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.

Demonstration of combined zero-valent iron and electrical resistance heating for in situ trichloroethene remediation.

The effectiveness of in situ treatment using zero-valent iron (ZVI) for nonaqueous phase or significant sediment-associated contaminant mass can be limited by relatively low rates of mass transfer to bring contaminants in contact with the reactive media. For a field test in a trichloroethene (TCE) source area, combining moderate-temperature subsurface electrical resistance heating with in situ ZVI treatment was shown to accelerate TCE treatment by a factor of about 4 based on organic daughter products and a factor about 8 based on chloride concentrations. A mass-discharge-based analysis was used to evaluate reaction, dissolution, and volatilization processes at ambient groundwater temperature (~10 °C) and as temperature was increased up to about 50 °C. Increased reaction and contaminant dissolution were observed with increased temperature, but vapor- or aqueous-phase migration of TCE out of the treatment zone was minimal during the test because reactions maintained low aqueous-phase TCE concentrations.

Spatially-distinct redox conditions and degradation rates following field-scale bioaugmentation for RDX-contaminated groundwater remediation.

In situ bioaugmentation for cleanup of an hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)-contaminated groundwater plume was recently demonstrated. Results of a forced-gradient, field-scale cell transport test with Gordonia sp. KTR9 and Pseudomonas fluorescens strain I-C cells (henceforth "KTR9" and "Strain I-C") showed these strains were transported 13 m downgradient over 1 month. Abundances of xplA and xenB genes, respective indicators of KTR9 and Strain I-C, approached injection well cell densities at 6 m downgradient, whereas gene abundances (and conservative tracer) had begun to increase at 13 m downgradient at test conclusion. In situ push-pull tests were subsequently completed to measure RDX degradation rates in the bioaugmented wells under ambient gradient conditions. Time-series monitoring of RDX, RDX end-products, conservative tracer, xplA and xenB gene copy numbers and XplA and XenB protein abundance were used to assess the efficacy of bioaugmentation and to estimate the apparent first-order RDX degradation rates during each test. A collective evaluation of redox conditions, RDX end-products, varied RDX degradation kinetics, and biomarkers indicated that Strain I-C and KTR9 rapidly degraded RDX. Results showed bioaugmentation is a viable technology for accelerating RDX cleanup in the demonstration site aquifer and may be applicable to other sites. Full-scale implementation considerations are discussed.

A thermodynamically-based model for predicting microbial growth and community composition coupled to system geochemistry: Application to uranium bioreduction.

'Bioimmobilization' of redox-sensitive heavy metals and radionuclides is being investigated as a way to remediate contaminated groundwater and sediments. In one approach, growth-limiting substrates are added to the subsurface to stimulate the activity of targeted groups of indigenous microorganisms and create conditions favorable for the microbially-mediated reductive precipitation ('bioreduction') of targeted contaminants. We present a theoretical framework for modeling this process that modifies conventional geochemical reaction path modeling to include thermodynamic descriptions for microbial growth and may be called biogeochemical reaction path modeling. In this approach, the actual microbial community is represented by a synthetic microbial community consisting of a collection of microbial groups; each with a unique growth equation that couples a specific pair of energy yielding redox reactions. The growth equations and their computed standard-state free energy yields are appended to the thermodynamic database used in conventional geochemical reaction path modeling, providing a direct coupling between chemical species participating in both microbial growth and geochemical reactions. To compute the biogeochemical reaction paths, growth substrates are reacted incrementally with the defined geochemical environment and the coupled equations are solved simultaneously to predict reaction paths that display changing microbial biomass, community composition (i.e. the fraction of total biomass in each microbial group), and the aqueous and mineral composition of the system, including aqueous speciation and oxidation state of the targeted contaminants. The approach, with growth equations derived from the literature using well-known bioenergetics principles, was used to predict the results of a laboratory microcosm experiment and an in situ field experiment that investigated the bioreduction of uranium. Predicted effects of ethanol or acetate addition on uranium concentration and speciation, major ion geochemistry, mineralogy, microbial biomass and community composition were in qualitative agreement with experimental observations although the available data precluded rigorous model testing. While originally developed for use in better understanding of bioimmobilization of heavy metals and radionuclides, the modeling approach is potentially useful for exploring the coupling of microbial growth and geochemical reactions in a variety of other basic and applied biotechnology research settings.