Occurrence of Rhodococcus sp. RR1 prmA and Rhodococcus jostii RHA1 prmA across microbial communities and their enumeration during 1,4-dioxane biodegradation.

1,4-Dioxane, a likely human carcinogen, is a co-contaminant at many chlorinated solvent contaminated sites. Conventional treatment technologies, such as carbon sorption or air stripping, are largely ineffective, and so many researchers have explored bioremediation for site clean-up. An important step towards this involves examining the occurrence of the functional genes associated with 1,4-dioxane biodegradation. The current research explored potential biomarkers for 1,4-dioxane in three mixed microbial communities (wetland sediment, agricultural soil, impacted site sediment) using monooxygenase targeted amplicon sequencing, followed by quantitative PCR (qPCR). A BLAST analysis of the sequencing data detected only two of the genes previously associated with 1,4-dioxane metabolism or co-metabolism, namely propane monooxygenase (prmA) from Rhodococcus jostii RHA1 and Rhodococcus sp. RR1. To investigate this further, qPCR primers and probes were designed, and the assays were used to enumerate prmA gene copies in the three communities. Gene copies of Rhodococcus RR1 prmA were detected in all three, while gene copies of Rhodococcus jostii RHA1 prmA were detected in two of the three sample types (except impacted site sediment). Further, there was a statistically significant increase in RR1 prmA gene copies in the microcosms inoculated with impacted site sediment following 1,4-dioxane biodegradation compared to the control microcosms (no 1,4-dioxane) or to the initial copy numbers before incubation. Overall, the results indicate the importance of Rhodococcus associated prmA, compared to other 1,4-dioxane degrading associated biomarkers, in three different microbial communities. Also, the newly designed qPCR assays provide a platform for others to investigate 1,4-dioxane biodegradation potential in mixed communities and should be of particular interest to those considering bioremediation as a potential 1,4-dioxane remediation approach.

In silico analysis of soil, sediment and groundwater microbial communities to predict biodegradation potential.

This study examined soil, sediment and groundwater microbial communities for a set of key functional genes important for contaminant biodegradation. This involved PICRUSt2 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) predictions based on 16S rRNA gene amplicon datasets from three separate studies with different inocula and incubation conditions, as follows: aerobic soils, oxygen-limited microcosms containing sediments and groundwater, as well as methanogenic microcosms with different inocula. PICRUSt2 predicts functional profiles of microbial communities based on marker gene (16S rRNA gene) data. The relative abundances of genera previously associated with the biodegradation of chlorinated solvents/metabolites and/or 1,4-dioxane were also determined. Predicted values for each functional gene varied between the three datasets. In all, values were high for propane monooxygenase and low for soluble methane monooxygenase. Common phylotypes associated with propane monooxygenase in two of the three datasets included Mycobacterium, Rhodococcus and Pseudonocardia. Toluene monooxygenase predicted values were greater in the oxygen-limited microcosms compared to the other two datasets. The methanogenic microcosms exhibited the highest predicted values for particulate methane/ammonia monooxygenase. The most common genera detected, previously reported as chlorinated solvents/metabolites and/or 1,4-dioxane degraders, included Pseudomonas, Sphingomonas, Rhodococcus and Rhodanobacter. Eighteen of the queried genera were not detected. As expected, more potential anaerobic degrading genera were found in the oxygen-limited and methanogenic microcosms compared to the aerobic soils. The results provide key insights as to which genes and genera may be important for biodegradation over a range of inocula and redox conditions.

Predicting the occurrence of monooxygenases and their associated phylotypes in soil microcosms.

Cometabolic oxidation involves the oxidation of chemicals often by monooxygenases or dioxygenases and can be a removal process for environmental contaminants such as trichloroethene (TCE) or 1,4-dioxane. Information on the occurrence of these genes and their associated microorganisms in environmental samples has the potential to enhance our understanding of contaminant removal. The overall aims were to 1) ascertain which genes encoding for monooxygenases (from methanotrophs, ammonia oxidizing bacteria and toluene/phenol oxidizers) and other key enzymes are present in soil microcosms and 2) determine which phylotypes are associated with those genes. The approach involved a predictive tool called PICRUSt2 and 16S rRNA gene amplicon datasets from two previous soil microcosm studies. The following targets from the KEGG database were examined: pmo/amo, mmo, dmp/pox/tomA, tmo/tbu/tou, bssABC (and downstream genes), tod, xylM, xylA, gst, dhaA, catE, dbfA1, dbfA2 and phenol 2-monooxygenase. A large number of phylotypes were associated with pmo/amo, while mmo was linked to only five. Several phylotypes were associated with both pmo/amo and mmo. The most dominant microorganism predicted for mmoX was Mycobacterium (also predicted for pmo/amo). A large number of phylotypes were associated with all six genes from the dmp/pox/tomA KEGG group. The taxonomic associations predicted for the tmo/tbu/tou KEGG group were more limited. In both datasets, Geobacter was a key phylotype for benzylsuccinate synthase. The dioxygenase-mediated toluene degradation pathway encoded by todC1C2BA was largely absent, as were the genes (xylM, xylA) encoding for xylene monooxygenase. All other genes investigated were predicted to be present and were associated with a number of microorganisms. Overall, the analysis predicted the genes encoding for sMMO (mmo), T3MO/T3MO/ToMO (tmo/tbu/tou) and benzylsuccinate synthase (bssABC) are present for a limited number of phylotypes compared to those encoding for pMMO/AMO (pmo/amo) and phenol monooxygenase/T2MO (dmp/poxA/tomA). These findings suggest in soils contaminant removal via pMMO/AMO or phenol monooxygenase/T2MO may be common because of the occurrence of these enzymes with a large number of phylotypes.

Predicted functional genes for the biodegradation of xenobiotics in groundwater and sediment at two contaminated naval sites.

The goals of this study were to predict the genes associated with the biodegradation of organic contaminants and to examine microbial community structure in samples from two contaminated sites. The approach involved a predictive bioinformatics tool (PICRUSt2) targeting genes from twelve KEGG xenobiotic biodegradation pathways (benzoate, chloroalkane and chloroalkene, chlorocyclohexane and chlorobenzene, toluene, xylene, nitrotoluene, ethylbenzene, styrene, dioxin, naphthalene, polycyclic aromatic hydrocarbons, and metabolism of xenobiotics by cytochrome P450). Further, the predicted phylotypes associated with functional genes early in each pathway were determined. Phylogenetic analysis indicated a greater diversity in the sediment compared to the groundwater samples. The most abundant genera for sediments/microcosms included Pseudomonas, Methylotenera, Rhodococcus, Stenotrophomonas, and Brevundimonas, and the most abundant for the groundwater/microcosms included Pseudomonas, Cupriavidus, Azospira, Rhodococcus, and unclassified Burkholderiaceae. Genes from all twelve of the KEGG pathways were predicted to occur. Seven pathways contained less than twenty-five genes. The predicted genes were lowest for xenobiotics metabolism by cytochrome P450 and ethylbenzene biodegradation and highest for benzoate biodegradation. Notable trends include the occurrence of the first genes for trinitrotoluene and 2,4-dinitrotoluene degradation. Also, the complete path from toluene to benzoyl-CoA was predicted. Twenty-two of the dioxin pathway genes were predicted, including genes within the first steps. The following phylotypes were associated with the greatest number of pathways: unclassified Burkholderiaceae, Burkholderia-Caballeronia-Paraburkholderia, Pseudomonas, Rhodococcus, unclassified Betaproteobacteria, and Polaromonas. This work illustrates the value of PICRUSt2 for predicting biodegradation potential and suggests that a subset of phylotypes could be important for the breakdown of organic contaminants or their metabolites. KEY POINTS: • The approach is a low-cost alternative to shotgun sequencing. • The genes and phylotypes encoding for xenobiotic degradation were predicted. • A subset of phylotypes were associated with many pathways.

Diversity and abundance of the functional genes and bacteria associated with RDX degradation at a contaminated site pre- and post-biostimulation.

Bioremediation is becoming an increasingly popular approach for the remediation of sites contaminated with the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). Multiple lines of evidence are often needed to assess the success of such approaches, with molecular studies frequently providing important information on the abundance of key biodegrading species. Towards this goal, the current study utilized shotgun sequencing to determine the abundance and diversity of functional genes (xenA, xenB, xplA, diaA, pnrB, nfsI) and species previously associated with RDX biodegradation in groundwater before and after biostimulation at an RDX-contaminated Navy Site. For this, DNA was extracted from four and seven groundwater wells pre- and post-biostimulation, respectively. From a set of 65 previously identified RDX degraders, 31 were found within the groundwater samples, with the most abundant species being Variovorax sp. JS1663, Pseudomonas fluorescens, Pseudomonas putida, and Stenotrophomonas maltophilia. Further, 9 RDX-degrading species significantly (p<0.05) increased in abundance following biostimulation. Both the sequencing data and qPCR indicated that xenA and xenB exhibited the highest relative abundance among the six genes. Several genes (diaA, nsfI, xenA, and pnrB) exhibited higher relative abundance values in some wells following biostimulation. The study provides a comprehensive approach for assessing biomarkers during RDX bioremediation and provides evidence that biostimulation generated a positive impact on a set of key species and genes. KEY POINTS: • A co-occurrence network indicated diverse RDX degraders. • >30 RDX-degrading species were detected. • Nine RDX-degrading species increased following biostimulation. • Sequencing and high-throughput qPCR indicated that xenA and xenB were most abundant.

Identification of the phylotypes involved in cis-dichloroethene and 1,4-dioxane biodegradation in soil microcosms.

Co-contamination with chlorinated compounds and 1,4-dioxane has been reported at many sites. Recently, there has been an increased interest in bioremediation because of the potential to degrade multiple contaminants concurrently. Towards improving bioremediation efficacy, the current study examined laboratory microcosms (inoculated separately with two soils) to determine the phylotypes and functional genes associated with the biodegradation of two common co-contaminants (cis-dichloroethene [cDCE] and 1,4-dioxane). The impact of amending microcosms with lactate on cDCE and 1,4-dioxane biodegradation was also investigated. The presence of either lactate or cDCE did not impact 1,4-dioxane biodegradation one of the two soils. Lactate appeared to improve the initiation of the biological removal of cDCE in microcosms inoculated with either soil. Stable isotope probing (SIP) was then used to determine which phylotypes were actively involved in carbon uptake from cDCE and 1,4-dioxane in both soil communities. The most enriched phylotypes for 13C assimilation from 1,4-dioxane included Rhodopseudomonas and Rhodanobacter. Propane monooxygenase was predicted (by PICRUSt2) to be dominant in the 1,4-dioxane amended microbial communities and propane monooxygenase gene abundance values correlated with other enriched (but less abundant) phylotypes for 13C-1,4-dioxane assimilation. The dominant enriched phylotypes for 13C assimilation from cDCE included Bacteriovorax, Pseudomonas and Sphingomonas. In the cDCE amended soil microcosms, PICRUSt2 predicted the presence of DNA encoding glutathione S-transferase (a known cDCE upregulated enzyme). Overall, the work demonstrated concurrent removal of cDCE and 1,4-dioxane by indigenous soil microbial communities and the enhancement of cDCE removal by lactate. The data generated on the phylotypes responsible for carbon uptake (as determined by SIP) could be incorporated into diagnostic molecular methods for site characterization. The results suggest concurrent biodegradation of cDCE and 1,4-dioxane should be considered for chlorinated solvent site remediation.

Diversity of nitrogen cycling genes at a Midwest long-term ecological research site with different management practices.

Nitrogen fertilizer results in the release of nitrous oxide (N2O), a concern because N2O is an ozone-depleting substance and a greenhouse gas. Although the reduction of N2O to nitrogen gas can control emissions, the factors impacting the enzymes involved have not been fully explored. The current study investigated the abundance and diversity of genes involved in nitrogen cycling (primarily denitrification) under four agricultural management practices (no tillage [NT], conventional tillage [CT], reduced input, biologically-based). The work involved examining soil shotgun sequencing data for nine genes (napA, narG, nirK, nirS, norB, nosZ, nirA, nirB, nifH). For each gene, relative abundance values, diversity and richness indices, and taxonomic classification were determined. Additionally, the genes associated with nitrogen metabolism (defined by the KEGG hierarchy) were examined. The data generated were statistically compared between the four management practices. The relative abundance of four genes (nifH, nirK, nirS, and norB) were significantly lower in the NT treatment compared to one or more of the other soils. The abundance values of napA, narG, nifH, nirA, and nirB were not significantly different between NT and CT. The relative abundance of nirS was significantly higher in the CT treatment compared to the others. Diversity and richness values were higher for four of the nine genes (napA, narG, nirA, nirB). Based on nirS/nirK ratios, CT represents the highest N2O consumption potential in four soils. In conclusion, the microbial communities involved in nitrogen metabolism were sensitive to different agricultural practices, which in turn, likely has implications for N2O emissions. KEY POINTS: • Four genes were less abundant in NT compared to one or more of the others soils (nifH, nirK, nirS, norB). • The most abundant sequences for many of the genes classified within the Proteobacteria. • Higher diversity and richness indices were observed for four genes (napA, narG, nirA, nirB). • Based on nirS/nirK ratios, CT represents the highest N2O consumption potential.

Anaerobic 1,4-dioxane biodegradation and microbial community analysis in microcosms inoculated with soils or sediments and different electron acceptors.

1,4-Dioxane, a probable human carcinogen, is a co-contaminant at many chlorinated solvent-contaminated sites. Although numerous 1,4-dioxane-degrading aerobic bacteria have been isolated, almost no information exists on the microorganisms able to degrade this chemical under anaerobic conditions. Here, the potential for 1,4-dioxane biodegradation was examined using multiple inocula and electron acceptor amendments. The inocula included uncontaminated agricultural soils and river sediments as well as sediments from two 1,4-dioxane contaminated sites. Five separate experiments involved the examination of triplicate live microcosms and abiotic controls for approximately 1 year. Compound-specific isotope analysis (CSIA) was used to further investigate biodegradation in a subset of the microcosms. Also, DNA was extracted from microcosms exhibiting 1,4-dioxane biodegradation for microbial community analysis using 16S rRNA gene amplicon high-throughput sequencing. Given the long incubation periods, it is likely that electron acceptor depletion occurred and methanogenic conditions eventually dominated. The iron/EDTA/humic acid or sulfate amendments did not result in 1,4-dioxane biodegradation in the majority of cases. 1,4-dioxane biodegradation was most commonly observed in the nitrate amended and no electron acceptor treatments. Notably, both contaminated site sediments illustrated removal in the samples compared to the abiotic controls in the no electron acceptor treatment. However, it is important to note that the degradation was slow (with concentration reductions occurring over approximately 1 year). In two of the three cases examined, CSIA provided additional evidence for 1,4-dioxane biodegradation. In one case, the reduction in 1,4-dioxane in the samples comparing the controls was likely too low for the method to detect a significant 13C/12C enrichment. Further research is required to determine the value of measuring 2H/1H for generating evidence for the biodegradation of this chemical. The microbial community analysis indicated that the phylotypes unclassified Comamonadaceae and 3 genus incertae sedis were more abundant in 1,4-dioxane-degrading microcosms compared to the live controls (no 1,4-dioxane) in microcosms inoculated with contaminated and uncontaminated sediment, respectively. The relative abundance of known 1,4-dioxane degraders was also investigated at the genus level. The soil microcosms were dominated primarily by Rhodanobacter with lower relative abundance values for Pseudomonas, Mycobacterium, and Acinetobacter. The sediment communities were dominated by Pseudomonas and Rhodanobacter. Overall, the current study indicates 1,4-dioxane biodegradation under anaerobic and, likely methanogenic conditions, is feasible. Therefore, natural attenuation may be an appropriate cleanup technology at sites where time is not a limitation.

Enrichment of novel Actinomycetales and the detection of monooxygenases during aerobic 1,4-dioxane biodegradation with uncontaminated and contaminated inocula.

1,4-Dioxane, a co-contaminant at many chlorinated solvent sites, is a problematic groundwater pollutant because of risks to human health and characteristics which make remediation challenging. In situ 1,4-dioxane bioremediation has recently been shown to be an effective remediation strategy. However, the presence/abundance of 1,4-dioxane degrading species across different environmental samples is generally unknown. Here, the objectives were to identify which 1,4-dioxane degrading functional genes are present and which genera may be using 1,4-dioxane and/or metabolites to support growth across different microbial communities. For this, laboratory sample microcosms and abiotic control microcosms (containing media) were inoculated with four uncontaminated soils and sediments from two contaminated sites. Live control microcosms were treated in the same manner, except 1,4-dioxane was not added. 1,4-Dioxane decreased in live microcosms with all six inocula, but not in the abiotic controls, suggesting biodegradation occurred. A comparison of live sample microcosms and live controls (no 1,4-dioxane) indicated nineteen genera were enriched following exposure to 1,4-dioxane, suggesting a growth benefit for 1,4-dioxane biodegradation. The three most enriched were Mycobacterium, Nocardioides, and Kribbella (classifying as Actinomycetales). There was also a higher level of enrichment for Arthrobacter, Nocardia, and Gordonia (all three classifying as Actinomycetales) in one soil, Hyphomicrobium (Rhizobiales) in another soil, Clavibacter (Actinomycetales) and Bartonella (Rhizobiales) in another soil, and Chelativorans (Rhizobiales) in another soil. Although Arthrobacter, Mycobacterium, and Nocardia have previously been linked to 1,4-dioxane degradation, Nocardioides, Gordonia, and Kribbella are potentially novel degraders. The analysis of the functional genes associated with 1,4-dioxane demonstrated three genes were present at higher relative abundance values, including Rhodococcus sp. RR1 prmA, Rhodococcus jostii RHA1 prmA, and Burkholderia cepacia G4 tomA3. Overall, this study provides novel insights into the identity of the multiple genera and functional genes associated with aerobic degradation of 1,4-dioxane in mixed communities.

Influence of terminal electron-accepting conditions on the soil microbial community and degradation of organic contaminants of emerging concern.

Better understanding of the fate and persistence of trace organic contaminants of emerging concern (CEC) in agricultural soils is critical for assessing the risks associated with using treated wastewater effluent to irrigate crops and land application of wastewater biosolids. This study reports on the influence of prevailing terminal electron-accepting processes (TEAPs, i.e., aerobic, nitrate-reducing, iron(III)-reducing, and sulfate-reducing conditions) and exposure to a mixture of nine trace CEC (90 ng/g each) on both the microbial community structure and CEC degradation in agricultural soil. DNA analysis revealed significant differences in microbial community composition following establishment of different TEAPs, but no significant change upon exposure to the mixture of CEC. The largest community shift was observed after establishing nitrate-reducing conditions and the smallest shift for sulfate-reducing conditions. Two of the CEC (atrazine and sulfamethoxazole) showed significant degradation in both bioactive and abiotic (i.e., sterilized) conditions, with half-lives ranging from 1 to 64 days for different TEAPs, while six of the CEC (amitriptyline, atenolol, trimethoprim, and three organophosphate flame retardants) only degraded in bioactive samples, with half-lives ranging from 27 to 90 days; carbamazepine did not degrade appreciably within 90 days in any of the incubations. Amplicon sequence variants (ASVs) from Firmicutes Hydrogenispora, Gemmatimonadetes Gemmatimonadaceae, and Verrucomicrobia OPB34 soil group were identified as potentially responsible for the biodegradation of organophosphate flame retardants, and ASVs from other taxa groups were suspected to be involved in biodegrading the other target CEC. These results demonstrate that CEC fate and persistence in agricultural soils is influenced by the prevailing TEAPs and their influence on the microbial community, suggesting the need to incorporate these factors into contaminant fate models to improve risk assessment predictions.

High throughput quantification of the functional genes associated with RDX biodegradation using the SmartChip real-time PCR system.

The explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a contaminant at many military sites. RDX bioremediation as a clean-up approach has been gaining popularity because of cost benefits compared to other methods. RDX biodegradation has primarily been linked to six functional genes (diaA, nfsI, pnrB, xenA, xenB, xplA). However, current methods for gene quantification have the risk of false negative results because of low theoretical primer coverage. To address this, the current study designed new primer sets using the EcoFunPrimer tool based on sequences collected by the Functional Gene Pipeline and Repository and these were verified based on residues and motifs. The primers were also designed to be compatible with the SmartChip Real-Time PCR system, a massively parallel singleplex PCR platform (high throughput qPCR), that enables quantitative gene analysis using 5,184 simultaneous reactions on a single chip with low volumes of reagents. This allows multiple genes and/or multiple primer sets for a single gene to be used with multiple samples. Following primer design, the six genes were quantified in RDX-contaminated groundwater (before and after biostimulation), RDX-contaminated sediment, and uncontaminated samples. The final 49 newly designed primer sets improved upon the theoretical coverage of published primer sets, and this corresponded to more detections in the environmental samples. All genes, except diaA, were detected in the environmental samples, with xenA and xenB being the most predominant. In the sediment samples, nfsI was the only gene detected. The new approach provides a more comprehensive tool for understanding RDX biodegradation potential at contaminated sites.

Carbamazepine, triclocarban and triclosan biodegradation and the phylotypes and functional genes associated with xenobiotic degradation in four agricultural soils.

Pharmaceuticals and personal care products (PPCPs) are released into the environment due to their poor removal during wastewater treatment. Agricultural soils subject to irrigation with wastewater effluent and biosolids application are possible reservoirs for these chemicals. This study examined the impact of the pharmaceutical carbamazepine (CBZ), and the antimicrobial agents triclocarban (TCC) and triclosan (TCS) on four soil microbial communities using shotgun sequencing (HiSeq Illumina) with the overall aim of determining possible degraders as well as the functional genes related to general xenobiotic degradation. The biodegradation of CBZ and TCC was slow, with ≤50% decrease during the 80-day incubation period. In contrast, TCS biodegradation was rapid, with ~80% removal in 25 days. For each chemical, when all four soils were considered together, between three and ten phylotypes (from multiple phyla) were more abundant in the soil samples compared to the live controls. The genera of a number of previously reported CBZ, TCC or TCS degrading isolates were present; Rhodococcus (CBZ), Streptomyces (CBZ), Pseudomonas (CBZ, TCC, TCS), Sphingomonas (TCC, TCS), Methylobacillus (TCS) and Stenotrophomonas (TCS) were among the most abundant (chemical previously reported to be degraded is shown in parenthesis). From the analysis of xenobiotic degrading pathways, genes from five KEGG (Kyoto Encyclopedia of Genes and Genomes) Orthology pathways were the most dominant, including those associated with aminobenzoate, benzoate (most common), chlorocyclohexane/chlorobenzene, dioxin and nitrotoluene biodegradation. Several phylotypes including Bradyrhizobium, Mycobacterium, Rhodopseudomonas, Pseudomonas, Cupriavidus, and Streptomyces were common genera associated with these pathways. Overall, the data suggest several phylotypes are likely involved in the biodegradation of these PPCPs with Pseudomonas being an important genus.

Abundance of Chlorinated Solvent and 1,4-Dioxane Degrading Microorganisms at Five Chlorinated Solvent Contaminated Sites Determined via Shotgun Sequencing.

Shotgun sequencing was used for the quantification of taxonomic and functional biomarkers associated with chlorinated solvent bioremediation in 20 groundwater samples (five sites), following bioaugmentation with SDC-9. The analysis determined the abundance of (1) genera associated with chlorinated solvent degradation, (2) reductive dehalogenase (RDases) genes, (3) genes associated with 1,4-dioxane removal, (4) genes associated with aerobic chlorinated solvent degradation, and (5) D. mccartyi genes associated with hydrogen and corrinoid metabolism. The taxonomic analysis revealed numerous genera previously linked to chlorinated solvent degradation, including Dehalococcoides, Desulfitobacterium, and Dehalogenimonas. The functional gene analysis indicated vcrA and tceA from D. mccartyi were the RDases with the highest relative abundance. Reads aligning with both aerobic and anaerobic biomarkers were observed across all sites. Aerobic solvent degradation genes, etnC or etnE, were detected in at least one sample from each site, as were pmoA and mmoX. The most abundant 1,4-dioxane biomarker detected was Methylosinus trichosporium OB3b mmoX. Reads aligning to thmA or Pseudonocardia were not found. The work illustrates the importance of shotgun sequencing to provide a more complete picture of the functional abilities of microbial communities. The approach is advantageous over current methods because an unlimited number of functional genes can be quantified.

Diclofenac, carbamazepine and triclocarban biodegradation in agricultural soils and the microorganisms and metabolic pathways affected.

The incomplete elimination of pharmaceuticals and personal care products (PPCPs) during wastewater treatment has resulted in their detection in the environment. PPCP biodegradation is a potential removal mechanism; however, the microorganisms and pathways involved in soils are generally unknown. Here, the biodegradation of diclofenac (DCF), carbamazepine (CBZ) and triclocarban (TCC) in four agricultural soils at concentrations typically detected in soils and biosolids (50 ng g-1) was examined. Rapid DCF removal (<7 days) was observed under aerobic conditions, but only limited biodegradation was noted under other redox conditions. CBZ and TCC degradation under aerobic conditions was slow (half-lives of 128-241 days and 165-190 days for CBZ and TCC). Phylotypes in the Proteobacteria, Gemmatimonadales and Actinobacteria were significantly more abundant during DCF biodegradation compared to the controls (no DCF). For CBZ, those in the Bacteroidetes, Actinobacteria, Proteobacteria and Verrucomicrobia were enriched compared to the controls. Actinobacteria and Proteobacteria were also enriched during TCC biodegradation. Such differences could indicate these microorganisms are associated with the biodegradation of these compounds, as they appear to be benefiting from their removal. The impact of these PPCPs on the KEGG pathways associated with metabolism was also examined. Four pathways were positively impacted during DCF biodegradation (propanoate, lysine, fatty acid & benzoate metabolism). These pathways are likely common in soils, explaining the rapid removal of DCF. There was limited impact of CBZ on the metabolic pathways. TCC removal was linked to genes associated with the degradation of simple and complex substrates. The results indicate even low concentrations of PPCPs significantly affect soil communities. The recalcitrant nature of TCC and CBZ suggests soils receiving biosolids could accumulate these chemicals, representing risks concerning crop uptake.

Integrated methodological approach reveals microbial diversity and functions in aerobic groundwater microcosms adapted to vinyl chloride.

Vinyl chloride (VC), a known human carcinogen, is often formed in groundwater (GW) by incomplete reductive dechlorination of chlorinated ethenes. An integrated microbial ecology approach involving bacterial enrichments and isolations, carbon stable-isotope probing (SIP) and metagenome and genome sequencing was applied to ethene-fed GW microcosms that rapidly transitioned to aerobic growth on VC. Actinobacteria, Proteobacteria and Bacteroidetes dominated the microbial communities in ethene- and VC-grown cultures. SIP with 13C2-VC demonstrated that Nocardioides spp. significantly participated in carbon uptake from VC (52.1%-75.7% enriched in heavy fractions). Sediminibacterium, Pedobacter and Pseudomonas spp. also incorporated 13C from VC into genomic DNA. Ethene- and VC-assimilating Nocardioides sp. strain XL1 was isolated. Sequencing revealed a large (∼300 kbp) plasmid harboring genes encoding alkene monooxygenase and epoxyalkane: coenzyme M transferase, enzymes known to participate in aerobic VC and ethene biodegradation. The plasmid was 100% identical to pNOCA01 found in VC-assimilating Nocardioides sp. strain JS614. Metagenomic analysis of enrichment cultures indicated other bacteria implicated in carbon uptake from VC possessed the genetic potential to detoxify epoxides via epoxide hydrolase or glutathione S-transferase (Pseudomonas) and/or metabolize VC epoxide breakdown products and downstream VC metabolites. This study provides new functional insights into aerobic VC metabolism within a GW microbial community.

Most probable number with visual based LAMP for the quantification of reductive dehalogenase genes in groundwater samples.

The remediation of chlorinated solvent contaminated sites frequently involves bioaugmentation with mixed cultures containing Dehalococcoides mccartyi. Their activity is then examined by quantifying reductive dehalogenase (RDase) genes. Recently, we described a rapid, low cost approach, based on loop mediated isothermal amplification (LAMP), which allowed for the visual detection of RDase genes from groundwater. In that study, samples were concentrated (without DNA extraction), incubated in a water bath (avoiding the use of a thermal cycler) and amplification was visualized by the addition of SYBR green (post incubation). Despite having a detection limit less than the threshold recommended for effective remediation, the application of the assay was limited because of the semi-quantitative nature of the data. Moreover, the assay was prone to false positives due to the aerosolization of amplicons. In this study, deoxyuridine triphosphate (dUTP) and uracil DNA glycosylase (UNG) were incorporated into the assay to reduce the probability of false positives. Optimization experiments revealed a UNG concentration of 0.2units per reaction was adequate for degrading trace levels of AUGC based contamination (~1.4×104 gene copies/reaction) without significant changes to the detection limit (~100 gene copies/reaction). Additionally, the optimized assay was used with the most probable number (MPN) method to quantify RDase genes (vcrA and tceA) in multiple groundwater samples from a chlorinated solvent contaminated site. Using this approach, gene concentrations were significantly correlated to concentrations obtained using traditional methods (qPCR and DNA templates). Although the assay underestimated RDase genes concentrations, a strong correlation (R2=0.78 and 0.94) was observed between the two data sets. The regression equations obtained will be valuable to determine gene copies in groundwater using the newly developed, low cost and time saving method.

Development and application of a rapid, user-friendly, and inexpensive method to detect Dehalococcoides sp. reductive dehalogenase genes from groundwater.

TaqMan probe-based quantitative polymerase chain reaction (qPCR) specific to the biomarker reductive dehalogenase (RDase) genes is a widely accepted molecular biological tool (MBT) for determining the abundance of Dehalococcoides sp. in groundwater samples from chlorinated solvent-contaminated sites. However, there are significant costs associated with this MBT. In this study, we describe an approach that requires only low-cost laboratory equipment (a bench top centrifuge and a water bath) and requires less time and resources compared to qPCR. The method involves the concentration of biomass from groundwater, without DNA extraction, and loop-mediated isothermal amplification (LAMP) of the cell templates. The amplification products are detected by a simple visual color change (orange/green). The detection limits of the assay were determined using groundwater from a contaminated site. In addition, the assay was tested with groundwater from three additional contaminated sites. The final approach to detect RDase genes, without DNA extraction or a thermal cycler, was successful to 1.8 × 105 gene copies per L for vcrA and 1.3 × 105 gene copies per L for tceA. Both values are below the threshold recommended for effective in situ dechlorination.

Most probable number - loop mediated isothermal amplification (MPN-LAMP) for quantifying waterborne pathogens in <25min.

We are reporting a most probable number approach integrated to loop mediated isothermal technique (MPN-LAMP) focusing on Gram-negative Escherichia coli and Gram-positive Enterococcus faecalis bacterial cells without nucleic acids extraction. LAMP assays for uidA from E. coli and gelE from E. faecalis were successfully performed directly on cells up to single digit concentration using a commercial real time PCR instrument. Threshold time values of LAMP assays of bacterial cells, heat treated bacterial cells (95°C for 5min), and their purified genomic DNA templates were similar, implying that amplification could be achieved directly from bacterial cells at 63°C. Viability of bacterial cells was confirmed by using propidium monoazide in a LAMP assay with E. faecalis. To check its functionality on a microfluidic platform, MPN-LAMP assays targeting <10CFU of bacteria were also translated onto polymeric microchips and monitored by a low-cost fluorescence imaging system. The overall system provided signal-to-noise (SNR) ratios up to 800, analytical sensitivity of <10CFU, and time to positivity of about 20min. MPN-LAMP assays were performed for cell concentrations in the range of 105CFU to <10CFU. MPN values from LAMP assays confirmed that the amplifications were from <10CFU. The method described here, applicable directly on cells at 63°C, eliminates the requirement of complex nucleic acids extraction steps, facilitating the development of sensitive, rapid, low-cost, and field-deployable systems. This rapid MPN-LAMP approach has the potential to replace conventional MPN method for waterborne pathogens.

Direct loop mediated isothermal amplification on filters for quantification of Dehalobacter in groundwater.

Nucleic acid amplification of biomarkers is increasingly used to monitor microbial activity and assess remedial performance in contaminated aquifers. Previous studies described the use of filtration, elution, and direct isothermal amplification (i.e. no DNA extraction and purification) as a field-able means to quantify Dehalococcoides spp. in groundwater. This study expands previous work with direct loop mediated isothermal amplification (LAMP) for the detection and quantification of Dehalobacter spp. in groundwater. Experiments tested amplification of DNA with and without crude lysis and varying concentrations of humic acid. Three separate field-able methods of biomass concentration with eight aquifer samples were also tested, comparing direct LAMP with traditional DNA extraction and quantitative PCR (qPCR). A new technique was developed where filters were amplified directly within disposable Gene-Z chips. The direct filter amplification (DFA) method eliminated an elution step and provided a detection limit of 102Dehalobacter cells per 100mL. LAMP with crudely lysed Dehalobacter had a negligible effect on threshold time and sensitivity compared to lysed samples. The LAMP assay was more resilient than traditional qPCR to humic acid in sample, amplifying with up to 100mg per L of humic acid per reaction compared to 1mg per L for qPCR. Of the tested field-able concentrations methods, DFA had the lowest coefficient of variation among Dehalobacter spiked groundwater samples and lowest threshold time indicating high capture efficiency and low inhibition. While demonstrated with Dehalobacter, the DFA method can potentially be used for a number of applications requiring field-able, rapid (<60min) and highly sensitive quantification of microorganisms in environmental water samples.

The identification of carbamazepine biodegrading phylotypes and phylotypes sensitive to carbamazepine exposure in two soil microbial communities.

Carbamazepine (CBZ), an antiepileptic drug, has been introduced into agricultural soils via irrigation with treated wastewater and biosolids application. Such contamination is problematic because CBZ is persistent and the risks to ecosystems or human health are unknown. The current study examined CBZ biodegradation in two agricultural soils (soil 1 and 2) and the effects on the soil microbial communities during CBZ exposure. The experimental design involved three CBZ concentrations (50, 500, 5000ng/g), under aerobic as well as anaerobic conditions. CBZ concentrations were determined using solid phase extraction and LC MS/MS. The effect of CBZ on the soil microbial community was investigated using high throughput sequencing and a computational approach to predict functional composition of the metagenomes (phylogenetic investigation of communities by reconstruction of unobserved states, PICRUSt). The most significant CBZ biodegradation occurred in soil 1 under aerobic conditions. In contrast, CBZ biodegradation was limited under anaerobic conditions in soil 1 and under both conditions in soil 2. For soil 1, several phylotypes were enriched following CBZ degradation compared to the controls, including unclassified Sphingomonadaceae, Xanthomonadaceae and Rhodobacteraceae, as well as Sphingomonas, Aquicella and Microvirga. These phylotypes are considered putative CBZ degraders as they appear to be benefiting from CBZ biodegradation. PICRUSt revealed that soil 1 contained a greater abundance of xenobiotic degrading genes compared to soil 2, and thus, this analysis method offers a potential valuable approach for predicting CBZ attenuation in soils. PICRUSt analysis also implicated Sphingomonadaceae and Xanthomonadaceae in drug metabolism. Interestingly, numerous phylotypes decreased in abundance following CBZ exposure and these varied with soil type, concentration, duration of exposure, and the availability of oxygen. For three phylotypes (Flavobacterium, 3 genus incertae sedis and unclassified Bacteroidetes), the relative abundance was reduced in both soils, indicating a notable sensitivity to CBZ for these microorganisms.