Metabolic Engineering to Reduce Toxicity Related to the Aerobic Degradation of Chlorinated Ethenes  
Lingyun Rui, University of Connecticut, Storrs, CT 
Young Man Kwon, University of Connecticut, Storrs, CT 
Thomas K. Wood, University of Connecticut, Storrs, CT
Valerie A. Pferdeort, Colorado State University, Fort Collins, CO Kenneth F. Reardon, Colorado State University, Fort Collins, CO

Cleaning Up with Genomics: Application of Molecular Biology to Bioremediation
Dr. Derek Lovely, University of Massachusetts, Amherst, MA

Specific Tools for Monitoring Perchlorate Bioremediation
Dr. John Coates, University of California - Berkeley, Berkeley, CA  
Susan M. O’Connor, University of California, Berkeley, Berkeley, CA 
Nora B. Sutton, Claremont McKenna College, Claremont, CA

Biostimulation or Bioaugmentation?  Decision Making Based on Scientific Data: Remediation Decisions Guided by Real Time PCR Quantification of Reductively Dechlorinating Dehalococcoides Populations 
Dr. Kirsti Ritalahti, Georgia Institute of Technology, Atlanta, GA
Frank E. Löeffler, Georgia Institute of Technology, Atlanta, GA 
Steven S. Koenigsberg, Regenesis Bioremediation Products, San Clemente, CA 

Let’s Ask the Microbes What matters?  A Field Guide to the Biochemical Responses of Degrader Populations to Environmental Conditions
David C. White, University of Tennessee, Knoxville, TN
Aaron D. Peacock, University of Tennessee, Knoxville, TN
Edward Sobek, Microbial Insights, Inc.,Rockford, TN

An Overview of Environmental Biotechnology

Terry C. Hazen, Lawrence Berkeley National Laboratory, MS 70A-3317, One Cyclotron Rd., Berkeley, CA  94720, Tel: 510-486-6223, Fax: 510-4867152

Environmental biotechnology encompasses a wide range of characterization, monitoring and control or remediation technologies that are based on biological processes.  Recent breakthroughs in our understanding of biogeochemical processes are leading to exciting new and cost effective ways to monitor and manipulate the environment.  Bioremediation has proven to be one of the most cost effective and environmentally sound remediation technologies available at sites where it will work.  Though not a “new” technology, given that petroleum land farming is about 50 years old, there are a number of exciting and relevant technologies derived from molecular biology that have tremendous implications for the future of this branch of environmental biotechnology.  This should not be all that surprising considering that microbes are the dominant life on earth (>10³⁰ cells and >10¹⁷ g) and have had >3.7 billion years to evolve.  Bioventing, biopiles, biofilters, bioreactors, biosparging, prepared beds, reactive barriers, and intrinsic bioremediation (natural attenuation) are all become widely used for bioremediation of soil, air, and groundwater.  Emerging technologies in include, bioimmobilization, biocurtains, bioaugmentation, and treatment trains, especially as applied to mixed waste, metals, and radionuclides in the environment.  Examples of various new techniques for biostimulation and bioaugmentation and their efficacy will be discussed.  The possibilities for genetically modified organisms will be considered along with the reasons that they have not been used for bioremediation up until now.  Monitoring techniques that inventory and monitor terminal electron acceptors and electron donors, enzyme probes that measure functional activity in the environment, functional genomic microarrays, phylogenetic microarrays, metabolomics, proteomics, and quantitative PCR are also being rapidly adapted for studies in environmental biotechnology.

Metabolic Engineering to Reduce Toxicity Related to the Aerobic Degradation of Chlorinated Ethenes 

Lingyun Rui, Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269-3222

Young Man Kwon, Department of Chemical Engineering, University of Connecticut, Storrs, CT 06269-3222

Thomas K. Wood, Department of Chemical Engineering, University of Connecticut, Storrs, CT 06269-3222, Tel #: 860-486-2483, Fax #: 860-486-2959

Valerie A. Pferdeort, Department of Chemical Engineering, Colorado State University, Fort Collins, CO 80523-1370

Kenneth F. Reardon, Department of Chemical Engineering, Colorado State University, Fort Collins, CO 80523-1370

Chlorinated ethenes are some of the most–frequently detected organic contaminants in groundwater, soil, and at U.S. EPA Superfund-designated waste sites.  Toluene o-monooxygenase (TOM), encoded by tomA012345 of the soil bacterium Burkholderia cepacia G4, was evolved by us for their degradation using DNA shuffling (J. Bacteriol. 184: 344-349, 2002) and saturation mutagenesis.  TOM is a multi-component enzyme (six genes) consisting of a three-component hydroxylase with a catalytic oxygen-bridged binuclear iron center, a NADH-ferredoxin oxidoreductase, and a mediating protein involved in electron transfer between the hydroxylase and reductase.  After directed evolution identified position 106 of the a-subunit of the hydroxylase as critical, saturation mutagenesis was implemented and the combined mutagenesis resulted in a 6-12 fold enhancement in 1-naphthol production (from naphthalene oxidation) as well as a 3-fold enhancement in the degradation of chlorinated aliphatics (e.g., trichloroethylene, chloroform).  To enhance further the degradation of chlorinated ethenes by reducing the toxicity of the chlorinated epoxides formed in the TOM reaction, a novel glutathione S-transferase (GST) from Rhodococcus sp. strain AD45 was identified that is active with cis-DCE epoxide (IsoILR1), and it was cloned into TOM-expressing strains.  GST reduces toxicity by adding glutathione (g-glutamylcysteinylglycine) across the chlorinated ethene epoxide bond formed by TOM.  Simultaneous expression of this GST and TOM (total of 7 genes) in an Escherichia coli dual-plasmid system led to an increase in cis-1,2-dichloroethene degradation as well as to an increase in chloride ion generation.  By overexpressing E. coli mutant g-glutamylcysteine synthetase (GSHI*), the rate-limiting enzyme for producing glutathione, a 7-fold increase in the intracellular glutathione concentration was achieved along with a 3.5-fold improvement in the cis-DCE degradation rate (2.1 ± 0.1 versus 0.6 ± 0.1 nmoles cis-DCE/min/mg protein) in the presence of IsoILR1 and TOM-Green.  Several epoxide hydrolases have also been cloned to reduce toxicity.  The physiological changes in E. coli TG1 resulting from these genetic manipulations were also studied through a proteomic analysis.  Significant changes in the E. coli proteome were noted upon expression of TOM and GST, and when cells were exposed to trichloroethylene.

Cleaning Up with Genomics: Application of Molecular Biology to Bioremediation 

Derek R. Lovley, Department of Microbiology, University of Massachusetts-Amherst, Amherst, MA  01003, Phone: 413-545-9651, Fax: 413-545-1578, Email: dlovley@microbio.umass.edu

It is becoming increasingly apparent that the microorganisms which are important in the bioremediation of organic and metal contaminants in subsurface environments can be recovered in pure culture in the laboratory.  This, coupled with readily available techniques for whole-genome sequencing, and high-throughput physiological analysis with whole-genome DNA microarrays, proteomic, and genetic strategies is rapidly providing the tools necessary to understand the physiology of microorganism important in bioremediation and to predict their metabolic response under various environmental conditions.  Advances in techniques for evaluating the genetic potential of microorganisms in contaminated environments and for monitoring in situ gene expression make it conceivable to couple in silico models of pure culture metabolism with environmental data to predict the likely outcome of various bioremediation strategies prior to conducting field studies.  For example, numerous molecular studies have demonstrated that Geobacter species are the predominant organisms in subsurface environments in which metal-reducing microorganisms play an important role in the anaerobic degradation of organic contaminants or the reductive precipitation of uranium.  Genome-enabled analysis of the physiology of several Geobacter species available in pure culture has significantly changed the basic concepts of how these species function in the subsurface.  Environmental genomic analysis of as-yet-uncultured Geobacter species in subsurface sediments undergoing bioremediation indicated that the genetic potential and gene arrangement of the as-yet-uncultured Geobacters have some significant similarities and differences from pure cultures that have been studied in depth.  Analysis of in situ gene expression in subsurface sediments is providing insights into the metabolic state and rates of metabolism of Geobacter, which in turn can aid in a rationale strategy for modifying subsurface conditions to best promote the growth and activity of these organisms.  The genome-enabled approach is changing bioremediation from a largely empirical practice into a science.

Specific Tools for Monitoring Perchlorate Bioremediation 

John D. Coates, Associate Professor, Department of Plant and Microbial Biology, 271 Koshland Hall, University of California, Berkeley, Berkeley, CA 94720, Tel:  510-643-8455, Fax:  510-642-4995, Email:  jcoates@nature.berkeley.edu
Susan M. O’Connor, Department of Plant and Microbial Biology, 111 Koshland Hall, University of California, Berkeley, Berkeley, CA 94720, Tel: 510-643-4734, Fax: 510-642-4995, Email:  smoc@nature.berkeley.edu
Nora B. Sutton, Joint Science Department, Claremont McKenna College, Claremont, CA 91711, Tel: 909-621-8298, Fax: 909-621-8588, Email:  nsutton04@mckenna.edu

Recently we demonstrated the ubiquity, diversity, and metabolic versatility of microorganisms which grow anaerobically with chlorate or perchlorate ((per)chlorate).  We identified two taxonomic groups, the Dechloromonas and the Dechlorosoma species, which represent the dominant (per)chlorate-reducing bacteria (ClRB) in the environment.  We also demonstrated that chlorite dismutation is a key step in the reductive pathway common to all ClRB and is mediated by the enzyme, chlorite dismutase (CD).  Biochemical and genetic analyses suggested that CD is highly conserved amongst the ClRB.  As such, this enzyme makes an ideal target for a probe specific for these organisms.  Polyclonal antibodies were raised against the purified CD from Dechloromonas agitata strain CKB.  The obtained antisera was deproteinated and the antigen binding activity was assessed using dot-blot analysis.  Titer values obtained indicated that the anitsera had a high affinity for purified CD and activity was observed in dilutions as low as 1 x 10^-6 of the antisera.  The antisera was active against both cell lysates and whole cells, but only if the cells were grown anaerobically with (per)chlorate.  In addition to D. agitata, dot-blot analysis employed with several diverse ClRB representing the alpha, beta , and gamma subclasses of the Proteobacteria tested positive regardless of phylogenetic affiliation.  The dot-blot response for the respective ClRB varied suggesting that some differences may exist in the anitgenic sites of the CD in these organisms.  In general, no reactions were observed with closely related non-(per)chlorate-reducing organisms.  These studies have resulted in the development of a highly specific and sensitive immuno-probe based on the commonality of the CD in ClRB.  From these data a rapid (45 min) ELISA microtiter assay was developed which can be used to assess ClRB in environmental samples regardless of their phylogenetic affiliations.  This has important implications with regards to the successful monitoring of ClRB in an engineered bioremediative strategy.

Biostimulation or Bioaugmentation?  Decision Making Based on Scientific Data:  Remediation Decisions Guided by Real Time PCR Quantification of Reductively Dechlorinating Dehalococcoides Populations 

Kirsti M. Ritalahti, Georgia Institute of Technology, Civil and Environmental Engineering,  311 Ferst Dr., ES&T Bldg. Room 3355, Atlanta, GA, 30332, Tel: 404-894-5009, Fax: 404-894-8266, Email: krita@ce.gatech.edu

Frank E. Löeffler, Georgia Institute of Technology, Civil and Environmental Engineering, 311 Ferst Dr., ES&T Bldg. Room 3228, Atlanta, GA, 30332, Tel: 404-894-0279, Fax: 404-894-8266, Email: Frank.Loeffler@ce.gatech.edu

Steven S. Koenigsberg, Regenesis Bioremediation Products, 1011 Calle Sombra, San Clemente, CA  92673, Tel: 949-366-8000, Fax: 949-366-8090, Email: steve@regenesis.com

Complete transformation of toxic and carcinogenic compounds to benign products is an achievable goal at many contaminated groundwater sites.  Recent pilot studies in the field successfully demonstrated that bacterial populations with desirable physiological traits could be stimulated, if already present, by overcoming existing limitations (biostimulation).  In aquifers contaminated with chlorinated solvents, microbial reductive dechlorination activity is frequently limited by electron donor availability.  Treatments that increase fluxes of hydrogen and acetate are often sufficient to stimulate the reductive dechlorination process.  If dechlorination rates are insufficient, or dechlorinating organisms are not present at a site, they can be supplied as a pre-grown inoculum (bioaugmentation).  Bioaugmentation contributes heavily to overall remediation costs, and hence, should be applied only when the indigenous microflora fails to promote complete detoxification within acceptable time frames.  Hence, site assessment must include a qualitative and quantitative evaluation of the key dechlorinating populations.  Recent research shed light on the bacterial populations contributing to complete detoxification of chlorinated ethenes, and molecular tools that target indicator genes of key dechlorinating populations (including Dehalococcoides sp. strain BAV1) were designed.  Initially, positive-negative response PCR amplification of Dehalococcoides-specific genes allowed a qualitative assessment.  When applied as a nested PCR approach, this method has unsurpassed sensitivity, with detection limits of a few cells per liter of groundwater.  Although the real time PCR approach currently cannot match such low detection limits, this approach offers the benefit of quantifying the organisms of interest.  Enumeration is critical to predict dechlorination performance and decide on the most cost-effective strategy, although the minimum Dehalococcoides cell number per gram of aquifer material necessary to promote robust dechlorination activity has yet to be established.  In addition, real-time PCR has proven to be a valuable tool for monitoring treatment success, and for establishing cause-effect relationships.  Refining the quantitative real time PCR approach holds great promise to provide practicing engineers with key information required to implement the most promising and cost-efficient remediation strategy at a given contaminated site.

Let’s Ask the Microbes What matters?  A Field Guide to the Biochemical Responses of Degrader Populations to Environmental Conditions

David C. White, Center for Biomarker Analysis, University of Tennessee, 10515 Research Drive, Suite 300, Knoxville TN 37932-2575, Tel: 865-974-8001, Fax: 865-974-8027, Email Dwhite1@utk.edu

Aaron D. Peacock, Center for Biomarker Analysis, University of Tennessee, 10515 Research Drive, Suite 300, Knoxville TN 37932-2575, Tel: 865-974-8014, Fax: 865-974-8027, Email Apeacock@utk.edu

Edward Sobek, Microbial Insights, Inc., 2340 Stock Creek Blvd., Rockford, TN 37853-3044, Tel: 865-573-8188, Fax: 865-573-8133,  E-mail Esobek@microbe.com

Greg A. Davis, Microbial Insights, Inc., 2340 Stock Creek Blvd., Rockford, TN 37853-3044, Tel: 865-573-8188, Fax: 865-573-8133, Email Gdavis@microbe.com

Biomarkers recovered from subsurface microbial communities provide quantitative details of the viable biomass, community composition, indications of specific metabolic activities, and the nutritional/physiological status of the microbiota.  The active components of subsurface microbial communities are readily recovered from biofilms colonizing powdered activated carbon surfaces (over 600 m³/g) in solid phase samplers containing BioSep^® beads in a nomex coating.  Lipid and DNA biomarkers provide indications for the remediation potential, diagnostics as to what conditions best potentates the proposed remediation, and forensic analysis if the program proves unsuccessful based on in situ down-well exposures. Lipids are readily concentrated and purified from complex environmental matrices and are amenable to structural analysis by chromatography/mass spectrometry. Lipid extraction potentiates recovery of DNA. Viable bacteria possess intact polar phospholipid membranes assessed quantitatively with analysis of phospholipids ester-linked fatty acids (PLFA). PLFA are metabolically labile and act as quantitative measures of viable microbial biomass.  Community composition of multispecies microbial communities can be quantitatively discerned from the lipid composition. Presence of specific microbes (rDNA) or metabolic activities (functional genes) can be determined with DNA isolation, PCR amplification with appropriate primers, amplicon separation and sequencing with comparisons to comprehensive data bases. Specific patterns of lipids can indicate physiological and toxin induced stress. Lipid composition reflects conditions at specific in situ microniches of the bacteria such as in situ redox conditions reflected in respiratory unbiquinone/menaquinone ratios and the plasmalogen content, toxic exposures induce increased cyclopropane and specific trans monoenoic PLFA. Increases in poly -hydroxyalkanoic acid indicates unbalanced growth (division inhibited because some essential component is missing (phosphate, nitrate, trace metal, etc.), and accumulation of intercellular reductants. Specific lack of bioavailable phosphate induces ornithine-lipids. BioSep beads facilitate ¹³C incorporation into biomarkers.  Insights into in situ environments guide the corrective actions, bioaugmentation with missing microbial components, and maintenance of optimum conditions for subsurface remediation.

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