Archaeal 16S rRNA Gene Research Articles

Archaeal Community Structures in the Solfataric Acidic Hot Springs with Different Temperatures and Elemental Compositions

Archaeal 16S rRNA gene compositions and environmental factors of four distinct solfataric acidic hot springs in Kirishima, Japan were compared. The four ponds were selected by differences of temperature and total dissolved elemental concentration as follows: (1) Pond-A: 93°C and 1679 mg L−1, (2) Pond-B: 66°C and 2248 mg L−1, (3) Pond-C: 88°C and 198 mg L−1, and (4) Pond-D: 67°C and 340 mg L−1. In total, 431 clones of 16S rRNA gene were classified into 26 phylotypes. In Pond-B, the archaeal diversity was the highest among the four, and the members of the order Sulfolobales were dominant. The Pond-D also showed relatively high diversity, and the most frequent group was uncultured thermoacidic spring clone group. In contrast to Pond-B and Pond-D, much less diverse archaeal clones were detected in Pond-A and Pond-C showing higher temperatures. However, dominant groups in these ponds were also different from each other. The members of the order Sulfolobales shared 89% of total clones in Pond-A, and the uncultured crenarchaeal groups shared 99% of total Pond-C clones. Therefore, species compositions and biodiversity were clearly different among the ponds showing different temperatures and dissolved elemental concentrations.

Enrichment of nitrous oxide reducing bacteria from coastal marsh sediments

We attempted to recover organisms capable of respiratory nitrous oxide reduction with acetate as an electron donor from a variety of coastal marine sediments from Lavaca Bay area, Texas by use of liquid enrichment cultures. Putative positive cultures were analyzed by amplifying eubacterial and archaeal 16S rRNA gene fragments and analyzing their diversity by separating them by a denaturing gradient gel electrophoresis (DGGE). No Archaea was detected in our enrichments; however, positive enrichments from coastal salt marsh indicated the presence of putative nitrous oxide reducing bacteria. DGGE patterns of the amplified DNA were similar between enrichments, with ca. 7 obvious bands. The dominant bands were tentatively identified as members of the Gammaproteobacteria class, closely related to various denitrifying pseudomonads. Our results indicate that coastal marine environments may sustain a nitrous oxide reducing community, although nitrous oxide reduction is probably an opportunistic form of metabolism in that environment.

Prokaryotic Diversity of the Tucuruí Hydropower Plant Reservoir in the Brazilian Amazon

To perform the a culture-independent characterization of the microbial diversity observed within a water body in the Amazon, we constructed a library of bacterial and archaeal 16S rRNA genes isolated from environmental samples from the Tucurui Hydroelectric Plant reservoir. The sampling locations included the upstream compartment (MR), which is located 50 km upstream of the dam, and Itupiranga (ITU), which is located upstream on the Tocantins River at the inlet of the reservoir. The bacterial phyla Cyanobacteria (46%), Proteobacteria (12%), Actinobacteria (9%), Bacteroidetes (4%) and Verrucomicrobia (2%) were identified at the MR. Phyla including the Chloroflexi, Acidobacteria, Planctomycetes and TM6 candidate division were observed at a frequency of <2%. The phyla Cyanobacteria (62%), Proteobacteria (5%) and Actinobacteria (7%) were identified at ITU. The archaeal diversity was limited to the Euryarchaeota and Crenarchaeota phyla; the latter was found in greater abundance and mainly consisted of uncultured phylotypes. In both regions, 25% and 95% of the bacteria and archaea, respectively, that were identified were previously unclassified. The phylum Cyanobacteria was almost exclusively represented by the genus Synechococcus sp.; however, a wide variety of microorganisms from the phylum Proteobacteria were identified from the classes alpha, gamma, delta and beta proteobacteria, with the latter being the most abundant. This study demonstrates that high prokaryotic diversity exists in the Amazonian rivers in addition to the previously reported megadiversity of fauna and flora.

Changes in community structure of methanogenic archaea brought about by water-saving practice in paddy field soil

Changes in the community structure of methanogenic archaea in a paddy field soil brought about by an alternate wetting and drying (AWD) water-saving practice were shown by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative PCR analyses targeting archaeal 16S rRNA gene and methanogenic archaeal mcrA gene. Soil samples were collected four times from the field plots under the treatments of continuous flooding (CF) and AWD water-saving, where irrigation was conducted when soil water potential at 15 cm depth decreased to −20 kPa. The community analysis was performed over three rice cultivation periods in the wet season in 2008 (2008WS) and the dry and wet seasons in 2009 (2009DS and 2009WS, respectively). PCR-DGGE analysis showed that the archaeal 16S rRNA genes differed depending on water management. During the three rice cultivation periods, the community of the CF plots gradually changed with time, while the community of the AWD plots possessed a relatively large variation. Principal component analysis and sequencing analysis of selected 16S rRNA genes indicated that members of Methanosarcinales dominated in the CF plots while members of Methanocellales and uncultured group of “Thaumarchaeota” characterized the AWD plots. Numbers of archaeal 16S rRNA and methanogenic archaeal mcrA genes were significantly different between the CF and AWD plots in 2008WS, but not in 2009DS and 2009WS. Thus, the AWD water-saving practice did not reduce populations of the methanogenic archaea but moderately influenced the community structure in the paddy field soil.

Partaking of Archaea to biogeochemical cycling in oxygen‐deficient zones of meromictic saline Lake Faro (Messina, Italy)

We used a combination of molecular and microbiological approaches to determine the activity, abundance and diversity of archaeal populations inhabiting meromictic saline Lake Faro (Messina, Italy). Analysis of archaeal 16S rRNA, amoA, accA and hbd genes and transcripts revealed that sub- and anoxic layers of Lake Faro are primarily inhabited by the organisms related to the clusters of Marine Group I.1a of Thaumarchaeota frequently recovered from oxygen-depleted marine ecosystems. These organisms dominated the metabolically active archaea down to the bottom of the lake, indicating their adaptation to recurrent changes in the levels of water column hypoxia. The upper microaerobic layer of Lake Faro redoxcline has the maximal rates of dark primary production much lower than those of other previously studied pelagic redoxclines, but comparable to the values of meso- and bathypelagic areas of Mediterranean Sea. Application of bacterial inhibitors, especially azide, significantly declined the CO2 fixation rates in the low interface and monimolimnion, whereas archaea-specific inhibitor had effect only in upper part of the redoxcline. Based on these findings, we hypothesize that dark bicarbonate fixation in suboxic zone of Lake Faro results mainly from archaeal activity which is affected by the predicted lack in oxygen in lower layers.

Methanogenic Community Dynamics during Anaerobic Utilization of Agricultural Wastes

This work is devoted to the investigation of the methanogenic archaea involved in anaerobic digestion of cattle manure and maize straw on the basis of terminal restriction fragment length polymorphism (T-RFLP) analysis of archaeal 16S rRNA genes. The biological diversity and dynamics of methanogenic communities leading to anaerobic degradation of agricultural organic wastes with biogas production were evaluated in laboratory-scale digesters. T-RFLP analysis, along with the establishment of archaeal 16S rRNA gene clone libraries, showed that the methanogenic consortium consisted mainly of members of the generaMethanosarcinaandMethanoculleus,with a predominance ofMethanosarcinaspp. throughout the experiment.

Gold Nanoparticle Clusters in Quasinematic Layers of Liquid-Crystalline Dispersion Particles of Double-Stranded Nucleic Acids

The interaction between gold nanoparticles and particles of cholesteric liquid-crystalline dispersions formed by double-stranded DNA and poly(I)×poly(C) molecules is considered. It is shown that small-sized (~ 2 nm) gold nanoparticles induce two different structural processes. First, they facilitate the reorganization of the spatial cholesteric structure of the particles into a nematic one. This process is accompanied by a fast decrease in the amplitude of an abnormal band in the CD spectrum. Second, they induce cluster formation in a "free space" between neighboring nucleic acid molecules fixed in the structure of the quasinematic layers of liquid-crystalline particles. This process is accompanied by slow development of the surface plasmon resonance band in the visible region of the absorption spectrum. Various factors influencing these processes are outlined. Some assumptions concerning the possible mechanism(s) of fixation of gold nanoparticles between the neighboring double-stranded nucleic acid molecules in quasinematic layers are formulated.

Microbial community changes at a terrestrial volcanic CO2vent induced by soil acidification and anaerobic microhabitats within the soil column

CO2 capture and storage (CCS) in deep geological formations is one option currently evaluated to reduce greenhouse gas emissions. Consequently, the impact of a possible CO2 leakage from a storage site into surface environments has to be evaluated. During such a hypothetical leakage event, the CO2 migrates upwards along fractures entering surface soils, a scenario similar to naturally occurring CO2 vents. Therefore, such a natural analogue site at the Laacher See was chosen for an ecosystem study on the effects of high CO2 concentrations on soil chemistry and microbiology. The microbial activities revealed differences in their spatial distribution and temporal variability for CO2 -rich and reference soils. Furthermore, the abundance of several functional and group-specific gene markers revealed further differences, for example, a decrease in Geobacteraceae and an increase in sulphate-reducing prokaryotes in the vent centre. Molecular-biological fingerprinting of the microbial communities with DGGE indicated a shift in the environmental conditions within the Laacher See soil column leading to anaerobic and potentially acidic microenvironments. Furthermore, the distribution and phylogenetic affiliation of the archaeal 16S rRNA genes, the presence of ammonia-oxidizing Archaea and the biomarker analysis revealed a predominance of Thaumarchaeota as possible indicator organisms for elevated CO2 concentrations in soils.

Changes in microbial communities in a hybrid anaerobic reactor with organic loading rate and temperature

Poor understanding of the response of microbial communities to sudden changes in organic and hydraulic loads is one of the major reasons for the inability to prevent operational instabilities in anaerobic reactors. Effect of changes in hydraulic retention time (HRT) and organic loading rate (OLR) on reactor performance and its anaerobic microbial community were investigated in two anaerobic hybrid reactors operated at 37 and 55°C. HRT was reduced stepwise, while OLR was increased along with influent chemical oxygen demand at fixed HRT until the performance of reactor deteriorated. The profile of archaeal 16S rRNA gene amplicons, resolved by denaturing gradient gel electrophoresis, reflected system status during disturbances. The more diverse archaeal community in the reactor operated at 37°C showed better performance than the communities present at 55°C at higher OLR and shorter HRT, suggesting that higher diversity is indicative of more stable operation of reactors despite organic and hydraulic shocks.

Seasonal and vertical distribution of putative ammonia-oxidizing thaumarchaeotal communities in an oligotrophic lake

The discovery of Archaea carrying an amoA gene coding for the A-subunit of ammonia monooxygenase gave a boost to studies aimed at detecting this gene under diverse conditions. Despite numerous studies describing the archaeal amoA gene abundance and richness in different habitats, the understanding of the freshwater ecology of potentially archaeal ammonia oxidizers, recently positioned in the phylum Thaumarchaeota, is still lacking. In a seasonal and vertical study of deep oligotrophic Lake Lucerne, Switzerland, with high Thaumarchaeota-specific crenarchaeol concentrations, we showed that all archaeal 16S rRNA genes found belong to the thaumarchaeotal phylum. The abundances of archaeal 16S rRNA and amoA genes remained in the same order of magnitude (average 6×mL(-1) ) and displayed matching seasonal dynamics within 16 monthly collected samples at three different water depths (r(2) =0.80, slope of 1.06). The Thaumarchaeota in this lake form a distinct cluster in both the 16S rRNA and amoA gene phylogenies, are affiliated to other thaumarchaeotal freshwater sequences within group 1.1a Archaea, and fall in the low saline cluster of the amoA phylogeny. In accordance with temperature and conductivity, the Thaumarchaeota in the surface water showed a significantly different seasonality and lower abundance than their counterparts in the deeper waters. This study indicates that the ecology of Thaumarchaeota, with their potential in nitrogen cycling, may differ per water depth in deep freshwater ecosystems.

Impact of Long-Term Diesel Contamination on Soil Microbial Community Structure

Microbial community composition and diversity at a diesel-contaminated railway site were investigated by pyrosequencing of bacterial and archaeal 16S rRNA gene fragments to understand the interrelationships among microbial community composition, pollution level, and soil geochemical and physical properties. To this end, 26 soil samples from four matrix types with various geochemical characteristics and contaminant concentrations were investigated. The presence of diesel contamination significantly impacted microbial community composition and diversity, regardless of the soil matrix type. Clean samples showed higher diversity than contaminated samples (P < 0.001). Bacterial phyla with high relative abundances in all samples included Proteobacteria, Firmicutes, Actinobacteria, Acidobacteria, and Chloroflexi. High relative abundances of Archaea, specifically of the phylum Euryarchaeota, were observed in contaminated samples. Redundancy analysis indicated that increased relative abundances of the phyla Chloroflexi, Firmicutes, and Euryarchaeota correlated with the presence of contamination. Shifts in the chemical composition of diesel constituents across the site and the abundance of specific operational taxonomic units (OTUs; defined using a 97% sequence identity threshold) in contaminated samples together suggest that natural attenuation of contamination has occurred. OTUs with sequence similarity to strictly anaerobic Anaerolineae within the Chloroflexi, as well as to Methanosaeta of the phylum Euryarchaeota, were detected. Anaerolineae and Methanosaeta are known to be associated with anaerobic degradation of oil-related compounds; therefore, their presence suggests that natural attenuation has occurred under anoxic conditions. This research underscores the usefulness of next-generation sequencing techniques both to understand the ecological impact of contamination and to identify potential molecular proxies for detection of natural attenuation.

Changes in the microbial community structure of bacteria, archaea and fungi in response to elevated CO2 and warming in an Australian native grassland soil

The microbial community structure of bacteria, archaea and fungi is described in an Australian native grassland soil after more than 5 years exposure to different atmospheric CO2 concentrations ([CO2]) (ambient, +550 ppm) and temperatures (ambient, + 2°C) under different plant functional types (C3 and C4 grasses) and at two soil depths (0-5 cm and 5-10 cm). Archaeal community diversity was influenced by elevated [CO2], while under warming archaeal 16S rRNA gene copy numbers increased for C4 plant Themeda triandra and decreased for the C3 plant community (P < 0.05). Fungal community diversity resulted in three groups based upon elevated [CO2], elevated [CO2] plus warming and ambient [CO2]. Overall bacterial community diversity was influenced primarily by depth. Specific bacterial taxa changed in richness and relative abundance in response to climate change factors when assessed by a high-resolution 16S rRNA microarray (PhyloChip). Operational taxonomic unit signal intensities increased under elevated [CO2] for both Firmicutes and Bacteroidetes, and increased under warming for Actinobacteria and Alphaproteobacteria. For the interaction of elevated [CO2] and warming there were 103 significant operational taxonomic units (P < 0.01) representing 15 phyla and 30 classes. The majority of these operational taxonomic units increased in abundance for elevated [CO2] plus warming plots, while abundance declined in warmed or elevated [CO2] plots. Bacterial abundance (16S rRNA gene copy number) was significantly different for the interaction of elevated [CO2] and depth (P < 0.05) with decreased abundance under elevated [CO2] at 5-10 cm, and for Firmicutes under elevated [CO2] (P < 0.05). Bacteria, archaea and fungi in soil responded differently to elevated [CO2], warming and their interaction. Taxa identified as significantly climate-responsive could show differing trends in the direction of response ('+' or '-') under elevated CO2 or warming, which could then not be used to predict their interactive effects supporting the need to investigate interactive effects for climate change. The approach of focusing on specific taxonomic groups provides greater potential for understanding complex microbial community changes in ecosystems under climate change.

Effect of winter-flooding on methanogenic archaeal community structure in paddy field under organic farming

Paddy field is a major emission source of methane. Methane is the terminal product of anaerobic decomposition of organic matter and generated by methanogenic archaea under flooded conditions in paddy fields. This study aimed to reveal the effect of winter flooding on methanogenic archaeal community structure in paddy fields of Andosols under organic farming. Soil samples were collected from experimental paddy fields in the Field Science Center, Tohoku University, for two years. They were under flooding conditions during winter with organic farming, under non-flooding conditions during winter with organic farming and under non-flooding conditions during winter with conventional farming (non-organic farming). Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of methanogenic archaeal 16S rRNA gene revealed that the DGGE patterns were nearly the same irrespective of the treatment and sampling times. Twenty-three bands were observed from each treatment and 4, 13 and 6 sequences were closely related to Methanomicrobiales, Methanosarcinales and Methanocellales, respectively. Real-time quantitative PCR analysis indicated that the abundance of methanogenic archaeal 16S rRNA gene and mcrA gene, encoding α subunit of methyl-coenzyme M reductase, was not significantly different among the paddy fields. This study first revealed a methanogenic archaeal community in an Andosol paddy field and showed that the community was not affected by winter flooding under organic farming.

Composition of the archaeal community involved in methane production during the decomposition of Microcystis blooms in the laboratory

We investigated the microbial processes involved in methane (CH(4)) production from Microcystis bloom scums at different temperatures. A Microcystis slurry was collected from Lake Taihu and incubated in airtight bottles at 15, 25, and 35°C. The production of CH(4) was monitored, and the emission rate was calculated. The dynamics of the methanogenic community were analyzed by terminal restriction fragment length polymorphism analysis of archaeal 16S rRNA genes. Phylogenetic information for the methanogens was obtained by cloning and sequencing selected samples. Significant CH(4) emission from the Microcystis scums was delayed by approximately 12days by the natural oxygen depletion process, and CH(4) production was enhanced at higher temperatures. Phylogenetic analysis indicated that the archaeal community was composed of Methanomicrobiales, Methanobacteriaceae, and a novel cluster of Archaea. An apparent succession of the methanogenic community was demonstrated, with a predominance of Methanobacteriaceae at higher temperatures. Higher temperatures enhanced the methanogenic transformation of the Microcystis biomass and the phylogenetic dominance of hydrogenotrophic methanogens, suggesting that H(2) and CO(2) might be the primary substrates for CH(4) production during Microcystis decomposition without the participation of lake sediment. This work provides insight into the microbial components involved in Microcystis biomass fermentation in controlled systems.

Archaea in the foregut of macropod marsupials: PCR and amplicon sequence-based observations

To investigate, using culture-independent techniques, the presence and diversity of methanogenic archaea in the foregut of kangaroos. DNA was extracted from forestomach contents of 42 kangaroos (three species), three sheep and three cattle. Four qualitative and quantitative PCR assays targeting the archaeal domain (16S rRNA gene) or the functional methanogenesis gene, mcrA, were used to determine the presence and population density of archaea in kangaroos and whether they were likely to be methanogens. All ruminal samples were positive for archaea, produced PCR product of expected size, contained high numbers of archaea and high numbers of cells with mcrA genes. Kangaroos were much more diverse and contradictory. Fourteen kangaroos had detectable archaea with numbers 10- to 1000-fold fewer than sheep and cattle. Many kangaroos that did not possess archaea were positive for the mcrA gene and had detectable numbers of cells with this gene and vice versa. DNA sequence analysis of kangaroos' archaeal 16S rRNA gene clones show that many methanogens were related to Methanosphaera stadmanae. Other sequences were related to non-methanogenic archaea (Thermoplasma sp.), and a number of kangaroos had mcrA gene sequences related to methane oxidising archaea (ANME). Discrepancies between qualitative and quantitative PCR assays for archaea and the mcrA gene suggest that the archaeal communities are very diverse and it is possible that novel species exist. Archaea (in general) were below detectable limits in many kangaroos, especially Red kangaroos; when present they are in lower numbers than in ruminants, and the archaea are not necessarily methanogenic. The determination of why this is the case in the kangaroo foregut could assist in reducing emissions from other ecosystems in the future.

Prokaryotic ultramicroforms in a Sphagnum peat bog of upper Volga catchment

The waters of small lakes located in swampy catchment areas of upper Volga contain considerable amounts of ultrasmall microbial cells that pass through 0.22-μm-pore-size filters. As shown in our previous study [1], most of these cells represent the bacterial genera Herbaspirillum, Herminiimonas, Curvibacter, and Burkholderia of the class Betaproteobacteria, as well as euryarchaea of the uncharacterized clade LDS. The aim of the present study was to investigate the possible effect of the waters draining swampy areas on the composition of the filterable microbial fraction in lakes fed by swampy catchments. To address this question molecular identification was performed of prokaryotic ultramicroforms in the peat of the ombrotrophic Sphagnum bog Obukhovskoe, located, like the lakes studied previously [1], in the Mologa-Sheksna catchment area. The number of filterable microorganisms in 1 g wet peat was 3.8 × 106 cells, or 0.5% of total microbial cell number in the peat. From the DNA of the filterable cell fraction, 100 clones of bacterial and 77 clones of archaeal 16S rRNA genes were obtained. The bacterial clone library contained 16S rRNA gene sequences representing the classes Beta- and Gammaproteobacteria (the genera Janthinobacterium and Pseudomonas, respectively) and the phylum Bacteroidetes (the genera Chryseobacterium and Epilithonimonas) and differed significantly from the clone library of bacterial ultramicroforms of lake water. By contrast, the pools of filterable archaea in bogs and lakes were essentially similar. They were represented by the euryarchaeal clade LDS and methanogens of the orders Methanobacteriales and Methanosarcinales. Additionally, the pool of filterable archaea of the bog included methanogens of the order Methanomicrobiales and representatives of the uncharacterized euryarchaeal clade RC-V (Rice Cluster V) and of the phylum Thaumarchaeota.

Next-Generation Sequencing of Microbial Communities in the Athabasca River and Its Tributaries in Relation to Oil Sands Mining Activities

The Athabasca oil sands deposit is the largest reservoir of crude bitumen in the world. Recently, the soaring demand for oil and the availability of modern bitumen extraction technology have heightened exploitation of this reservoir and the potential unintended consequences of pollution in the Athabasca River. The main objective of the present study was to evaluate the potential impacts of oil sands mining on neighboring aquatic microbial community structure. Microbial communities were sampled from sediments in the Athabasca River and its tributaries as well as in oil sands tailings ponds. Bacterial and archaeal 16S rRNA genes were amplified and sequenced using next-generation sequencing technology (454 and Ion Torrent). Sediments were also analyzed for a variety of chemical and physical characteristics. Microbial communities in the fine tailings of the tailings ponds were strikingly distinct from those in the Athabasca River and tributary sediments. Microbial communities in sediments taken close to tailings ponds were more similar to those in the fine tailings of the tailings ponds than to the ones from sediments further away. Additionally, bacterial diversity was significantly lower in tailings pond sediments. Several taxonomic groups of Bacteria and Archaea showed significant correlations with the concentrations of different contaminants, highlighting their potential as bioindicators. We also extensively validated Ion Torrent sequencing in the context of environmental studies by comparing Ion Torrent and 454 data sets and by analyzing control samples.

Temperature increases from 55 to 75 °C in a two-phase biogas reactor result in fundamental alterations within the bacterial and archaeal community structure

Agricultural biogas plants were operated in most cases below their optimal performance. An increase in the fermentation temperature and a spatial separation of hydrolysis/acetogenesis and methanogenesis are known strategies in improving and stabilizing biogas production. In this study, the dynamic variability of the bacterial and archaeal community was monitored within a two-phase leach bed biogas reactor supplied with rye silage and straw during a stepwise temperature increase from 55 to 75°C within the leach bed reactor (LBR), using TRFLP analyses. To identify the terminal restriction fragments that were obtained, bacterial and archaeal 16S rRNA gene libraries were constructed. Above 65°C, the bacterial community structure changed from being Clostridiales-dominated toward being dominated by members of the Bacteroidales, Clostridiales, and Thermotogales orders. Simultaneously, several changes occurred, including a decrease in the total cell count, degradation rate, and biogas yield along with alterations in the intermediate production. A bioaugmentation with compost at 70°C led to slight improvements in the reactor performance; these did not persist at 75°C. However, the archaeal community within the downstream anaerobic filter reactor (AF), operated constantly at 55°C, altered by the temperature increase in the LBR. At an LBR temperature of 55°C, members of the Methanobacteriales order were prevalent in the AF, whereas at higher LBR temperatures Methanosarcinales prevailed. Altogether, the best performance of this two-phase reactor was achieved at an LBR temperature of below 65°C, which indicates that this temperature range has a favorable effect on the microbial community responsible for the production of biogas.

PCR biases distort bacterial and archaeal community structure in pyrosequencing datasets.

As 16S rRNA gene targeted massively parallel sequencing has become a common tool for microbial diversity investigations, numerous advances have been made to minimize the influence of sequencing and chimeric PCR artifacts through rigorous quality control measures. However, there has been little effort towards understanding the effect of multi-template PCR biases on microbial community structure. In this study, we used three bacterial and three archaeal mock communities consisting of, respectively, 33 bacterial and 24 archaeal 16S rRNA gene sequences combined in different proportions to compare the influences of (1) sequencing depth, (2) sequencing artifacts (sequencing errors and chimeric PCR artifacts), and (3) biases in multi-template PCR, towards the interpretation of community structure in pyrosequencing datasets. We also assessed the influence of each of these three variables on α- and β-diversity metrics that rely on the number of OTUs alone (richness) and those that include both membership and the relative abundance of detected OTUs (diversity). As part of this study, we redesigned bacterial and archaeal primer sets that target the V3–V5 region of the 16S rRNA gene, along with multiplexing barcodes, to permit simultaneous sequencing of PCR products from the two domains. We conclude that the benefits of deeper sequencing efforts extend beyond greater OTU detection and result in higher precision in β-diversity analyses by reducing the variability between replicate libraries, despite the presence of more sequencing artifacts. Additionally, spurious OTUs resulting from sequencing errors have a significant impact on richness or shared-richness based α- and β-diversity metrics, whereas metrics that utilize community structure (including both richness and relative abundance of OTUs) are minimally affected by spurious OTUs. However, the greatest obstacle towards accurately evaluating community structure are the errors in estimated mean relative abundance of each detected OTU due to biases associated with multi-template PCR reactions.

Molecular quantification of environmental DNA using microfluidics and digital PCR

Real-time PCR has been widely used to evaluate gene abundance in natural microbial habitats. However, PCR-inhibitory substances often reduce the efficiency of PCR, leading to the underestimation of target gene copy numbers. Digital PCR using microfluidics is a new approach that allows absolute quantification of DNA molecules. In this study, digital PCR was applied to environmental samples, and the effect of PCR inhibitors on DNA quantification was tested. In the control experiment using λ DNA and humic acids, underestimation of λ DNA at 1/4400 of the theoretical value was observed with 6.58ngμL−1 humic acids. In contrast, digital PCR provided accurate quantification data with a concentration of humic acids up to 9.34ngμL−1. The inhibitory effect of paddy field soil extract on quantification of the archaeal 16S rRNA gene was also tested. By diluting the DNA extract, quantified copy numbers from real-time PCR and digital PCR became similar, indicating that dilution was a useful way to remedy PCR inhibition. The dilution strategy was, however, not applicable to all natural environmental samples. For example, when marine subsurface sediment samples were tested the copy number of archaeal 16S rRNA genes was 1.04×103copies/g-sediment by digital PCR, whereas real-time PCR only resulted in 4.64×102copies/g-sediment, which was most likely due to an inhibitory effect. The data from this study demonstrated that inhibitory substances had little effect on DNA quantification using microfluidics and digital PCR, and showed the great advantages of digital PCR in accurate quantifications of DNA extracted from various microbial habitats.