Investigation of Microbial Populations in the Extremely Metal-Contaminated Coeur d'Alene River Sediments

The deposition of mine tailings generated from 125 years of sulfidic ore mining resulted in the enrichment of Coeur d'Alene River (CdAR) sediments with significant amounts of toxic heavy metals. A review of literature suggests that microbial populations play a pivotal role in the biogeochemical cycling of elements in such mining-impacted sedimentary environments. To assess the indigenous microbial communities associated with metal-enriched sediments of the CdAR, high-density 16S microarray (PhyloChip) and clone libraries specific to bacteria (16S rRNA), ammonia oxidizers (amoA), and methanogens (mcrA) were analyzed. PhyloChip analysis provided a comprehensive assessment of bacterial populations and detected the largest number of phylotypes in Proteobacteria followed by Firmicutes and Actinobacteria. Furthermore, PhyloChip and clone libraries displayed considerable metabolic diversity in indigenous microbial populations by capturing several chemolithotrophic groups such as ammonia oxidizers, iron-reducers and -oxidizers, methanogens, and sulfate-reducers in the CdAR sediments. Twenty-two phylotypes detected on PhyloChip could not be classified even at phylum level thus suggesting the presence of novel microbial populations in the CdAR sediments. Clone libraries demonstrated very limited diversity of ammonia oxidizers and methanogens in the CdAR sediments as evidenced by the fact that only Nitrosospira- and Methanosarcina-related phylotypes were retrieved in amoA and mcrA clone libraries, respectively.     

Molecular analysis of prokaryotic diversity in the deep subsurface of the former Homestake gold mine,South Dakota,USA

A culture-independent molecular phylogenetic analysis was carried out to study the prokaryotic diversity in two soil samples collected from the subsurface (1.34 km depth) of the former Homestake gold mine, Lead, South Dakota, USA at two sites, the Ross shaft and number 6 Winze. Microbial community analyses were performed by cloning and sequencing of 16S rRNA genes retrieved directly from soil samples. Geochemical characterization of soils revealed high amount of toxic metals such as As, Cd, Co, Cr, Cu, Ni, Pb, Zn, and U at both the sites. Phylogenetic analyses showed that soil samples were predominantly composed of phylotypes related to phylum Proteobacteria. Other phyla detected in libraries were Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Chlorobi, Firmicutes, Gemmatimonadetes, Nitrospirae, Planctomycetes, Verrucomicrobia, and candidate divisions OP10 and TM7. The majority (>95%) of the phylotypes retrieved in the libraries were most closely related to environmental sequences from yet-uncultured bacteria representing a hitherto unidentified diversity. The archaeal communities at both the sites exhibited lower diversity and were most closely affiliated to uncultivated species within the Crenarchaeota. Results showed the existence of diverse microbial populations in deep subsurface environment of the Homestake gold mine. Statistical analyses demonstrated that each site harbored phylogenetically distinct microbial populations that were more diverse at Ross site compare to winze site.     

Microbial and Mineralogical Characterizations of Soils Collected from the Deep Biosphere of the Former Homestake Gold Mine, South Dakota

A microbial census on deep biosphere (1.34 km depth) microbial communities was performed in two soil samples collected from the Ross and number 6 Winze sites of the former Homestake gold mine, Lead, South Dakota using high-density 16S microarrays (PhyloChip). Soil mineralogical characterization was carried out using X-ray diffraction, X-ray photoelectron, and Mössbauer spectroscopic techniques which demonstrated silicates and iron minerals (phyllosilicates and clays) in both samples. Microarray data revealed extensive bacterial diversity in soils and detected the largest number of taxa in Proteobacteria phylum followed by Firmicutes and Actinobacteria. The archael communities in the deep gold mine environments were less diverse and belonged to phyla Euryarchaeota and Crenarchaeota. Both the samples showed remarkable similarities in microbial communities (1,360 common OTUs) despite distinct geochemical characteristics. Fifty-seven phylotypes could not be classified even at phylum level representing a hitherto unidentified diversity in deep biosphere. PhyloChip data also suggested considerable metabolic diversity by capturing several physiological groups such as sulfur-oxidizer, ammonia-oxidizers, iron-oxidizers, methane-oxidizers, and sulfate-reducers in both samples. High-density microarrays revealed the greatest prokaryotic diversity ever reported from deep subsurface habitat of gold mines.     

Bacterial Diversity of Terrestrial Crystalline Volcanic Rocks, Iceland

Bacteria inhabiting crystalline rocks from two terrestrial Icelandic volcanic lava flows of similar age and from the same geographical region, but differing in porosity and mineralogy, were characterised. Microarray (PhyloChip) and clone library analysis of 16S rRNA genes revealed the presence of a diverse assemblage of bacteria in each lava flow. Both methods suggested a more diverse community at the Dómadalshraun site (rhyolitic/andesitic lava flow) than that present at the Hnausahraun site (basaltic lava flow). Proteobacteria dominated the clone library at the Dómadalshraun site, while Acidobacteria was the most abundant phylum in the Hnausahraun site. Although analysis of similarities of denaturing gradient gel electrophoresis profiles suggested a strong correlation of community structure with mineralogy, rock porosity may also play an important role in shaping the bacterial community in crystalline volcanic rocks. Clone sequences were most similar to uncultured microorganisms, mainly from soil environments. Of these, Antarctic soils and temperate rhizosphere soils were prominent, as were clones retrieved from Hawaiian and Andean volcanic soils. The novel diversity of these Icelandic microbial communities was supported by the finding that up to 46% of clones displayed <85% sequence identities to sequences currently deposited in the RDP database.     

Characterization of monooxygenase gene diversity in benzene‐amended soils

Aim: To understand soil benzene monooxygenase gene diversity by clone library construction and microarray profiling. Methods and Results: A primer set was designed, and benzene monooxygenase gene diversity was characterized in two benzene‐amended soils. The dominant sequence types in the clone libraries were distinct between the two soils, and both sequences were assigned to novel clusters. Monooxygenase gene richness and diversity increased after benzene degradation. Oligonucleotide probes for microarray analysis were designed to detect a number of sequenced clones and reported monooxygenase genes. The microarray detected several genes that were not detected in the clone libraries of the same samples. Six probes were detected in more than one soil. Conclusions: The primer set designed in this study successfully detected diverse benzene monooxygenase genes. The level of diversity may have increased because the degradation of benzene differed from soil to soil. Microarrays have great potential in the comprehensive detection of gene richness as well as the elucidation of key genes for degradation. Significance and Impact of the Study: This study introduces a new primer set that may be used to identify diverse benzene monooxygenase genes in the environment; moreover, it demonstrates the potential of microarray technology in the profiling of environmental samples.     

PhyloChip hybridization uncovered an enormous bacterial diversity in the rhizosphere of different potato cultivars: many common and few cultivar-dependent taxa

The phylogenetic composition of bacterial communities in the rhizosphere of three potato cultivars grown at two distant field sites was analysed. Ribosomal gene fragments amplified from total community DNA were hybridized to PhyloChips. A total of 2432 operational taxonomic units (OTUs) were detected by the PhyloChips, of which 65% were found in the rhizosphere of all cultivars at both field sites. From all detected OTUs, 9% revealed a cultivar-dependent abundance at the one or the other field site and 4% at both sites. Differential abundance on the three cultivars was mainly observed for OTUs belonging to the Pseudomonadales, Actinomycetales and Enterobacteriales. More than 40% of OTUs belonging to Bradyrhizobiales, Sphingomonadales, Burkholderiales, Rhodocyclales, Xanthomonadales and Actinomycetales differed significantly in their abundance between the sites. A sequence analysis of six 16S rRNA gene clone libraries corresponded well with the taxonomic community structure evidenced by the PhyloChip hybridization. Most ribotypes matched OTUs detected by the PhyloChip. Those OTUs that responded to the potato cultivar at both field sites might be of interest in view of cultivar-specific effects on bacterial biocontrol strains and pathogens.     

Bacterial Diversity of Weathered Terrestrial Icelandic Volcanic Glasses

The diversity of microbial communities inhabiting two terrestrial volcanic glasses of contrasting mineralogy and age was characterised. Basaltic glass from a <0.8 Ma hyaloclastite deposit (Valafell) harboured a more diverse Bacteria community than the younger rhyolitic glass from ～150-300 AD (D?madalshraun lava flow). Actinobacteria dominated 16S rRNA gene clone libraries from both sites, however, Proteobacteria, Acidobacteria and Cyanobacteria were also numerically abundant in each. A significant proportion (15-34%) of the sequenced clones displayed <85% sequence similarities with current database sequences, thus suggesting the presence of novel microbial diversity in each volcanic glass. The majority of clone sequences shared the greatest similarity to uncultured organisms, mainly from soil environments, among these clones from Antarctic environments and Hawaiian and Andean volcanic deposits. Additionally, a large number of clones within the Cyanobacteria and Proteobacteria were more similar to sequences from other lithic environments, included among these Icelandic clones from crystalline basalt and rhyolite, however, no similarities to sequences reported from marine volcanic glasses were observed. PhyloChip analysis detected substantially greater numbers of phylotypes at both sites than the corresponding clone libraries, but nonetheless also identified the basaltic glass community as the richer, containing approximately 29% unique phylotypes compared to rhyolitic glass.     

Phylogenetic comparisons of bacterial communities from serpentine and nonserpentine soils

I present the results of a culture-independent survey of soil bacterial communities from serpentine soils and adjacent nonserpentine comparator soils using a variety of newly developed phylogenetically based statistical tools. The study design included site-based replication of the serpentine-to-nonserpentine community comparison over a regional scale ( approximately 100 km) in Northern California and Southern Oregon by producing 16S rRNA clone libraries from pairs of samples taken on either side of the serepentine-nonserpentine edaphic boundary at three geographical sites. At the division level, the serpentine and nonserpentine communities were similar to each other and to previous data from forest soils. Comparisons of both richness and Shannon diversity produced no significant differences between any of the libraries, but the vast majority of phylogenetically based tests were significant, even with only 50 sequences per library. These results suggest that most samples were distinct, consisting of a collection of lineages generally not found in other samples. The pattern of results showed that serpentine communities tended to be more similar to each other than they were to nonserpentine communities, and these differences were at a lower taxonomic scale. Comparisons of two nonserpentine communities generally showed differences, and some results suggest that the geographical site may control community composition as well. These results show the power of phylogenetic tests to discern differences between 16S rRNA libraries compared to tests that discard DNA data to bin sequences into operational taxonomic units, and they stress the importance of replication at larger scales for inferences regarding microbial biogeography.     

Analysis of Facultative Lithotroph Distribution and Diversity on Volcanic Deposits by Use of the Large Subunit of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase

A 492- to 495-bp fragment of the gene coding for the large subunit of the form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) (rbcL) was amplified by PCR from facultatively lithotrophic aerobic CO-oxidizing bacteria, colorless and purple sulfide-oxidizing microbial mats, and genomic DNA extracts from tephra and ash deposits from Kilauea volcano, for which atmospheric CO and hydrogen have been previously documented as important substrates. PCR products from the mats and volcanic sites were used to construct rbcL clone libraries. Phylogenetic analyses showed that the rbcL sequences from all isolates clustered with form IC rbcL sequences derived from facultative lithotrophs. In contrast, the microbial mat clone sequences clustered with sequences from obligate lithotrophs representative of form IA rbcL. Clone sequences from volcanic sites fell within the form IC clade, suggesting that these sites were dominated by facultative lithotrophs, an observation consistent with biogeochemical patterns at the sites. Based on phylogenetic and statistical analyses, clone libraries differed significantly among volcanic sites, indicating that they support distinct lithotrophic assemblages. Although some of the clone sequences were similar to known rbcL sequences, most were novel. Based on nucleotide diversity and average pairwise difference, a forested site and an 1894 lava flow were found to support the most diverse and least diverse lithotrophic populations, respectively. These indices of diversity were not correlated with rates of atmospheric CO and hydrogen uptake but were correlated with estimates of respiration and microbial biomass.     

Molecular analysis of carbon monoxide-oxidizing bacteria associated with recent Hawaiian volcanic deposits

Genomic DNA extracts from four sites at Kilauea Volcano were used as templates for PCR amplification of the large subunit (coxL) of aerobic carbon monoxide dehydrogenase. The sites included a 42-year-old tephra deposit, a 108-year-old lava flow, a 212-year-old partially vegetated ash-and-tephra deposit, and an approximately 300-year-old forest. PCR primers amplified coxL sequences from the OMP clade of CO oxidizers, which includes isolates such as Oligotropha carboxidovorans, Mycobacterium tuberculosis, and Pseudomonas thermocarboxydovorans. PCR products were used to create clone libraries that provide the first insights into the diversity and phylogenetic affiliations of CO oxidizers in situ. On the basis of phylogenetic and statistical analyses, clone libraries for each site were distinct. Although some clone sequences were similar to coxL sequences from known organisms, many sequences appeared to represent phylogenetic lineages not previously known to harbor CO oxidizers. On the basis of average nucleotide diversity and average pairwise difference, a forested site supported the most diverse CO-oxidizing populations, while an 1894 lava flow supported the least diverse populations. Neither parameter correlated with previous estimates of atmospheric CO uptake rates, but both parameters correlated positively with estimates of microbial biomass and respiration. Collectively, the results indicate that the CO oxidizer functional group associated with recent volcanic deposits of the remote Hawaiian Islands contains substantial and previously unsuspected diversity.     

Seasonal and spatial diversity of microbial communities in marine sediments of the South China Sea

This study was conducted to characterize the diversity of microbial communities in marine sediments of the South China Sea by means of 16S rRNA gene clone libraries. The results revealed that the sediment samples collected in summer harboured a more diverse microbial community than that collected in winter, Deltaproteobacteria dominated 16S rRNA gene clone libraries from both seasons, followed by Gammaproteobacteria, Acidobacteria, Nitrospirae, Planctomycetes, Firmicutes. Archaea phylotypes were also found. The majority of clone sequences shared greatest similarity to uncultured organisms, mainly from hydrothermal sediments and cold seep sediments. In addition, the sedimentary microbial communities in the coastal sea appears to be much more diverse than that of the open sea. A spatial pattern in the sediment samples was observed that the sediment samples collected from the coastal sea and the open sea clustered separately, a novel microbial community dominated the open sea. The data indicate that changes in environmental conditions are accompanied by significant variations in diversity of microbial communities at the South China Sea.     

Phylogenetic Comparisons of Bacterial Communities from Serpentine and Nonserpentine Soils

I present the results of a culture-independent survey of soil bacterial communities from serpentine soils and adjacent nonserpentine comparator soils using a variety of newly developed phylogenetically based statistical tools. The study design included site-based replication of the serpentine-to-nonserpentine community comparison over a regional scale (~100 km) in Northern California and Southern Oregon by producing 16S rRNA clone libraries from pairs of samples taken on either side of the serepentine-nonserpentine edaphic boundary at three geographical sites. At the division level, the serpentine and nonserpentine communities were similar to each other and to previous data from forest soils. Comparisons of both richness and Shannon diversity produced no significant differences between any of the libraries, but the vast majority of phylogenetically based tests were significant, even with only 50 sequences per library. These results suggest that most samples were distinct, consisting of a collection of lineages generally not found in other samples. The pattern of results showed that serpentine communities tended to be more similar to each other than they were to nonserpentine communities, and these differences were at a lower taxonomic scale. Comparisons of two nonserpentine communities generally showed differences, and some results suggest that the geographical site may control community composition as well. These results show the power of phylogenetic tests to discern differences between 16S rRNA libraries compared to tests that discard DNA data to bin sequences into operational taxonomic units, and they stress the importance of replication at larger scales for inferences regarding microbial biogeography.     

Analysis of facultative lithotroph distribution and diversity on volcanic deposits by use of the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase

A 492- to 495-bp fragment of the gene coding for the large subunit of the form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) (rbcL) was amplified by PCR from facultatively lithotrophic aerobic CO-oxidizing bacteria, colorless and purple sulfide-oxidizing microbial mats, and genomic DNA extracts from tephra and ash deposits from Kilauea volcano, for which atmospheric CO and hydrogen have been previously documented as important substrates. PCR products from the mats and volcanic sites were used to construct rbcL clone libraries. Phylogenetic analyses showed that the rbcL sequences from all isolates clustered with form IC rbcL sequences derived from facultative lithotrophs. In contrast, the microbial mat clone sequences clustered with sequences from obligate lithotrophs representative of form IA rbcL. Clone sequences from volcanic sites fell within the form IC clade, suggesting that these sites were dominated by facultative lithotrophs, an observation consistent with biogeochemical patterns at the sites. Based on phylogenetic and statistical analyses, clone libraries differed significantly among volcanic sites, indicating that they support distinct lithotrophic assemblages. Although some of the clone sequences were similar to known rbcL sequences, most were novel. Based on nucleotide diversity and average pairwise difference, a forested site and an 1894 lava flow were found to support the most diverse and least diverse lithotrophic populations, respectively. These indices of diversity were not correlated with rates of atmospheric CO and hydrogen uptake but were correlated with estimates of respiration and microbial biomass.     

High-Density Universal 16S rRNA Microarray Analysis Reveals Broader Diversity than Typical Clone Library When Sampling the Environment

Molecular approaches aimed at detection of a broad-range of prokaryotes in the environment routinely rely on classifying heterogeneous 16S rRNA genes amplified by polymerase chain reaction (PCR) using primers with broad specificity. The general method of sampling and categorizing DNA has been to clone then sequence the PCR products. However, the number of clones required to adequately catalog the majority of taxa in a sample is unwieldy. Alternatively, hybridizing target sequences to a universal 16S rRNA gene microarray may provide a more rapid and comprehensive view of prokaryotic community composition. This study investigated the breadth and accuracy of a microarray in detecting diverse 16S rRNA gene sequence types compared to clone-and-sequencing using three environmental samples: urban aerosol, subsurface soil, and subsurface water. PCR products generated from universal 16S rRNA gene-targeted primers were classified by using either the clone-and-sequence method or by hybridization to a novel high-density microarray of 297,851 probes complementary to 842 prokaryotic subfamilies. The three clone libraries comprised 1391 high-quality sequences. Approximately 8% of the clones could not be placed into a known subfamily and were considered novel. The microarray results confirmed the majority of clone-detected subfamilies and additionally demonstrated greater amplicon diversity extending into phyla not observed by the cloning method. Sequences matching operational taxonomic units within the phyla Nitrospira, Planctomycetes, and TM7, which were uniquely detected by the array, were verified with specific primers and subsequent amplicon sequencing. Subfamily richness detected by the array corresponded well with nonparametric richness predictions extrapolated from clone libraries except in the water community where clone-based richness predictions were greatly exceeded. It was concluded that although the microarray is unreliable in identifying novel prokaryotic taxa, it reveals greater diversity in environmental samples than sequencing a typically sized clone library. Furthermore, the microarray allowed samples to be rapidly evaluated with replication, a significant advantage in studies of microbial ecology.     

In-depth Characterization via Complementing Culture-Independent Approaches of the Microbial Community in an Acidic Hot Spring of the Colombian Andes

The microbial community of a Colombian high mountain hot spring, El Coquito, was analyzed using three different culture-independent assessments of 16S ribosomal RNA genes: clone libraries, pyrosequencing of the V5–V6 hypervariable region, and microarray. This acidic spring had a diverse community composed mainly of Bacteria that shared characteristics with those from other hot springs and extreme acidic environments. The microbial community was dominated by Proteobacteria, Firmicutes, and Planctomycetes and contained chemotrophic bacteria potentially involved in cycling of ferrous and sulfur-containing minerals and phototrophic organisms, most of which were eukaryotic micro-algae. Despite the presence of a large proportion of novel, unclassified sequences, the taxonomic profiles obtained with each strategy showed similarities at higher taxonomic levels. However, some groups, such as Spirochaetes and Aquificae, were identified using only one methodology, and more taxa were detected with the gene array, which also shared more groups with the pyrosequencing data. Overall, the combined use of different approaches provided a broader view of the microbial community in this acidic hot spring.     

Bacterial diversity in Cr(VI) and Cr(III)-contaminated industrial wastewaters

The bacterial community structure of a chromium water bath, a chromium drainage waste system, a chromium pretreatment tank, and a trivalent chromium precipitation tank from the Hellenic Aerospace Industry S.A. was assessed using 16S rRNA libraries and a high-density DNA microarray (PhyloChip). 16S rRNA libraries revealed a bacterial diversity consisting of 14 distinct operational taxonomic units belonging to five bacterial phyla: Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Actinobacteria, and Bacteroidetes. However, employing a novel microarray-based approach (PhyloChip), a high bacterial diversity consisting of 30 different phyla was revealed, with representatives of 181 different families. This made it possible to identify a core set of genera present in all wastewater treatment stages examined, consisting of members of Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Epsilonproteobacteria, and Bacteroidetes. In the chromium pretreatment tank, where the concentration of Cr(VI) is high (2.3 mg/l), we identified the presence of Pseudomonadales, Actinomycetales, and Enterobacteriales in abundance. In the chromium precipitation tank, where the concentration of Cr(III) is high, the dominant bacteria consortia were replaced by members of Rhodocyclales and Chloroflexi. The bacterial community structure changed significantly with changes in the chromium concentration. This in-depth analysis should prove useful for the design and development of improved bioremediation strategies.     

Molecular Analysis of Carbon Monoxide-Oxidizing Bacteria Associated with Recent Hawaiian Volcanic Deposits

Genomic DNA extracts from four sites at Kilauea Volcano were used as templates for PCR amplification of the large subunit (coxL) of aerobic carbon monoxide dehydrogenase. The sites included a 42-year-old tephra deposit, a 108-year-old lava flow, a 212-year-old partially vegetated ash-and-tephra deposit, and an approximately 300-year-old forest. PCR primers amplified coxL sequences from the OMP clade of CO oxidizers, which includes isolates such as Oligotropha carboxidovorans, Mycobacterium tuberculosis, and Pseudomonas thermocarboxydovorans. PCR products were used to create clone libraries that provide the first insights into the diversity and phylogenetic affiliations of CO oxidizers in situ. On the basis of phylogenetic and statistical analyses, clone libraries for each site were distinct. Although some clone sequences were similar to coxL sequences from known organisms, many sequences appeared to represent phylogenetic lineages not previously known to harbor CO oxidizers. On the basis of average nucleotide diversity and average pairwise difference, a forested site supported the most diverse CO-oxidizing populations, while an 1894 lava flow supported the least diverse populations. Neither parameter correlated with previous estimates of atmospheric CO uptake rates, but both parameters correlated positively with estimates of microbial biomass and respiration. Collectively, the results indicate that the CO oxidizer functional group associated with recent volcanic deposits of the remote Hawaiian Islands contains substantial and previously unsuspected diversity.     

High bacterial diversity in nearshore and oceanic biofilms and their influence on larval settlement by Hydroides elegans (Polychaeta)

Settlement of many benthic marine invertebrates is stimulated by bacterial biofilms, although it is not known if patterns of settlement reflect microbial communities that are specific to discrete habitats. Here, we characterized the taxonomic and functional gene diversity (16S rRNA gene amplicon and metagenomic sequencing analyses), as well as the specific bacterial abundances, in biofilms from diverse nearby and distant locations, both inshore and offshore, and tested them for their ability to induce settlement of the biofouling tubeworm Hydroides elegans, an inhabitant of bays and harbours around the world. We found that compositions of the bacterial biofilms were site specific, with the greatest differences between inshore and offshore sites. Further, biofilms were highly diverse in their taxonomic and functional compositions across inshore sites, while relatively low diversity was found at offshore sites. Hydroides elegans settled on all biofilms tested, with settlement strongly correlated with bacterial abundance. Bacterial density in biofilms was positively correlated with biofilm age. Our results suggest that the localized distribution of H. elegans is not determined by ‘selection’ to locations by specific bacteria, but it is more likely linked to the prevailing local ecology and oceanographic features that affect the development of dense biofilms and the occurrence of larvae.     

Prokaryotic diversity of arctic ice shelf microbial mats

The prokaryotic diversity and respiratory activity of microbial mat communities on the Markham Ice Shelf and Ward Hunt Ice Shelf in the Canadian high Arctic were analysed. All heterotrophic isolates and > 95% of bacterial 16S rRNA gene clone library sequences from both ice shelves grouped within the phyla Bacteroidetes , Proteobacteria and Actinobacteria . Clone library analyses showed that the bacterial communities were diverse and varied significantly between the two ice shelves, with the Markham library having a higher estimated diversity (Chao1 = 243; 105 operational taxonomic units observed in 189 clones) than the Ward Hunt library (Chao1 = 106; 52 operational taxonomic units observed in 128 clones). Archaeal 16S rRNA gene clone libraries from both ice shelves were dominated by a single Euryarchaeota sequence, which appears to represent a novel phylotype. Analyses of community activity by radiorespiration assays detected metabolism in mat samples from both ice shelves at temperatures as low as −10°C. These findings provide the first insight into the prokaryotic biodiversity of Arctic ice shelf communities and underscore the importance of these cryo-ecosystems as a rich source of microbiota that are adapted to extreme cold.     

Could nested PCR be applicable for the study of microbial diversity?

PCR-based methods for rRNA gene analysis have been widely used to study diversity of microbiology. However, the analysis would be difficult when the DNA content in samples is too low to be amplified by conventional PCR. Nested PCR comes up with the advantage of higher sensitivity. It can detect target DNA at several-fold lower concentrations than conventional PCR. However, the amplification bias and factors that potentially affect measurement of sample diversity associated with nested PCR method has received little attention. Here, nested PCR was compared to reconditioning PCR which is based on conventional PCR and it would reduce the formation of heteroduplex. We investigated the use of both nested and reconditioning PCR methods to construct clone libraries of 16S rRNA genes from four swimming pool water samples. Abundances of OTUs (operational taxonomic units) were correlated between the libraries (r ² = 0.88, P < 0.0005), and some OTUs had equivalent abundances in the two libraries using the Chi-square test. Differences in taxonomic groups, as well as diversity and richness estimators, were compared by paired t-test and the Wilcoxon test, respectively. There were no significant differences between clone libraries using these two PCR methods. The results of ∫-Libshuff analysis suggested that nested PCR have no particular biases in revealing OTU diversity of a bacterial community. Thus, nested PCR produce congruent pictures with reconditioning PCR in the microbial community analysis.