Monitoring  Bioremediation by Analyzing In Situ Gene Expression
Dawn Holmes, University of Massachusetts, Amherst, MA
Regina A. O’Neil, University of Massachusetts, Amherst, MA
Kelly P. Nevin, University of Massachusetts, Amherst, MA
Derek R. Lovley, University of Massachusetts, Amherst, MA

Innovative Methods for Evaluating the Feasibility of Bioremediation and Natural Attenuation
Susan L. Boyle, Haley & Aldrich, Inc., Rochester, NY  
Glenn M. White, and Paul M. Tornatore, Haley & Aldrich, Inc. 

Quantitative Detection of and Bioaugmentation with Reductive Dechlorinators
Stephen S. Koenigsberg, Regenesis Bioremediation Products, San Clemente, CA 
Erin Rasch, Regenesis Bioremediation Products, San Clemente, CA 

Functional and Taxonomic Microarrays for Profiling Natural Microbial Populations Involved in Bioremediation
Charles W. Greer, National Research Council Canada, Montreal, Quebec, Canada
David F. Juck, National Research Council Canada, Montreal, Quebec, Canada
Sylvie Sanschagrin, National Research Council Canada, Montreal, Quebec, Canada
Diane Labbé, National Research Council Canada, Montreal, Quebec, Canada
John R. Lawrence, Environment Canada, Saskatoon, Saskatchewan, Canada,
Lyle G. Whyte, McGill University, Macdonald Campus, Ste-Anne-de-Bellevue, Quebec

Kinetics and Microbial Ecology of Perchlorate-Reducing Bacteria: Implications for Remediation
Robert Nerenberg, University of Notre Dame, Notre Dame, IN
Yasunori Kawagoshi, Kumamoto University
Bruce E. Rittmann, Northwestern University  fax: 574-631-9236

Preliminary Knowledge Base of the Association between Molecular Techniques and Other Site Parameters for Better Evaluating Reductive Dechlorination Potential
Greg Davis, Microbial Insights, Inc. Rockford, TN
Aaron Peacock, Microbial Insights, Inc. Rockford, TN
and Center for Biomarker Analysis, Knoxville, TN
Dora Ogles, Microbial Insights, Inc. Rockford, TN
Debra McElroy, Microbial Insights, Inc. Rockford, TN
Josh Streufert, Microbial Insights, Inc. Rockford, TN

Monitoring Bioremediation by in situ Gene Expression Analysis 

Dawn E. Holmes, University of Massachusetts, Dept of Microbiology, Morrill IV N Science Center, Amherst, MA, 01003, Tel: 413-577-0447, Fax: 413-545-1578, Email: dholmes@microbio.umass.edu
Regina A. O’Neil, University of Massachusetts, Dept of Microbiology, Morrill IV N Science Center, Amherst, MA, 01003, Tel: 413-577-2939, Fax: 413-545-1578, Email: rtarallo@microbio.umass.edu
Kelly P. Nevin, University of Massachusetts, Dept of Microbiology, Morrill IV N Science Center, Amherst, MA, 01003, Tel: 413-577-0447, Fax: 413-545-1578, Email: knevin@microbio.umass.edu
Derek R. Lovley, University of Massachusetts, Dept of Microbiology, Morrill IV N Science Center, Amherst, MA, 01003, Tel: 413-545-9651, Fax: 413-545-1578, Email: dlovley@microbio.umass.edu

Investigations into the mechanisms for dissimilatory Fe(III) reduction in subsurface environments may be greatly simplified by the fact that microorganisms in the Geobacteraceae are the predominant Fe(III)-reducing microorganisms in a diversity of subsurface environments in which Fe(III) reduction is important. The finding that Geobacteraceae can account for ca. 40-90 % of the total microbial community under Fe(III)-reducing conditions in some subsurface environments suggests that subsurface Fe(III)-reducing communities may provide the type of low-diversity community that is most amenable to environmental expression studies. In this study, the ability to monitor the in situ metabolic state of the microbial community via mRNA analysis was evaluated with mRNA extracted from 3 different subsurface Fe(III)-reducing subsurface environments where Geobacteraceae predominate:  1) a U(VI) contaminated field site in Rifle, CO that was supplemented with acetate to stimulate U(VI) and Fe(III) reduction,; 2) the Fe(III)-reduction zone of a petroleum contaminated aquifer at a USGS Groundwater Toxics Site in Bemidji, Minnesota:  and 3) a runoff recharge pool from a highway in Plymouth, MA that has been exposed to calcium magnesium acetate (CMA) as the primary road deicing agent every winter for the past 7 years. In situ gene expression studies were first carried out on three genes that are unique to micro-organisms within the Geobacteraceae; nifD, which encodes the dinitrogenase gene involved in nitrogen fixation, citrate synthase, a key enzyme in Geobacteraceae central metabolism that is distinct from other prokaryotes, and ompB, a gene with homology to previously described multicopper oxidases, that is thought to be involved in the reduction of insoluble Fe(III)-oxides. nifD was selected because it has been suggested that there might be a need for nitrogen fixation in acetate-amended or petroleum-contaminated subsurface sediments where the influx of organic carbon into these otherwise nutrient-poor subsurface environments can result in a limitation of fixed nitrogen relative to the availability of carbon substrates. Analysis of levels of nifD mRNA in sediments collected from the petroleum-contaminated site did, in fact, demonstrate that Geobacteraceae were expressing nitrogen fixation genes. Expression of nifD was repressed when ammonium was added to the sediments. A diversity of Geobacteraceae citrate synthase, ompB, recA, and nifD genes have also been identified in all three subsurface environments and their levels of expression have been monitored. In addition, BAC and small insert libraries from genomic DNA extracted from these sites are currently being assembled in order to identify other significant genes that are unique to the Geobacteraceae for future in situ gene expression studies. These results demonstrate that monitoring the in situ metabolic state of the microbial community as well as estimating rates of metabolism is feasible via mRNA analysis.  This will help move bioremediation from a primarily empirical practice to more of a science.

Innovative Methods for Evaluating the Feasibility of Bioremediation and Natural Attenuation

Susan L Boyle, Senior Engineer, Haley & Aldrich, Inc., 200 Town Centre Dr., Suite 2, Rochester, NY 14623-4264, Tel: 585-321-4222, Fax: 585-359-4650, Email: sboyle@HaleyAldrich.com
Glenn M. White, and Paul M. Tornatore, Haley & Aldrich, Inc. 

Recently, the utilization of biotechnology in contaminated site investigation and remediation has exploded.  Applications of biotechnology that previously were used only in high-end genetic laboratories and universities are now being applied to the environmental cleanup industry – with excellent results and many opportunities for continued advancements.  These new tools allow the environmental practitioner to evaluate site aquifer conditions in a more efficient, more accurate, and less costly manner than conventional aquifer characterization techniques.

Previously, when environmental practitioners were interested in evaluating the potential for biodegradation in an aquifer few choices existed beyond utilization of conventional aquifer characterization techniques, evaluation of natural attenuation geochemical datasets, and performance of laboratory biodegradation microcosms.  Today, several new sampling and analytical tools exist that have the potential to revolutionize how biofeasibility evaluations are performed.  This paper presents some of these new tools, case study data from sites where these tools were implemented, and summarizes where the state of the science is heading.

One of the tools to be discussed is the “Bio-Trap” sampling device, also known as a down-well microcosm.  The Bio-Traps contain “BioSep® beads that absorb organics and provide a porous surface that allows for the rapid development of bio-films.  The Bio-Traps can either be installed “non-baited” to assess baseline biological conditions in the aquifer or can be impregnated with a variety of electron donors or acceptors to assess microbial response.

The bio-films are extracted from the traps at a specialty laboratory where a variety of analyses are performed, including:  DNA (both PCR and DGGE), functional gene identification, phospholipid fatty acids (PLFA) and other genetic biomarkers.

Site-specific results for use of Bio-Traps for biofeasibility studies on chlorinated solvent, BTEX, and MTBE sites will be discussed.  Data from the down-well microcosms (BioTraps) will be compared with laboratory biodegradation microcosms performed with site soils and groundwater.  Utilization of Compound Specific Isotope Analysis (CSIA) will also be discussed.  

Quantitative Detection of and Bioaugmentation with Reductive Dechlorinators

Stephen S. Koenigsberg, Ph.D., Vice President for Research and Development, Regenesis Bioremediation Products, 1011 Calle Sombra, San Clemente, CA  92673, Tel: 949-366-8000, Fax: 949-366-8090, Email: skoenigsberg@regenesis.com
Erin Rasch, Applications Engineer, Regenesis Bioremediation Products, 1011 Calle Sombra, San Clemente, CA  92673, Tel: 949-366-8000 x128, Fax: 949-366-8090, Email: erasch@regenesis.com

With the recent discovery that the microorganisms responsible for the complete biodegradation of chlorinated solvents may not be present at all sites, bioaugmentation with mixed cultures capable of degrading a wide range of contaminants is now being successfully used at a variety of sites to increase the rate of reductive dechlorination and reduce the time that it takes to obtain site closure.  By using a “differential diagnosis” approach, bioaugmentation can be used to close sites which may have otherwise not achieved complete reductive dechlorination within a reasonable timeframe. Regenesis’s Bio-Dechlor INOCULUM^TM product, an enriched natural microbial consortium containing various species of dechlorinating microorganisms, has now been applied at fourteen sites located within eight states. Bio-Dechlor INOCULUM^TM includes Dehalococcoides strain BAV-1, an organism that can complete the final stages of chlorinated ethene degradation from the dichloroethene step through to ethene. The BAV-1 strain is unique because it can metabolically degrade chloroethene (vinyl chloride) to ethene. This is considered to be a superior feature relative to all other known strains that can only degrade vinyl chloride co-metabolically. The difference is that the BAV-1 strain will derive energy from the degradation process whereas organisms that perform the task co-metabolically cannot.

Real Time Polymerase Chain Reaction (RT-PCR) is a technique in which the number of organisms in a sample can be determined by measuring the amount fluorescence of produced during the PCR reaction. Available commercially as Bio-Dechlor CENSUS^SM, this technique is now being used not only to determine if the necessary microorganisms are present within the aquifer in the appropriate numbers and if the addition of external organisms is required, but also to track the changes induced in the microbial community by the addition of a carbon source or the addition of organisms. Recent advances in this technology have now allowed us to target functional genes, or gene sequences that are specific to organisms able to perform the desired reductive dechlorination tasks, as opposed to only genes on the 16S rRNA sequence that are common to all organisms with this phylogenic group. DNA-based methods such as Denaturing Gradient Gel Electrophoresis (DGGE) and lipid analyses such as Phospholipid Fatty Acid analysis (PLFA) can also be used to observe the effects of bioaugmentation or biostimulation on the microbial community as a whole, often more quickly than the effects of the application can be observed through VOC analysis only. The integration of these techniques and others into existing monitoring programs gives us a greater understanding of what is occurring within the subsurface and allows us to answer questions that we were once unable to. The full nature of our experience with these techniques will be presented.

Functional and Taxonomic Microarrays for Profiling Natural Microbial Populations Involved in Bioremediation

Charles W. Greer, Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Ave., Montreal, Quebec.  H4P 2R2, Canada, Tel: 514-496-6182, Fax: 514-496-6265, Email: charles.greer@nrc-cnrc.gc.ca
David F. Juck, Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Ave., Montreal, Quebec.  H4P 2R2, Canada, Tel: 514-496-5297, Fax: 514-496-6265, Email: david.juck@nrc-cnrc.gc.ca
Sylvie Sanschagrin, Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Ave., Montreal, Quebec.  H4P 2R2, Canada, Tel: 514-496-5122, Fax: 514-496-6265, Email: sylvie.sanschagrin@nrc-cnrc.gc.ca
Diane Labbé, Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Ave., Montreal, Quebec,  H4P 2R2, Canada, Tel: 514-496-5006, Fax: 514-496-6265, Email: diane.labbe@nrc-cnrc.gc.ca
John R. Lawrence, National Water Research Institute, Environment Canada, 11 Innovation Blvd., Saskatoon, Saskatchewan. S7N 3H5, Canada, Tel: 306-975-5789, Fax: 306-975-5143, Email: John.Lawrence@ec.gc.ca
Lyle G. Whyte, Department of Natural Resource Sciences, McGill University, Macdonald Campus, 21,111 Lakeshore Road, Ste-Anne-de-Bellevue, Quebec. H9X 3V9,  Canada, Tel: 514-398-7889, Fax: 514-398-7990, Email: whyte@nrs.mcgill.ca

Two types of microarrays were developed to profile indigenous microorganisms in a variety of environments.  The functional gene microarray uses gene probe amplicons derived from various bacterial catabolic pathways for organic pollutants and biogeochemical cycles. The taxonomic microarray has oligonucleotide probes derived from the 16S rDNA gene and the cpn60, chaperonin gene.  These two microarrays have been used to monitor changes in the indigenous microbial population during a bioremediation project at Eureka, in the high Arctic.  Treatment of the hydrocarbon-contaminated soils was started in 2000 and consists of applying nutrients (solid and liquid fertilizers) and tilling the soil during the summer. The effect of this treatment on the composition and activity of the indigenous microbial population has been monitored on a yearly basis in the active and supra-permafrost layers of treated and untreated soils. Total petroleum hydrocarbon (TPH) concentrations in the treated soil have been reduced by 63% in the active zone, and by 75% in the supra-permafrost zone. Although total heterotrophic or diesel-degrading bacterial population sizes have not changed significantly during the treatment, the ndoB (naphthalene degradation) and alkB (alkane degradation) genotypes increased in 2001 and 2002, respectively. Mineralization assays, using radiolabeled naphthalene and hexadecane, demonstrated a corresponding increase, and functional gene microarray analysis indicated that hydrocarbon degrader genotypes increased, which was supported by a quantitative analysis of target gene signal intensity. Analysis with the taxonomic microarray indicated that the composition of the indigenous population had changed during the monitoring period, and specific microorganisms such as Pseudomonas, Rhodococcus and Vibrio increased significantly, paralleling the increase in corresponding catabolic genes associated with these same microorganisms.  The use of functional gene and taxonomic microarrays provides a new method to rapidly evaluate the composition and functional capacity of indigenous microbial communities, and to monitor their responses to, and recovery from, stress.  

Kinetics and Microbial Ecology of Perchlorate-Reducing Bacteria: Implications for Remediation 

Robert Nerenberg, Department of Civil Engineering and Geological Sciences, University of Notre Dame, 156 Fitzpatrick Hall, Notre Dame, IN 46556, Tel: 574-631-4098, Fax: 574-631-9236
Yasunori Kawagoshi, Kumamoto University, Department of Architecture and Civil Engineering, Kumamoto, Japan, Tel. +81-96-342-3549
Bruce E. Rittmann, Northwestern University, Department of Civil and Environmental Engineering, 2145 Sheridan Road, Evanston, IL 60208-3109, Tel: 847-491-8790, Fax: 847-491-4011

Perchlorate bioremediation at trace levels may be more difficult than at high levels, due to slower microbial reduction rates and smaller selective pressure for perchlorate-reducing bacteria (PCRB).  We used kinetic and microbial ecology tests to investigate this.  For kinetic studies, we used Dechloromonas sp. PC1, a PCRB isolated from a perchlorate-reducing bioreactor.  With hydrogen as an electron donor, PC1’s doubling time was around 24 hours and its apparent K_m was 150 µg/L.  K_m was significantly lower than values reported in the literature, but high enough to significantly slow reduction rates at µg/L perchlorate levels.  At high perchlorate concentrations, chlorate accumulated as a transitory intermediate, a novel finding for PCRB.  Kinetics suggests perchlorate alone cannot sustain PCRB below 12 µg/L, and that nitrate or oxygen, used concurrently with perchlorate, is needed as a primary electron acceptor.  We studied the effect of perchlorate on the microbial ecology of a mixed denitrifying community using four identical reactors, inoculated with a denitrifying culture and continuously supplied with 5 mgN/L nitrate and 8 mg/L oxygen.  At steady state, the effluent contained no measurable oxygen and 0.05 mgN/L.  Subsequently, 0, 100, 1000, and 10,000 µg/L perchlorate were added to the influents of the reactors.  Perchlorate reduction initially was small, but greatly improved over several weeks, suggesting enrichment for PCRB.  This was confirmed by denaturing gradient gel electrophoresis (DGGE), fluorescent in-situ hybridization (FISH), and perchlorate-reduction activity tests.  A Dechloromonas strain was present at 14%, 22%, 31%, and 49% of total bacteria in reactors with 0, 100, 1,000, and 10,000 mg/L perchlorate, respectively.  Results suggest that perchlorate gave PCRB an advantage in competing for oxygen and nitrate, producing PCRB biomass far in excess of the expected yield on perchlorate.  Our kinetics and microbial ecology results suggest oxygen and nitrate play a key role in trace perchlorate remediation.

Preliminary Knowledge Base of the Association between Molecular Techniques and Other Site Parameters for Better Evaluating Reductive Dechlorination Potential

Greg Davis, Microbial Insights, Inc. 2340 Stock Creek Blvd., Rockford, TN 37853
Aaron Peacock, Microbial Insights, Inc. 2340 Stock Creek Blvd., Rockford, TN 37853
and Center for Biomarker Analysis, 10515 Research Drive, Suite 300, Knoxville, TN 37932-2575
Dora Ogles, Microbial Insights, Inc. 2340 Stock Creek Blvd., Rockford, TN 37853
Debra McElroy, Microbial Insights, Inc. 2340 Stock Creek Blvd., Rockford, TN 37853
Josh Streufert, Microbial Insights, Inc. 2340 Stock Creek Blvd., Rockford, TN 37853

Substantial research has been conducted to understand the microbiology of reductive dechlorination. This research has lead to the isolation of genera known to dechlorinate (e.g. Dehalococcoides, Dehalobacter, etc.) as well as the isolation of specific functional genes (tceA, BAV1) associated with certain steps in the dechlorination process. Recently, modern molecular techniques (such as Bio-Dechlor CENSUS^sm) have been applied to quantify the abundance of specific bacterial groups involved in reductive dechlorination and also to quantify functional genes of interest.  Microbial Insights has pioneered this field, and has realized that creating a database that includes these microbial indicators along with associated contaminant concentrations and other geochemical parameters could provide essential information for effective site management and decision making. Preliminary results on the distribution of known dechlorinating bacteria in association with other site specific parameters have been combined into this Knowledge Database.  This presentation will discuss the trends that have been observed thus far, based on data from over 1,000 samples from a wide range of sites.

Top