Seasonal Dynamics of Methanotrophic Bacteria in a Boreal Oil Sands End Pit Lake.

Base Mine Lake (BML) is the first full-scale demonstration end pit lake for the oil sands mining industry in Canada. We examined aerobic methanotrophic bacteria over all seasons for 5 years in this dimictic lake. Methanotrophs comprised up to 58% of all bacterial reads in 16S rRNA gene amplicon sequencing analyses (median 2.8%), and up to 2.7 × 104 cells mL-1 of water (median 0.5 × 103) based on qPCR of pmoA genes. Methanotrophic activity and populations in the lake water were highest during fall turnover and remained high through the winter ice-covered period into spring turnover. They declined during summer stratification, especially in the epilimnion. Three methanotroph genera (Methylobacter, Methylovulum, and Methyloparacoccus) cycled seasonally, based on both relative and absolute abundance measurements. Methylobacter and Methylovulum populations peaked in winter/spring, when methane oxidation activity was psychrophilic. Methyloparacoccus populations increased in the water column through summer and fall, when methane oxidation was mesophilic, and also predominated in the underlying tailings sediment. Other, less abundant genera grew primarily during summer, possibly due to distinct CH4/O2 microniches created during thermal stratification. These data are consistent with temporal and spatial niche differentiation based on temperature, CH4 and O2. This pit lake displays methane cycling and methanotroph population dynamics similar to natural boreal lakes. IMPORTANCE The study examined methanotrophic bacteria in an industrial end pit lake, combining molecular DNA methods (both quantitative and descriptive) with biogeochemical measurements. The lake was sampled over 5 years, in all four seasons, as often as weekly, and included sub-ice samples. The resulting multiseason and multiyear data set is unique in its size and intensity, and allowed us to document clear and consistent seasonal patterns of growth and decline of three methanotroph genera (Methylobacter, Methylovulum, and Methyloparacoccus). Laboratory experiments suggested that one major control of this succession was niche partitioning based on temperature. The study helps to understand microbial dynamics in engineered end pit lakes, but we propose that the dynamics are typical of boreal stratified lakes and widely applicable in microbial ecology and limnology. Methane-oxidizing bacteria are important model organisms in microbial ecology and have implications for global climate change.

Feedstock variability impacts the bioconversion of sugar and lignin streams derived from corn stover by Clostridium tyrobutyricum and engineered Pseudomonas putida.

Feedstock variability represents a challenge in lignocellulosic biorefineries, as it can influence both lignocellulose deconstruction and microbial conversion processes for biofuels and biochemicals production. The impact of feedstock variability on microbial performance remains underexplored, and predictive tools for microbial behaviour are needed to mitigate risks in biorefinery scale-up. Here, twelve batches of corn stover were deconstructed via deacetylation, mechanical refining, and enzymatic hydrolysis to generate lignin-rich and sugar streams. These batches and their derived streams were characterised to identify their chemical components, and the streams were used as substrates for producing muconate and butyrate by engineered Pseudomonas putida and wildtype Clostridium tyrobutyricum, respectively. Bacterial performance (growth, product titers, yields, and productivities) differed among the batches, but no strong correlations were identified between feedstock composition and performance. To provide metabolic insights into the origin of these differences, we evaluated the effect of twenty-three isolated chemical components on these microbes, including three components in relevant bioprocess settings in bioreactors, and we found that growth-inhibitory concentrations were outside the ranges observed in the streams. Overall, this study generates a foundational dataset on P. putida and C. tyrobutyricum performance to enable future predictive models and underscores their resilience in effectively converting fluctuating lignocellulose-derived streams into bioproducts.

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.

Dissecting the dominant hot spring microbial populations based on community-wide sampling at single-cell genomic resolution.

With advances in DNA sequencing and miniaturized molecular biology workflows, rapid and affordable sequencing of single-cell genomes has become a reality. Compared to 16S rRNA gene surveys and shotgun metagenomics, large-scale application of single-cell genomics to whole microbial communities provides an integrated snapshot of community composition and function, directly links mobile elements to their hosts, and enables analysis of population heterogeneity of the dominant community members. To that end, we sequenced nearly 500 single-cell genomes from a low diversity hot spring sediment sample from Dewar Creek, British Columbia, and compared this approach to 16S rRNA gene amplicon and shotgun metagenomics applied to the same sample. We found that the broad taxonomic profiles were similar across the three sequencing approaches, though several lineages were missing from the 16S rRNA gene amplicon dataset, likely the result of primer mismatches. At the functional level, we detected a large array of mobile genetic elements present in the single-cell genomes but absent from the corresponding same species metagenome-assembled genomes. Moreover, we performed a single-cell population genomic analysis of the three most abundant community members, revealing differences in population structure based on mutation and recombination profiles. While the average pairwise nucleotide identities were similar across the dominant species-level lineages, we observed differences in the extent of recombination between these dominant populations. Most intriguingly, the creek's Hydrogenobacter sp. population appeared to be so recombinogenic that it more closely resembled a sexual species than a clonally evolving microbe. Together, this work demonstrates that a randomized single-cell approach can be useful for the exploration of previously uncultivated microbes from community composition to population structure.

GAL08, an Uncultivated Group of Acidobacteria, Is a Dominant Bacterial Clade in a Neutral Hot Spring.

GAL08 are bacteria belonging to an uncultivated phylogenetic cluster within the phylum Acidobacteria. We detected a natural population of the GAL08 clade in sediment from a pH-neutral hot spring located in British Columbia, Canada. To shed light on the abundance and genomic potential of this clade, we collected and analyzed hot spring sediment samples over a temperature range of 24.2-79.8°C. Illumina sequencing of 16S rRNA gene amplicons and qPCR using a primer set developed specifically to detect the GAL08 16S rRNA gene revealed that absolute and relative abundances of GAL08 peaked at 65°C along three temperature gradients. Analysis of sediment collected over multiple years and locations revealed that the GAL08 group was consistently a dominant clade, comprising up to 29.2% of the microbial community based on relative read abundance and up to 4.7 × 105 16S rRNA gene copy numbers per gram of sediment based on qPCR. Using a medium quality threshold, 25 single amplified genomes (SAGs) representing these bacteria were generated from samples taken at 65 and 77°C, and seven metagenome-assembled genomes (MAGs) were reconstructed from samples collected at 45-77°C. Based on average nucleotide identity (ANI), these SAGs and MAGs represented three separate species, with an estimated average genome size of 3.17 Mb and GC content of 62.8%. Phylogenetic trees constructed from 16S rRNA gene sequences and a set of 56 concatenated phylogenetic marker genes both placed the three GAL08 bacteria as a distinct subgroup of the phylum Acidobacteria, representing a candidate order (Ca. Frugalibacteriales) within the class Blastocatellia. Metabolic reconstructions from genome data predicted a heterotrophic metabolism, with potential capability for aerobic respiration, as well as incomplete denitrification and fermentation. In laboratory cultivation efforts, GAL08 counts based on qPCR declined rapidly under atmospheric levels of oxygen but increased slightly at 1% (v/v) O2, suggesting a microaerophilic lifestyle.

Analysis of microbial communities in natural halite springs reveals a domain-dependent relationship of species diversity to osmotic stress.

Microbial species diversity may peak at certain optimal environmental conditions and decrease toward more extreme conditions. Indeed, bell-shaped relationships of species diversity against pH and temperature have been demonstrated, but diversity patterns across other environmental conditions are less well reported. In this study, we investigated the impact of salinity on the diversity of microorganisms from all three domains in a large set of natural springs with salinities ranging from freshwater to halite saturated. Habitat salinity was found to be linearly and inversely related to diversity of all three domains. The relationship was strongest in the bacteria, where salinity explained up to 44% of the variation in different diversity metrics (OTUs, Shannon index, and Phylogenetic Diversity). However, the relationship was weaker for Eukarya and Archaea. The known salt-in strategist Archaea of the Halobacteriaceae even showed the opposite trend, with increasing diversity at higher salinity. We propose that high energetic requirements constrain species diversity at high salinity but that the diversity of taxa with energetically less expensive osmotolerance strategies is less affected. Declining diversity with increasing osmotic stress may be a general rule for microbes as well as plants and animals, but the strength of this relationship varies greatly across microbial taxa.