Genome streamlining in Parcubacteria transitioning from soil to groundwater.

BACKGROUND: To better understand the influence of habitat on the genetic content of bacteria, with a focus on members of Candidate Phyla Radiation (CPR) bacteria, we studied the effects of transitioning from soil via seepage waters to groundwater on genomic composition of ultra-small Parcubacteria, the dominating CPR class in seepage waters, using genome resolved metagenomics. RESULTS: Bacterial metagenome-assembled genomes (MAGs), (318 total, 32 of Parcubacteria) were generated from seepage waters and compared directly to groundwater counterparts. The estimated average genome sizes of members of major phyla Proteobacteria, Bacteroidota and Cand. Patescibacteria (Candidate Phyla Radiation - CPR bacteria) were significantly higher in soil-seepage water as compared to their groundwater counterparts. Seepage water Parcubacteria (Paceibacteria) exhibited 1.18-fold greater mean genome size and 2-fold lower mean proportion of pseudogenes than those in groundwater. Bacteroidota and Proteobacteria also showed a similar trend of reduced genomes in groundwater compared to seepage. While exploring gene loss and adaptive gains in closely related CPR lineages in groundwater, we identified a membrane protein, and a lipoglycopeptide resistance gene unique to a seepage Parcubacterium genome. A nitrite reductase gene was also identified and was unique to the groundwater Parcubacteria genomes, likely acquired from other planktonic microbes via horizontal gene transfer. CONCLUSIONS: Overall, our data suggest that bacteria in seepage waters, including ultra-small Parcubacteria, have significantly larger genomes and higher metabolic enrichment than their groundwater counterparts, highlighting possible genome streamlining of the latter in response to habitat selection in an oligotrophic environment.

Genetic basis of clarithromycin resistance in Bacillus anthracis.

The high-consequence pathogen Bacillus anthracis causes human anthrax and often results in lethal infections without the rapid administration of effective antimicrobial treatment. Antimicrobial resistance profiling is therefore critical to inform post-exposure prophylaxis and treatment decisions, especially during emergencies such as outbreaks or where intentional release is suspected. Whole-genome sequencing using a rapid long-read sequencer can uncover antimicrobial resistance patterns if genetic markers of resistance are known. To identify genomic markers associated with antimicrobial resistance, we isolated B. anthracis derived from the avirulent Sterne strain with elevated minimal inhibitory concentrations to clarithromycin. Mutants were characterized both phenotypically through broth microdilution susceptibility testing and observations during culturing, as well as genotypically with whole-genome sequencing. We identified two different in-frame insertions in the L22 ribosomal protein-encoding gene rplV, which were subsequently confirmed to be involved in clarithromycin resistance through the reversion of the mutant gene to the parent (drug-susceptible) sequence. Detection of the rplV insertions was possible with rapid long-read sequencing, with a time-to-answer within 3 h. The mutations associated with clarithromycin resistance described here will be used in conjunction with known genetic markers of resistance for other antimicrobials to strengthen the prediction of antimicrobial resistance in B. anthracis.IMPORTANCEThe disease anthrax, caused by the pathogen Bacillus anthracis, is extremely deadly if not treated quickly and appropriately. Clarithromycin is an antibiotic recommended for the treatment and post-exposure prophylaxis of anthrax by the Centers for Disease Control and Prevention; however, little is known about the ability of B. anthracis to develop resistance to clarithromycin or the mechanism of that resistance. The characterization of clarithromycin-resistant isolates presented here provides valuable information for researchers and clinicians in the event of a release of the resistant strain. Additionally, knowledge of the genetic basis of resistance provides a foundation for susceptibility prediction through rapid genome sequencing to inform timely treatment decisions.

Differential contribution of nitrifying prokaryotes to groundwater nitrification.

The ecophysiology of complete ammonia-oxidizing bacteria (CMX) of the genus Nitrospira and their widespread occurrence in groundwater suggests that CMX bacteria have a competitive advantage over ammonia-oxidizing bacteria (AOB) and archaea (AOA) in these environments. However, the specific contribution of their activity to nitrification processes has remained unclear. We aimed to disentangle the contribution of CMX, AOA and AOB to nitrification and to identify the environmental drivers of their niche differentiation at different levels of ammonium and oxygen in oligotrophic carbonate rock aquifers. CMX ammonia monooxygenase sub-unit A (amoA) genes accounted on average for 16 to 75% of the total groundwater amoA genes detected. Nitrification rates were positively correlated to CMX clade A associated phylotypes and AOB affiliated with Nitrosomonas ureae. Short-term incubations amended with the nitrification inhibitors allylthiourea and chlorate suggested that AOB contributed a large fraction to overall ammonia oxidation, while metaproteomics analysis confirmed an active role of CMX in both ammonia and nitrite oxidation. Ecophysiological niche differentiation of CMX clades A and B, AOB and AOA was linked to their requirements for ammonium, oxygen tolerance, and metabolic versatility. Our results demonstrate that despite numerical predominance of CMX, the first step of nitrification in oligotrophic groundwater appears to be primarily governed by AOB. Higher growth yields at lower ammonia turnover rates and energy derived from nitrite oxidation most likely enable CMX to maintain consistently high populations.

Redefining the phylogenetic and metabolic diversity of phylum Omnitrophota.

The candidate phylum Omnitrophica-recently termed Omnitrophota, and originally known as OP3-is an understudied bacterial clade that has primarily been found in aquatic ecosystems. To characterize the diversity and ecology of this phylum, we reconstructed 55 Omnitrophota metagenome-assembled genomes (MAGs) from a well-characterized groundwater system within central Germany and placed them within the context of publicly available genomes. Seven clades were identified, four of which contained novel genomes obtained from our groundwater system. All clades exhibited the capacity for type IV pili, type II secretion systems, glycogen storage, and carbohydrate degradation. Only the characterized Cand. Omnitrophus magneticus genome exhibited functions associated with magnetosome construction. Clades were characterized by sets of traits rather than unique pathways, which were then used to infer ecological strategies. These lifestyles consisted of mixotrophs, obligate fermenters, and versatile respiratory heterotrophs. Patterns in 16S rRNA gene amplicons from a 6 years, monthly sampled groundwater time-series dataset reflected the persistent and widespread occurrence of Clade 7 Wood-Ljungdahl utilizing mixotrophs and highlight this group as a core member of the groundwater community. Overall, this study uncovered, characterized, and contextualized the metabolic and phylogenetic diversity within phylum Omnitrophota, and predicts that environmental populations may mediate both nitrogen and sulfur cycling, along with organic matter production and degradation within aquatic ecosystems.

Bolstering fitness via CO2 fixation and organic carbon uptake: mixotrophs in modern groundwater.

Current understanding of organic carbon inputs into ecosystems lacking photosynthetic primary production is predicated on data and inferences derived almost entirely from metagenomic analyses. The elevated abundances of putative chemolithoautotrophs in groundwaters suggest that dark CO2 fixation is an integral component of subsurface trophic webs. To understand the impact of autotrophically fixed carbon, the flux of CO2-derived carbon through various populations of subsurface microbiota must first be resolved, both quantitatively and temporally. Here we implement novel Stable Isotope Cluster Analysis to render a time-resolved and quantitative evaluation of 13CO2-derived carbon flow through a groundwater community in microcosms stimulated with reduced sulfur compounds. We demonstrate that mixotrophs, not strict autotrophs, were the most abundant active organisms in groundwater microcosms. Species of Hydrogenophaga, Polaromonas, Dechloromonas, and other metabolically versatile mixotrophs drove the production and remineralization of organic carbon. Their activity facilitated the replacement of 43% and 80% of total microbial carbon stores in the groundwater microcosms with 13C in just 21 and 70 days, respectively. The mixotrophs employed different strategies for satisfying their carbon requirements by balancing CO2 fixation and uptake of available organic compounds. These different strategies might provide fitness under nutrient-limited conditions, explaining the great abundances of mixotrophs in other oligotrophic habitats, such as the upper ocean and boreal lakes.

The economical lifestyle of CPR bacteria in groundwater allows little preference for environmental drivers.

BACKGROUND: The highly diverse Cand. Patescibacteria are predicted to have minimal biosynthetic and metabolic pathways, which hinders understanding of how their populations differentiate in response to environmental drivers or host organisms. Their mechanisms employed to cope with oxidative stress are largely unknown. Here, we utilized genome-resolved metagenomics to investigate the adaptive genome repertoire of Patescibacteria in oxic and anoxic groundwaters, and to infer putative host ranges. RESULTS: Within six groundwater wells, Cand. Patescibacteria was the most dominant (up to 79%) super-phylum across 32 metagenomes sequenced from DNA retained on 0.2 and 0.1 µm filters after sequential filtration. Of the reconstructed 1275 metagenome-assembled genomes (MAGs), 291 high-quality MAGs were classified as Cand. Patescibacteria. Cand. Paceibacteria and Cand. Microgenomates were enriched exclusively in the 0.1 µm fractions, whereas candidate division ABY1 and Cand. Gracilibacteria were enriched in the 0.2 µm fractions. On average, Patescibacteria enriched in the smaller 0.1 µm filter fractions had 22% smaller genomes, 13.4% lower replication measures, higher proportion of rod-shape determining proteins, and of genomic features suggesting type IV pili mediated cell-cell attachments. Near-surface wells harbored Patescibacteria with higher replication rates than anoxic downstream wells characterized by longer water residence time. Except prevalence of superoxide dismutase genes in Patescibacteria MAGs enriched in oxic groundwaters (83%), no major metabolic or phylogenetic differences were observed. The most abundant Patescibacteria MAG in oxic groundwater encoded a nitrate transporter, nitrite reductase, and F-type ATPase, suggesting an alternative energy conservation mechanism. Patescibacteria consistently co-occurred with one another or with members of phyla Nanoarchaeota, Bacteroidota, Nitrospirota, and Omnitrophota. Among the MAGs enriched in 0.2 µm fractions,, only 8% Patescibacteria showed highly significant one-to-one correlation, mostly with Omnitrophota. Motility and transport related genes in certain Patescibacteria were highly similar to genes from other phyla (Omnitrophota, Proteobacteria and Nanoarchaeota). CONCLUSION: Other than genes to cope with oxidative stress, we found little genomic evidence for niche adaptation of Patescibacteria to oxic or anoxic groundwaters. Given that we could detect specific host preference only for a few MAGs, we speculate that the majority of Patescibacteria is able to attach multiple hosts just long enough to loot or exchange supplies.

Inclusion of Oxford Nanopore long reads improves all microbial and viral metagenome-assembled genomes from a complex aquifer system.

Assembling microbial and viral genomes from metagenomes is a powerful and appealing method to understand structure-function relationships in complex environments. To compare the recovery of genomes from microorganisms and their viruses from groundwater, we generated shotgun metagenomes with Illumina sequencing accompanied by long reads derived from the Oxford Nanopore Technologies (ONT) sequencing platform. Assembly and metagenome-assembled genome (MAG) metrics for both microbes and viruses were determined from an Illumina-only assembly, ONT-only assembly, and a hybrid assembly approach. The hybrid approach recovered 2× more mid to high-quality MAGs compared to the Illumina-only approach and 4× more than the ONT-only approach. A similar number of viral genomes were reconstructed using the hybrid and ONT methods, and both recovered nearly fourfold more viral genomes than the Illumina-only approach. While yielding fewer MAGs, the ONT-only approach generated MAGs with a high probability of containing rRNA genes, 3× higher than either of the other methods. Of the shared MAGs recovered from each method, the ONT-only approach generated the longest and least fragmented MAGs, while the hybrid approach yielded the most complete. This work provides quantitative data to inform a cost-benefit analysis of the decision to supplement shotgun metagenomic projects with long reads towards the goal of recovering genomes from environmentally abundant groups.

Integrated Omics Elucidate the Mechanisms Driving the Rapid Biodegradation of Deepwater Horizon Oil in Intertidal Sediments Undergoing Oxic-Anoxic Cycles.

Crude oil buried in intertidal sands may be exposed to alternating oxic and anoxic conditions but the effect of this tidally induced biogeochemical oscillation remains poorly understood, limiting the effectiveness of remediation and managing efforts after oil spills. Here, we used a combination of metatranscriptomics and genome-resolved metagenomics to study microbial activities in oil-contaminated sediments during oxic-anoxic cycles in laboratory chambers that closely emulated in situ conditions. Approximately 5-fold higher reductions in the total petroleum hydrocarbons were observed in the oxic as compared to the anoxic phases with a relatively constant ratio between aerobic and anaerobic oil decomposition rates even after prolonged anoxic conditions. Metatranscriptomics analysis indicated that the oxic phases promoted oil biodegradation in subsequent anoxic phases by microbially mediated reoxidation of alternative electron acceptors like sulfide and by providing degradation-limiting nitrogen through biological nitrogen fixation. Most population genomes reconstructed from the mesocosm samples represented uncultured taxa and were present typically as members of the rare biosphere in metagenomic data from uncontaminated field samples, implying that the intertidal communities are adapted to changes in redox conditions. Collectively, these results have important implications for enhancing oil spill remediation efforts in beach sands and coastal sediments and underscore the role of uncultured taxa in such efforts.

The core seafloor microbiome in the Gulf of Mexico is remarkably consistent and shows evidence of recovery from disturbance caused by major oil spills.

The microbial ecology of oligotrophic deep ocean sediments is understudied relative to their shallow counterparts, and this lack of understanding hampers our ability to predict responses to current and future perturbations. The Gulf of Mexico has experienced two of the largest accidental marine oil spills, the 1979 Ixtoc-1 blowout and the 2010 Deepwater Horizon (DWH) discharge. Here, microbial communities were characterized for 29 sites across multiple years in > 700 samples. The composition of the seafloor microbiome was broadly consistent across the region and was well approximated by the overlying water depth and depth within the sediment column, while geographic distance played a limited role. Biogeographical distributions were employed to generate predictive models for over 4000 OTU that leverage easy-to-obtain geospatial variables which are linked to measured sedimentary oxygen profiles. Depth stratification and putative niche diversification are evidenced by the distribution of taxa that mediate the microbial nitrogen cycle. Furthermore, these results demonstrate that sediments impacted by the DWH spill had returned to near baseline conditions after 2 years. The distributions of benthic microorganisms in the Gulf can be constrained, and moreover, deviations from these predictions may pinpoint impacted sites and aid in future response efforts or long-term stability studies.

"Candidatus Macondimonas diazotrophica", a novel gammaproteobacterial genus dominating crude-oil-contaminated coastal sediments.

Modeling crude-oil biodegradation in sediments remains a challenge due in part to the lack of appropriate model organisms. Here we report the metagenome-guided isolation of a novel organism that represents a phylogenetically narrow (>97% 16S rRNA gene identity) group of previously uncharacterized, crude-oil degraders. Analysis of available sequence data showed that these organisms are highly abundant in oiled sediments of coastal marine ecosystems across the world, often comprising ~30% of the total community, and virtually absent in pristine sediments or seawater. The isolate genome encodes functional nitrogen fixation and hydrocarbon degradation genes together with putative genes for biosurfactant production that apparently facilitate growth in the typically nitrogen-limited, oiled environment. Comparisons to available genomes revealed that this isolate represents a novel genus within the Gammaproteobacteria, for which we propose the provisional name "Candidatus Macondimonas diazotrophica" gen. nov., sp. nov. "Ca. M. diazotrophica" appears to play a key ecological role in the response to oil spills around the globe and could be a promising model organism for studying ecophysiological responses to oil spills.

Anaerobic degradation of hexadecane and phenanthrene coupled to sulfate reduction by enriched consortia from northern Gulf of Mexico seafloor sediment.

To advance understanding of the fate of hydrocarbons released from the Deepwater Horizon oil spill and deposited in marine sediments, this study characterized the microbial populations capable of anaerobic hydrocarbon degradation coupled with sulfate reduction in non-seep sediments of the northern Gulf of Mexico. Anaerobic, sediment-free enrichment cultures were obtained with either hexadecane or phenanthrene as sole carbon source and sulfate as a terminal electron acceptor. Phylogenetic analysis revealed that enriched microbial populations differed by hydrocarbon substrate, with abundant SSU rRNA gene amplicon sequences from hexadecane cultures showing high sequence identity (up to 98%) to Desulfatibacillum alkenivorans (family Desulfobacteraceae), while phenanthrene-enriched populations were most closely related to Desulfatiglans spp. (up to 95% sequence identity; family Desulfarculaceae). Assuming complete oxidation to CO2, observed stoichiometric ratios closely resembled the theoretical ratios of 12.25:1 for hexadecane and 8.25:1 for phenanthrene degradation coupled to sulfate reduction. Phenanthrene carboxylic acid was detected in the phenanthrene-degrading enrichment cultures, providing evidence to indicate carboxylation as an activation mechanism for phenanthrene degradation. Metagenome-assembled genomes (MAGs) revealed that phenanthrene degradation is likely mediated by novel genera or families of sulfate-reducing bacteria along with their fermentative syntrophic partners, and candidate genes linked to the degradation of aromatic hydrocarbons were detected for future study.

Degradation of Deepwater Horizon oil buried in a Florida beach influenced by tidal pumping.

After Deepwater Horizon oil reached the Florida coast, oil was buried in Pensacola Beach (PB) sands to ~70cm depth, resulting in Total Petroleum Hydrocarbon (TPH) concentrations up to ~2kg per meter of beach. This study followed the decomposition of the buried oil and the factors influencing its degradation. The abundance of bacteria in oiled sand increased by 2 orders of magnitude within one week after oil burial, while diversity decreased by ~50%. Half-lives of aliphatic and aromatic hydrocarbons reached 25 and 22days, respectively. Aerobic microbial oil decomposition, promoted by tidal pumping, and human cleaning activities effectively removed oil from the beach. After one year, concentrations of GC-amenable hydrocarbons at PB were similar to those in the uncontaminated reference beach at St. George Island/FL, and microbial populations that disappeared after the oil contamination had reestablished. Yet, oxihydrocarbons can be found at PB to the present day.

Hydrocarbon-Degrading Bacteria Exhibit a Species-Specific Response to Dispersed Oil while Moderating Ecotoxicity.

The Deepwater Horizon blowout in April 2010 represented the largest accidental marine oil spill and the largest release of chemical dispersants into the environment to date. While dispersant application may provide numerous benefits to oil spill response efforts, the impacts of dispersants and potential synergistic effects with crude oil on individual hydrocarbon-degrading bacteria are poorly understood. In this study, two environmentally relevant species of hydrocarbon-degrading bacteria were utilized to quantify the response to Macondo crude oil and Corexit 9500A-dispersed oil in terms of bacterial growth and oil degradation potential. In addition, specific hydrocarbon compounds were quantified in the dissolved phase of the medium and linked to ecotoxicity using a U.S. Environmental Protection Agency (EPA)-approved rotifer assay. Bacterial treatment significantly and drastically reduced the toxicity associated with dispersed oil (increasing the 50% lethal concentration [LC50] by 215%). The growth and crude oil degradation potential of Acinetobacter were inhibited by Corexit by 34% and 40%, respectively; conversely, Corexit significantly enhanced the growth of Alcanivorax by 10% relative to that in undispersed oil. Furthermore, both bacterial strains were shown to grow with Corexit as the sole carbon and energy source. Hydrocarbon-degrading bacterial species demonstrate a unique response to dispersed oil compared to their response to crude oil, with potentially opposing effects on toxicity. While some species have the potential to enhance the toxicity of crude oil by producing biosurfactants, the same bacteria may reduce the toxicity associated with dispersed oil through degradation or sequestration.

Sedimentation Pulse in the NE Gulf of Mexico following the 2010 DWH Blowout.

The objective of this study was to investigate the impacts of the Deepwater Horizon (DWH) oil discharge at the seafloor as recorded in bottom sediments of the DeSoto Canyon region in the northeastern Gulf of Mexico. Through a close coupling of sedimentological, geochemical, and biological approaches, multiple independent lines of evidence from 11 sites sampled in November/December 2010 revealed that the upper ~1 cm depth interval is distinct from underlying sediments and results indicate that particles originated at the sea surface. Consistent dissimilarities in grain size over the surficial ~1 cm of sediments correspond to excess (234)Th depths, which indicates a lack of vertical mixing (bioturbation), suggesting the entire layer was deposited within a 4-5 month period. Further, a time series from four deep-sea sites sampled up to three additional times over the following two years revealed that excess (234)Th depths, accumulation rates, and (234)Th inventories decreased rapidly, within a few to several months after initial coring. The interpretation of a rapid sedimentation pulse is corroborated by stratification in solid phase Mn, which is linked to diagenesis and redox change, and the dramatic decrease in benthic formanifera density that was recorded in surficial sediments. Results are consistent with a brief depositional pulse that was also reported in previous studies of sediments, and marine snow formation in surface waters closer to the wellhead during the summer and fall of 2010. Although sediment input from the Mississippi River and advective transport may influence sedimentation on the seafloor in the DeSoto Canyon region, we conclude based on multidisciplinary evidence that the sedimentation pulse in late 2010 is the product of marine snow formation and is likely linked to the DWH discharge.

Deep Characterization of the Microbiomes of Calophya spp. (Hemiptera: Calophyidae) Gall-Inducing Psyllids Reveals the Absence of Plant Pathogenic Bacteria and Three Dominant Endosymbionts.

Bacteria associated with sap-feeding insect herbivores include not only symbionts that may increase their hosts' fitness but also harmful plant pathogens. Calophya spp. gall-inducing psyllids (Hemiptera: Calophyidae) are being investigated for their potential as biological control agents of the noxious weed, Brazilian peppertree (Schinus terebinthifolia), in Florida. Although there are no examples of plant pathogen transmission by members of the family Calophyidae, several insects in the superfamily Psylloidea are known to transmit pathogenic bacteria in the genera Candidatus Liberibacter and Candidatus Phytoplasma. To determine whether Calophya spp. harbor potentially harmful plant pathogenic bacteria, we sequenced small subunit (SSU) ribosomal RNA (rRNA) gene amplicons generated from individuals from four Calophya spp. populations: All microbial SSU gene sequences fell into the bacterial domain, with 98-99% belonging to the Proteobacteria. The Calophya microbiomes contained a relatively simple community, with 49-79 operational taxonomic units (OTUs; 97%) detected, and only 5-8 OTUs with greater than 1% abundance. Candidatus Carsonella showed the highest relative abundance, with OTUs from this candidate genus representing between 51-65% of all recovered sequences. The next most abundant clade observed was an unclassified Enterobacteriacae group closely related to bacteria from the genera Buchnera and Blochmannia that ranged from 20-31% in relative abundance. Wolbachia populations were the third most abundant group and represented 7-27% of the diversity in microbial OTUs. No SSU rRNA gene sequences from putative pathogenic bacteria from the genera Ca. Liberibacter or Ca. Phytoplasma were detected in the microbiomes of the four Calophya populations. The probability that infected psyllids were present in our colonies, but were not sampled, was extremley low (1.39 x 10(-10)). As far as we are aware, our study is the first to characterize the microbiome of a candidate biological control agent, and coupled with previous work demonstrating a high degree of host specificity and absence of plant viruses, suggests that releasing Calophya spp. in United States poses minimal risk to non-target plants.

Microbial community successional patterns in beach sands impacted by the Deepwater Horizon oil spill.

Although petroleum hydrocarbons discharged from the Deepwater Horizon (DWH) blowout were shown to have a pronounced impact on indigenous microbial communities in the Gulf of Mexico, effects on nearshore or coastal ecosystems remain understudied. This study investigated the successional patterns of functional and taxonomic diversity for over 1 year after the DWH oil was deposited on Pensacola Beach sands (FL, USA), using metagenomic and 16S rRNA gene amplicon techniques. Gamma- and Alphaproteobacteria were enriched in oiled sediments, in corroboration of previous studies. In contrast to previous studies, we observed an increase in the functional diversity of the community in response to oil contamination and a functional transition from generalist populations within 4 months after oil came ashore to specialists a year later, when oil was undetectable. At the latter time point, a typical beach community had reestablished that showed little to no evidence of oil hydrocarbon degradation potential, was enriched in archaeal taxa known to be sensitive to xenobiotics, but differed significantly from the community before the oil spill. Further, a clear succession pattern was observed, where early responders to oil contamination, likely degrading aliphatic hydrocarbons, were replaced after 3 months by populations capable of aromatic hydrocarbon decomposition. Collectively, our results advance the understanding of how natural benthic microbial communities respond to crude oil perturbation, supporting the specialization-disturbance hypothesis; that is, the expectation that disturbance favors generalists, while providing (microbial) indicator species and genes for the chemical evolution of oil hydrocarbons during degradation and weathering.

Temperature response of denitrification and anaerobic ammonium oxidation rates and microbial community structure in Arctic fjord sediments.

The temperature dependency of denitrification and anaerobic ammonium oxidation (anammox) rates from Arctic fjord sediments was investigated in a temperature gradient block incubator for temperatures ranging from -1 to 40°C. Community structure in intact sediments and slurry incubations was determined using Illumina SSU rRNA gene sequencing. The optimal temperature (Topt ) for denitrification was 25-27°C, whereas anammox rates were optimal at 12-17°C. Both denitrification and anammox exhibited temperature responses consistent with a psychrophilic community, but anammox bacteria may be more specialized for psychrophilic activity. Long-term (1-2 months) warming experiments indicated that temperature increases of 5-10°C above in situ had little effect on the microbial community structure or the temperature response of denitrification and anammox. Increases of 25°C shifted denitrification temperature responses to mesophilic with concurrent community shifts, and anammox activity was eliminated above 25°C. Additions of low molecular weight organic substrates (acetate and lactate) caused increases in denitrification rates, corroborating the hypothesis that the supply of organic substrates is a more dominant control of respiration rates than low temperature. These results suggest that climate-related changes in sinking particulate flux will likely alter rates of N removal more rapidly than warming.

Watershed-scale fungal community characterization along a pH gradient in a subsurface environment cocontaminated with uranium and nitrate.

The objective of this study was to characterize fungal communities in a subsurface environment cocontaminated with uranium and nitrate at the watershed scale and to determine the potential contribution of fungi to contaminant transformation (nitrate attenuation). The abundance, distribution, and diversity of fungi in subsurface groundwater samples were determined using quantitative and semiquantitative molecular techniques, including quantitative PCR of eukaryotic small-subunit rRNA genes and pyrosequencing of fungal internal transcribed spacer (ITS) regions. Potential bacterial and fungal denitrification was assessed in sediment-groundwater slurries amended with antimicrobial compounds and in fungal pure cultures isolated from the subsurface. Our results demonstrate that subsurface fungal communities are dominated by members of the phylum Ascomycota, and a pronounced shift in fungal community composition occurs across the groundwater pH gradient at the field site, with lower diversity observed under acidic (pH <4.5) conditions. Fungal isolates recovered from subsurface sediments, including cultures of the genus Coniochaeta, which were detected in abundance in pyrosequence libraries of site groundwater samples, were shown to reduce nitrate to nitrous oxide. Denitrifying fungal isolates recovered from the site were classified and found to be distributed broadly within the phylum Ascomycota and within a single genus of the Basidiomycota. Potential denitrification rate assays with sediment-groundwater slurries showed the potential for subsurface fungi to reduce nitrate to nitrous oxide under in situ acidic pH conditions.