggested great community diversity differences between them. The taxa of bacteria in two AD sludge samples were considerably different, but the abundances of the functional KEGG pathways took on similar levels. The numbers of identified pathogens were significantly lower in the digested sludge than in the feedstock, but the PICRUSt results showed the difference in “human diseases” abundances in the level-1 pathway between the two sludge samples was small.     

Microbial community structure of a pilot-scale thermophilic anaerobic digester treating poultry litter

The microbial community structure of a stable pilot-scale thermophilic continuous stirred tank reactor digester stabilized on poultry litter was investigated. This 40-m³ digester produced biogas with 57 % methane, and chemical oxygen demand removal of 54 %. Bacterial and archaeal diversity were examined using both cloning and pyrosequencing that targeted 16S rRNA genes. The bacterial community was dominated by phylum Firmicutes, constituting 93 % of the clones and 76 % of the pyrotags. Of the Firmicutes, class Clostridia (52 % pyrotags) was most abundant followed by class Bacilli (13 % pyrotags). The bacterial libraries identified 94 operational taxonomic units (OTUs) and pyrosequencing identified 577 OTUs at the 97 % minimum similarity level. Fifteen OTUs were dominant (≥2 % abundance), and nine of these were novel unclassified Firmicutes. Several of the dominant OTUs could not be classified more specifically than Clostridiales, but were most similar to plant biomass degraders, including Clostridium thermocellum. Of the rare pyrotag OTUs (<0.5 % abundance), 75 % were Firmicutes. The dominant methanogen was Methanothermobacter which has hydrogenotrophic metabolism, and accounted for >99 % of the archaeal clones. Based on the primary methanogen, as well as digester chemistry (high VA and ammonia levels), we propose that bacterial acetate oxidation is the primary pathway in this digester for the control of acetate levels.