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FINAL REPORT Groundwater Chemistry and Microbial Ecology Effects on Explosives Biodegradation

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FINAL REPORT Groundwater Chemistry and Microbial Ecology Effects on Explosives Biodegradation FINAL REPORT Groundwater Chemistry and Microbial Ecology Effects on Explosives Biodegradation SERDP Project ER-1378 SEPTEMBER 2008 Dr. Mark E. Fuller Dr. Robert J. Steffan Shaw Environmental, Inc. This report was prepared under contract to the Department of Defense Strategic Environmental Research and Development Program (SERDP). The publication of this report does not indicate endorsement by the Department of Defense, nor should the contents be construed as reflecting the official policy or position of the Department of Defense. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the Department of Defense. Final Report Table of Contents List of Abbreviations ····················································································································ii List of Tables ·······························································································································iv List of Figures·····························································································································vii Acknowledgements·······················································································································x I. EXECUTIVE SUMMARY ·······································································································1 II. PROJECT OBJECTIVES·········································································································3 III. PROJECT BACKGROUND···································································································6 IV. PROJECT ACCOMPLISHMENTS SUMMARY·································································12 1. BACTERIAL STRAINS AND PRIMERS. ............................................................................ 12 1.1 IDENTIFYING GENES OF INTEREST AND DEVELOPING PRIMERS ....................... 12 1.2 SEQUENCING OF KNOWN DEGRADATIVE STRAINS ................................................ 16 1.3 DEGRADATION OF RDX BY KNOWN DEGRADATIVE STRAINS............................... 16 1.4 SELECTION OF UNIVERSAL PRIMER SET FOR DGGE ............................................. 18 2. DEVELOPMENT OF MOLECULAR PROTOCOLS. .......................................................... 20 2.1 PROCEDURES TO ADDRESS AND MINIMIZE CONTAMINATION............................ 20 2.2 DNA EXTRACTION FROM GROUNDWATER ............................................................... 22 2.3 OPTIMIZATION OF MOLECULAR TECHNIQUES....................................................... 25 3. APPLICATION OF MOLECULAR PROTOCOLS. ............................................................. 36 3.1 WEST VIRGINIA ORDNANCE WORKS (WVOW)........................................................... 36 3.2 PICATINNY ARSENAL..................................................................................................... 56 3.3 MOLECULAR ANALYSIS OF GROUNDWATER FROM MULTIPLE SITES ................ 84 4. STABLE ISOTOPE PROBING.............................................................................................. 93 4.1 METHOD DEVELOPMENT ............................................................................................ 94 4.2 SIP OF COLUMN EFFLUENT AND GROUNDWATER SAMPLES............................... 97 V. CONCLUSIONS··················································································································106 VI. REFERENCES ···················································································································110 Appendix 1 – Draft manuscripts with additional experimental details. Appendix 2 – Technical presentations. Appendix 3 – Standard protocols for DNA collection, PCR, and DGGE. i Final Report List of Abbreviations BLAST basic local alignment search tool BSA bovine serum albumin BSM basal salts medium 12C carbon (normal isotope signature) 13C stable carbon isotope CsCl2 cesium chloride CW cheese whey 2,4-DNT / 2,6-DNT 2,4- and 2,6-dinitrotoluene 2A-DNT/4A-DNT 2-amino-4,6- and 4-amino-2,6-dinitrotoluene DGGE denaturing gradient gel electrophoresis Dicumarol 3,3’-methylene-bis(4-hydroxycoumarin) DNA deoxyribonucleic acid DNX hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine DO dissolved oxygen DoD Department of Defense EPA Environmental Protection Agency EOD explosive ordnance disposal GW groundwater HA health advisory HMX octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine HPLC high performance liquid chromatography Kow soil organic water partition coefficient MMR Massachusetts Military Reservation MNX hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine N nitrogen 15N stable nitrogen isotope NCBI National Center for Biotechnology Information NDAB 4-nitro-2,4-diazabutanal NG nitroglycerine (glycerol trinitrate) NH4 ammonia NH4Cl ammonium chloride NOP Nebraska Ordnance Plant OB/OD open burn/open detonation ORP oxidation-reduction potential PBS phosphate-buffered saline PCB polychlorinated biphenyl PCD Pueblo Chemical Depot PCR polymerase chain reaction PETN pentaerythritoltetranitrate PVDF polyvinylidine fluoride qPCR quantitative polymerase chain reaction RDX hexahydro-1,3,5-trinitro-1,3,5-triazine SAB Scientific Advisory Board ii Final Report SERDP Strategic Environmental Research and Development Program SIP stable isotope probing tetryl 2,4,6-trinitrophenylmethylnitramine TKN Total Kjeldahl Nitrogen TNT 2,4,6-trinitrotoluene TNX hexahydro-1,3,5-trinitroso-1,3,5-triazine tRFLP terminal restriction fragment length polymorphism UXO unexploded ordnance WVOW West Virginia Ordnance Works YE yeast extract iii Final Report List of Tables Table II-1. Explosive compounds found in the soil, sediment, and groundwater at DoD installations that that were examined during this research. .................................................... 7 Table II-2. Bacterial strains with known abilities to degrade RDX................................................ 9 Table 1.1-1. Bacterial strains and/or genes that were selected for study for this project. ............ 12 Table 1.1-2. Primers designed to amplify genes associated with explosive biotransformation. .. 13 Table 1.2-1. In-house 16S rRNA sequences of known explosive-degrading strains.................... 14 Table 1.2-2. Identification of known degradative strains based on in-house DGGE and sequencing............................................................................................................................. 15 Table 1.3-1. Screening of known degradative strains for RDX degradation under various conditions.............................................................................................................................. 17 Table 1.4-1. Universal primers evaluated for use during DGGE. ................................................ 19 Table 1.4-2. Evaluation of bias/selectivity of universal primers. ................................................. 19 Table 2.1-1. Identification of contaminants that appeared in negative controls........................... 21 Table 2.2-1. Reproducibility of DNA extraction from groundwater............................................ 25 Table 2.3.3-1. Reproducibility of internal PCR/DGGE and external DNA sequencing. ............. 31 Table 3.1-1. Field sample information for WVOW site sediment cores. ..................................... 38 Table 3.1-2. Recipes for the two WVOW artificial groundwaters. .............................................. 39 Table 3.1-4. Identification of bands from initial WVOW DGGE analysis. ................................. 51 Table 3.1-5. Identification of bands from subsequent WVOW DGGE analysis.......................... 52 Table 3.1-6. Description of the genera detected in the WVOW enrichments. ............................. 55 Table 3.1-7. Detection of putative RDX-degradative genes in the WVOW enrichments............ 56 Table 3.2.1-1. Identification of bands from initial molecular analysis of Picatinny Arsenal column effluent. .................................................................................................................... 61 Table 3.2.1-2. Identification of bands from molecular analysis of Picatinny Arsenal column effluent. ................................................................................................................................. 64 Table 3.2.1-3. Description of the genera detected in Picatinny Arsenal column effluent. ........... 66 iv Final Report Table 3.2.1-4. Results of screening for putative RDX-degradative genes in Picatinny Arsenal column effluent. .................................................................................................................... 67 Table 3.2.2-1. Identities of Picatinny Arsenal column and Picatinny Arsenal groundwater isolates based on sequencing of 16S rRNA genes. ........................................................................... 69 Table 3.2.2-2. Degradation of RDX by isolates from Picatinny Arsenal columns and Picatinny Arsenal groundwater............................................................................................................. 71 Table 3.2.3-1. Degradation of RDX in enrichments derived from Picatinny Arsenal column effluent. ................................................................................................................................. 75 Table 3.2.3-2. Degradation of RDX in enrichments derived from Picatinny Arsenal groundwater.
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