Genomics, Metagenomics and Stable Isotope Probing: Applications to Understanding MTBE and TBA Biodegradation
Mike Hyman, North Carolina State University, Raleigh, NC  

Adaptive Evolution: A Strategy for Better Understanding and Optimizing Environmental Biotechnologies
Zarath M. Summers, University of Massachusetts Amherst, Amherst, MA

Achieving National Paradigm Shifts:  How ITRC Increases Regulatory Efficiency by Educating on Innovative Environmental Technologies 
Anna Willett, Interstate Technology and Regulatory Council (ITRC), Washington DC

Use of Advanced Passive Soil Gas Technology for Site Conceptualization and Closure Strategies
Harry O`Neill, Beacon Environmental Services, Bel Air, MD

Soil Water Potential Effects on Bio-inspired Sensor Response
C.M. Reynolds, USA Engineering Research & Development Center, Cold Regions Research and Engineering Laboratory, Hanover, NH  

Advanced Diagnostic Tools and Applications to Site Design, Management and Expedited Closure
Stephen S. Koenigsberg, ENVIRON International Corporation, 18100 Von Karman Avenue, Ste 600 , Irvine , CA   92612 , Tel: 949-798-3604, Email: skoenigsberg@environcorp.com

Site remediation has evolved from energy intensive, mechanically driven remediation processes to more effective and cost efficient in situ processes.  In many cases, in-situ remediation is best served if integrated with advanced diagnostics that employ molecular biological tools (MBTs), compound specific isotope analysis (CSIA) and advanced geotechnical protocols.  These Diagnostic protocols, taken alone or in combination, can help with site assessment and subsequently assure the best in-situ remedy from among the available options.  As an extension of this, the diagnostics can further be used for optimization of on-going remedial operations. 

While advanced diagnostics can assist in site assessment and the design and management of in situ remediation options, there are also important extensions of this work to site closure, with an emphasis on monitored natural attenuation (MNA).  This paper will set forth arguments for the necessity of expedited site closure in the current climate and review the basic elements of a program built for that objective.  Two cases will be examined in detail that relied on the use of MBTs to achieve MNA as a remedy for the sites in question.

The larger of the two sites that received and MNA ruling had soil and groundwater concentrations of chlorinated solvents greater than the Georgia Risk Reduction Standards (RRS).  Applications of phospholipid fatty acid analysis (PLFA) and nucleic acid diagnostics were employed to assess the status of natural attenuation prepare a petition for MNA.  With the results of these analyses the site was put into an MNA status.  This ruling was in concert with excavation of the source and an ozone treatment of source groundwater.  The MBT data was used to successfully establish that downgradient bioremediation barriers could be held in abeyance subject to the impacts of the source treatments and if permanently avoided an average cost savings of $1.2 MM will be realized.

The lesser of the two sites that received an MNA ruling was in Tennessee at an industrial facility contaminated with petroleum hydrocarbons. MNA wa s s elected as the long term groundwater remedy for the site in order to meet the onsite and offsite groundwater cleanup goal s s et in the Consent Order and the decision was driven by the MBT results. Specifically, in addition to the same PLFA and nucleic acid analyses used in Georgia , a special application of stable isotope analysis (SIP) was employed.  U sin g non-radioactive 13C isotopes of benzene, we demonstrated that label was incorporated into the organisms from the site, thus providing conclusive evidence supporting natural attenuation and, as a result, MNA wa s s elected as the remedy.  By showing the regulatory agency that the overall plume is decrea sin g in concentration and that natural attenuation is an active process, hydrauli c c ontainment was not required at the property boundary leading to an  overall cost savings of approximately $450,000.  

Genomics, Metagenomics and Stable Isotope Probing: Applications to Understanding MTBE and TBA Biodegradation
Michael Hyman, Department of Microbiology, North Carolina State University , Raleigh NC 27695 , USA , Tel: 919-515-7814, Fax: 919-515-7867, Email: michael_hyman@ncsu.edu

The oxygenate methyl tertiary butyl ether (MTBE), and its major metabolite, tertiary butyl alcohol (TBA), are common ground water contaminants associated with gasoline. Under anaerobic conditions MTBE can be converted to TBA. Under mildly reductive conditions TBA can be further biodegraded whereas under more strongly reducing conditions TBA can be persistent. In contrast, both MTBE and TBA can be rapidly biodegraded under aerobic conditions and aerobic approaches are often preferred as treatment options for these compounds.

Aerobic biodegradation of MTBE and TBA occurs through two distinct processes. A growing number of strains are known that grow on MTBE or TBA. The best-characterized MTBE/TBA-metabolizing bacterium is Methylibium petroleiphilum PM1. MTBE and TBA can also be extensively cometabolically degraded by bacteria that grow on other gasoline components such as normal or branched alkanes. The best-characterized MTBE/TBA cometabolizing strain is Mycobacterium vaccae JOB5. In our recent studies we have obtained genome sequences for two MTBE/TBA-oxidizing bacteria. One TBA-oxidizing strain, S1B1 is closely related to M. petroleiphilum PM1. Our genome analysis shows a region of a large plasmid that has been implicated in MTBE/TBA oxidation by strain PM1 is highly conserved in strain S1B1. In contrast, none of these genes are found in the second genome, that of M. vaccae JOB5. We have also used ¹³C stable isotope probing to identify organisms that metabolize ¹³C₄-TBA in Bio-GAC reactors. Our analysis indicates that all of the strains identified by SIP are closely related to strains S1B1 and PM1. Furthermore, an analysis of the ¹³C-labelled metagenome from reactor samples indicates that at least 4 of the key genes associated with MTBE/TBA oxidation by strains PM1 and S1B1 are found in these samples. These results will be interpreted in terms of their immediate and long term impacts on our understanding or MTBE and TBA biodegradation. 

Adaptive Evolution: a Strategy for Better Understanding and Optimizing Environmental Biotechnologies
Student Presenter
Zarath M. Summers, University of Massachusetts, 422A Morrill IV North, 639 North Pleasant St, Amherst, MA, 01003, USA, Tel: 413-577-3069, Email: zsummers@microbio.umass.edu
Derek R. Lovley, University of Massachusetts, 400 Morrill IV North, 639 North Pleasant St, Amherst, MA, 01003, USA, Tel: 413-545-9648, Email: dlovley@microbio.umass.edu

Adaptive evolution studies can provide new insights into the physiology and ecology of environmentally relevant organisms by identifying which mutations in genes and regulatory pathways are associated with adaptations producing a more beneficial phenotype. Genome-scale in silico modeling of the metabolism of Geobacter species has indicated that these organisms have a metabolic potential far beyond that previously demonstrated in culture.  Some of these potential metabolic features may have practical benefit. For example, lactate is a convenient electron donor to add to the subsurface in order to promote in situ uranium bioremediation, but lactate is not a common electron donor in natural anaerobic sedimentary environments.  Geobacter sulfurreducens does not naturally grow well on lactate, but with the appropriate selective pressure a strain that could rapidly grow on lactate was developed. In a similar manner, strains of G. sulfurreducens have been evolved that can metabolize sugars and glycerol, substrates that the wild-type strain does not utilize.  This has important implications not only for bioremediation applications, but also for the conversion of various wastes to electricity in Geobacter-based microbial fuel cells.  Effective subsurface bioremediation and electricity production with Geobacter also requires higher rates of extracellular electron transfer than is typical for Geobacter in pristine subsurface environments. In order create a strain of G. sulfurreducens capable of more rapid extracellular electron transfer; cells were repeatedly transferred as rapidly as possible with Fe(III) oxide as the electron acceptor.  A strain was developed which can transfer electrons to Fe(III) oxide 10 times faster than wild type. Strains are currently being adapted for other applications, such as serving as syntrophs in the conversion of wastes and hydrocarbons to methane. Whole-genome resequencing facilitates the identification of the mutations associated with these adaptations, which should aid in the further optimization of application-specific strains for carrying out complex cellular processes.

Achieving National Paradigm Shifts: How ITRC Increases Regulatory Efficiency by Educating on Innovative Environmental Technologies
Anna Willett,  Interstate Technology & Regulatory Council (ITRC), 444 North Capitol Street NW, Suite 445, Washington, DC 20001, Tel: 202-624-3686, Email: awillett@sso.org

The Interstate Technology and Regulatory Council (ITRC) is a public-private coalition working to achieve regulatory acceptance of innovative environmental technologies.  ITRC’s national network of professionals works to eliminate barriers to the use of new environmental technologies and approaches, so that compliance costs are reduced and clean up efficacy is maximized.  ITRC works to broaden and deepen technical knowledge and expedite quality regulatory decision-making, while protecting human health and the environment.  With private and public sector members from all 50 states and the District of Columbia , ITRC truly provides a national perspective.   

ITRC achieves its mission through its technical teams, which are composed of state and federal environmental regulators, federal agency representatives, industry experts, community stakeholders, and academia.  ITRC technical teams are formed around a specific emerging environmental contaminant, innovative technology, or new clean up process.  The teams produce documents ranging from technical overviews and case studies of innovative technologies to technical/regulatory guidance documents for applying technologies.  Technical experts from the teams then provide web-based and classroom training on their team’s topic.  Over the past 10 years, ITRC has published nearly 100 documents and reached nearly 50,000 participants through training courses on 40 topics.  ITRC’s products are propagated throughout the state environmental agencies by ITRC’s state liaison network, which includes state environmental agency employees who advocate and communicate with their states regarding ITRC products. 

Advanced diagnostic techniques for subsurface monitoring are being used across the country and, in some cases, are the basis for regulatory decision-making.  However, there is no nation-wide practical decision framework on how to best use these techniques during a regulated clean up process.  Over the past two years, ITRC has produced overview and guidance documents and associated web-based training on methods for evaluating contaminant mass loading from a source and the attenuation capacity of an aquifer.  Working from this foundation, ITRC is planning to launch a team focused on advanced diagnostics and their use during the assessment and clean up process.  The guidance and training that this team produces can accelerate the proper and practical use of advanced diagnostics on a national scale, greatly reducing costs and energy input during all phases of the clean up process.

Use of Advanced Passive Soil Gas Technology for Site Conceptualization and Closure Strategies    

Harry O`Neill, Beacon Environmental Services, 323 Williams Street, Suite D, Bel Air, MD Tel: 410-838-8780, Email: harry.oneill@beacon-usa.com 
Joseph E. Odencrantz, Ph.D, Beacon Environmental Services, 2121 Yacht Yankee, Newport Beach, CA 92660, Tel: 949-644-8602, Email: joe.odencrantz@beacon-usa.com

Passive soil gas (PSG) testing is a powerful investigatory tool that involves engineered sorbents in contact with the subsurface for a period of several days to several weeks. The time-weighted capture of volatile organic compounds (VOCs) combined with a grid-pattern of placement allows for a detailed assessment of the spatial variability of contaminant sources as well as the extent of contamination over a wide-range of geologic materials and molecular-weights of the target VOCs. Many sites fail to have an adequate realization of contaminant distribution that results in failed remedial efforts, poor risk evaluations and an incomplete site conceptual model (SCM). Advanced PSG technology involves both a rigorous analytical laboratory methodology for determining the mass of VOCs captured on the sorbents and a sufficient density of samplers to ensure adequate spatial variability assessment for an important step in gaining a complete SCM.

ASTM D 5314-92(2006) is the standard guide for soil gas monitoring in the vadose zone and ASTM WK20609 is a working standard passive soil gas practice that outlines procedures for PSG sample collection, analysis, and reporting for a multitude of applications. These applications include vapor intrusion evaluation, monitoring, spatial variability assessment and source identification that can all be used to help identify the most rapid steps to achieve site closure. As vapor intrusion issues are clearly becoming a regulatory necessity for almost all sites, it is clear that useful vapor testing at sites should be maximized to determine as much information as possible. A proper PSG survey can assist the site investigator sort out the path of least resistance to site closure.

As the implementation of advanced diagnostic tools is utilized at sites across the world, it is important to ensure that there are no weaknesses in any of them so that the potential for surprises is minimized. The monitoring of the effectiveness of remediation systems is often limited to an examination of data collected from permanent monitoring or pumping points in the vadose zone or groundwater system. These evaluations often do not give a representative picture of what is occurring in areas away from the immediate vicinity of remedial efforts. PSG sampling on a regular basis in a grid-pattern in and around the remediation systems gives a reliable snapshot of the performance both in close proximity to and away from active remedial areas.

Soil Water Potential Effects on Bio-inspired Sensor Response
C.M. Reynolds, USA Engineering Research & Development Center, Cold Regions Research and Engineering Laboratory, 72 Lyme Road, Hanover, NH, USA  03755, Tel: 603-646-4394, Fax: 603-646-4785, Email: charles.m.reynold@us.army.mil
K.L. Foley, USA Engineering Research & Development Center, Cold Regions Research and Engineering Laboratory, 72 Lyme Road, Hanover, NH, USA  03755, Tel: 603-646-4563, Fax: 603-646-4785, Email: karen.l.foley@usace.army.mil
D.B. Ringelberg, USA Engineering Research & Development Center, Cold Regions Research and Engineering Laboratory, 72 Lyme Road, Hanover, NH, USA  03755, Tel: 603-646-4394, Fax: 603-646-4785, Email: david.b.ringelberg@usace.army.mil
J.E. Anderson, USA Engineering Research & Development Center, Topographic Engineering Center, 7701 Telegraph Road, Alexandria, VA 22315, Tel: 703-428-6698, Fax: 703-428-8176, Email: john.anderson@usace.army.mil

Bio-inspired sensors often perform poorly or not at all in soil.  We hypothesized that there are soil conditions that favor bio-inspired sensor performance and, because many bio-inspired sensors mimic enzymes, we can better predict their performance in soil by understanding soil enzymes.  Near surface soils undergo diurnal moisture and temperature fluctuations.  We exploited the concept that soil water potential is comprised of matric, osmotic, and gravitational components, and used the osmotic potential of an ionic solution to mimic soil water potential, which can be dominated by matric contributions.  We related soil water potential to ionic strength of a simple salt solution by generating a water retention curve for soil, which expressed matric water potential to water content, and a water potential curve for salt solutions, which expressed osmotic water potential as a function of salt concentrations.  Activities of alkaline phosphatase and ß-glucosaminidase were measured in increasing salt concentrations and expressed as 1st order decay.  When related to soil water potential, enzyme activity profiles observed in the salt solutions were similar to those measured in soils at different water potentials.  Many bio-inspired sensors, such as molecularly imprinted polymers (MIPs), are analogues of enzymes.  We used a similar approach to measure a MIP response as a function of water potential.  Using a system having constant concentration of a target molecule, we measured a linear decrease in MIP performance from 0.1M to 5.0M NaCl.  The results obtained represent a unique dataset that relates the response of a biomimitic sensor to low water potentials representative of surface soils.  This approach has application in predicting times when, based on ephemeral soil conditons, biomimitic sensor response would be maximal.

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