Development of a Viable Fluorescent Stain Method for Monitoring Bacterial Transport

Fuller et al.Aerobic RDX bioaugmentation1

Laboratory evaluation of bioaugmentation for aerobic treatment of RDX in groundwater

Mark E. Fuller1*, Paul B. Hatzinger1, Charles W. Condee1, Christina Andaya1, Mandy M. Michalsen2, Fiona H. Crocker3, Karl J. Indest3, Carina M. Jung3, Dawn E. Hancock3, and Jonathan D. Istok4

SUPPLEMENTAL INFORMATION

Strain Selection

The aerobic conditions of the UMCD aquifer are likely to support the activity of the aerobic actinomycete bacteria that degrade RDX via the denitration pathway using the XplA/XplB enzymes (1-3). Nitrate concentrations above 4 mM and microaerophilic to anaerobic conditions inhibit the rates of RDX degradation in these strains (1-4). For this reason, and because spatially variable redox conditions may be created in the aquifer following substrate additions (e.g. due to preferential flow of added substrate in the heterogeneous aquifer), we included two facultative anaerobic bacteria that degrade RDX via xenobiotic reductases under microaerophilic to anoxic conditions, and are not inhibited by nitrate in our strain selection (5).

Cell suspension studies.The growth of seven aerobic and two facultative anaerobic candidate microbial strains (Table S3) was optimized by varying the type and concentration of nine carbon and four nitrogen sources.Fructose at 50 mM and ammonium sulfate at 18 mM provided the optimal growth conditions that would support maximum biomass yields necessary for large scale production of culture inoculum for field demonstration phases.

The nine strains were then examined for their ability to survive and degrade RDX in UMCD groundwater. Following growth in the optimal medium, the cells were washed once with AGW and resuspended in AGW to an absorbance of 1.0 (600 nm). The cultures were starved for 24 h at 15°C to reduce residual nitrogen levels and then RDX and fructose dissolved in AGW were added to achieve final concentration of 5.5 μM (1.2 mg/L) and 1 mM, respectively. Cell viability and RDX concentrations were monitored periodically. Strains KTR9, RHA1 pGKT2 and I-C maintained 100% of cell viability and RDX was completely degraded in 1 day with KTR9 and RHA1 and 91% degraded by I-C in 7 days (Figure S1). These three strains did not clump during the starvation and so these strains were selected for bioaugmentation in UMCD sediment microcosms.

Microcosm testing. Microcosms were constructed in 20 mL vials, and consisted of 2 g of UMCD sediment (2 mm sieved) plus 1 ml of AGW amended with RDX at 1.1 mg L-1 (5.5 µM), 1 mM fructose (180 mg L-1) and 1 x 106 cells ml-1 of each bacterial culture. Bacterial cultures were starved in AGW for 24 h at 15°C before inoculation. Uninoculated microcosms were prepared without the addition of cells. Microcosms were incubated at 15°C and three replicates of each treatment were periodically sacrificed for analysis of RDX concentrations and cell viability. The UMCD sediment initially contained a viable population of 102 to 103 cells ml-1 that increased in by two to three orders of magnitude during the study (Figure S2). Strains KTR9, RHA1, and I-C survived and grew by an order of magnitude in the sediment microcosms (Figure S2), and were the dominant cultures in the microcosms.All of the RDX was consumed within 1 day in the inoculated microcosms but was much slower in the uninoculated microcosms RDX. After a lag period of 4 days, approximately 15% of the RDX was degraded in the uninoculated microcosms (Figure S3). The three strains survived and remained active in UMCD sediments for 7 days and bioaugmentation of UMCD sediment with RDX‐degrading bacteria stimulated the rapid degradation of RDX.

Based on these results, strains KTR9, RHA1, and I-C were selected for the repacked column experiments.

REFERENCES

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2. Thompson, K.T., F.H. Crocker, and H.L. Fredrickson, Mineralization of the cyclic nitramine explosive hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine by Gordonia and Williamsia spp.Applied and Environmental Microbiology, 2005. 71(8265‐8272).

3. Coleman, N.V., D.R. Nelson, and T. Duxbury, Aerobic biodegradation of hexahydro‐1,3,5‐ trinitro‐1,3,5‐triazine (RDX) as a nitrogen source by a Rhodococcus sp., strain DN22. Soil Biology & Biochemistry, 1998. 30(8‐9): p. 1159‐1167.

4. Fuller, M.E., J. Hawari, and N. Perreault, Microaerophilic degradation of hexahydro‐ 1,3,5‐trinitro‐1,3,5‐triazine (RDX) by three Rhodococcus strains. Letters In Applied Microbiology, 2010. 51(3): p. 313‐318.

5. Fuller, M.E., et al., Transformation of RDX and other energetic compounds by xenobiotic reductases XenA and XenB. Applied Microbiology and Biotechnology, 2009. 84(3): p.

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Table S1. Geochemical analysis of UMCD groundwater and recipe for UMCD artificial groundwater.

Table S2. Particle size analysis of UMCD aquifer sediment used in the repacked columns.

Table S3.List of all RDX‐degrading microbial strains included in the initial screening.

Table S4. Primers used to quantify genes specific to inoculum strains

Crocker, F.H. et al. 2012 Identification of Microbial Gene Biomarkersfor in situ RDX Biodegradation.Strategic Environmental Research and Development Program, Project ER-1609, Final Report. Technical Report ERDC/EL TR-12-33. Vicksburg, MS: U.S.Army Engineer Research and Development Center.

Dionisi, H.M. et al. 2003. Power analysis for real-time PCR quantification of genes in activated sludge and analysis of the variability introduced by DNA extraction. Applied and Environmental Microbiology 69:6597-6604.

Fuller, M. E., K. McClay, M. Higham, P. B. Hatzinger, and R. J. Steffan. 2010. Hexahydro-

1,3,5-trinitro-1,3,5-triazine (RDX) bioremediation in groundwater: are known

RDX-degrading bacteria the dominant players? Bioremed J 14: 121-134.

FAM: 6-carboxyfluorescein; TAMRA: carboxytetramethylrhodamine
Figure S1.Survival of RDX‐degrading strains in artificial groundwater amended with RDX (5.5 µM) and fructose (1 mM). Panel A: KTR9 ( ); RHA1 ( ); I-C ( ); IIB ( ). Panel B: KTR4 ( ); DN22 ( ); 11Y ( ); G. polyisoprenivorans pGKT2 ( ); Nocardiodes TW2 pGKT2 ( ).

Figure S2. Bacterial viable cell numbers in UMCD microcosms on LB + 50 µg/L kanamycin agar plates (A) or cetrimide:nalidixic acid agar plates (B) from uninoculated and inoculated UMCD microcosms. Bacterial counts from the uninoculated microcosms are total bacterial counts on each medium (). Bacterial counts from the inoculated microcosms represent the individual strains: KTR9 Km+ (), RHA1 pGKT2 (), or I-C () as indicated in each panel.

Figure S3. RDX concentrations in UMCD uninoculated microcosms () ormicrocosms inoculated with strains KTR9 Km+, RHA1 pGKT2, andI‐C ().

Figure S4. Illustration of the column setup.

Figure S5. RDX concentrations (□) in the effluent of repacked column during biostimulation with fructose before bioaugmentation (Phase 2). Start and end of the fructose additions are indicated by the dashed vertical lines (---).

Figure S6. RDX concentrations (as C/C0) in a parallel repacked column experiment run for a longer duration. Column was initially bioaugmented with 2 PV of approximately 108 cells/ml of each of the three RDX degrading strains. The column was operated under flow velocities of both 1 and 10 ft/d and with periodic biostimulation with fructose (conditions as denoted in the figure legend).

Figure S7. Change in culture densities (relative to the initial OD550) of pilot-scale cultures of the three RDX degrading strains (A, KTR9; B, RHA1; C, IC) over time during incubation at 4°C (solid lines) and 37°C (dashed lines).