An investigation of soil and groundwater metagenomes for genes encoding soluble and particulate methane monooxygenase, toluene-4-monoxygenase, propane monooxygenase and phenol hydroxylase.

Soil and groundwater were investigated for the genes encoding soluble and particulate methane monooxygenase/ammonia monooxygenase (sMMO, pMMO/AMO), toluene 4-monooxygenase (T4MO), propane monooxygenase (PMO) and phenol hydroxylase (PH). The objectives were (1) to determine which subunits were present, (2) to examine the diversity of the phylotypes associated with the biomarkers and (3) to identify which metagenome associated genomes (MAGs) contained these subunits. All T4MO and PH subunits were annotated in the groundwater metagenomes, while few were annotated in the soil metagenomes. The majority of the soil metagenomes included only four sMMO subunits. Only two groundwater metagenomes contained five sMMO subunits. Gene counts for the pMMO subunits varied between samples. The majority of the soil metagenomes were annotated for all four PMO subunits, while three out of eight groundwater metagenomes contained all four PMO subunits. A comparison of the blast alignments for the sMMO alpha chain (mmoX) indicated the phylotypes differed between the soil and groundwater metagenomes. For the pMMO/AMO alpha subunit (pmoA/amoA), Nitrosospira was important for the soil metagenomes, while Methylosinus and Methylocystis were dominant for the groundwater metagenomes. The majority of pmoA alignments from both metagenomes were from uncultured bacteria. High quality MAGs were obtained from the groundwater data. Four MAGs (Methylocella and Cypionkella) contained sMMO subunits. Another three MAGs, within the order Pseudomonadales, contained all three pMMO subunits. All PH subunits were detected in seven MAGs (Azonexus, Rhodoferax, Aquabacterium). In those seven, all contained catechol 2,3-dioxagenase, and Aquabacterium also contained catechol 1,2-dioxygenase. T4MO subunits were detected in eight MAGs (Azonexus, Rhodoferax, Siculibacillus) and all, except one, contained all six subunits. Four MAGs (Rhodoferax and Azonexus) contained all subunits for PH and T4MO, as well as catechol 2,3-dixoygenase. The detection of T4MO and PH in groundwater metagenomes and MAGs has important implications for the potential oxidation of groundwater contaminants.

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 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.

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.

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.

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.

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.

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.

Loop-Mediated Isothermal Amplification (LAMP) for Rapid Detection and Quantification of Dehalococcoides Biomarker Genes in Commercial Reductive Dechlorinating Cultures KB-1 and SDC-9.

Real-time quantitative PCR (qPCR) protocols specific to the reductive dehalogenase (RDase) genes vcrA, bvcA, and tceA are commonly used to quantify Dehalococcoides spp. in groundwater from chlorinated solvent-contaminated sites. In this study, loop-mediated isothermal amplification (LAMP) was developed as an alternative approach for the quantification of these genes. LAMP does not require a real-time thermal cycler (i.e., amplification is isothermal), allowing the method to be performed using less-expensive and potentially field-deployable detection devices. Six LAMP primers were designed for each of three RDase genes (vcrA, bvcA, and tceA) using Primer Explorer V4. The LAMP assays were compared to conventional qPCR approaches using plasmid standards, two commercially available bioaugmentation cultures, KB-1 and SDC-9 (both contain Dehalococcoides species). DNA was extracted over a growth cycle from KB-1 and SDC-9 cultures amended with trichloroethene and vinyl chloride, respectively. All three genes were quantified for KB-1, whereas only vcrA was quantified for SDC-9. A comparison of LAMP and qPCR using standard plasmids indicated that quantification results were similar over a large range of gene concentrations. In addition, the quantitative increase in gene concentrations over one growth cycle of KB-1 and SDC-9 using LAMP was comparable to that of qPCR. The developed LAMP assays for vcrA and tceA genes were validated by comparing quantification on the Gene-Z handheld platform and a real-time thermal cycler using DNA isolated from eight groundwater samples obtained from an SDC-9-bioaugmented site (Tulsa, OK). These assays will be particularly useful at sites subject to bioaugmentation with these two commonly used Dehalococcoides species-containing cultures.

Microbial community characterization and functional gene quantification in RDX-degrading microcosms derived from sediment and groundwater at two naval sites.

The explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) has long been recognized as a problematic environmental pollutant, and efforts to remediate contaminated soils, sediments, and groundwater have been going on for decades. In recent years, much interest has focused on using bioremediation to clean up these sites. The current study investigated the microorganisms (16S rRNA genes, Illumina) and functional genes (xenA, xenB, and xplA) linked to RDX biodegradation in microcosms composed of sediment or groundwater from two Navy sites. For this, experiments included sediment samples from three depths (5 to 30 ft) from two wells located in one Navy site. In addition, the groundwater upstream and downstream of an emulsified oil biobarrier was examined from another Navy site. Further, for the groundwater experiments, the effect of glucose addition was explored. For the sediment experiments, the most enriched phylotypes during RDX degradation varied over time, by depth and well locations. However, several trends were noted, including the enrichment of Pseudomonas, Rhodococcus, Arthrobacter, and Sporolactobacillus in the sediment microcosms. For the groundwater-based experiments, Pseudomonas, unclassified Rhodocyclaceae, Sphingomonas, and Rhodococcus were also highly abundant during RDX degradation. The abundance of both xplA and xenA significantly increased during RDX degradation compared to the control microcosms for many treatments (both groundwater and sediment microcosms). In a limited number of microcosms, the copy number of the xenB gene increased. Phylotype data were correlated with functional gene data to highlight potentially important biomarkers for RDX biodegradation at these two Navy sites.

Elucidating carbon uptake from vinyl chloride using stable isotope probing and Illumina sequencing.

Vinyl chloride (VC), a known human carcinogen, is a common and persistent groundwater pollutant at many chlorinated solvent contaminated sites. The remediation of such sites is challenging because of the lack of knowledge on the microorganisms responsible for in situ VC degradation. To address this, the microorganisms involved in carbon assimilation from VC were investigated in a culture enriched from contaminated site groundwater using stable isotope probing (SIP) and high-throughput sequencing. The mixed culture was added to aerobic media, and these were amended with labeled ((13)C-VC) or unlabeled VC ((12)C-VC). The cultures were sacrificed on days 15, 32, and 45 for DNA extraction. DNA extracts and SIP ultracentrifugation fractions were subject to sequencing as well as quantitative PCR (qPCR) for a functional gene linked to VC-assimilation (etnE). The gene etnE encodes for epoxyalkane coenzyme M transferase, a critical enzyme in the pathway for VC degradation. The relative abundance of phylotypes was compared across ultracentrifugation fractions obtained from the (13)C-VC- and (12)C-VC-amended cultures. Four phylotypes were more abundant in the heavy fractions (those of greater buoyant density) from the (13)C-VC-amended cultures compared to those from the (12)C-VC-amended cultures, including Nocardioides, Brevundimonas, Tissierella, and Rhodoferax. Therefore, both a previously identified VC-assimilating genus (Nocardioides) and novel microorganisms were responsible for carbon uptake. Enrichment of etnE with time was observed in the heavy fractions, and etnE sequences illustrated that VC-assimilators harbor similar Nocardioides-like etnE. This research provides novel data on the microorganisms able to assimilate carbon from VC.

A comparative study of microbial communities in four soil slurries capable of RDX degradation using illumina sequencing.

The nitramine explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) has contaminated many military sites. Recently, attempts to remediate these sites have focused on biostimulation to promote RDX biodegradation. Although many RDX degrading isolates have been obtained in the laboratory, little is known about the potential of microorganisms to degrade this chemical while existing in a soil community. The current study examined and compared the RDX degrading communities in four soil slurries to elucidate the potential of natural systems to degrade this chemical. These soils were selected as they had no previous exposure to RDX, therefore their microbial communities offered an excellent baseline to determine changes following RDX degradation. High throughput sequencing was used to determine which phylotypes experienced an increase in relative abundance following RDX degradation. For this, total genomic DNA was sequenced from (1) the initial soil, (2) soil slurry microcosms following RDX degradation and (3) control soil slurry microcosms without RDX addition. The sequencing data provided valuable information on which phylotypes increased in abundance following RDX degradation compared to control microcosms. The most notable trend was the increase in abundance of Brevundimonas and/or unclassified Bacillaceae 1 in the four soils studied. Although isolates of the family Bacillaceae 1 have previously been linked to RDX degradation, isolates of the genus Brevundimonas have not been previously associated with RDX degradation. Overall, the data suggest these two phylotypes have key roles in RDX degradation in soil communities.

The effect of the potential fuel additive isobutanol on benzene, toluene, ethylbenzene, and p-xylene degradation in aerobic soil microcosms.

Isobutanol is being considered as a fuel additive; however, the effect of this chemical on gasoline degradation (following a spill) has yet to be fully explored. To address this, the current study investigated the effect of isobutanol on benzene, toluene, ethylbenzene and p-xylene (BTEX) degradation in 14 sets of experiments in saturated soils. This involved four hydrocarbons for three soils (12 experiments) and two extra experiments with a lower level of isobutanol (for toluene only). Each soil and hydrocarbon combination involved four abiotic control microcosms and 12 sample microcosms (six with and six without isobutanol). The time for complete degradation of each hydrocarbon varied between treatments. Both toluene and ethylbenzene were rapidly degraded (5-13 days for toluene and 3-13 days for ethylbenzene). In contrast, the time for complete degradation for benzene ranged from 5 to 47 days. The hydrocarbon p-xylene was the most recalcitrant chemical (time for removal ranged from 14 to 86 days) and, in several microcosms, no p-xylene degradation was observed. The effect of isobutanol on hydrocarbon degradation was determined by comparing degradation lag times with and without isobutanol addition. From the 14 treatments, isobutanol only affected degradation lag times in three cases. In two cases (benzene and p-xylene), an enhancement of degradation (reduced lag times) was observed in the presence of isobutanol. In contrast, toluene degradation in one soil was inhibited (increased lag time). These results indicate that co-contamination with isobutanol should not inhibit aerobic BTEX degradation rates.

DNA extraction-free quantification of Dehalococcoides spp. in groundwater using a hand-held device.

Nucleic acid amplification of biomarkers is increasingly used to measure microbial activity and predict remedial performance in sites with trichloroethene (TCE) contamination. Field-based genetic quantification of microorganisms associated with bioremediation may help increase accuracy that is diminished through transport and processing of groundwater samples. Sterivex cartridges and a previously undescribed mechanism for eluting biomass was used to concentrate cells. DNA extraction-free loop mediated isothermal amplification (LAMP) was monitored in real-time with a point of use device (termed Gene-Z). A detection limit of 10(5) cells L(­1) was obtained, corresponding to sensitivity between 10 to 100 genomic copies per reaction for assays targeting the Dehalococcoides spp. specific 16S rRNA gene and vcrA gene, respectively. The quantity of Dehalococcoides spp. genomic copies measured from two TCE contaminated groundwater samples with conventional means of quantification including filtration, DNA extraction, purification, and qPCR was comparable to the field ready technique. Overall, this method of measuring Dehalococcoides spp. and vcrA genes in groundwater via direct amplification without intentional DNA extraction and purification is demonstrated, which may provide a more accurate mechanism of predicting remediation rates.

Presence, diversity and enumeration of functional genes (bssA and bamA) relating to toluene degradation across a range of redox conditions and inoculum sources.

The study investigates two functional genes for toluene degradation across three redox conditions (nitrate and sulfate amended and methanogenic). The genes targeted include benzylsuccinate synthase α-subunit (bssA) and a gene recently identified as being a strong indicator of anaerobic aromatic degradation, called 6-oxocylcohex-1-ene-1-carbonyl-CoA hydrolase (bamA). In all, sixteen different anaerobic toluene degrading microcosms were investigated using several primers sets targeting bssA and one primer set targeting bamA. One bssA primer set (7772f/8546r) was the most successful in producing a strong amplicon (eight from sixteen) with the other bssA primers sets producing strong amplicons in six or less samples. In contrast, the bamA primer set (bam-sp9 and bam-asp1) produced a strong amplicon in DNA extracted from all except one microcosm. Partial bssA and bamA sequences were obtained for a number of samples and compared to those available in GenBank. The partial bssA sequences (from nitrate amended and methanogenic microcosms) were most similar to Thauera sp. DNT-1, Thauera aromatica, Aromatoleum aromaticum EbN1 and bssA clones from a study involving sulfate reducing toluene degradation. The bamA sequences obtained could be placed into five previously defined clades (bamA-clade 1, Georgfuchsia/Azoarcus, Magnetospirillum/Thauera Syntrophus and Geobacter clades), with the placement generally depending on redox conditions. Gene numbers were also correlated with toluene degradation and the final gene number for both genes differed considerably between the range of redox conditions. The work is the first in depth investigation of bamA diversity over a range of redox conditions and inoculum sources.

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.

Effect of isobutanol on toluene biodegradation in nitrate amended, sulfate amended and methanogenic enrichment microcosms.

Isobutanol is an alternate fuel additive that is being considered because of economic and lower emission benefits. However, future gasoline spills could result in co-contamination of isobutanol with gasoline components such as benzene, toluene, ethyl-benzene and xylene. Hence, isobutanol could affect the degradability of gasoline components thereby having an effect on contaminant plume length and half-life. In this study, the effect of isobutanol on the biodegradation of a model gasoline component (toluene) was examined in laboratory microcosms. For this, toluene and isobutanol were added to six different toluene degrading laboratory microcosms under sulfate amended, nitrate amended or methanogenic conditions. While toluene biodegradation was not greatly affected in the presence of isobutanol in five out of the six different experimental sets, toluene degradation was completely inhibited in one set of microcosms. This inhibition occurred in sulfate amended microcosms constructed with inocula from wastewater treatment plant activated sludge. Our data suggest that toluene degrading consortia are affected differently by isobutanol addition. These results indicate that, if co-contamination occurs, in some cases the in situ half-life of toluene could be significantly extended.

Diversity of five anaerobic toluene-degrading microbial communities investigated using stable isotope probing.

Time-series DNA-stable isotope probing (SIP) was used to identify the microbes assimilating carbon from [(13)C]toluene under nitrate- or sulfate-amended conditions in a range of inoculum sources, including uncontaminated and contaminated soil and wastewater treatment samples. In all, five different phylotypes were found to be responsible for toluene degradation, and these included previously identified toluene degraders as well as novel toluene-degrading microorganisms. In microcosms constructed from granular sludge and amended with nitrate, the putative toluene degraders were classified in the genus Thauera, whereas in nitrate-amended microcosms constructed from a different source (agricultural soil), microorganisms in the family Comamonadaceae (genus unclassified) were the key putative degraders. In one set of sulfate-amended microcosms (agricultural soil), the putative toluene degraders were identified as belonging to the class Clostridia (genus Desulfosporosinus), while in other sulfate-amended microcosms, the putative degraders were in the class Deltaproteobacteria, within the family Syntrophobacteraceae (digester sludge) or Desulfobulbaceae (contaminated soil) (genus unclassified for both). Partial benzylsuccinate synthase gene (bssA, the functional gene for anaerobic toluene degradation) sequences were obtained for some samples, and quantitative PCR targeting this gene, along with SIP, was further used to confirm anaerobic toluene degradation by the identified species. The study illustrates the diversity of toluene degraders across different environments and highlights the utility of ribosomal and functional gene-based SIP for linking function with identity in microbial communities.

Anaerobic methyl tert-butyl ether-degrading microorganisms identified in wastewater treatment plant samples by stable isotope probing.

Anaerobic methyl tert-butyl ether (MTBE) degradation potential was investigated in samples from a range of sources. From these 22 experimental variations, only one source (from wastewater treatment plant samples) exhibited MTBE degradation. These microcosms were methanogenic and were subjected to DNA-based stable isotope probing (SIP) targeted to both bacteria and archaea to identify the putative MTBE degraders. For this purpose, DNA was extracted at two time points, subjected to ultracentrifugation, fractioning, and terminal restriction fragment length polymorphism (TRFLP). In addition, bacterial and archaeal 16S rRNA gene clone libraries were constructed. The SIP experiments indicated bacteria in the phyla Firmicutes (family Ruminococcaceae) and Alphaproteobacteria (genus Sphingopyxis) were the dominant MTBE degraders. Previous studies have suggested a role for Firmicutes in anaerobic MTBE degradation; however, the putative MTBE-degrading microorganism in the current study is a novel MTBE-degrading phylotype within this phylum. Two archaeal phylotypes (genera Methanosarcina and Methanocorpusculum) were also enriched in the heavy fractions, and these organisms may be responsible for minor amounts of MTBE degradation or for the uptake of metabolites released from the primary MTBE degraders. Currently, limited information exists on the microorganisms able to degrade MTBE under anaerobic conditions. This work represents the first application of DNA-based SIP to identify anaerobic MTBE-degrading microorganisms in laboratory microcosms and therefore provides a valuable set of data to definitively link identity with anaerobic MTBE degradation.