Final Report New Jersey Department of Environmental Protection Project Title: Applying Innovative Diagnostic Tools at New Jersey Publicly Funded Sites Investigators: Donna E. Fennell1 and Max Häggblom2 1 Department of Environmental Sciences 2 Department of Biochemistry and Microbiology Rutgers University Mailing Address: Department of Environmental Sciences 14 College Farm Road New Brunswick, NJ 08901 Phone: 848-932-5748 Fax: 732-932-88644 Email: [email protected] Project Manager: Robert Mueller Total budget Year 1: $83,500 Project Duration: 30 Jun 2011 to NCE requested to 31 Dec 2013 Reporting Period: 15 July 2012 – 31 December 2013 Contract number: SR11-025 Acknowledgements This study was funded by the New Jersey Department of Environmental Protection (NJDEP). This project supported the research of Dr. Weimin Sun, Post-Doctoral Associate, in the Department of Environmental Sciences at Rutgers University. We thank Ms. Lora McGuinness and Professor Lee J. Kerkhof of Rutgers University for assistance with stable isotope probing analyses. We acknowledge Mr. Shuai Luo, a Rutgers University undergraduate student, who assisted with laboratory work. We thank Mr. Eric Raes of Engineering and Land Planning Associates, Inc. for enabling access to contaminated sites and for sharing information. We thank Mr. Robert (Bob) Mueller for his support of this project and for establishing site contacts. 1 Table of Contents Acknowledgements 1 Executive Summary 3 Introduction/Problem Statement 4 Project Objectives 4 Project Design and Methods 5 Quality Assurance 8 Results and Discussion 9 Conclusions and Future Work 20 Recommendations and Application and Use by NJDEP 21 Expenditures 22 References 24 Project Publications and Presentations 27 2 Executive Summary This project demonstrated use of environmental molecular diagnostics (EMDs) for detecting microbial biodegradation of contaminants and identifying bacteria responsible for contaminant biodegradation or biotransformation at three contaminated sites in New Jersey. Three site studies were performed. First, sediment cores were collected from the contaminated aquifer at the former Pagan-Martinez site (Jersey City, NJ) to aid an on-going bioremediation effort. The former Pagan-Martinez site has perchloroethene and trichloroethene contamination along with fuel oil contamination. A work plan was developed for the site and microcosms were established using the aquifer core materials. After six months, no dechlorination of PCE was detected in the microcosms from the site. The site has since undergone bioaugmentation and biostimulation. An ongoing effort involves community analysis of the original aquifer sediments using terminal restriction fragment length polymorphism and sequencing. Methods to target metabolically active bacteria have been developed using isotope incorporation (e.g., 13C) into newly biosynthesized macromolecules. This is termed stable isotope probing (SIP). The 13C DNA can be recovered separately from the unlabeled DNA from other organisms. In the second project, stable isotope probing (SIP) was used to identify dehalorespiring bacteria in sediments from the organochloride-contaminated Hackensack River, NJ. Certain dehalorespiring bacteria do not assimilate carbon from the chlorinated compounds they use as electron acceptors. To label DNA from dechlorinators using SIP, site materials were exposed to 13C-based carbon sources that are assimilated by the bacteria (i.e., 13C-acetate and 13C-labeled lactate which is fermented to 13C-labeled acetate and hydrogen). The bacterial community in active Hackensack River microcosms included Dehalococcoides, Dehalogenimonas, Desulfobacterium, Geobacteraceae and Desulfuromonas as identified in the clone library derived from 13C-heavy fraction. Dehalococcoides was detected when 13C-labeled lactate was added to microcosms, but not when 13C-labeled acetate alone was added. This indicated that both hydrogen and acetate were needed to stimulate dechlorination by Dehalococcoides. This method could also be used to guide and monitor the bioremediation effort at the Pagan-Martinez site after bioaugmentation (since no dechlorination was observed in the native sediments). Finally in the third effort supported by this project, bacteria involved in the biodegradation of aniline at a chemical industrial contaminated site were identified. Results from microcosms operated over many months as part of an earlier project indicated loss of aniline in site sediments. However, the biodegradation of aniline was not conclusively demonstrated because intermediates were not identified and loss from sorption could not be ruled out. Results using SIP with 13C-labeled aniline conclusively demonstrated that aniline biodegradation occurred and the specific bacteria responsible for biodegradation under different redox conditions were identified. The use of this molecular method therefore provided evidence for bioremediation and specifically identified bacterial targets for monitoring further bioremediation efforts. This study applied EMD techniques at contaminated sites in New Jersey. Results have greatly enhanced enriched the findings that would normally be obtained using traditional site investigation tools. 3 Introduction/Problem Statement Management of contaminated sites encompasses everything from site characterization to ultimate selection, implementation, and completion of a site remedial action plan and then finally, site case closure. The investigation begins with characterization that includes specifying the contaminants and media (e.g., groundwater aquifer, sediments or soils) of interest, assessing exposure pathways, and determining remediation goals. The estimated volumes or areas of the site to be remediated must be defined. Finally, detail concerning the equipment, methods, and locations to be evaluated for each possible site remediation approach alternative (e.g., potential natural recovery processes, potential remediation plan, need for monitoring and/or institutional controls, etc.) must be determined. To the extent possible the time frame(s) in which alternatives are expected to achieve cleanup levels are also estimated. Remediation of contaminated sites may be performed using physical-chemical processes such as pump and treat with sorption or volatilization, dredging and landfilling, or chemical oxidation. More sustainable remediation options include bioremediation approaches to degrade or transform pollutants to benign end products. The Environmental Protection Agency’s definition of bioremediation is “a managed or spontaneous process in which (micro)biological processes are used to degrade or transform contaminants to less toxic or nontoxic forms, thereby remedying or eliminating environmental contamination” (USEPA). To assess the potential for application of bioremediation to a site or to select the type of bioprocess to be used in bioremediation, a variety of assessment protocols have been developed that provide guidance for monitored natural attenuation (MNA) or enhanced bioremediation based on geochemical indicators such as pH, prevailing redox conditions as indicated by the presence of various electron acceptors, and presence of transformation products of the original contaminants (for example see rating protocols from USEPA [http://www.afcee.af.mil/shared/media/document/AFD-071210-020.pdf; accessed online April 2014] and the Department of Defense [www.serdp.org/content/download/3728/59365/file/Rabitt_Protocol.pdf; accessed online April 2014]). To directly assess potential natural or enhanced biological processes for contaminant removal, powerful new environmental molecular diagnostics (EMDs) that detect and quantify the microorganisms, genes and enzymes responsible for specific processes are also recommended for use in site monitoring and management [http://www.itrcweb.org/teampublic_EMD.asp; accessed online April 2014]. These tools provide critical information to support other field data. Project Objectives The overarching goal of this project was to demonstrate applications for innovative environmental molecular diagnostics (EMDs) for informing bioremediation at contaminated sites in New Jersey. The specific objectives were: 1. Utilize emerging environmental molecular diagnostics (EMDs) (in-house and commercially available) to facilitate and guide site assessment to direct a bioremediation approach. 2. Assess a site that is or has undergone unsuccessful or stalled bioremediation and use diagnostic tools for re-assessment to re-screen and re-initiate remedial actions. 4 3. Provide data through EMD analyses to support site closure at one or more sites that have been successfully remediated or which are undergoing monitored natural attenuation. 4. Perform research in support of development and application of EMDs for novel bioremediation approaches for emerging contaminants or for those with no commercially available EMDs. In this final report we report results and findings from three NJ contaminated sites: 1. Microbial investigation of the former Pagan-Martinez site, Jersey City, NJ 2. Stable isotope probing to identify dechlorinating bacteria in the highly contaminated sediments of the Hackensack River, NJ 3. Stable isotope probing investigation of aniline degraders under different redox conditions at a large Figure 1. Sample collection at industrial site in NJ the Pagan-Martinez site in Jersey City, NJ. Project Methods Collection of Environmental Samples Aquifer materials. Sediment cores and groundwater samples were collected from