Rebecca A. Daly

Evaluating rRNA as an indicator of microbial activity in environmental communities: limitations and uses

Microbes exist in a range of metabolic states (for example, dormant, active and growing) and analysis of ribosomal RNA (rRNA) is frequently employed to identify the ‘active’ fraction of microbes in environmental samples. While rRNA analyses are no longer commonly used to quantify a population’s growth rate in mixed communities, due to rRNA concentration not scaling linearly with growth rate uniformly across taxa, rRNA analyses are still frequently used toward the more conservative goal of identifying populations that are currently active in a mixed community. Yet, evidence indicates that the general use of rRNA as a reliable indicator of metabolic state in microbial assemblages has serious limitations. This report highlights the complex and often contradictory relationships between rRNA, growth and activity. Potential mechanisms for confounding rRNA patterns are discussed, including differences in life histories, life strategies and non-growth activities. Ways in which rRNA data can be used for useful characterization of microbial assemblages are presented, along with questions to be addressed in future studies.

Airway Microbiota and Pathogen Abundance in Age- Stratified Cystic Fibrosis Patients

Bacterial communities in the airways of cystic fibrosis (CF) patients are, as in other ecological niches, influenced by autogenic and allogenic factors. However, our understanding of microbial colonization in younger versus older CF airways and the association with pulmonary function is rudimentary at best. Using a phylogenetic microarray, we examine the airway microbiota in age stratified CF patients ranging from neonates (9 months) to adults (72 years). From a cohort of clinically stable patients, we demonstrate that older CF patients who exhibit poorer pulmonary function possess more uneven, phylogenetically-clustered airway communities, compared to younger patients. Using longitudinal samples collected form a subset of these patients a pattern of initial bacterial community diversification was observed in younger patients compared with a progressive loss of diversity over time in older patients. We describe in detail the distinct bacterial community profiles associated with young and old CF patients with a particular focus on the differences between respective “early” and “late” colonizing organisms. Finally we assess the influence of Cystic Fibrosis Transmembrane Regulator (CFTR) mutation on bacterial abundance and identify genotype-specific communities involving members of the Pseudomonadaceae, Xanthomonadaceae, Moraxellaceae and Enterobacteriaceae amongst others. Data presented here provides insights into the CF airway microbiota, including initial diversification events in younger patients and establishment of specialized communities of pathogens associated with poor pulmonary function in older patient populations.

Bacterial community structure corresponds to performance during cathodic nitrate reduction.

Microbial fuel cells (MFCs) have applications other than electricity production, including the capacity to power desirable reactions in the cathode chamber. However, current knowledge of the microbial ecology and physiology of biocathodes is minimal, and as a result more research dedicated to understanding the microbial communities active in cathode biofilms is required. Here we characterize the microbiology of denitrifying bacterial communities stimulated by reducing equivalents generated from the anodic oxidation of acetate. We analyzed biofilms isolated from two types of cathodic denitrification systems: (1) a loop format where the effluent from the carbon oxidation step in the anode is subjected to a nitrifying reactor which is fed to the cathode chamber and (2) an alternative non-loop format where anodic and cathodic feed streams are separated. The results of our study indicate the superior performance of the loop reactor in terms of enhanced current production and nitrate removal rates. We hypothesized that phylogenetic or structural features of the microbial communities could explain the increased performance of the loop reactor. We used PhyloChip with 16S rRNA (cDNA) and fluorescent in situ hybridization to characterize the active bacterial communities. Our study results reveal a greater richness, as well as an increased phylogenetic diversity, active in denitrifying biofilms than was previously identified in cathodic systems. Specifically, we identified Proteobacteria, Firmicutes and Chloroflexi members that were dominant in denitrifying cathodes. In addition, our study results indicate that it is the structural component, in terms of bacterial richness and evenness, rather than the phylogenetic affiliation of dominant bacteria, that best corresponds to cathode performance.

Microbial metabolisms in a 2.5-km-deep ecosystem created by hydraulic fracturing in shales

Hydraulic fracturing is the industry standard for extracting hydrocarbons from shale formations. Attention has been paid to the economic benefits and environmental impacts of this process, yet the biogeochemical changes induced in the deep subsurface are poorly understood. Recent single-gene investigations revealed that halotolerant microbial communities were enriched after hydraulic fracturing. Here, the reconstruction of 31 unique genomes coupled to metabolite data from the Marcellus and Utica shales revealed that many of the persisting organisms play roles in methylamine cycling, ultimately supporting methanogenesis in the deep biosphere. Fermentation of injected chemical additives also sustains long-term microbial persistence, while thiosulfate reduction could produce sulfide, contributing to reservoir souring and infrastructure corrosion. Extensive links between viruses and microbial hosts demonstrate active viral predation, which may contribute to the release of labile cellular constituents into the extracellular environment. Our analyses show that hydraulic fracturing provides the organismal and chemical inputs for colonization and persistence in the deep terrestrial subsurface.

New roles in hemicellulosic sugar fermentation for the uncultivated Bacteroidetes family BS11

Ruminants have co-evolved with their gastrointestinal microbial communities that digest plant materials to provide energy for the host. Some arctic and boreal ruminants have already shown to be vulnerable to dietary shifts caused by changing climate, yet we know little about the metabolic capacity of the ruminant microbiome in these animals. Here, we use meta-omics approaches to sample rumen fluid microbial communities from Alaskan moose foraging along a seasonal lignocellulose gradient. Winter diets with increased hemicellulose and lignin strongly enriched for BS11, a Bacteroidetes family lacking cultivated or genomically sampled representatives. We show that BS11 are cosmopolitan host-associated bacteria prevalent in gastrointestinal tracts of ruminants and other mammals. Metagenomic reconstruction yielded the first four BS11 genomes; phylogenetically resolving two genera within this previously taxonomically undefined family. Genome-enabled metabolic analyses uncovered multiple pathways for fermenting hemicellulose monomeric sugars to short-chain fatty acids (SCFA), metabolites vital for ruminant energy. Active hemicellulosic sugar fermentation and SCFA production was validated by shotgun proteomics and rumen metabolites, illuminating the role BS11 have in carbon transformations within the rumen. Our results also highlight the currently unknown metabolic potential residing in the rumen that may be vital for sustaining host energy in response to a changing vegetative environment.

Chemical and pathogen-induced inflammation disrupt the murine intestinal microbiome

BackgroundSalmonella is one of the most significant food-borne pathogens to affect humans and agriculture. While it is well documented that Salmonella infection triggers host inflammation, the impacts on the gut environment are largely unknown. A CBA/J mouse model was used to evaluate intestinal responses to Salmonella-induced inflammation. In parallel, we evaluated chemically induced inflammation by dextran sodium sulfate (DSS) and a non-inflammation control. We profiled gut microbial diversity by sequencing 16S ribosomal ribonucleic acid (rRNA) genes from fecal and cecal samples. These data were correlated to the inflammation marker lipocalin-2 and short-chain fatty acid concentrations.ResultsWe demonstrated that inflammation, chemically or biologically induced, restructures the chemical and microbial environment of the gut over a 16-day period. We observed that the ten mice within the Salmonella treatment group had a variable Salmonella relative abundance, with three high responding mice dominated by >46% Salmonella at later time points and the remaining seven mice denoted as low responders. These low- and high-responding Salmonella groups, along with the chemical DSS treatment, established an inflammation gradient with chemical and low levels of Salmonella having at least 3 log-fold lower lipocalin-2 concentration than the high-responding Salmonella mice. Total short-chain fatty acid and individual butyrate concentrations each negatively correlated with inflammation levels. Microbial communities were also structured along this inflammation gradient. Low levels of inflammation, regardless of chemical or biological induction, enriched for Akkermansia spp. in the Verrucomicrobiaceae and members of the Bacteroidetes family S24-7. Relative to the control or low inflammation groups, high levels of Salmonella drastically decreased the overall microbial diversity, specifically driven by the reduction of Alistipes and Lachnospiraceae in the Bacteroidetes and Firmicutes phyla, respectively. Conversely, members of the Enterobacteriaceae and Lactobacillus were positively correlated to high levels of Salmonella-induced inflammation.ConclusionsOur results show that enteropathogenic infection and intestinal inflammation are interrelated factors modulating gut homeostasis. These findings may prove informative with regard to prophylactic or therapeutic strategies to prevent disruption of microbial communities, or promote their restoration.

Trends and Future Challenges in Sampling the Deep Terrestrial Biosphere

Research in the deep terrestrial biosphere is driven by interest in novel biodiversity and metabolisms, biogeochemical cycling, and the impact of human activities on this ecosystem. As this interest continues to grow, it is important to ensure that when subsurface investigations are proposed, materials recovered from the subsurface are sampled and preserved in an appropriate manner to limit contamination and ensure preservation of accurate microbial, geochemical, and mineralogical signatures. On February 20th, 2014, a workshop on “Trends and Future Challenges in Sampling The Deep Subsurface” was coordinated in Columbus, Ohio by The Ohio State University and West Virginia University faculty, and sponsored by The Ohio State University and the Sloan Foundation’s Deep Carbon Observatory. The workshop aims were to identify and develop best practices for the collection, preservation, and analysis of terrestrial deep rock samples. This document summarizes the information shared during this workshop.

Utilizing novel diversity estimators to quantify multiple dimensions of microbial biodiversity across domains

BackgroundMicrobial ecologists often employ methods from classical community ecology to analyze microbial community diversity. However, these methods have limitations because microbial communities differ from macro-organismal communities in key ways. This study sought to quantify microbial diversity using methods that are better suited for data spanning multiple domains of life and dimensions of diversity. Diversity profiles are one novel, promising way to analyze microbial datasets. Diversity profiles encompass many other indices, provide effective numbers of diversity (mathematical generalizations of previous indices that better convey the magnitude of differences in diversity), and can incorporate taxa similarity information. To explore whether these profiles change interpretations of microbial datasets, diversity profiles were calculated for four microbial datasets from different environments spanning all domains of life as well as viruses. Both similarity-based profiles that incorporated phylogenetic relatedness and naïve (not similarity-based) profiles were calculated. Simulated datasets were used to examine the robustness of diversity profiles to varying phylogenetic topology and community composition.ResultsDiversity profiles provided insights into microbial datasets that were not detectable with classical univariate diversity metrics. For all datasets analyzed, there were key distinctions between calculations that incorporated phylogenetic diversity as a measure of taxa similarity and naïve calculations. The profiles also provided information about the effects of rare species on diversity calculations. Additionally, diversity profiles were used to examine thousands of simulated microbial communities, showing that similarity-based and naïve diversity profiles only agreed approximately 50% of the time in their classification of which sample was most diverse. This is a strong argument for incorporating similarity information and calculating diversity with a range of emphases on rare and abundant species when quantifying microbial community diversity.ConclusionsFor many datasets, diversity profiles provided a different view of microbial community diversity compared to analyses that did not take into account taxa similarity information, effective diversity, or multiple diversity metrics. These findings are a valuable contribution to data analysis methodology in microbial ecology.

High-resolution sequencing reveals unexplored archaeal diversity in freshwater wetland soils

Summary Despite being key contributors to biogeochemical processes, archaea are frequently outnumbered by bacteria, and consequently are underrepresented in combined molecular surveys. Here, we demonstrate an approach to concurrently survey the archaea alongside the bacteria with high‐resolution 16S rRNA gene sequencing, linking these community data to geochemical parameters. We applied this integrated analysis to hydric soils sampled across a model methane‐emitting freshwater wetland. Geochemical profiles, archaeal communities, and bacterial communities were independently correlated with soil depth and water cover. Centimeters of soil depth and corresponding geochemical shifts consistently affected microbial community structure more than hundreds of meters of lateral distance. Methanogens with diverse metabolisms were detected across the wetland, but displayed surprising OTU‐level partitioning by depth. Candidatus Methanoperedens spp. archaea thought to perform anaerobic oxidation of methane linked to iron reduction were abundant. Domain‐specific sequencing also revealed unexpectedly diverse non‐methane‐cycling archaeal members. OTUs within the underexplored Woesearchaeota and Bathyarchaeota were prevalent across the wetland, with subgroups and individual OTUs exhibiting distinct occupancy and abundance distributions aligned with environmental gradients. This study adds to our understanding of ecological range for key archaeal taxa in a model freshwater wetland, and links these taxa and individual OTUs to hypotheses about processes governing biogeochemical cycling.

Methanogenesis in oxygenated soils is a substantial fraction of wetland methane emissions

The current paradigm, widely incorporated in soil biogeochemical models, is that microbial methanogenesis can only occur in anoxic habitats. In contrast, here we show clear geochemical and biological evidence for methane production in well-oxygenated soils of a freshwater wetland. A comparison of oxic to anoxic soils reveal up to ten times greater methane production and nine times more methanogenesis activity in oxygenated soils. Metagenomic and metatranscriptomic sequencing recover the first near-complete genomes for a novel methanogen species, and show acetoclastic production from this organism was the dominant methanogenesis pathway in oxygenated soils. This organism, Candidatus Methanothrix paradoxum, is prevalent across methane emitting ecosystems, suggesting a global significance. Moreover, in this wetland, we estimate that up to 80% of methane fluxes could be attributed to methanogenesis in oxygenated soils. Together, our findings challenge a widely held assumption about methanogenesis, with significant ramifications for global methane estimates and Earth system modeling.Methane production is traditionally not found in oxygenated soils, a paradigm incorporated in global greenhouse gas modelling efforts. Here the authors show geochemical and biological evidence of active methanogenesis in bulk-oxic wetland soils, attributing up to 80% of the total methane budget for the site.