Microbial Functional Diversity Correlates with Species Diversity along a Temperature Gradient.

Microbial community diversity is often correlated with physical environmental stresses like acidity, salinity, and temperature. For example, species diversity usually declines with increasing temperature above 20°C. However, few studies have examined whether the genetic functional diversity of community metagenomes varies in a similar way as species diversity along stress gradients. Here, we investigated bacterial communities in thermal spring sediments ranging from 21 to 88°C, representing communities of 330 to 3,800 bacterial and archaeal species based on 16S rRNA gene amplicon analysis. Metagenomes were sequenced, and Pfam abundances were used as a proxy for metagenomic functional diversity. Significant decreases in both species diversity and Pfam diversity were observed with increasing temperatures. The relationship between Pfam diversity and species diversity followed a power function with the steepest slopes in the high-temperature, low-diversity region of the gradient. Species additions to simple thermophilic communities added many new Pfams, while species additions to complex mesophilic communities added relatively fewer new Pfams, indicating that species diversity does not approach saturation as rapidly as Pfam diversity does. Many Pfams appeared to have distinct temperature ceilings of 60 to 80°C. This study suggests that temperature stress limits both taxonomic and functional diversity of microbial communities, but in a quantitatively different manner. Lower functional diversity at higher temperatures is probably due to two factors, including (i) the absence of many enzymes not adapted to thermophilic conditions, and (ii) the fact that high-temperature communities are comprised of fewer species with smaller average genomes and, therefore, contain fewer rare functions. IMPORTANCE Only recently have microbial ecologists begun to assess quantitatively how microbial species diversity correlates with environmental factors like pH, temperature, and salinity. However, still, very few studies have examined how the number of distinct biochemical functions of microbial communities, termed functional diversity, varies with the same environmental factors. Our study examined 18 microbial communities sampled across a wide temperature gradient and found that increasing temperature reduced both species and functional diversity, but in different ways. Initially, functional diversity increased sharply with increasing species diversity but eventually plateaued, following a power function. This pattern has been previously predicted in theoretical models, but our study validates this predicted power function with field metagenomic data. This study also presents a unique overview of the distribution of metabolic functions along a temperature gradient, demonstrating that many functions have temperature "ceilings" above which they are no longer found.

Role of Extracellular Carbonic Anhydrase in Dissolved Inorganic Carbon Uptake in Alkaliphilic Phototrophic Biofilm.

Alkaline Soda Lakes are extremely productive ecosystems, due to their high dissolved inorganic carbon (DIC) concentrations. Here, we studied the dynamics of the carbonate system, in particular, the role of extracellular carbonic anhydrase (eCA) of an alkaliphilic phototrophic biofilm composed of bacteria enriched from soda lake benthic mats. By using measurements with microsensors and membrane inlet mass spectrometry, combined with mathematical modeling, we show how eCA controls DIC uptake. In our experiments, the activity of eCA varied four-fold, and was controlled by the bicarbonate concentration during growth: a higher bicarbonate concentration led to lower eCA activity. Inhibition of eCA decreased both the net and the gross photosynthetic productivities of the investigated biofilms. After eCA inhibition, the efflux of carbon dioxide (CO2) from the biofilms increased two- to four-fold. This could be explained by the conversion of CO2, leaking from cyanobacterial cells, by eCA, to bicarbonate. Bicarbonate is then taken up again by the cyanobacteria. In suspensions, eCA reduced the CO2 leakage to the bulk medium from 90 to 50%. In biofilms cultivated at low bicarbonate concentration (~0.13 mM), the oxygen production was reduced by a similar ratio upon eCA inhibition. The role of eCA in intact biofilms was much less significant compared to biomass suspensions, as CO2 loss to the medium is reduced due to mass transfer resistance.

Assessing species biomass contributions in microbial communities via metaproteomics.

Microbial community structure can be analyzed by quantifying cell numbers or by quantifying biomass for individual populations. Methods for quantifying cell numbers are already available (e.g., fluorescence in situ hybridization, 16-S rRNA gene amplicon sequencing), yet high-throughput methods for assessing community structure in terms of biomass are lacking. Here we present metaproteomics-based methods for assessing microbial community structure using protein abundance as a measure for biomass contributions of individual populations. We optimize the accuracy and sensitivity of the method using artificially assembled microbial communities and show that it is less prone to some of the biases found in sequencing-based methods. We apply the method to communities from two different environments, microbial mats from two alkaline soda lakes, and saliva from multiple individuals. We show that assessment of species biomass contributions adds an important dimension to the analysis of microbial community structure.

Fast and Simple Analysis of MiSeq Amplicon Sequencing Data with MetaAmp.

Microbial community profiling by barcoded 16S rRNA gene amplicon sequencing currently has many applications in microbial ecology. The low costs of the parallel sequencing of multiplexed samples, combined with the relative ease of data processing and interpretation (compared to shotgun metagenomes) have made this an entry-level approach. Here we present the MetaAmp pipeline for processing of SSU rRNA gene and other non-coding or protein-coding amplicon sequencing data by investigators that are inexperienced with bioinformatics procedures. It accepts single-end or paired-end sequences in fasta or fastq format from various sequencing platforms. It includes read quality control, and merging of forward and reverse reads of paired-end reads. It makes use of UPARSE, Mothur, and the SILVA database for clustering, removal of chimeric reads, taxonomic classification, and generation of diversity metrics. The pipeline has been validated with a mock community of known composition. MetaAmp provides a convenient web interface as well as command line interface. It is freely available at: http://ebg.ucalgary.ca/metaamp. Since its launch 2 years ago, MetaAmp has been used >2,800 times, by many users worldwide.

Robust, high-productivity phototrophic carbon capture at high pH and alkalinity using natural microbial communities.

BACKGROUND: Bioenergy with carbon capture and storage (BECCS) has come to be seen as one of the most viable technologies to provide the negative carbon dioxide emissions needed to constrain global temperatures. In practice, algal biotechnology is the only form of BECCS that could be realized at scale without compromising food production. Current axenic algae cultivation systems lack robustness, are expensive and generally have marginal energy returns. RESULTS: Here it is shown that microbial communities sampled from alkaline soda lakes, grown as biofilms at high pH (up to 10) and high alkalinity (up to 0.5 kmol m-3 NaHCO3 and NaCO3) display excellent (>1.0 kg m-3 day-1) and robust (>80 days) biomass productivity, at low projected overall costs. The most productive biofilms contained >100 different species and were dominated by a cyanobacterium closely related to Phormidium kuetzingianum (>60%). CONCLUSION: Frequent harvesting and red light were the key factors that governed the assembly of a stable and productive microbial community.

Actinocrinis puniceicyclus gen. nov., sp. nov., an actinobacterium isolated from an acidic spring.

An aerobic, mildly acidophilic actinobacterium was isolated from the Ochre Beds bog in Kootenay National Park, Canada. Cells of isolate OB1T were Gram-stain-positive, non-motile, pink- to purple-pigmented filaments. The pH range for growth was pH 3.5-6.5 (optimum pH 5.5), and the temperature range was 13-30°C. The major cellular fatty acids were i-C16 : 0 (28.5 %), i-C15 : 0 (14.6 %) and ai-C15 : 0 (14.3 %), and the major polar lipid was phosphohexose. The major quinone was menaquinone-11 (MK-11), and the peptidoglycan type was A1γ. The DNA G+C content was 70.2 %. Along with growth on complex media including yeast extract, proteose peptone, casamino acids and tryptic soy broth, growth occured on mono- and disaccharides (glucose, sucrose, galactose and xylose) and polysaccharides (starch, gellan, pectin, xylan and alginate). Anaerobic growth was not observed. The cells did not fix atmospheric nitrogen. On the basis of comparative 16S rRNA gene sequence analysis, this isolate belonged to the family Actinospicaceae, in the suborder Catenulisporineae of the order Actinomycetales. The most closely related species was Actinospica robiniae. However, the 16S rRNA gene sequence identity to this bacterium was low (92.8 %) and there were several chemotaxonomic differences from this species. We therefore propose a novel genus and species, Actinocrinis puniceicyclus gen. nov., sp. nov., with strain OB1T (=DSM 45618T=ATCC BAA-2771T) as the type strain.

Use of highly alkaline conditions to improve cost-effectiveness of algal biotechnology.

Phototrophic microorganisms have been proposed as an alternative to capture carbon dioxide (CO2) and to produce biofuels and other valuable products. Low CO2 absorption rates, low volumetric productivities, and inefficient downstream processing, however, currently make algal biotechnology highly energy intensive, expensive, and not economically competitive to produce biofuels. This mini-review summarizes advances made regarding the cultivation of phototrophic microorganisms at highly alkaline conditions, as well as other innovations oriented toward reducing the energy input into the cultivation and processing stages. An evaluation, in terms of energy requirements and energy return on energy invested, is performed for an integrated high-pH, high-alkalinity growth process that uses biofilms. Performance in terms of productivity and expected energy return on energy invested is presented for this process and is compared to previously reported life cycle assessments (LCAs) for systems at near-neutral pH. The cultivation of alkaliphilic phototrophic microorganisms in biofilms is shown to have a significant potential to reduce both energy requirements and capital costs.

Anaerobic carboxydotrophic bacteria in geothermal springs identified using stable isotope probing.

Carbon monoxide (CO) is a potential energy and carbon source for thermophilic bacteria in geothermal environments. Geothermal sites ranging in temperature from 45 to 65°C were investigated for the presence and activity of anaerobic CO-oxidizing bacteria. Anaerobic CO oxidation potentials were measured at up to 48.9 µmoles CO g(-1) (wet weight) day(-1) within five selected sites. Active anaerobic carboxydotrophic bacteria were identified using (13)CO DNA stable isotope probing (SIP) combined with pyrosequencing of 16S rRNA genes amplified from labeled DNA. Bacterial communities identified in heavy DNA fractions were predominated by Firmicutes, which comprised up to 95% of all sequences in (13)CO incubations. The predominant bacteria that assimilated (13)C derived from CO were closely related (>98% 16S rRNA gene sequence identity) to genera of known carboxydotrophs including Thermincola, Desulfotomaculum, Thermolithobacter, and Carboxydocella, although a few species with lower similarity to known bacteria were also found that may represent previously unconfirmed CO-oxidizers. While the distribution was variable, many of the same OTUs were identified across sample sites from different temperature regimes. These results show that bacteria capable of using CO as a carbon source are common in geothermal springs, and that thermophilic carboxydotrophs are probably already quite well known from cultivation studies.

Draft Genome Sequence of the Moderately Halophilic Methanotroph Methylohalobius crimeensis Strain 10Ki.

Methylohalobius crimeensis strain 10Ki is a moderately halophilic aerobic methanotroph isolated from a hypersaline lake in the Crimean Peninsula, Ukraine. This organism has the highest salt tolerance of any cultured methanotroph. Here, we present a draft genome sequence of this bacterium.

Stable-Isotope Probing Identifies Uncultured Planctomycetes as Primary Degraders of a Complex Heteropolysaccharide in Soil.

The exopolysaccharides (EPSs) produced by some bacteria are potential growth substrates for other bacteria in soil. We used stable-isotope probing (SIP) to identify aerobic soil bacteria that assimilated the cellulose produced by Gluconacetobacter xylinus or the EPS produced by Beijerinckia indica. The latter is a heteropolysaccharide comprised primarily of l-guluronic acid, d-glucose, and d-glycero-d-mannoheptose. (13)C-labeled EPS and (13)C-labeled cellulose were purified from bacterial cultures grown on [(13)C]glucose. Two soils were incubated with these substrates, and bacteria actively assimilating them were identified via pyrosequencing of 16S rRNA genes recovered from (13)C-labeled DNA. Cellulose C was assimilated primarily by soil bacteria closely related (93 to 100% 16S rRNA gene sequence identities) to known cellulose-degrading bacteria. However, B. indica EPS was assimilated primarily by bacteria with low identities (80 to 95%) to known species, particularly by different members of the phylum Planctomycetes. In one incubation, members of the Planctomycetes made up >60% of all reads in the labeled DNA and were only distantly related (<85% identity) to any described species. Although it is impossible with SIP to completely distinguish primary polysaccharide hydrolyzers from bacteria growing on produced oligo- or monosaccharides, the predominance of Planctomycetes suggested that they were primary degraders of EPS. Other bacteria assimilating B. indica EPS included members of the Verrucomicrobia, candidate division OD1, and the Armatimonadetes. The results indicate that some uncultured bacteria in soils may be adapted to using complex heteropolysaccharides for growth and suggest that the use of these substrates may provide a means for culturing new species.

Selective Pressure of Temperature on Competition and Cross-Feeding within Denitrifying and Fermentative Microbial Communities.

In coastal marine sediments, denitrification and fermentation are important processes in the anaerobic decomposition of organic matter. Microbial communities performing these two processes were enriched from tidal marine sediments in replicated, long term chemostat incubations at 10 and 25°C. Whereas denitrification rates at 25°C were more or less stable over time, at 10°C denitrification activity was unstable and could only be sustained either by repeatedly increasing the amount of carbon substrates provided or by repeatedly decreasing the dilution rate. Metagenomic and transcriptomic sequencing was performed at different time points and provisional whole genome sequences (WGS) and gene activities of abundant populations were compared across incubations. These analyses suggested that a temperature of 10°C selected for populations related to Vibrionales/Photobacterium that contributed to both fermentation (via pyruvate/formate lyase) and nitrous oxide reduction. At 25°C, denitrifying populations affiliated with Rhodobacteraceae were more abundant. The latter performed complete denitrification, and may have used carbon substrates produced by fermentative populations (cross-feeding). Overall, our results suggest that a mixture of competition-for substrates between fermentative and denitrifying populations, and for electrons between both pathways active within a single population -, and cross feeding-between fermentative and denitrifying populations-controlled the overall rate of denitrification. Temperature was shown to have a strong selective effect, not only on the populations performing either process, but also on the nature of their ecological interactions. Future research will show whether these results can be extrapolated to the natural environment.

Methanotrophic bacteria in warm geothermal spring sediments identified using stable-isotope probing.

We investigated methanotrophic bacteria in sediments of several warm geothermal springs ranging in temperature from 22 to 45 °C. Methane oxidation was measured at potential rates up to 141 µmol CH4 d(-1) g(-1) sediment. Active methanotrophs were identified using (13) CH4 stable-isotope probing (SIP) incubations performed at close to in situ temperatures for each site. Quantitative (q) PCR of pmoA genes identified the position of the heavy ((13) C-labelled) DNA fractions in density gradients, and 16S rRNA gene pyrotag sequencing of the heavy fractions was performed to identify the active methanotrophs. Methanotroph communities identified in heavy fractions of all samples were predominated by species similar (≥ 95% 16S rRNA gene identities) to previously characterized Gammaproteobacteria and Alphaproteobacteria methanotrophs. Among the five hottest samples (45 °C), members of the Gammaproteobacteria genus Methylocaldum dominated in two cases, while three others were dominated by an OTU closely related (96.8% similarity) to the Alphaproteobacteria genus Methylocapsa. These results suggest that diverse methanotroph groups are adapted to warm environments, including the Methylocapsa-Methylocella-Methyloferula group, which has previously only been detected in cooler sites.

Distribution and diversity of Verrucomicrobia methanotrophs in geothermal and acidic environments.

Recently, methanotrophic members of the phylum Verrucomicrobia have been described, but little is known about their distribution in nature. We surveyed methanotrophic bacteria in geothermal springs and acidic wetlands via pyrosequencing of 16S rRNA gene amplicons. Putative methanotrophic Verrucomicrobia were found in samples covering a broad temperature range (22.5-81.6°C), but only in acidic conditions (pH 1.8-5.0) and only in geothermal environments, not in acidic bogs or fens. Phylogenetically, three 16S rRNA gene sequence clusters of putative methanotrophic Verrucomicrobia were observed. Those detected in high-temperature geothermal samples (44.1-81.6°C) grouped with known thermoacidiphilic 'Methylacidiphilum' isolates. A second group dominated in moderate-temperature geothermal samples (22.5-40.1°C) and a representative mesophilic methanotroph from this group was isolated (strain LP2A). Genome sequencing verified that strain LP2A possessed particulate methane monooxygenase, but its 16S rRNA gene sequence identity to 'Methylacidiphilum infernorum' strain V4 was only 90.6%. A third group clustered distantly with known methanotrophic Verrucomicrobia. Using pmoA-gene targeted quantitative polymerase chain reaction, two geothermal soil profiles showed a dominance of LP2A-like pmoA sequences in the cooler surface layers and 'Methylacidiphilum'-like pmoA sequences in deeper, hotter layers. Based on these results, there appears to be a thermophilic group and a mesophilic group of methanotrophic Verrucomicrobia. However, both were detected only in acidic geothermal environments.

Humboldt's spa: microbial diversity is controlled by temperature in geothermal environments.

Over 200 years ago Alexander von Humboldt (1808) observed that plant and animal diversity peaks at tropical latitudes and decreases toward the poles, a trend he attributed to more favorable temperatures in the tropics. Studies to date suggest that this temperature-diversity gradient is weak or nonexistent for Bacteria and Archaea. To test the impacts of temperature as well as pH on bacterial and archaeal diversity, we performed pyrotag sequencing of 16S rRNA genes retrieved from 165 soil, sediment and biomat samples of 36 geothermal areas in Canada and New Zealand, covering a temperature range of 7.5-99 °C and a pH range of 1.8-9.0. This represents the widest ranges of temperature and pH yet examined in a single microbial diversity study. Species richness and diversity indices were strongly correlated to temperature, with R(2) values up to 0.62 for neutral-alkaline springs. The distributions were unimodal, with peak diversity at 24 °C and decreasing diversity at higher and lower temperature extremes. There was also a significant pH effect on diversity; however, in contrast to previous studies of soil microbial diversity, pH explained less of the variability (13-20%) than temperature in the geothermal samples. No correlation was observed between diversity values and latitude from the equator, and we therefore infer a direct temperature effect in our data set. These results demonstrate that temperature exerts a strong control on microbial diversity when considered over most of the temperature range within which life is possible.

Methanotrophic bacteria in oilsands tailings ponds of northern Alberta.

We investigated methanotrophic bacteria in slightly alkaline surface water (pH 7.4-8.7) of oilsands tailings ponds in Fort McMurray, Canada. These large lakes (up to 10 km(2)) contain water, silt, clay and residual hydrocarbons that are not recovered in oilsands mining. They are primarily anoxic and produce methane but have an aerobic surface layer. Aerobic methane oxidation was measured in the surface water at rates up to 152 nmol CH4 ml(-1) water d(-1). Microbial diversity was investigated via pyrotag sequencing of amplified 16S rRNA genes, as well as by analysis of methanotroph-specific pmoA genes using both pyrosequencing and microarray analysis. The predominantly detected methanotroph in surface waters at all sampling times was an uncultured species related to the gammaproteobacterial genus Methylocaldum, although a few other methanotrophs were also detected, including Methylomonas spp. Active species were identified via (13)CH4 stable isotope probing (SIP) of DNA, combined with pyrotag sequencing and shotgun metagenomic sequencing of heavy (13)C-DNA. The SIP-PCR results demonstrated that the Methylocaldum and Methylomonas spp. actively consumed methane in fresh tailings pond water. Metagenomic analysis of DNA from the heavy SIP fraction verified the PCR-based results and identified additional pmoA genes not detected via PCR. The metagenome indicated that the overall methylotrophic community possessed known pathways for formaldehyde oxidation, carbon fixation and detoxification of nitrogenous compounds but appeared to possess only particulate methane monooxygenase not soluble methane monooxygenase.

Detection of autotrophic verrucomicrobial methanotrophs in a geothermal environment using stable isotope probing.

Genomic analysis of the methanotrophic verrucomicrobium "Methylacidiphilum infernorum" strain V4 has shown that most pathways conferring its methanotrophic lifestyle are similar to those found in proteobacterial methanotrophs. However, due to the large sequence divergence of its methane monooxygenase-encoding genes (pmo), "universal" pmoA polymerase chain reaction (PCR) primers do not target these bacteria. Unlike proteobacterial methanotrophs, "Methylacidiphilum" fixes carbon autotrophically, and uses methane only for energy generation. As a result, techniques used to detect methanotrophs in the environment such as (13)CH(4)-stable isotope probing (SIP) and pmoA-targeted PCR do not detect verrucomicrobial methanotrophs, and they may have been overlooked in previous environmental studies. We developed a modified SIP technique to identify active methanotrophic Verrucomicrobia in the environment by labeling with (13)CO(2) and (13)CH(4), individually and in combination. Testing the protocol in "M. infernorum" strain V4 resulted in assimilation of (13)CO(2) but not (13)CH(4), verifying its autotrophic lifestyle. To specifically detect methanotrophs (as opposed to other autotrophs) via (13)CO(2)-SIP, a quantitative PCR (qPCR) assay specific for verrucomicrobial-pmoA genes was developed and used in combination with SIP. Incubation of an acidic, high-temperature geothermal soil with (13)CH(4) + (12)CO(2) caused little shift in the density distribution of verrucomicrobial-pmoA genes relative to controls. However, labeling with (13)CO(2) in combination with (12)CH(4) or (13)CH(4) induced a strong shift in the distribution of verrucomicrobial-pmoA genes towards the heavy DNA fractions. The modified SIP technique demonstrated that the primary methanotrophs active in the soil were autotrophs and belonged to the Verrucomicrobia. This is the first demonstration of autotrophic, non-proteobacterial methanotrophy in situ, and provides a tool to detect verrucomicrobial methanotrophs in other ecosystems.