RDX degradation in bioaugmented model aquifer columns under aerobic and low oxygen conditions.

Degradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in laboratory columns following biostimulation and bioaugmentation was investigated using sediment and groundwater from a contaminated aquifer at a US Navy facility. No RDX degradation was observed following aerobic biostimulation with either fructose or lactate (both 0.1 mM) prior to bioaugmentation. Replicate columns were then bioaugmented with either Gordonia sp. KTR9, Pseudomonas fluorescens I-C (Ps I-C), or both strains. Under aerobic conditions (influent dissolved oxygen (DO) >6 mg/L), RDX was degraded following the addition of fructose, and to a lesser extent with lactate, in columns bioaugmented with KTR9. No degradation was observed in columns bioaugmented with only Ps I-C under aerobic conditions, consistent with the known anaerobic RDX degradation pathway for this strain. When influent DO was reduced to <2 mg/L, good RDX degradation was observed in the KTR9-bioaugmented column, and some degradation was also observed in the Ps I-C-bioaugmented column. After DO levels were kept below 1 mg/L for more than a month, columns bioaugmented with KTR9 became unresponsive to fructose addition, while RDX degradation was still observed in the Ps I-C-bioaugmented columns. These results indicate that bioaugmentation with the aerobic RDX degrader KTR9 could be effective at sites where site geology or geochemistry allow higher DO levels to be maintained. Further, inclusion of strains capable of anoxic RDX degradation such as Ps I-C may facilitate bimodal RDX removal when DO levels decrease.

Evaluation of Biostimulation and Bioaugmentation To Stimulate Hexahydro-1,3,5-trinitro-1,3,5,-triazine Degradation in an Aerobic Groundwater Aquifer.

Hexahydro-1,3,5-trinitro-1,3,5,-triazine (RDX) is a toxic and mobile groundwater contaminant common to military sites. This study compared in situ RDX degradation rates following bioaugmentation with Gordonia sp. strain KTR9 (henceforth KTR9) to rates under biostimulation conditions in an RDX-contaminated aquifer in Umatilla, OR. Bioaugmentation was achieved by injecting site groundwater (6000 L) amended with KTR9 cells (10(8) cells mL(-1)) and low carbon substrate concentrations (<1 mM fructose) into site wells. Biostimulation (no added cells) was performed by injecting groundwater amended with low (<1 mM fructose) or high (>15 mM fructose) carbon substrate concentrations in an effort to stimulate aerobic or anaerobic microbial activity, respectively. Single-well push-pull tests were conducted to measure RDX degradation rates for each treatment. Average rate coefficients were 1.2 day(-1) for bioaugmentation and 0.7 day(-1) for high carbon biostimulation; rate coefficients for low carbon biostimulation were not significantly different from zero (p values ≥0.060). Our results suggest that bioaugmentation with KTR9 is a feasible strategy for in situ biodegradation of RDX and, at this site, is capable of achieving RDX concentration reductions comparable to those obtained by high carbon biostimulation while requiring ~97% less fructose. Bioaugmentation has potential to minimize substrate quantities and associated costs, as well as secondary groundwater quality impacts associated with anaerobic biostimulation processes (e.g., hydrogen sulfide, methane production) during full-scale RDX remediation.

Relating Carbon and Nitrogen Isotope Effects to Reaction Mechanisms during Aerobic or Anaerobic Degradation of RDX (Hexahydro-1,3,5-Trinitro-1,3,5-Triazine) by Pure Bacterial Cultures.

UNLABELLED: Kinetic isotopic fractionation of carbon and nitrogen during RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) biodegradation was investigated with pure bacterial cultures under aerobic and anaerobic conditions. Relatively large bulk enrichments in (15)N were observed during biodegradation of RDX via anaerobic ring cleavage (ε(15)N = -12.7‰ ± 0.8‰) and anaerobic nitro reduction (ε(15)N = -9.9‰ ± 0.7‰), in comparison to smaller effects during biodegradation via aerobic denitration (ε(15)N = -2.4‰ ± 0.2‰). (13)C enrichment was negligible during aerobic RDX biodegradation (ε(13)C = -0.8‰ ± 0.5‰) but larger during anaerobic degradation (ε(13)C = -4.0‰ ± 0.8‰), with modest variability among genera. Dual-isotope ε(13)C/ε(15)N analyses indicated that the three biodegradation pathways could be distinguished isotopically from each other and from abiotic degradation mechanisms. Compared to the initial RDX bulk δ(15)N value of +9‰, δ(15)N values of the NO2 (-) released from RDX ranged from -7‰ to +2‰ during aerobic biodegradation and from -42‰ to -24‰ during anaerobic biodegradation. Numerical reaction models indicated that N isotope effects of NO2 (-) production were much larger than, but systematically related to, the bulk RDX N isotope effects with different bacteria. Apparent intrinsic ε(15)N-NO2 (-) values were consistent with an initial denitration pathway in the aerobic experiments and more complex processes of NO2 (-) formation associated with anaerobic ring cleavage. These results indicate the potential for isotopic analysis of residual RDX for the differentiation of degradation pathways and indicate that further efforts to examine the isotopic composition of potential RDX degradation products (e.g., NOx) in the environment are warranted. IMPORTANCE: This work provides the first systematic evaluation of the isotopic fractionation of carbon and nitrogen in the organic explosive RDX during degradation by different pathways. It also provides data on the isotopic effects observed in the nitrite produced during RDX biodegradation. Both of these results could lead to better understanding of the fate of RDX in the environment and help improve monitoring and remediation technologies.

Application of (13)C and (15)N stable isotope probing to characterize RDX degrading microbial communities under different electron-accepting conditions.

This study identified microorganisms capable of using the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) or its metabolites as carbon and/or nitrogen sources under different electron-accepting conditions using (13)C and (15)N stable isotope probing (SIP). Mesocosms were constructed using groundwater and aquifer solids from an RDX-contaminated aquifer. The mesocosms received succinate as a carbon source and one of four electron acceptors (nitrate, manganese(IV), iron(III), or sulfate) or no additional electron acceptor (to stimulate methanogenesis). When RDX degradation was observed, subsamples from each mesocosm were removed and amended with (13)C3- or ring-(15)N3-, nitro-(15)N3-, or fully-labeled (15)N6-RDX, followed by additional incubation and isolation of labeled nucleic acids. A total of fifteen 16S rRNA sequences, clustering in α- and γ-Proteobacteria, Clostridia, and Actinobacteria, were detected in the (13)C-DNA fractions. A total of twenty seven sequences were derived from different (15)N-DNA fractions, with the sequences clustered in α- and γ-Proteobacteria, and Clostridia. Interestingly, sequences identified as Desulfosporosinus sp. (in the Clostridia) were not only observed to incorporate the labeled (13)C or (15)N from labeled RDX, but also were detected under each of the different electron-accepting conditions. The data suggest that (13)C- and (15)N-SIP can be used to characterize microbial communities involved in RDX biodegradation, and that the dominant pathway of RDX biodegradation may differ under different electron-accepting conditions.

Laboratory evaluation of bioaugmentation for aerobic treatment of RDX in groundwater.

The potential for bioaugmentation with aerobic explosive degrading bacteria to remediate hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) contaminated aquifers was demonstrated. Repacked aquifer sediment columns were used to examine the transport and RDX degradation capacity of the known RDX degrading bacterial strains Gordonia sp. KTR9 (modified with a kanamycin resistance gene) Pseudomonas fluorescens I-C, and a kanamycin resistant transconjugate Rhodococcus jostii RHA1 pGKT2:Km+. All three strains were transported through the columns and eluted ahead of the conservative bromide tracer, although the total breakthrough varied by strain. The introduced cells responded to biostimulation with fructose (18 mg L(-1), 0.1 mM) by degrading dissolved RDX (0.5 mg L(-1), 2.3 µM). The strains retained RDX-degrading activity for at least 6 months following periods of starvation when no fructose was supplied to the column. Post-experiment analysis of the soil indicated that the residual cells were distributed along the length of the column. When the strains were grown to densities relevant for field-scale application, the cells remained viable and able to degrade RDX for at least 3 months when stored at 4 °C. These results indicate that bioaugmentation may be a viable option for treating RDX in large dilute aerobic plumes.

Large-scale production of bacterial consortia for remediation of chlorinated solvent-contaminated groundwater.

Chlorinated solvents such as perchloroethylene (PCE) and trichloroethylene (TCE) continue to be significant groundwater contaminants throughout the USA. In many cases efficient bioremediation of aquifers contaminated with these chemicals requires the addition of exogenous microorganisms, specifically members of the genus Dehalococcoides (DHC). This process is referred to as bioaugmentation. In this study a fed-batch fermentation process was developed for producing large volumes (to 3,200 L) of DHC-containing consortia suitable for treating contaminated aquifers. Three consortia enriched from three different sites were grown anaerobically with sodium lactate as an electron donor and PCE or TCE as an electron acceptor. DHC titers in excess of 10(11) DHC/L could be reproducibly obtained at all scales tested and with all three of the enrichment cultures. The mean specific DHC growth rate for culture SDC-9 was 0.036 +/- 0.005 (standard error, SE)/h with a calculated mean doubling time of 19.3 +/- 2.7 (SE) h. Finished cultures could be concentrated approximately tenfold by membrane filtration and stored refrigerated (4 degrees C) for more that 40 days without measurable loss of activity. Dehalogenation of PCE by the fermented cultures was affected by pH with no measurable activity at pH <5.0.

Bioaugmentation for chlorinated ethenes using Dehalococcoides sp.: comparison between batch and column experiments.

Batch and column experiments were performed to evaluate the transport, growth and dechlorination activity of Dehalococcoides sp. (DHC) during bioaugmentation for chlorinated ethenes. Batch experiments showed that the reductive dechlorination of trichloroethene (TCE), cis-1,2-dichloroethene (DCE), and vinyl chloride (VC), as well as growth of the DHC, were well described by the Monod kinetic model. The measured maximum utilization rate coefficients for TCE, DCE, and VC were 1.3x10(-12), 5.2x10(-13), and 1.4x10(-12)mmol Cl(-) (cellh)(-1), respectively. Results of the column experiments showed that dechlorination occurred throughout the length of the column, and that extractable DHC concentrations associated with the soil phase throughout the column were negligible relative to the aqueous phase concentrations. Dechlorination rates relative to aqueous DHC concentrations in the column were approximately 200-times greater than in the batch experiments. Additional batch experiments performed using column effluent water confirmed this result. Incorporation of these enhanced dechlorination kinetics in the transport model provided a reasonable prediction of the column data. Overall results of this study suggest that aqueous phase (as opposed to soil phase) DHC concentrations can be used to estimate dechlorination activity in saturated soils, and DHC dechlorination activity in porous media may be substantially greater than DHC dechlorination activity measured in batch experiments.