Clone Library Construction Research Articles

Impacts of Mariculture on the Diversity of Bacterial Communities within Intertidal Sediments in the Northeast of China

Mariculture is one of the major seafood supplies worldwide and has caused serious environmental concerns on the coastal zone. Its rapid development has been shown to disrupt the sediment ecosystems and thus influence the benthic bacterial communities. Bacterial diversity and community structure within both adjacent farms and non-cultured zones intertidal sediments along the coasts of Qinhuangdao and Dalian, China, were investigated using full-length 16S rRNA gene-based T-RFLP analyses and clone library construction. Richness and Shannon-Wiener index were significantly increased at sites adjacent the mariculture farm with mean values of 29 and 2.97 from peak profiles of T-RFLP result. Clustering analyses suggested that impacts of mariculture on bacterial diversity of sediment were significantly larger than those resulted from temporal and spatial scales. Upon comparisons of RFLP patterns from 602 clones from libraries of the selected five samples, 137 OTUs were retrieved. Members of γ- and δ-Proteobacteria, Bacilli, Flavobacteria, and Actinobacteria were recorded in all libraries. In addition, γ-Proteobacteria were dominant in all samples (21.7~45.0%). Redundancy analysis revealed that the distribution of bacterial composition seemed to be determined by the variables of salinity, PO4 (3-)-P, NH4 (+)-N, and Chlorophyll a content. The phyla of γ-Proteobacteria, Clostridia, Flavobacteria, Bacilli, and Planctomycetes were principal components to contribute to the bacterial differences of clone libraries. Our finding demonstrated that these phyla could display variations of bacterial composition linked to environmental disturbance resulted from mariculture.

Bacterial community structure in cooling water and biofilm in an industrial recirculating cooling water system

Microbial fouling is a serious problem in open recirculating cooling water systems. The bacterial communities that cause it have not been fully understood. In this study, we analyzed the community structure of free-living bacteria and particle-attached bacteria in cooling water, and bacteria in biofilm collected from the wall of the water reservoir in an industrial recirculating cooling water system by construction of a 16S rRNA gene clone library. Based on amplified ribosomal DNA restriction analysis, clones of all three libraries were clustered into 45 operational taxonomic units (OTUs). Thirteen OTUs displaying 91-96% sequence similarity to a type strain might be novel bacterial species. Noted differences in community structure were observed among the three libraries. The relative species richness of the free-living bacteria in cooling water was much lower than that of particle-attached bacteria and bacteria in biofilm. The majority of the free-living bacterial community (99.0%) was Betaproteobacteria. The predominant bacteria in the particle-attached bacterial community were Alphaproteobacteria (20.5%), Betaproteobacteria (27.8%) and Planctomycetes (42.0%), while those in the biofilm bacterial community were Alphaproteobacteria (47.9%), Betaproteobacteria (11.7%), Acidobacteria (13.1%) and Gemmatimonadetes (11.3%). To control microbial fouling in industrial recirculating cooling water systems, additional physiological and ecological studies of these species will be essential.

Endophytic and mycorrhizal fungi associated with roots of endangered native orchids from the Atlantic Forest, Brazil

The composition and diversity of fungal communities associated with three endangered orchid species, Hadrolaelia jongheana, Hoffmannseggella caulescens, and Hoffmannseggella cinnabarina, found in different vegetation formations of the Atlantic Forest were determined by constructing clone libraries and by applying diversity and richness indices. Our results demonstrated the presence of Basidiomycetes. Sebacinales (81.61%) and Cantharellales (12.10%) were the dominant orders and are potential candidates for orchid mycorrhizal fungi. The Ascomycetes identified included the Helotiales (29.31%), Capnodiales (18.10%), and Sordariales (10.34%), among others. These orders may represent potentially endophytic fungi. A Shannon-Wiener diversity index (H') analysis showed a relatively high fungal community diversity associated with these tropical orchids. This diversity may offer greater flexibility in terms of the adaptation of the plants to changing environmental conditions and the potential facilitation of reintroduction programs. The Simpson diversity index values showed that all of the libraries included dominant species, and a LIBSHUFF analysis showed that the fungal communities were structurally different from each other, suggesting an influence of local factors on this diversity. This study offers important information for the development of conservation strategies for threatened and endemic species of Brazilian flora in an important and threatened hotspot.

Development of genus‐specific primers for better understanding the diversity and population structure of Sphingomonas in soils

Genus Sphingomonas has received increasing attentions due to its somewhat unique metabolic versatilities in the contaminated environment. However, due to the lack of genus-specific primers, the ecological significance of Sphingomonas in polluted soils has been rarely documented by 16S rDNA finger-printing methods. In this study, three genus-specific primer sets targeted at the 16S rRNA gene of Sphingomonas were developed and their specificities were tested with four contaminated soils from Shenfu petroleum-wastewater irrigation zone by constructing clone libraries, amplified ribosomal DNA restriction analysis (ARDRA) and sequencing the represented ARDRA patterns. Meanwhile, the newly designed primer sets and a previously reported primer set were compared, and the results showed that the newly developed primer set SA/429f-933r could detect a larger spectrum (90%) of Sphingomonas strains with higher specificity. Despite the superiority of primer set SA/429f-933r in specifically detecting Sphingomonas from contaminated soils, we cannot blink the fact that different primer sets preferentially amplified different dominant species. Therefore, two or more primer sets are recommended for evaluating the diversity and population structure of genus Sphingomonas. Additionally, a proportion (9.7%) of the cloned sequences discovered in this study were different from known Sphingomonas sequences, suggesting that new Sphingomonas sequences might present in soils from Shenfu irrigation zone.

Significance of Anaerobes and Oral Bacteria in Community-Acquired Pneumonia

BackgroundMolecular biological modalities with better detection rates have been applied to identify the bacteria causing infectious diseases. Approximately 10–48% of bacterial pathogens causing community-acquired pneumonia are not identified using conventional cultivation methods. This study evaluated the bacteriological causes of community-acquired pneumonia using a cultivation-independent clone library analysis of the 16S ribosomal RNA gene of bronchoalveolar lavage specimens, and compared the results with those of conventional cultivation methods.MethodsPatients with community-acquired pneumonia were enrolled based on their clinical and radiological findings. Bronchoalveolar lavage specimens were collected from pulmonary pathological lesions using bronchoscopy and evaluated by both a culture-independent molecular method and conventional cultivation methods. For the culture-independent molecular method, approximately 600 base pairs of 16S ribosomal RNA genes were amplified using polymerase chain reaction with universal primers, followed by the construction of clone libraries. The nucleotide sequences of 96 clones randomly chosen for each specimen were determined, and bacterial homology was searched. Conventional cultivation methods, including anaerobic cultures, were also performed using the same specimens.ResultsIn addition to known common pathogens of community-acquired pneumonia [Streptococcus pneumoniae (18.8%), Haemophilus influenzae (18.8%), Mycoplasma pneumoniae (17.2%)], molecular analysis of specimens from 64 patients with community-acquired pneumonia showed relatively higher rates of anaerobes (15.6%) and oral bacteria (15.6%) than previous reports.ConclusionOur findings suggest that anaerobes and oral bacteria are more frequently detected in patients with community-acquired pneumonia than previously believed. It is possible that these bacteria may play more important roles in community-acquired pneumonia.

Spatial Distribution and Factors Shaping the Niche Segregation of Ammonia-Oxidizing Microorganisms in the Qiantang River, China

Ammonia oxidation is performed by both ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). However, the current knowledge of the distribution, diversity, and relative abundance of these two microbial groups in freshwater sediments is insufficient. We examined the spatial distribution and analyzed the possible factors leading to the niche segregation of AOA and AOB in the sediments of the Qiantang River, using clone library construction and quantitative PCR for both archaeal and bacterial amoA genes. pH and NH4(+)-N content had a significant effect on AOA abundance and AOA operational taxonomy unit (OTU) numbers. pH and organic carbon content influenced the ratio of AOA/AOB OTU numbers significantly. The influence of these factors showed an obvious spatial trend along the Qiantang River. This result suggested that AOA may contribute more than AOB to the upstream reaches of the Qiantang River, where the pH is lower and the organic carbon and NH4(+)-N contents are higher, but AOB were the principal driver of nitrification downstream, where the opposite environmental conditions were present.

Microbial life associated with low‐temperature alteration of ultramafic rocks in the Leka ophiolite complex

Water-rock interactions in ultramafic lithosphere generate reduced chemical species such as hydrogen that can fuel subsurface microbial communities. Sampling of this environment is expensive and technically demanding. However, highly accessible, uplifted oceanic lithospheres emplaced onto continental margins (ophiolites) are potential model systems for studies of the subsurface biosphere in ultramafic rocks. Here, we describe a microbiological investigation of partially serpentinized dunite from the Leka ophiolite (Norway). We analysed samples of mineral coatings on subsurface fracture surfaces from different depths (10-160 cm) and groundwater from a 50-m-deep borehole that penetrates several major fracture zones in the rock. The samples are suggested to represent subsurface habitats ranging from highly anaerobic to aerobic conditions. Water from a surface pond was analysed for comparison. To explore the microbial diversity and to make assessments about potential metabolisms, the samples were analysed by microscopy, construction of small subunit ribosomal RNA gene clone libraries, culturing and quantitative-PCR. Different microbial communities were observed in the groundwater, the fracture-coating material and the surface water, indicating that distinct microbial ecosystems exist in the rock. Close relatives of hydrogen-oxidizing Hydrogenophaga dominated (30% of the bacterial clones) in the oxic groundwater, indicating that microbial communities in ultramafic rocks at Leka could partially be driven by H2 produced by low-temperature water-rock reactions. Heterotrophic organisms, including close relatives of hydrocarbon degraders possibly feeding on products from Fischer-Tropsch-type reactions, dominated in the fracture-coating material. Putative hydrogen-, ammonia-, manganese- and iron-oxidizers were also detected in fracture coatings and the groundwater. The microbial communities reflect the existence of different subsurface redox conditions generated by differences in fracture size and distribution, and mixing of fluids. The particularly dense microbial communities in the shallow fracture coatings seem to be fuelled by both photosynthesis and oxidation of reduced chemical species produced by water-rock reactions.

Transient shifts in bacterial communities associated with the temperate gorgonian Paramuricea clavata in the Northwestern Mediterranean Sea.

BackgroundBacterial communities that are associated with tropical reef-forming corals are being increasingly recognized for their role in host physiology and health. However, little is known about the microbial diversity of the communities associated with temperate gorgonian corals, even though these communities are key structural components of the ecosystem. In the Northwestern Mediterranean Sea, gorgonians undergo recurrent mass mortalities, but the potential relationship between these events and the structure of the associated bacterial communities remains unexplored. Because microbial assemblages may contribute to the overall health and disease resistance of their host, a detailed baseline of the associated bacterial diversity is required to better understand the functioning of the gorgonian holobiont.Methodology/Principal FindingsThe bacterial diversity associated with the gorgonian Paramuricea clavata was determined using denaturing gradient gel electrophoresis, terminal-restriction fragment length polymorphism and the construction of clone libraries of the bacterial 16S ribosomal DNA. Three study sites were monitored for 4 years to assess the variability of communities associated with healthy colonies. Bacterial assemblages were highly dominated by one Hahellaceae-related ribotype and exhibited low diversity. While this pattern was mostly conserved through space and time, in summer 2007, a deep shift in microbiota structure toward increased bacterial diversity and the transient disappearance of Hahellaceae was observed.Conclusion/SignificanceThis is the first spatiotemporal study to investigate the bacterial diversity associated with a temperate shallow gorgonian. Our data revealed an established relationship between P. clavata and a specific bacterial group within the Oceanospirillales. These results suggest a potential symbiotic role of Hahellaceae in the host-microbe association, as recently suggested for tropical corals. However, a transient imbalance in bacterial associations can be tolerated by the holobiont without apparent symptoms of disease. The subsequent restoration of the Hahellaceae-dominated community is indicative of the specificity and resilience of the bacteria associated with the gorgonian host.

Diversity, dynamic and abundance of Geobacteraceae species in paddy soil following slurry incubation

Microbially-mediated Fe(III) reduction is of environmental significance in wetland ecosystems such as rice fields. Despite a number of incubation experiments showing the dynamic structure and activity of microbial communities in rice paddy soils amended with different substrates, little is known regarding the succession of Fe(III)-reducing bacterial populations in non-amended, natural paddy soils upon flooding. In this study, a 30-d laboratory incubation experiment was conducted to examine the diversity, dynamic and abundance of representative Fe(III)-reducing bacterial family Geobacteraceae in anaerobic natural paddy soil slurry incubations. The Logistic model showed that the microbial Fe(III) reduction rate in paddy slurry reached the highest level (1.36 mg g−1 d−1) after 3.3-d flooding, and the accumulated Fe(II) level stabilized at 8.14 mg g−1 on day 20. Quantitative, real-time PCR assay showed that the absolute abundances of Geobacteraceae and total bacterial populations varied in similar trends. Both decreased from day 1–10 and peaked on day 20 (13.98 × 106 and 5.29 × 108 copies of 16S rDNA g−1 dry soil, respectively), followed by large decreases on day 30 (1.94 × 106 and 0.62 × 108 copies of 16S rDNA g−1 dry soil, respectively). The relative abundance of Geobacteraceae, i.e., the proportion of Geobacteraceae to total bacteria reached the highest level (∼4%) following 5-d flooding, and then slightly fluctuated at 2.6%–3.9% till the end of the experiment. Clone library construction and sequencing analysis showed that the Geobacteraceae mainly consisted of Geobacter spp. which promoted bacterial Fe(III) reduction in paddy slurries upon flooding. UniFrac principal coordinate analysis revealed the succession of Geobacteraceae species, with the highest diversity observed in the initial stage (1 h–1 d) and the dominant successional members in the late stage (20–30 d). These results indicated that Geobacteraceae species contributed to Fe(III) reduction in flood paddy soils, and that the structure of Geobacteraceae population was maintained through the common occurrence of generalized species and the succession of specialized species.

An anion-exchange method to concentrate dissolved DNA from aquifer water

A rapid DNA isolation method was developed to concentrate dissolved DNA (dDNA) in aquifer water for molecular analysis. The aquifer dDNA from the Eastern Snake River Plain Aquifer (ESRPA) was extracted and concentrated using a new method with an anion-exchange Mustang® Q membrane. The concentration of aquifer dDNA in this study ranged from 60 to 264.5ngl−1 in ESRPA aquifer wells. DNA stability in ESRPA aquifer water was also tested in this study. The dDNA extracted from aquifer water samples was used for PCR amplification of bacterial 16S rRNA genes for terminal restriction fragment length polymorphism (T-RFLP) analysis and construction of 16S rRNA gene clone libraries. The ureC gene, IncP, IncQ and IncW plasmid genes were also PCR amplified from dDNA samples. Based on the results, dDNA is relatively stable in aquifer water and can be concentrated by Q membrane method for molecular analysis. The quality of isolated dDNA was suitable as a PCR template.

Bacterial diversity of the rock-water interface in an East Antarctic freshwater ecosystem, Lake Tawani(P)†

Schirmacher Oasis is one of the few ice-free plateaus in East Antarctica that maintains a unique distribution of over 120 microbial-rich, dynamic freshwater lakes, most of which are unexplored. In this study, we describe the bacterial diversity of the rock-water interface in Lake Tawani(P) using culture-independent Bacterial Tag Encoded FLX Amplicon Pyrosequencing (bTEFAP), clone library construction, and culture-based analysis targeting the eubacterial 16S rRNA gene. Lake Tawani(P)was formed in a fossil valley by the accumulation of snow and glacial melt through surface channels into a low-catchment depression. Overall this lake exhibited thirteen bacterial phyla and one-hundred and twelve genera. The Qiime bioinformatics analysis on the bTEFAP alone exhibited higher coverage of the bacterial composition in Lake Tawani(P) than the clone library construction or culture-based methodology. Particularly due to the higher sensitivity of the bTEFAP approach, we detected and differentiated members of the phyla: Chloroflexi, Gemmatimonadetes, Planctomycetes, Nitrospira, and Candidate Division TM7 that other methods were unable to reveal. Nevertheless we found that the use of multiple approaches identified a more complete bacterial community than by using any single approach. Investigating the bacterial diversity of the Schirmacher Oasis lakes, especially those connected through surface channels and encompassed by valleys, will help unravel the dynamic nature of these unique seasonal, freshwater lakes, which potentially harbors highly adapted bacterial taxa with defined ecological functions.

Bacterial biodiversity in deep-sea sediments from two regions of contrasting surface water productivity near the Crozet Islands, Southern Ocean

The relationship between surface-derived particulate organic matter (POM) and deep-sea sediment bacterial abundance, community structure and composition was investigated in two different sediment layers from two zones of contrasting surface water productivity in the southern Indian Ocean. Bacterial sediment communities from high chlorophyll (HC) and low chlorophyll (LC) sites were characterized and compared using direct counts, clone library construction, denaturing gradient gel electrophoresis (DGGE) and fluorescence in situ hybridization (FISH). Of the 1566 bacterial clones generated from the sediment communities, 1010 matched published 16S rDNA sequences at ≥97% identity. A comparison of surface sediment clone libraries showed that at least one third of all identified operational taxonomic units (OTUs) were common to both HC and LC sites. DGGE community profiles were consistent (82% similar) and evenness of the major phylogenetic groups was 96% similar between surface sediment communities, where gamma- and alpha-Proteobacteria were dominant. Sediment communities shared similarly high biodiversity, while species richness was marginally higher at the LC site. Intra-site shifts in bacterial abundance and composition were observed with increasing sediment depth. Despite the differences in organic matter input between sites, the consistency observed between HC and LC sediment communities pointed to (1) the extent of remineralisation by mega and meio-fauna as a potential factor affecting the quantity and quality of POM available to sediment bacteria, (2) sampling during the early ‘nutrient assimilation phase’ of the bacterial response to freshly deposited POM or (3) that the action of bacteria in the water column could affect the quantity and quality of POM available to sediment bacteria. Although factors other than these may explain the observed similarities, this first comparison of such deep-sea sediment communities in relation to surface-derived productivity may be useful in further elucidating the role of sediment bacteria in carbon remineralisation in the deep-sea environment.

Methyl Fluoride Affects Methanogenesis Rather than Community Composition of Methanogenic Archaea in a Rice Field Soil

The metabolic pathways of methane formation vary with environmental conditions, but whether this can also be linked to changes in the active archaeal community structure remains uncertain. Here, we show that the suppression of aceticlastic methanogenesis by methyl fluoride (CH3F) caused surprisingly little differences in community composition of active methanogenic archaea from a rice field soil. By measuring the natural abundances of carbon isotopes we found that the effective dose for a 90% inhibition of aceticlastic methanogenesis in anoxic paddy soil incubations was <0.75% CH3F (v/v). The construction of clone libraries as well as t-RFLP analysis revealed that the active community, as indicated by mcrA transcripts (encoding the α subunit of methyl-coenzyme M reductase, a key enzyme for methanogenesis), remained stable over a wide range of CH3F concentrations and represented only a subset of the methanogenic community. More precisely, Methanocellaceae were of minor importance, but Methanosarcinaceae dominated the active population, even when CH3F inhibition only allowed for aceticlastic methanogenesis. In addition, we detected mcrA gene fragments of a so far unrecognised phylogenetic cluster. Transcription of this phylotype at methyl fluoride concentrations suppressing aceticlastic methanogenesis suggests that the respective organisms perform hydrogenotrophic methanogenesis. Hence, the application of CH3F combined with transcript analysis is not only a useful tool to measure and assign in situ acetate usage, but also to explore substrate usage by as yet uncultivated methanogens.

Eukaryotic microorganisms in cold environments: examples from Pyrenean glaciers

Little is known about the viability of eukaryotic microorganisms preserved in icy regions. Here we report on the diversity of microbial eukaryotes in ice samples derived from four Pyrenean glaciers. The species composition of eukaryotic communities in these glaciers is unknown mostly because of the presence of a multi-year ice cap, and it is not clear whether they harbor the same populations. The recent deglaciation of these areas is allowing an easy access to glacial layers that correspond to the “Little Ice Age” although some isolated deposits are attributed to previous glacial cycles. In this study, we use molecular 18S rRNA-based approaches to characterize some of the microbial eukaryotic populations associated with Pyrenean glaciers. Firstly, we performed a chemical and microscopical characterization of ice samples. Secondly, molecular analyses revealed interesting protist genetic diversity in glaciers. In order to understand the microbial composition of the ice samples the eukaryotic communities resident in the glacial samples were examined by amplifying community DNA and constructing clone libraries with 18S rRNA primers. After removal of potential chimeric sequences and dereplication of identical sequences, phylogenetic analysis demonstrated that several different protists could be identified. Protist diversity was more phylum rich in Aneto and Monte Perdido glaciers. The dominant taxonomic groups across all samples (>1% of all sequences) were Viridiplantae and Rhizaria. Significant variations in relative abundances of protist phyla between higher and lower glaciers were observed. At the genus level, significant differences were also recorded for the dominant genera Chloromonas, Raphidonema, Heteromita, Koliella, and Bodomorpha. In addition, protist community structure showed significant differences between glaciers. The relative abundances of protist groups at different taxonomic levels correlated with the altitude and area of glaciers and with pH of ice, but little or no relationships to other chemical characteristics were found.

Comprehensive Analysis of Secondary Dental Root Canal Infections: A Combination of Culture and Culture-Independent Approaches Reveals New Insights

Persistence of microorganisms or reinfections are the main reasons for failure of root canal therapy. Very few studies to date have included culture-independent methods to assess the microbiota, including non-cultivable microorganisms. The aim of this study was to combine culture methods with culture-independent cloning methods to analyze the microbial flora of root-filled teeth with periradicular lesions. Twenty-one samples from previously root-filled teeth were collected from patients with periradicular lesions. Microorganisms were cultivated, isolated and biochemically identified. In addition, ribosomal DNA of bacteria, fungi and archaea derived from the same samples was amplified and the PCR products were used to construct clone libraries. DNA of selected clones was sequenced and microbial species were identified, comparing the sequences with public databases. Microorganisms were found in 12 samples with culture-dependent and -independent methods combined. The number of bacterial species ranged from 1 to 12 in one sample. The majority of the 26 taxa belonged to the phylum Firmicutes (14 taxa), followed by Actinobacteria, Proteobacteria and Bacteroidetes. One sample was positive for fungi, and archaea could not be detected. The results obtained with both methods differed. The cloning technique detected several as-yet-uncultivated taxa. Using a combination of both methods 13 taxa were detected that had not been found in root-filled teeth so far. Enterococcus faecalis was only detected in two samples using culture methods. Combining the culture-dependent and –independent approaches revealed new candidate endodontic pathogens and a high diversity of the microbial flora in root-filled teeth with periradicular lesions. Both methods yielded differing results, emphasizing the benefit of combined methods for the detection of the actual microbial diversity in apical periodontitis.

Co-occurrence of nitrite-dependent anaerobic methane oxidizing and anaerobic ammonia oxidizing bacteria in two Qinghai-Tibetan saline lakes

Nitrite-dependent anaerobic methane-oxidizing (n-damo) bacteria and anaerobic ammonia oxidizing (anammox) bacteria are two groups of microorganisms involved in global carbon and nitrogen cycling. In order to test whether the n-damo and anammox bacteria co-occur in natural saline environments, the DNA and cDNA samples obtained from the surficial sediments of two saline lakes (with salinity of 32 and 84 g/L, respectively) on the Tibetan Plateau were PCR-amplified with the use of anammox- and n-damo-specific primer sets, followed by clone library construction and phylogenetic analysis. DNA and cDNA-based clones affiliated with n-damo and anammox bacteria were successfully retrieved from the two samples, indicating that these two groups of bacteria can co-occur in natural saline environments with salinity as high as 84 g/L. Our finding has great implications for our understanding of the global carbon and nitrogen cycle in nature.

Effect of nitrogen deficiency during SBR operation on PHA storage and microbial diversity

In this study, changes in microbial diversity and polyhydroxyalkanoate storage ability of activated sludge under aerobic dynamic feeding conditions were investigated. Two sequencing batch reactors were operated with and without nitrogen limitation, by applying a moderate sludge retention time. Polymer storage abilities of the biomasses were improved significantly under dynamic conditions, in terms of specific polymer storage rate, polymer storage yield and polymer content of activated sludge. Moreover, aerobic dynamic feeding conditions together with nutrient limitation further improved the storage ability of the mixed population. Polymer storage yields of the biomass enriched under nitrogen-sufficient and nitrogen-deficient conditions were 0.43 and 0.61 Cmmol PHA/Cmmol substrate, respectively. This study also contributes to the knowledge of activated sludge microbiology, providing detailed information about temporal changes in community structure under dynamic conditions. Microbial community structure was determined by 16S rDNA clone library construction. Also changes in communities under different operating conditions were monitored by DGGE analysis based on bacterial 16S rDNA. The beta subclass of Proteobacteria was the most abundant phylum in both reactors during the operation periods. Changes in the community structure occurred in terms of relative abundance of the operational taxonomic units (OTUs) rather than the OTU types present in the system.

Microbial community structure and activity linked to contrasting biogeochemical gradients in bog and fen environments of the Glacial Lake Agassiz Peatland.

The abundances, compositions, and activities of microbial communities were investigated at bog and fen sites in the Glacial Lake Agassiz Peatland of northwestern Minnesota. These sites contrast in the reactivity of dissolved organic matter (DOM) and the presence or absence of groundwater inputs. Microbial community composition was characterized using pyrosequencing and clone library construction of phylogenetic marker genes. Microbial distribution patterns were linked to pH, concentrations of dissolved organic carbon and nitrogen, C/N ratios, optical properties of DOM, and activities of laccase and peroxidase enzymes. Both bacterial and archaeal richness and rRNA gene abundance were >2 times higher on average in the fen than in the bog, in agreement with a higher pH, labile DOM content, and enhanced enzyme activities in the fen. Fungi were equivalent to an average of 1.4% of total prokaryotes in gene abundance assayed by quantitative PCR. Results revealed statistically distinct spatial patterns between bacterial and fungal communities. Fungal distribution did not covary with pH and DOM optical properties and was vertically stratified, with a prevalence of Ascomycota and Basidiomycota near the surface and much higher representation of Zygomycota in the subsurface. In contrast, bacterial community composition largely varied between environments, with the bog dominated by Acidobacteria (61% of total sequences), while the Firmicutes (52%) dominated in the fen. Acetoclastic Methanosarcinales showed a much higher relative abundance in the bog, in contrast to the dominance of diverse hydrogenotrophic methanogens in the fen. This is the first quantitative and compositional analysis of three microbial domains in peatlands and demonstrates that the microbial abundance, diversity, and activity parallel with the pronounced differences in environmental variables between bog and fen sites.

Two-step denaturing gradient gel electrophoresis (2S-DGGE), a gel-based strategy to capture full-length 16S rRNA gene sequences

Obtaining full-length 16S rRNA gene sequences is important for generating accurate taxonomy assignments of bacteria, which normally is realized via clone library construction. However, the application of clone library has been hindered due to its limitations in sample throughput and in capturing minor populations (<1% of total microorganisms). To overcome these limitations, a new strategy, two-step denaturing gradient gel electrophoresis (2S-DGGE), is developed to obtain full-length 16S rRNA gene sequences. 2S-DGGE can compare microbial communities based on its first-round DGGE profiles and generate partial 16S rRNA gene sequences (8-534bp, Escherichia coli numbering). Then, strain-specific primers can be designed based on sequence information of bacteria of interest to PCR amplify their remaining 16S rRNA gene sequences (515-1541bps, E. coli numbering). The second-round DGGE can confirm DNA sequence purity of these PCR products. Finally, the full-length 16S rRNA gene sequences can be obtained through combining the two partial DNA sequences. By employing 2S-DGGE, taxonomies of a group of dehalogenating bacteria have been assigned based on their full-length 16S rRNA gene sequences, several of which existed in dehalogenating microcosms as minor populations. In all, 2S-DGGE can be utilized as a medium throughput method for taxonomic identification of interested/minor populations from single or multiple microbial consortia.

Changes in the species composition of a thermotolerant community of acidophilic chemolithotrophic microorganisms upon switching to the oxidation of a new energy substrate

Construction and analysis of the 16S rDNA clone libraries was used to investigate the species composition of two thermotolerant communities of acidophilic chemolithotrophic microorganisms (ACM) isolated from the pulp of laboratory reactors used for oxidation of different gold-containing ore concentrates. The first community was formed during oxidation of the pyrite-arsenopyrite ore concentrate from the Kyuchus deposit. The clones of the bacterial component of this community belonged to the genera Sulfobacillus (32 clones) and Leptospirillum (33 clones). The Sulfobacillus clones belonged to three groups: Sb. thermosulfidooxidans, Sb. benefaciens, and Sb. thermotolerans. All Leptospirillum clones were closely related to L. ferriphilum. All clones of the archaeal component belonged to Ferroplasma acidiphilum. The microorganisms of this community were used as inoculum for biooxidation of a different mineral concentrate, the pyrrhotite-containing pyrite-arsenopyrite ore concentrate from the Olympiadinskoe deposit, and the structure of the community formed in the process was investigated. The clones of the bacterial component of the second community also belonged to the genera Sulfobacillus (14 clones) and Leptospirillum (48 clones). The Sulfobacillus clones belonged to the species Sb. thermosulfidooxidans (13 clones) and Sb. thermotolerans (1 clone). All Leptospirillum clones were closely related to L. ferriphilum. All clones of the archaeal component belonged to Ferroplasma acidiphilum. During the adaptation of the community to a new oxidized mineral substrate, both the composition and the ratio of the constituent microbial species changed.