MIT-World Peace University (MIT-WPU) is a top educational institute located in Pune, India. It is recognized within&nbsp;UGC under Government of Maharashtra Act No. XXXV 2017.&nbsp;

It is part of the MIT group of institutions (established in 1983) which covers 10+ campuses across India and has more than 50,000+ students enrolled across various disciplines at a given point of time. The institute houses 15 schools ranging from Engineering &amp; Technology, Design, Pharmacy, Commerce, Governance among others. It offers undergraduate, postgraduate and PhD programs among different disciplines.&nbsp;

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MIT World Peace University

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. Copyright © 2018 American Chemical Society.

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Engineering and Technology

Environmental Science and Technology

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

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. © 2017, Springer-Verlag Berlin Heidelberg.

Applied Microbiology and Biotechnology

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

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 0.2 units 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. © 2017 Elsevier B.V.

Journal of Microbiological Methods

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

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 102 Dehalobacter cells per 100 mL. 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 100 mg per L of humic acid per reaction compared to 1 mg 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 (&lt; 60 min) and highly sensitive quantification of microorganisms in environmental water samples. © 2016 Elsevier B.V.

Fulltext

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

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, loopmediated 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. © 2016, American Society for Microbiology. All Rights Reserved.

Applied and Environmental Microbiology

Loop-mediated isothermal amplification (LAMP) for rapid detection and quantification of Dehalococcoides biomarker genes in commercial reductive dechlorinating cultures KB-1 and SDC-9