Prokaryotic microbial ecology as an ecosurveillance tool for eukaryotic pathogen colonisation: Meiothermus and Naegleria fowleri.

Naegleria fowleri has been detected in drinking water distribution systems (DWDS) in Australia, Pakistan and the United States and is the causative agent of the highly fatal disease primary amoebic meningoencephalitis. Previous small scale field studies have shown that Meiothermus may be a potential biomarker for N. fowleri. However, correlations between predictive biomarkers in small sample sizes often breakdown when applied to larger more representative datasets. This study represents one of the largest and most rigorous temporal investigations of Naegleria fowleri colonisation in an operational DWDS in the world and measured the association of Meiothermus and N. fowleri over a significantly larger space and time in the DWDS. A total of 232 samples were collected from five sites over three-years (2016-2018), which contained 29 positive N. fowleri samples. Two specific operational taxonomic units assigned to M. chliarophilus and M. hypogaeus, were significantly associated with N. fowleri presence. Furthermore, inoculation experiments demonstrated that Meiothermus was required to support N. fowleri growth in field-collected biofilms. This validates Meiothermus as prospective biological tool to aid in the identification and surveillance of N. fowleri colonisable sites.

Krumholzibacteriota and Deltaproteobacteria contain rare genetic potential to liberate carbon from monoaromatic compounds in subsurface coal seams.

Biogenic methane in subsurface coal seam environments is produced by diverse consortia of microbes. Although this methane is useful for global energy security, it remains unclear which microbes can liberate carbon from the coal. Most of this carbon is relatively resistant to biodegradation, as it is contained within aromatic rings. Thus, to explore for coal-degrading taxa in the subsurface, this study reconstructed relevant metagenome-assembled genomes (MAGs) from coal seams by using a key genomic marker for the anaerobic degradation of monoaromatic compounds as a guide: the benzoyl-CoA reductase gene (bcrABCD). Three MAGs were identified with this genetic potential. The first represented a novel taxon from the Krumholzibacteriota phylum, which this study is the first to describe. This Krumholzibacteriota MAG contained a full set of genes for benzoyl-CoA dearomatization, in addition to other genes for anaerobic catabolism of monoaromatics. Analysis of Krumholzibacteriota MAGs from other environments revealed that this genetic potential may be common, and thus, Krumholzibacteriota may be important organisms for the liberation of recalcitrant carbon in a broad range of environments. Moreover, the assembly and characterization of two Syntrophorhabdus aromaticivorans MAGs from different continents and a Syntrophaceae sp. MAG implicate the Deltaproteobacteria class in coal seam monoaromatic degradation. Each of these taxa are potential rate-limiting organisms for subsurface coal-to-methane biodegradation. Their description here provides some understanding of their function within the coal seam microbiome and will help inform future efforts in coal bed methane stimulation, anoxic bioremediation of organic pollutants, and assessments of anoxic, subsurface carbon cycling and emissions.IMPORTANCESubsurface coal seams are highly anoxic, oligotrophic environments, where the main source of carbon is "locked away" within aromatic rings. Despite these challenges, many coal seams accumulate biogenic methane, implying that the coal seam microbiome is "unlocking" this carbon source in situ. For over two decades, researchers have endeavored to understand which organisms perform these processes. This study provides the first descriptions of organisms with this genetic potential from the coal seam environment. Here, we report metagenomic insights into carbon liberation from aromatic molecules and the degradation pathways involved and describe a Krumholzibacteriota, two Syntrophorhabdus aromaticivorans, and a Syntrophaceae MAG that contain this genetic potential. This is also the first time that the Krumholzibacteriota phylum has been implicated in anaerobic dearomatization of aromatic hydrocarbons. This potential is identified here in numerous MAGs from other terrestrial and marine subsurface habitats, implicating the Krumholzibacteriota in carbon-cycling processes across a broad range of environments.

Naegleria fowleri Detected in Grand Teton National Park Hot Springs.

The free-living thermophilic amoeba Naegleria fowleri (N. fowleri) causes the highly fatal disease primary amoebic meningoencephalitis. The environmental conditions that are favorable to the growth and proliferation of N. fowleri are not well-defined, especially in northern regions of the United States. In this study, we used culture-based methods and multiple molecular approaches to detect and analyzeN. fowleri and other Naegleria spp. in water, sediment, and biofilm samples from five hot spring sites in Grand Teton National Park, Wyoming, U.S.A. These results provide the first detections of N. fowleri in Grand Teton National Park and provide new insights into the distribution of pathogenic N. fowleri and other nonpathogenic Naegleria spp. in natural thermal water systems in northern latitudes.

Algal amendment enhances biogenic methane production from coals of different thermal maturity.

The addition of small amounts of algal biomass to stimulate methane production in coal seams is a promising low carbon renewable coalbed methane enhancement technique. However, little is known about how the addition of algal biomass amendment affects methane production from coals of different thermal maturity. Here, we show that biogenic methane can be produced from five coals ranging in rank from lignite to low-volatile bituminous using a coal-derived microbial consortium in batch microcosms with and without algal amendment. The addition of 0.1 g/l algal biomass resulted in maximum methane production rates up to 37 days earlier and decreased the time required to reach maximum methane production by 17-19 days when compared to unamended, analogous microcosms. Cumulative methane production and methane production rate were generally highest in low rank, subbituminous coals, but no clear association between increasing vitrinite reflectance and decreasing methane production could be determined. Microbial community analysis revealed that archaeal populations were correlated with methane production rate (p = 0.01), vitrinite reflectance (p = 0.03), percent volatile matter (p = 0.03), and fixed carbon (p = 0.02), all of which are related to coal rank and composition. Sequences indicative of the acetoclastic methanogenic genus Methanosaeta dominated low rank coal microcosms. Amended treatments that had increased methane production relative to unamended analogs had high relative abundances of the hydrogenotrophic methanogenic genus Methanobacterium and the bacterial family Pseudomonadaceae. These results suggest that algal amendment may shift coal-derived microbial communities towards coal-degrading bacteria and CO2-reducing methanogens. These results have broad implications for understanding subsurface carbon cycling in coal beds and the adoption of low carbon renewable microbially enhanced coalbed methane techniques across a diverse range of coal geology.

Methanogenic archaea in subsurface coal seams are biogeographically distinct: an analysis of metagenomically-derived mcrA sequences.

The production of methane as an end-product of organic matter degradation in the absence of other terminal electron acceptors is common, and has often been studied in environments such as animal guts, soils and wetlands due to its potency as a greenhouse gas. To date, however, the study of the biogeographic distribution of methanogens across coal seam environments has been minimal. Here, we show that coal seams are host to a diverse range of methanogens, which are distinctive to each geological basin. Based on comparisons to close relatives from other methanogenic environments, the dominant methanogenic pathway in these basins is hydrogenotrophic, with acetoclastic being a second major pathway in the Surat Basin. Finally, mcrA and 16S rRNA gene primer biases were predominantly seen to affect the detection of Methanocellales, Methanomicrobiales and Methanosarcinales taxa in this study. Subsurface coal methanogenic community distributions and pathways presented here provide insights into important metabolites and bacterial partners for in situ coal biodegradation.

In Situ Enhancement and Isotopic Labeling of Biogenic Coalbed Methane.

Subsurface microbial (biogenic) methane production is an important part of the global carbon cycle that has resulted in natural gas accumulations in many coal beds worldwide. Laboratory studies suggest that complex carbon-containing nutrients (e.g., yeast or algae extract) can stimulate methane production, yet the effectiveness of these nutrients within coal beds is unknown. Here, we use downhole monitoring methods in combination with deuterated water (D2O) and a 200-liter injection of 0.1% yeast extract (YE) to stimulate and isotopically label newly generated methane. A total dissolved gas pressure sensor enabled real-time gas measurements (641 days preinjection and for 478 days postinjection). Downhole samples, collected with subsurface environmental samplers, indicate that methane increased 132% above preinjection levels based on isotopic labeling from D2O, 108% based on pressure readings, and 183% based on methane measurements 266 days postinjection. Demonstrating that YE enhances biogenic coalbed methane production in situ using multiple novel measurement methods has immediate implications for other field-scale biogenic methane investigations, including in situ methods to detect and track microbial activities related to the methanogenic turnover of recalcitrant carbon in the subsurface.

Subsurface hydrocarbon degradation strategies in low- and high-sulfate coal seam communities identified with activity-based metagenomics.

Environmentally relevant metagenomes and BONCAT-FACS derived translationally active metagenomes from Powder River Basin coal seams were investigated to elucidate potential genes and functional groups involved in hydrocarbon degradation to methane in coal seams with high- and low-sulfate levels. An advanced subsurface environmental sampler allowed the establishment of coal-associated microbial communities under in situ conditions for metagenomic analyses from environmental and translationally active populations. Metagenomic sequencing demonstrated that biosurfactants, aerobic dioxygenases, and anaerobic phenol degradation pathways were present in active populations across the sampled coal seams. In particular, results suggested the importance of anaerobic degradation pathways under high-sulfate conditions with an emphasis on fumarate addition. Under low-sulfate conditions, a mixture of both aerobic and anaerobic pathways was observed but with a predominance of aerobic dioxygenases. The putative low-molecular-weight biosurfactant, lichysein, appeared to play a more important role compared to rhamnolipids. The methods used in this study-subsurface environmental samplers in combination with metagenomic sequencing of both total and translationally active metagenomes-offer a deeper and environmentally relevant perspective on community genetic potential from coal seams poised at different redox conditions broadening the understanding of degradation strategies for subsurface carbon.

Activity-based, genome-resolved metagenomics uncovers key populations and pathways involved in subsurface conversions of coal to methane.

Microbial metabolisms and interactions that facilitate subsurface conversions of recalcitrant carbon to methane are poorly understood. We deployed an in situ enrichment device in a subsurface coal seam in the Powder River Basin (PRB), USA, and used BONCAT-FACS-Metagenomics to identify translationally active populations involved in methane generation from a variety of coal-derived aromatic hydrocarbons. From the active fraction, high-quality metagenome-assembled genomes (MAGs) were recovered for the acetoclastic methanogen, Methanothrix paradoxum, and a novel member of the Chlorobi with the potential to generate acetate via the Pta-Ack pathway. Members of the Bacteroides and Geobacter also encoded Pta-Ack and together, all four populations had the putative ability to degrade ethylbenzene, phenylphosphate, phenylethanol, toluene, xylene, and phenol. Metabolic reconstructions, gene analyses, and environmental parameters also indicated that redox fluctuations likely promote facultative energy metabolisms in the coal seam. The active "Chlorobi PRB" MAG encoded enzymes for fermentation, nitrate reduction, and multiple oxygenases with varying binding affinities for oxygen. "M. paradoxum PRB" encoded an extradiol dioxygenase for aerobic phenylacetate degradation, which was also present in previously published Methanothrix genomes. These observations outline underlying processes for bio-methane from subbituminous coal by translationally active populations and demonstrate activity-based metagenomics as a powerful strategy in next generation physiology to understand ecologically relevant microbial populations.

Repetitive Sampling and Control Threshold Improve 16S rRNA Gene Sequencing Results From Produced Waters Associated With Hydraulically Fractured Shale.

Sequencing microbial DNA from deep subsurface environments is complicated by a number of issues ranging from contamination to non-reproducible results. Many samples obtained from these environments - which are of great interest due to the potential to stimulate microbial methane generation - contain low biomass. Therefore, samples from these environments are difficult to study as sequencing results can be easily impacted by contamination. In this case, the low amount of sample biomass may be effectively swamped by the contaminating DNA and generate misleading results. Additionally, performing field work in these environments can be difficult, as researchers generally have limited access to and time on site. Therefore, optimizing a sampling plan to produce the best results while collecting the greatest number of samples over a short period of time is ideal. This study aimed to recommend an adequate sampling plan for field researchers obtaining microbial biomass for 16S rRNA gene sequencing, applicable specifically to low biomass oil and gas-producing environments. Forty-nine different samples were collected by filtering specific volumes of produced water from a hydraulically fractured well producing from the Niobrara Shale. Water was collected in two different sampling events 24 h apart. Four to five samples were collected from 11 specific volumes. These samples along with eight different blanks were submitted for analysis. DNA was extracted from each sample, and quantitative polymerase chain reaction (qPCR) and 16S rRNA Illumina MiSeq gene sequencing were performed to determine relative concentrations of biomass and microbial community composition, respectively. The qPCR results varied across sampled volumes, while no discernible trend correlated contamination to volume of water filtered. This suggests that collecting a larger volume of sample may not result in larger biomass concentrations or better representation of a sampled environment. Researchers could prioritize collecting many low volume samples over few high-volume samples. Our results suggest that there also may be variability in the concentration of microbial communities present in produced waters over short (i.e., hours) time scales, which warrants further investigation. Submission of multiple blanks is also vital to determining how contamination or low biomass effects may influence a sample set collected from an unknown environment.

Robotic environmental DNA bio-surveillance of freshwater health.

Autonomous water sampling technologies may help to overcome the human resource challenges of monitoring biological threats to rivers over long time periods and across large geographic areas. The Monterey Bay Aquarium Research Institute has pioneered a robotic Environmental Sample Processor (ESP) that overcomes some of the constraints associated with traditional sampling since it can automate water sample filtration and preservation of the captured material. The ESP was originally developed for marine environment applications. Here we evaluated whether the ESP can provide reliable, timely information on environmental (e)DNA detections of human and fish pathogens and introduced fishes at U.S. Geological Survey streamgage sites in freshwater rivers. We compared eDNA collected via ESP at high frequency (e.g., every 3 h) with manual eDNA collections collected at lower frequency (e.g., weekly). We found that water samples filtered and preserved by ESPs successfully detected the DNA of human pathogens, fish pathogens and introduced fishes. Both ESP and manually collected samples provided similar information about target DNA presence. We suggest that the greatest current benefit of the ESP is the cost savings of high frequency, bio-surveillance at remote or hard to access sites. The full potential of robotic technologies like the ESP will be realized when they can more easily execute in situ analyses of water samples and rapidly transmit results to decision-makers.

Potential Role of Acetyl-CoA Synthetase (acs) and Malate Dehydrogenase (mae) in the Evolution of the Acetate Switch in Bacteria and Archaea.

Although many Archaea have AMP-Acs (acetyl-coenzyme A synthetase) and ADP-Acs, the extant methanogenic genus Methanosarcina is the only identified Archaeal genus that can utilize acetate via acetate kinase (Ack) and phosphotransacetylase (Pta). Despite the importance of ack as the potential urkinase in the ASKHA phosphotransferase superfamily, an origin hypothesis does not exist for the acetate kinase in Bacteria, Archaea, or Eukarya. Here we demonstrate that Archaeal AMP-Acs and ADP-Acs contain paralogous ATPase motifs previously identified in Ack, which demonstrate a novel relation between these proteins in Archaea. The identification of ATPase motif conservation and resulting structural features in AMP- and ADP-acetyl-CoA synthetase proteins in this study expand the ASKHA superfamily to include acetyl-CoA synthetase. Additional phylogenetic analysis showed that Pta and MaeB sequences had a common ancestor, and that the Pta lineage within the halophilc archaea was an ancestral lineage. These results suggested that divergence of a duplicated maeB within an ancient halophilic, archaeal lineage formed a putative pta ancestor. These results provide a potential scenario for the establishment of the Ack/Pta pathway and provide novel insight into the evolution of acetate metabolism for all three domains of life.

Quantification of changing Pseudomonas aeruginosa sodA, htpX and mt gene abundance in response to trace metal toxicity: a potential in situ biomarker of environmental health.

Sediment-dwelling prokaryotes play a vital role in determining the fate and speciation of metals, yet are also susceptible to the biological effects of trace metals. In this article, optimized DNA extraction and purification techniques and species-specific primers are used to assess the genetic incidence and abundance of metal detoxification and general stress genes of Pseudomonas aeruginosa to complement chemical analysis in inferring the severity of metal-contaminated sites along the Clark Fork River, Montana. Results show the highest incidence of candidate genes related to bacterial stress at the most polluted site, while multiple regression analysis demonstrated significant correlations (P<0.05, r(2)=0.9) between in situ metal concentrations (As, Cu and Zn), total gene incidence, and the incidence of metal detoxification genes. Furthermore, principal components plotting the incidence of genes related to metal resistance show clear separation of sites giving clear clusters on the basis of contamination. Quantification of three genes (sodA, htpX and mt) from surveyed sites found significantly higher (anova, P<0.05) copy numbers at the more contaminated sites compared with reference sites. The development of rapid microbial biomarker tools represents a significant advance in the field of environmental biomonitoring and the prediction of metal bioavailability.