Harmful algal blooms in Cayuga lake, NY: From microbiome analysis to eDNA monitoring.

The global increase in harmful algal blooms (HABs) has become a growing concern over the years, and New York State (NYS) is no exception. The Finger Lakes region in NYS has been identified as a hotspot for HABs, with Cayuga Lake having the highest number of blooms reported. The Cayuga Lake HABs Monitoring Program has been tracking cHABs (dominant bloom taxa, chlorophyll A, and microcystin levels) since 2018. However, limited research has been conducted on the microbiome of HABs in this region. In this study, the microbiome of HABs in the Cayuga Lake was surveyed and compared with non-HAB baseline samples. Using 16S rDNA community analysis, common bloom-forming cyanobacteria, were identified, with Microcystis being the dominant taxa in high toxin blooms. Further, this study evaluated the ability of Microcystis mcyA qPCR to detect elevated levels of potential toxigenic Microcystis in water samples using both benchtop and handheld qPCR devices. The results showed good performance of the qPCR assay as a screening for high toxin versus low/no toxin blooms. Additionally, the handheld qPCR device holds potential for in-field rapid (<1 h) screenings for high toxin blooms. This study provides insights into the microbiome of HABs in Cayuga Lake and offers a potential tool for rapid screening of high toxin blooms.

RNA Biomarker Trends across Type I and Type II Aerobic Methanotrophs in Response to Methane Oxidation Rates and Transcriptome Response to Short-Term Methane and Oxygen Limitation in Methylomicrobium album BG8.

Methanotrophs, which help regulate atmospheric levels of methane, are active in diverse natural and man-made environments. This range of habitats and the feast-famine cycles seen by many environmental methanotrophs suggest that methanotrophs dynamically mediate rates of methane oxidation. Global methane budgets require ways to account for this variability in time and space. Functional gene biomarker transcripts are increasingly studied to inform the dynamics of diverse biogeochemical cycles. Previously, per-cell transcript levels of the methane oxidation biomarker pmoA were found to vary quantitatively with respect to methane oxidation rates in the model aerobic methanotroph Methylosinus trichosporium OB3b. In the present study, these trends were explored for two additional aerobic methanotroph pure cultures grown in membrane bioreactors, Methylocystis parvus OBBP and Methylomicrobium album BG8. At steady-state conditions, per-cell pmoA mRNA transcript levels strongly correlated with per-cell methane oxidation across the three methanotrophs across many orders of magnitude of activity (R2 = 0.91). The inclusion of both type I and type II aerobic methanotrophs suggests a universal trend between in situ activity level and pmoA RNA biomarker levels which can aid in improving estimates of both subsurface and atmospheric methane. Additionally, genome-wide expression data (obtained by transcriptome sequencing [RNA-seq]) were used to explore transcriptomic responses of steady-state M. album BG8 cultures to short-term CH4 and O2 limitation. These limitations induced regulation of genes involved in central carbon metabolism (including carbon storage), cell motility, and stress response. IMPORTANCE Methanotrophs are naturally occurring microorganisms capable of oxidizing methane, having an impact on global net methane emissions. Additionally, they have also gained interest for their biotechnological applications in single-cell protein production, biofuels, and bioplastics. Having better ways of measuring methanotroph activity and understanding how methanotrophs respond to changing conditions is imperative for both optimization in controlled-growth applications and understanding in situ methane oxidation rates. In this study, we explored the applicability of methane oxidation biomarkers as a universal indicator of methanotrophic activity and explored methanotroph transcriptomic response to short-term changes in substrate availability. Our results contribute to better understanding the activity of aerobic methanotrophs, their core metabolic pathways, and their stress responses.

Long-term survival of Dehalococcoides mccartyi strains in mixed cultures under electron acceptor and ammonium limitation.

Few strains of Dehalococcoides mccartyi harbour and express the vinyl chloride reductase (VcrA) that catalyzes the dechlorination of vinyl chloride (VC), a carcinogenic soil and groundwater contaminant. The vcrA operon is found on a Genomic Island (GI) and, therefore, believed to participate in horizontal gene transfer (HGT). To try to induce HGT of the vcrA-GI, we blended two enrichment cultures in medium without ammonium while providing VC. We hypothesized that these conditions would select for a mutant strain of D. mccartyi that could both fix nitrogen and respire VC. However, after more than 4 years of incubation, we found no evidence for HGT of the vcrA-GI. Rather, we observed VC-dechlorinating activity attributed to the trichloroethene reductase TceA. Sequencing and protein modelling revealed a mutation in the predicted active site of TceA, which may have influenced substrate specificity. We also identified two nitrogen-fixing D. mccartyi strains in the KB-1 culture. The presence of multiple strains of D. mccartyi with distinct phenotypes is a feature of natural environments and certain enrichment cultures (such as KB-1), and may enhance bioaugmentation success. The fact that multiple distinct strains persist in the culture for decades and that we could not induce HGT of the vcrA-GI suggests that it is not as mobile as predicted, or that mobility is restricted in ways yet to be discovered to specific subclades of Dehalococcoides.

Stimulation of dissimilatory sulfate reduction in response to sulfate in microcosm incubations from two contrasting temperate peatlands near Ithaca, NY, USA.

Peatlands are responsible for over half of wetland methane emissions, yet major uncertainties remain regarding carbon flow, especially when increased availability of electron acceptors stimulates competing physiologies. We used microcosm incubations to study the effects of sulfate on microorganisms in two temperate peatlands, one bog and one fen. Three different electron donor treatments were used (13C-acetate, 13C-formate and a mixture of 12C short-chain fatty acids) to elucidate the responses of sulfate-reducing bacteria (SRB) and methanogens to sulfate stimulation. Methane production was measured and metagenomic sequencing was performed, with only the heavy DNA fraction sequenced from treatments receiving 13C electron donors. Our data demonstrate stimulation of dissimilatory sulfate reduction in both sites, with contrasting community responses. In McLean Bog (MB), hydrogenotrophic Deltaproteobacteria and acetotrophic Peptococcaceae lineages of SRB were stimulated, as were lineages with unclassified dissimilatory sulfite reductases. In Michigan Hollow Fen (MHF), there was little stimulation of Peptococcaceae populations, and a small stimulation of Deltaproteobacteria SRB populations only in the presence of formate as electron donor. Sulfate stimulated an increase in relative abundance of reads for both oxidative and reductive sulfite reductases, suggesting stimulation of an internal sulfur cycle. Together, these data indicate a stimulation of SRB activity in response to sulfate in both sites, with a stronger growth response in MB than MHF. This study provides valuable insights into microbial community responses to sulfate in temperate peatlands and is an important first step to understanding how SRB and methanogens compete to regulate carbon flow in these systems.

Relationship Between Peat Type and Microbial Ecology in Sphagnum-Containing Peatlands of the Adirondack Mountains, NY, USA.

Peatland microbial community composition varies with respect to a range of biological and physicochemical variables. While the extent of peat degradation (humification) has been linked to microbial community composition along vertical stratification gradients within peatland sites, across-site variations have been relatively unexplored. In this study, we compared microbial communities across ten pristine Sphagnum-containing peatlands in the Adirondack Mountains, NY, which represented three different peat types-humic fen peat, humic bog peat, and fibric bog peat. Using 16S amplicon sequencing and network correlation analysis, we demonstrate that microbial community composition is primarily linked to peat type, and that distinct taxa networks distinguish microbial communities in each type. Shotgun metagenomic sequencing of the active water table region (mesotelm) from two Sphagnum-dominated bogs-one with fibric peat and one with humic peat-revealed differences in primary carbon degradation pathways, with the fibric peat being dominated by carbohydrate metabolism and hydrogenotrophic methanogenesis, and the humic peat being dominated by aliphatic carbon metabolism and aceticlastic methanogenesis. Our results suggest that peat humification is a major factor driving microbial community dynamics across peatland ecosystems.

Linking microbial Sphagnum degradation and acetate mineralization in acidic peat bogs: from global insights to a genome-centric case study.

Ombrotrophic bogs accumulate large stores of soil carbon that eventually decompose to carbon dioxide and methane. Carbon accumulates because Sphagnum mosses slow microbial carbon decomposition processes, leading to the production of labile intermediate compounds. Acetate is a major product of Sphagnum degradation, yet rates of hydrogenotrophic methanogenesis far exceed rates of aceticlastic methanogenesis, suggesting that alternative acetate mineralization processes exist. Two possible explanations are aerobic respiration and anaerobic respiration via humic acids as electron acceptors. While these processes have been widely observed, microbial community interactions linking Sphagnum degradation and acetate mineralization remain cryptic. In this work, we use ordination and network analysis of functional genes from 110 globally distributed peatland metagenomes to identify conserved metabolic pathways in Sphagnum bogs. We then use metagenome-assembled genomes (MAGs) from McLean Bog, a Sphagnum bog in New York State, as a local case study to reconstruct pathways of Sphagnum degradation and acetate mineralization. We describe metabolically flexible Acidobacteriota MAGs that contain all genes to completely degrade Sphagnum cell wall sugars under both aerobic and anaerobic conditions. Finally, we propose a hypothetical model of acetate oxidation driven by changes in peat redox potential that explain how bogs may circumvent aceticlastic methanogenesis through aerobic and humics-driven respiration.

Methane Monooxygenase Gene Transcripts as Quantitative Biomarkers of Methanotrophic Activity in Methylosinus trichosporium OB3b.

Methanotrophic microorganisms are characterized by their ability to oxidize methane. Globally they have a significant impact on methane emissions by attenuating net methane fluxes to the atmosphere in natural and engineered systems, though the populations are dynamic in their activity level in soils and waters. Methanotrophs oxidize methane using methane monooxygenase (MMO) enzymes, and selected subunit genes of the most common MMOs, specifically pmoA and mmoX, are used as biomarkers for the presence and abundance of populations of bacterial methanotrophs. The relative expression of these biomarker genes is dependent on copper-to-biomass ratios. Empirically derived quantitative relationships between methane oxidation biomarker transcript amounts and methanotrophic activity could facilitate determination of methane oxidation rates. In this study, pure cultures of a model type II methanotroph, Methylosinus trichosporium OB3b, were grown in hollow-fiber membrane bioreactors (HFMBR) under different steady-state methane oxidation conditions. Methanotroph biomass (DNA based) and methane oxidation biomarker mRNA transcript amounts were determined using quantitative PCR (qPCR) and reverse transcription-PCR (RT-qPCR), respectively. Under both copper-present and copper-limited conditions, per-cell pmoA mRNA transcript levels positively correlated with measured per-cell methane oxidation rates across 3 orders of magnitude. These correlations, if maintained across different methanotrophs, could prove valuable for inferring in situ oxidation rates of methanotrophs and understanding the dynamics of their impact on net methane emissions.IMPORTANCE Methanotrophs are naturally occurring microorganisms capable of oxidizing methane and have an impact on global net methane emissions. The genes pmoA and mmoX are used as biomarkers for bacterial methanotrophs. Quantitative relationships between transcript amounts of these genes and methane oxidation rates could facilitate estimation of methanotrophic activity. In this study, a strong correlation was observed between per-cell pmoA transcript levels and per-cell methane oxidation rates for pure cultures of the aerobic methanotroph M. trichosporium OB3b grown in bioreactors. If similar relationships exist across different methanotrophs, they could prove valuable for inferring in situ oxidation rates of methanotrophs and better understanding their impact on net methane emissions.

Ecogenomics reveals community interactions in a long-term methanogenic bioreactor and a rapid switch to sulfate-reducing conditions.

The anaerobic digestion of wastes is globally important in the production of methane (CH4) as a biofuel. When sulfate is present, sulfate-reducing bacteria (SRB) are stimulated, competing with methanogens for common substrates, which decreases CH4 production and results in the formation of corrosive, odorous hydrogen sulfide gas (H2S). Here, we show that a population of SRB within a methanogenic bioreactor fed only butyrate for years immediately (within hours) responded to sulfate availability and shifted the microbial community dynamics within the bioreactor. By mapping shotgun metatranscriptomes to metagenome-assembled genomes, we shed light on the transcriptomic responses of key community members in response to increased sulfate provision. We link these short-term transcriptional responses to long-term niche partitioning using comparative metagenomic analyses. Our results suggest that sulfate provision supports a syntrophic butyrate oxidation community that disfavors poly-ß-hydroxyalkanoate storage and that hydrogenotrophic SRB populations effectively exclude obligately hydrogenotrophic, but not aceticlastic, methanogens when sulfate is readily available. These findings elucidate key ecological dynamics between SRB, methanogens and syntrophic butyrate-oxidizing bacteria, which can be applied to a variety of engineered and natural systems.

Fecal indicator bacteria, fecal source tracking markers, and pathogens detected in two Hudson River tributaries.

Volunteer monitoring in the Hudson River watershed since 2012 has identified that the Wallkill River and Rondout Creek tributary complex have elevated concentrations of the fecal indicator bacteria, enterococci. Concentrations of enterococci do not provide insight into the sources of pollution and are imperfect indicators of health risks. In 2017, the regular monthly volunteer monitoring campaign for culturable enterococci at 24 sites on the Wallkill and Rondout expanded to include: (1) culturable measurements of E. coli and quantification of E. coli and Enterococcus specific markers vis nanoscale qPCR, (2) microbial source tracking (MST) assays (avian, human, bovine, and equine) via real time PCR and nanoscale qPCR, and 3) quantification of 12 gastrointestinal pathogens including viruses, bacteria, and protozoa via nanoscale qPCR. Three human associated MST markers (HumM2, HF183, and B. theta) corroborated that human pollution was present in Rondout Creek and widespread in the Wallkill River. The presence of B. theta was associated with increased concentrations of culturable E. coli. Genes for adenovirus 40 and 41 conserved region, rotavirus A NSP3, E. coli eae and stx1, and Giardia lamblia 18S rRNA were detected in >45% of samples. Abundance of rotavirus A NSP3 genes was significantly correlated to the bovine marker gene, CowM3, though wild bird sources cannot be ruled out. This is the first study to investigate potential fecal pollution sources and pathogen concentrations in Hudson tributaries during the months of peak recreational use.

Detection of Organohalide-Respiring Enzyme Biomarkers at a Bioaugmented TCE-Contaminated Field Site.

RNA-based biomarkers have been successfully detected at field sites undergoing in situ bioremediation, but the detection of expressed enzymes is a more direct way to prove activity for a particular biocatalytic process of interest since they provide evidence of potential in situ activity rather than simply confirming presence and abundance of genes in a given population by measurement of DNA copies using qPCR. Here we successfully applied shotgun proteomics to field samples from a trichloroethene (TCE)-contaminated industrial site in southern Ontario, Canada that had been bio-augmented with the commercially available KB-1TM microbial culture. The KB-1TM culture contains multiple strains of Dehalococcoides mccartyi (D. mccartyi) as well as an organohalide respiring Geobacter species. The relative abundances of specific enzymatic proteins were subsequently compared to corresponding qPCR-derived levels of DNA and RNA biomarkers in the same samples. Samples were obtained from two wells with high hydraulic connectivity to the KB-1TM-bioaugemented enhanced in situ bioremediation system, and two control wells that showed evidence of low levels of native organohalide respiring bacteria (OHRB), Dehalococcoides and Geobacter. Enzymes involved in organohalide respiration were detected in the metaproteomes of all four field samples, as were chaperonins of D. mccartyi, chemotaxis proteins, and ATPases. The most highly expressed RDase in the bioaugmentation culture (VcrA) was the most highly detected enzyme overall in the bioaugmented groundwater samples. In one background groundwater well, we found high expression of the Geobacter pceA RDase. The DNA and RNA biomarkers detected using qPCR-based assays were a set of orthologs of Dehalococcoides reductive dehalogenases (VcrA, TceA, BvcA, dehalogenase "DET1545"), and the Ni-Fe uptake hydrogenase, HupL. Within a sample, RNA levels for key enzymes correlated with relative protein abundance. These results indicate that laboratory observations of TCE-bioremediation biomarker protein expression are recapitulated in field environmental systems and that both RNA and protein biomarker monitoring hold promise for activity monitoring of in situ populations of OHRB.

Microbial communities controlling methane and nutrient cycling in leach field soils.

Septic systems inherently rely on microbial communities in the septic tank and leach field to attenuate pollution from household sewage. Operating conditions of septic leach field systems, especially the degree of water saturation, are likely to impact microbial biogeochemical cycling, including carbon (C), nitrogen (N), and phosphorus (P), as well as greenhouse gas (GHG) emissions to the atmosphere. To study the impact of flooding on microbial methane (CH4) and nutrient cycling, two leach field soil columns were constructed. One system was operated as designed and the other was operated in both flooded and well-maintained conditions. CH4 emissions were significantly higher in flooded soils (with means between 0.047 and 0.33 g CH4 m-2 d-1) as compared to well-drained soils (means between -0.0025 and 0.004 g CH4 m-2 d-1). Subsurface CH4 profiles were also elevated under flooded conditions and peaked near the wastewater inlet. Gene abundances of mcrA, a biomarker for methanogens, were also greatest near the wastewater inlet. In contrast, gene abundances of pmoA, a biomarker for methanotrophs, were greatest in surface soils at the interface of CH4 produced subsurface and atmospheric oxygen. 16S rRNA, mcrA, and pmoA amplicon library sequencing revealed microbial community structure in the soil columns differed from that of the original soils and was driven largely by CH4 fluxes and soil VWC. Additionally, active microbial populations differed from those present at the gene level. Flooding did not appear to affect N or P removals in the soil columns (between 75 and 99% removal). COD removal was variable throughout the experiment, and was negatively impacted by flooding. Our study shows septic system leach field soils are dynamic environments where CH4 and nutrients are actively cycled by microbial populations. Our results suggest proper siting, installation, and routine maintenance of leach field systems is key to reducing the overall impact of these systems on water and air quality.

Performance Evaluation of Gravity-Fed Water Treatment Systems in Rural Honduras: Verifying Robust Reduction of Turbidity and Escherichia coli during Wet and Dry Weather.

This is the first study to document the reduction of turbidity and Escherichia coli throughout the processes of full-scale gravity-fed drinking water plants (GFWTPs) and their downstream distribution systems in rural Honduras. The GFWTPs, which in these cases were designed by AguaClara, use standard treatment processes: coagulation, sedimentation, filtration, and chlorination. During the dry season, we measured E. coli, turbidity, and chlorine residual at five GFWTPs with < 1,000 connections and at three alternative piped-water systems in neighboring communities. Samples were evaluated from the raw water, settled water, filtered water, post-chlorination in the distribution tank, and at a distant-piped household connection. During the dry season, the treated water and household connections serviced by the GFWTPs met World Health Organization (WHO) recommendations for E. coli (< 1 most probable number [MPN]/100 mL). Alternative plants with the same water sources had comparable or higher E. coli and turbidity measurements posttreatment. We examined the performance robustness of two GFWTPs during the transition into the rainy season. The turbidity of the filtered water met WHO recommendations (< 1 nephelometric turbidity units). Escherichia coli was not detected in treated water, indicating that the two GFWTPs can consistently remove particulates and E. coli from source waters containing varying levels of turbidity. During two sampling events during the rainy season, E. coli was detected at the household connection of a GFWTP system with intermittent service and a substandard chlorine residual (geometric mean = 1.0 MPN/100 mL). Strategies to avoid contamination or inactivate E. coli in the distribution system are needed to ensure safe drinking water at the points of delivery, especially for systems with intermittent service.

Physiological and proteomic analyses of Fe(III)-reducing co-cultures of Desulfotomaculum reducens MI-1 and Geobacter sulfurreducens PCA.

We established Fe(III)-reducing co-cultures of two species of metal-reducing bacteria, the Gram-positive Desulfotomaculum reducens MI-1 and the Gram-negative Geobacter sulfurreducens PCA. Co-cultures were given pyruvate, a substrate that D. reducens can ferment and use as electron donor for Fe(III) reduction. G. sulfurreducens relied upon products of pyruvate oxidation by D. reducens (acetate, hydrogen) for use as electron donor in the co-culture. Co-cultures reduced Fe(III) to Fe(II) robustly, and Fe(II) was consistently detected earlier in co-cultures than pure cultures. Notably, faster cell growth, and correspondingly faster pyruvate oxidation, was observed by D. reducens in co-cultures. Global comparative proteomic analysis was performed to observe differential protein abundance during co-culture vs. pure culture growth. Proteins previously associated with Fe(III) reduction in G. sulfurreducens, namely c-type cytochromes and type IV pili proteins, were significantly increased in abundance in co-cultures relative to pure cultures. D. reducens ribosomal proteins were significantly increased in co-cultures, likely a reflection of faster growth rates observed for D. reducens cells while in co-culture. Furthermore, we developed multiple reaction monitoring (MRM) assays to quantitate specific biomarker peptides. The assays were validated in pure and co-cultures, and protein abundance ratios from targeted MRM and global proteomic analysis correlate significantly.

Methane and nitrous oxide cycling microbial communities in soils above septic leach fields: Abundances with depth and correlations with net surface emissions.

Onsite septic systems use soil microbial communities to treat wastewater, in the process creating potent greenhouse gases (GHGs): methane (CH4) and nitrous oxide (N2O). Subsurface soil dispersal systems of septic tank overflow, known as leach fields, are an important part of wastewater treatment and have the potential to contribute significantly to GHG cycling. This study aimed to characterize soil microbial communities associated with leach field systems and quantify the abundance and distribution of microbial populations involved in CH4 and N2O cycling. Functional genes were used to target populations producing and consuming GHGs, specifically methyl coenzyme M reductase (mcrA) and particulate methane monooxygenase (pmoA) for CH4 and nitric oxide reductase (cnorB) and nitrous oxide reductase (nosZ) for N2O. All biomarker genes were found in all soil samples regardless of treatment (leach field, sand filter, or control) or depth (surface or subsurface). In general, biomarker genes were more abundant in surface soils than subsurface soils suggesting the majority of GHG cycling is occurring in near-surface soils. Ratios of production to consumption gene abundances showed a positive relationship with CH4 emissions (mcrA:pmoA, p < 0.001) but not with N2O emission (cnorB:nosZ, p > 0.05). Of the three measured soil parameters (volumetric water content (VWC), temperature, and conductivity), only VWC was significantly correlated to a biomarker gene, mcrA (p = 0.0398) but not pmoA or either of the N2O cycling genes (p > 0.05 for cnorB and nosZ). 16S rRNA amplicon library sequencing results revealed soil VWC, CH4 flux and N2O flux together explained 64% of the microbial community diversity between samples. Sequencing of mcrA and pmoA amplicon libraries revealed treatment had little effect on diversity of CH4 cycling organisms. Overall, these results suggest GHG cycling occurs in all soils regardless of whether or not they are associated with a leach field system.

Altered Microbiome Leads to Significant Phenotypic and Transcriptomic Differences in a Lipid Accumulating Chlorophyte.

Given the challenges facing the economically favorable production of products from microalgae, understanding factors that might impact productivity rates including growth rates and accumulation of desired products, for example, triacylglycerols (TAG) for biodiesel feedstock, remains critical. Although operational parameters such as media composition and reactor design can clearly effect growth rates, the role of microbe-microbe interactions is just beginning to be elucidated. In this study an oleaginous marine algae Chlorella spp. C596 culture is shown to be better described as a microbial community. Perturbations to this microbial community showed a significant impact on phenotypes including sustained differences in growth rate and TAG accumulation of 2.4 and 2.5 fold, respectively. Characterization of the associated community using Illumina 16S rRNA amplicon and random shotgun transcriptomic analyses showed that the fast growth rate correlated with two specific bacterial species ( Ruegeria and Rhodobacter spp). The transcriptomic response of the Chlorella species revealed that the slower growing algal consortium C596-S1 upregulated genes associated with photosynthesis and resource scavenging and decreased the expression of genes associated with transcription and translation relative to the initial C596-R1. Our studies advance the appreciation of the effects microbiomes can have on algal growth in bioreactors and suggest that symbiotic interactions are involved in a range of critical processes including nitrogen, carbon cycling, and oxidative stress.

Biomarkers' Responses to Reductive Dechlorination Rates and Oxygen Stress in Bioaugmentation Culture KB-1TM.

Using mRNA transcript levels for key functional enzymes as proxies for the organohalide respiration (OHR) rate, is a promising approach for monitoring bioremediation populations in situ at chlorinated solvent-contaminated field sites. However, to date, no correlations have been empirically derived for chlorinated solvent respiring, Dehalococcoides mccartyi (DMC) containing, bioaugmentation cultures. In the current study, genome-wide transcriptome and proteome data were first used to confirm the most highly expressed OHR-related enzymes in the bioaugmentation culture, KB-1TM, including several reductive dehalogenases (RDases) and a Ni-Fe hydrogenase, Hup. Different KB-1™ DMC strains could be resolved at the RNA and protein level through differences in the sequence of a common RDase (DET1545-like homologs) and differences in expression of their vinyl chloride-respiring RDases. The dominant strain expresses VcrA, whereas the minor strain utilizes BvcA. We then used quantitative reverse-transcriptase PCR (qRT-PCR) as a targeted approach for quantifying transcript copies in the KB-1TM consortium operated under a range of TCE respiration rates in continuously-fed, pseudo-steady-state reactors. These candidate biomarkers from KB-1TM demonstrated a variety of trends in terms of transcript abundance as a function of respiration rate over the range: 7.7 × 10-12 to 5.9 × 10-10 microelectron equivalents per cell per hour (µeeq/cell∙h). Power law trends were observed between the respiration rate and transcript abundance for the main DMC RDase (VcrA) and the hydrogenase HupL (R² = 0.83 and 0.88, respectively), but not transcripts for 16S rRNA or three other RDases examined: TceA, BvcA or the RDase DET1545 homologs in KB1TM. Overall, HupL transcripts appear to be the most robust activity biomarker across multiple DMC strains and in mixed communities including DMC co-cultures such as KB1TM. The addition of oxygen induced cell stress that caused respiration rates to decline immediately (>95% decline within one hour). Although transcript levels did decline, they did so more slowly than the respiration rate observed (transcript decay rates between 0.02 and 0.03 per hour). Data from strain-specific probes on the pangenome array strains suggest that a minor DMC strain in KB-1™ that harbors a bvcA homolog preferentially recovered following oxygen stress relative to the dominant, vcrA-containing strain.

Evaluating Human Sensory Perceptions and the Compartment Bag Test Assays as Proxies for the Presence and Concentration of Escherichia coli in Drinking Water in Western Kenya.

We compared the concentrations of Escherichia coli quantified with Colilert™ and the compartment bag test (CBT) in the source water and household stored drinking water (SDW) of 35 households in western Kenya. We also investigated the associations of the perceptions of organoleptic properties and overall quality with ≥ 1 MPN/100 mL E. coli in SDW. Participants who rated the taste or smell of their SDW "< 5" on a 1 = "poor" to 5 = "excellent" Likert scale were 8.71 or 7.04 times more likely, respectively, to have ≥ 1 MPN/100 mL E. coli. Organoleptic properties are innate, albeit imperfect, indicators of fecal pollution in water. Within their shared quantification range, concentrations of E. coli enumerated with Colilert and CBT were similar and had a significant correlation coefficient, 0.896 (95% confidence interval = 0.691-1.101). The methods had moderate agreement within the World Health Organization's health risk levels (Cohen's Kappa coefficient = 0.640). In low-resource settings, CBT provides comparable assessments of E. coli concentrations to Colilert.

Inferring Gene Networks for Strains of Dehalococcoides Highlights Conserved Relationships between Genes Encoding Core Catabolic and Cell-Wall Structural Proteins.

The interpretation of high-throughput gene expression data for non-model microorganisms remains obscured because of the high fraction of hypothetical genes and the limited number of methods for the robust inference of gene networks. Therefore, to elucidate gene-gene and gene-condition linkages in the bioremediation-important genus Dehalococcoides, we applied a Bayesian inference strategy called Reverse Engineering/Forward Simulation (REFS™) on transcriptomic data collected from two organohalide-respiring communities containing different Dehalococcoides mccartyi strains: the Cornell University mixed community D2 and the commercially available KB-1® bioaugmentation culture. In total, 49 and 24 microarray datasets were included in the REFS™ analysis to generate an ensemble of 1,000 networks for the Dehalococcoides population in the Cornell D2 and KB-1® culture, respectively. Considering only linkages that appeared in the consensus network for each culture (exceeding the determined frequency cutoff of ≥ 60%), the resulting Cornell D2 and KB-1® consensus networks maintained 1,105 nodes (genes or conditions) with 974 edges and 1,714 nodes with 1,455 edges, respectively. These consensus networks captured multiple strong and biologically informative relationships. One of the main highlighted relationships shared between these two cultures was a direct edge between the transcript encoding for the major reductive dehalogenase (tceA (D2) or vcrA (KB-1®)) and the transcript for the putative S-layer cell wall protein (DET1407 (D2) or KB1_1396 (KB-1®)). Additionally, transcripts for two key oxidoreductases (a [Ni Fe] hydrogenase, Hup, and a protein with similarity to a formate dehydrogenase, "Fdh") were strongly linked, generalizing a strong relationship noted previously for Dehalococcoides mccartyi strain 195 to multiple strains of Dehalococcoides. Notably, the pangenome array utilized when monitoring the KB-1® culture was capable of resolving signals from multiple strains, and the network inference engine was able to reconstruct gene networks in the distinct strain populations.

Comparative Proteomic Analysis of Desulfotomaculum reducens MI-1: Insights into the Metabolic Versatility of a Gram-Positive Sulfate- and Metal-Reducing Bacterium.

The proteomes of the metabolically versatile and poorly characterized Gram-positive bacterium Desulfotomaculum reducens MI-1 were compared across four cultivation conditions including sulfate reduction, soluble Fe(III) reduction, insoluble Fe(III) reduction, and pyruvate fermentation. Collectively across conditions, we observed at high confidence ~38% of genome-encoded proteins. Here, we focus on proteins that display significant differential abundance on conditions tested. To the best of our knowledge, this is the first full-proteome study focused on a Gram-positive organism cultivated either on sulfate or metal-reducing conditions. Several proteins with uncharacterized function encoded within heterodisulfide reductase (hdr)-containing loci were upregulated on either sulfate (Dred_0633-4, Dred_0689-90, and Dred_1325-30) or Fe(III)-citrate-reducing conditions (Dred_0432-3 and Dred_1778-84). Two of these hdr-containing loci display homology to recently described flavin-based electron bifurcation (FBEB) pathways (Dred_1325-30 and Dred_1778-84). Additionally, we propose that a cluster of proteins, which is homologous to a described FBEB lactate dehydrogenase (LDH) complex, is performing lactate oxidation in D. reducens (Dred_0367-9). Analysis of the putative sulfate reduction machinery in D. reducens revealed that most of these proteins are constitutively expressed across cultivation conditions tested. In addition, peptides from the single multiheme c-type cytochrome (MHC) in the genome were exclusively observed on the insoluble Fe(III) condition, suggesting that this MHC may play a role in reduction of insoluble metals.

Use of De Novo Transcriptome Libraries to Characterize a Novel Oleaginous Marine Chlorella Species during the Accumulation of Triacylglycerols.

Marine chlorophytes of the genus Chlorella are unicellular algae capable of accumulating a high proportion of cellular lipids that can be used for biodiesel production. In this study, we examined the broad physiological capabilities of a subtropical strain (C596) of Chlorella sp. "SAG-211-18" including its heterotrophic growth and tolerance to low salt. We found that the alga replicates more slowly at diluted salt concentrations and can grow on a wide range of carbon substrates in the dark. We then sequenced the RNA of Chlorella strain C596 to elucidate key metabolic genes and investigate the transcriptomic response of the organism when transitioning from a nutrient-replete to a nutrient-deficient condition when neutral lipids accumulate. Specific transcripts encoding for enzymes involved in both starch and lipid biosynthesis, among others, were up-regulated as the cultures transitioned into a lipid-accumulating state whereas photosynthesis-related genes were down-regulated. Transcripts encoding for two of the up-regulated enzymes-a galactoglycerolipid lipase and a diacylglyceride acyltransferase-were also monitored by reverse transcription quantitative polymerase chain reaction assays. The results of these assays confirmed the transcriptome-sequencing data. The present transcriptomic study will assist in the greater understanding, more effective application, and efficient design of Chlorella-based biofuel production systems.