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Adapted with permission from: Chandler, D.P., A. Kukhtin, R. Mokhiber, C. Kinckerbocker, D. Ogles, G. Rudy, J. Golova, P. Long and A. D. Peacock. 2010. Monitoring Microbial Community Structure and Dynamics during in situ U(VI) Bioremediation with a Field-Portable Microarray Analysis System, Environmental Science & Technology. 44:5516-5522. Copyright 2010 American Chemical Society.
The Rifle IFRC is funded by the U.S. Department of Energy, Office of Science, Biological and Environmental Research, Climate and Environmental Science Division, Subsurface Biogeochemistry Program. The Rifle IFRC web site includes a list of publications based on research at the Rifle IFRC.
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The Old Rifle Uranium Mill Tailings Remedial Action (UMTRA) Project site is a former ore processing facility located east of the city of Rifle in Garfield County, Colorado. Figure A.10.1 is a site location map. The site is located adjacent to the Colorado River. The site is bounded to the north, west and east by steep upward slopes of sedimentary rocks belonging to the Wasatch Formation and to the south by a steep downward slope to the river. Remediation was required for the uranium mill tailings and other radioactive material associated with the former operations. The U.S. Department of Energy (DOE) completed surface remediation at the site and the contaminated materials were taken to the Estes Gulch disposal cell. The surface remediation was conducted from 1992 to 1996. The cleanup level used for the remediation was 15 pico curies/gram Radium 226 + 228. The National Regulatory Commission concurred that the site was cleaned up and no supplemental standards exist. Groundwater at the site is still above the action level for uranium (DOE 1999). The Rifle IFRC is managed for the U.S. Department of Energy by Lawrence Berkeley National Laboratory, Earth Sciences Division. Additional information for the site can be found on the DOE Legacy Management web site.
Figure A.10-1. Location map of the Old Rifle Site.
Source: DOE 1999.
Groundwater contamination by uranium is a localized but worldwide problem. While uranium contamination can derive from natural sources, most groundwater is contaminated by uranium leaching from mining waste and mill tailings (Wall and Krumholtz 2006). There is currently no proven cost effective remediation strategy for uranium-contaminated aquifers (DOE 1999). Strategies such as soil washing, solidification, chemical immobilization, chemical reduction, and phytoremediation are being explored (El-Sabour 2007). Uranium can be removed from potable waters by ion-exchange or reverse osmosis, but these technologies are too expensive to be applied to a large subsurface aquifer. An additional background reference to experiments at the site is Williams et al. 2011.
Bio-immobilization has emerged as an attractive approach to controlling uranium groundwater contamination. The soluble form of uranium is U(VI), usually complexed with carbonate (Wall & Krumholtz 2006). Some metal-reducing bacteria, in particular Geobacter, can reduce U(VI) to U(IV), which forms insoluble uranite, UO2. There is also evidence that U(VI) is adsorbed by metal sulfides formed by sulfate-reducing bacteria. Acetate, ethanol, and glucose are the subsurface amendments most commonly used to drive bio-immobilization in situ. Issues to be resolved include the timing and amount of subsurface amendment to maximize bio-immobilization, and the long-term stability of bio-immobilized uranium. Microorganisms in the subsurface have a direct impact on the nature, extent, and fate of many contaminants. Microorganisms can create conditions that decrease contaminant mobility or directly transform contaminants into innocuous or immobile forms. However, there are presently very few readily available methods for assessing in situ microbial communityThe microorganisms present in a particular sample. structure, activityRefers to when a microorganism performs a specific function (e.g., sulfate reduction, metabolism of benzene) or remediation potential within a time frame that impacts treatment or remediation decisions.
The objective of this effort was to develop and validate a simple-to-use, field-portable, microarrayDetects and estimates the relative abundances of hundreds to thousands of genes simultaneously.-based system for monitoring microbial community structure and dynamics in groundwater and subsurface environments. The full details of this work are presented in Chandler et al. (2010).
The field-portable microarray study using the TruArray® was part of a series of biostimulationA remedial technique which provides the electron donor, electron acceptor, and/or nutrients to an existing subsurface microbial community to promote degradation. experiments designed to investigate the use of adding carbon substrates to the subsurface aquifer in order to reduce soluble U (VI) to insoluble U (IV) via microbial enzymatic mechanisms. Groundwater samples were acquired from a multi-level sampling transect of U02-D02-D06-D10 at three depth intervals (12 feet, 15 feet and 20 feet) and four phases of the field experiment (pre-injection, iron-reduction, iron-sulfate transition and sulfate-reduction). Figure A.10-2 shows these sampling locations. Background samples were also acquired for other boreholes and areas in the site. Nucleic acids were extracted and split for microarray and matched qPCR analyses. Akonni Biosystems processed each sample in triplicate (300 total arrays) according to the optimized procedures at an equivalent sample volume used for qPCR tests.
Replicate (n=33) negative control reactions were all negative by microarray analysis, indicating that if there were any contaminating DNA that found its way into the samples it did not affect the microarray signatures. Analysis of the background samples supported the hypothesis of a residual shift in microbial community structure as a consequence of previous donor injections in the same gallery, especially with respect to the Dechloromonas and nitrate-reducer signatures that were much more pronounced [that is Signal/Noise (S/N) ratios] in all of the current samples than in any previous sample. However, there were also many more dechlorinator and fermenter signals in the current background than in previous background samples, which may simply reflect an overall improvement in the assay performance.
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Figure A.10-2. Rifle monitoring well gallery.
Source: Data from http://gems.lm.doe.gov/imf/imf.jsp?site=rifleoldprocessing&title=Rifle%20Old,%20CO,%20Processing%20Site, map created 2013.
The heat map (Figure A.10-3) is divided into four panels that display array results for each of the geochemical conditions during the experiment. Each panel is further divided into 12-foot, 15-foot and 20-foot intervals that display array results with depth. The heat map showed the expected progression of microbial signatures from iron- to sulfate -reducers with changes in acetate amendment and in situ geochemical field conditions. Once acetate addition started there was an increase in both the nitrate-reducers and iron-reducing microbes. The microarray response for Geobacter (a known uranium reducer) was highly correlated with qPCR (Figure A.10-3 panel B) for the same target geneA segment of DNA containing the code for a protein, transfer RNA, or ribosomal RNA molecule (based on Madigan et al. 2010). (R2 = 0.84). Probes targeting Desulfobacter and Desulfitobacterium were the most reactive during the iron- to sulfate-reducing transition and into sulfate-reduction, with a consistent Desulfotomaculum signature throughout the field experiment and a general decrease in Geobacter signal to noise ratios during the onset of sulfate-reducing conditions. Nitrate reducers represented by Dechloromonas and Dechlorosoma signatures were consistently detected throughout the field experiment. The intensity of the microarray signatures were also correlated with depth (Figure A.10-3, panel C), where the 12- and 15-foot intervals showed a stronger response than the 20-foot interval. Microarray results and S/N ratios were in concordance with quantitative PCR data sets with the array data providing a more in depth community profile and the qPCR a more quantitative result of specific community constituents.
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Figure A.10-3. (A) Heat map, (B) TaqMan® qPCR data, (C) microarray signal intensity.
Reprinted with permission from Chandler, D.P., A. Kukhtin, R. Mokhiber, C. Kinckerbocker, D. Ogles, G. Rudy, J. Golova, P. Long and A. Peacock. 2010. Monitoring Microbial Community Structure and Dynamics during in situ U(VI) Bioremediation with a Field-Portable Microarray Analysis System, Environmental Science & Technology. 44:5516-5522. Copyright 2010 American Chemical Society.
This study has established some fundamental technology milestones that are generally applicable to environmental science, in that this was the first successful deployment of a low-cost, low-complexity, portable, array-based environmental monitoring system that can generate a community profile on site, within four hours of sample receipt. Method complexity, logistical burden and analysis time have been reduced so that field deployment of microarray technology and real-time monitoring of microbial community response to environmental conditions is possible. Results from the validation study showed that interpreting microarray field data probably requires several levels of detail, from fine-scale analysis of individual probe responses, to summed intensities over genera, to integrated intensities over boreholes and/or the entire site. Translating these analyses and results into simple, intelligible outputs for site engineers and decision makers can now be accomplished through relatively straightforward analysis macros and software upgrades. That the TruArray® Geobacter response was quantitatively and strongly correlated with qPCR data provide evidence that the asymmetric PCR portion of the protocol is relatively unbiased, and provide hope that S/N ratio values may someday be used as a proxy for in situ microbial abundance. The combined body of evidence presented here demonstrates that the field portable TruArray® is capable of monitoring real, ecologically significant changes in microbial community compositionDescription of the types or identities of microorganisms present in a sample. during in situ bioremediationThe treatment of environmental contamination through the use of techniques that rely on biodegradation. Bioremediation has two essential components: biostimulation and bioaugmentation..
There are several considerations when assessing costs for array technologies, however one of the most critical is volume. Because of the way arrays are manufactured the more arrays produced the less expensive (all other parameters equal). It is estimated that the arrays would cost from $200 to $750 each depending on the type of array required for the analyses.
Currently most array applications in the environmental remediation field have been for investigation or research use only. A significant challenge for microarrays from an Environmental Molecular Diagnostics (EMD) regulatory perspective would or will most likely consist of issues with the following:
At this point the methods, manufacture and use of array technologies can have a direct impact on the quality and reliability of the results. While it may not be critical for remediation per se the continued development of array technology for other environmental applications with direct impact on human health (such as food safety, drinking water, and biosecurity) will be dependent on manufacturing quality assurance (QA) and quality control (QC) and adequate uniform laboratory procedures.
Akonni Biosystems, http://www.akonni.com/
Chandler, D.P., A. Kukhtin, R. Mokhiber, C. Kinckerbocker, D. Ogles, G. Rudy, J. Golova, P. Long, and A. D. Peacock. 2010. "Monitoring Microbial Community Structure and Dynamics during in situ U(VI) Bioremediation with a Field-Portable Microarray Analysis System." Environmental Science & Technology 44:5516-5522.
DOE (United States Department of Energy). 1999. Final site observational work plan for the UMTRA project Old Rifle site GJO-99–88-TAR. U.S. Department of Energy, Grand Junction, Colo. http://www.lm.doe.gov/Rifle/Old_Processing/Sites.aspx
El-Sabour, A. 2007. "Remediation and bioremediation of uranium contaminated soils." Electronic Journal of Environmental, Agricultural and Food Chemistry 6(5):2009-2023.
Wall, J.D., and L.R. Krumholz. 2006. Annual Review of Microbiology 60:149-166.
Williams KH, Long , P.E., Davis, JA, Steefel CI, Wilkins MJ, N’Guessan AL, Yang L, Newcomer D, Spane FA, Kerkhof LJ, McGuinness L, Dayvault R, Lovely DR. 2011. "Acetate availability and its influence on sustainable bioremediation of uranium-contaminated groundwater." Geomicrobiology Journal 28(5–6):519–539.