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1999 Progress Report: A Contained Simulation of Field Application of Genetically Engineered Microorganisms (Gems) for the Bioremediation of PCB Contaminated Soils
EPA Grant Number: R825540C004Subproject: this is subproject number 004 , established and managed by the Center Director under grant R825540
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: HSRC (1989) - Great Lakes/Mid Atlantic HSRC
Center Director: Hunter, Ray
Title: A Contained Simulation of Field Application of Genetically Engineered Microorganisms (Gems) for the Bioremediation of PCB Contaminated Soils
Investigators: Tharakan, John , Liou, Raycharn , Martin, Edward , Quensen, John , Tiedje, James M. , Tsoi, Tamara V.
Institution: Howard University , Michigan State University
EPA Project Officer: Manty, Dale
Project Period:
Project Period Covered by this Report: January 1, 1998 through September 30, 1999
RFA: Hazardous Substance Research Centers - HSRC (1989)
Research Category: Hazardous Substance Research Centers
Description:
Objective:The overall goals are characterized by a shift from the discovery phase to the developmental phase, i.e. to capitalize on past research findings by converting the past discoveries into a field-suitable technology. These studies will also define needed parameters for HU larger scale test, e.g. inoculum density, physiological state and carrier conditions.
Objective of Task 1: Perform anaerobic biotreatment of Aroclor contaminated soils from contaminated sites.
Objective of Task 2: Perform bench scale testing of long term survival and feasibility of use of genetically constructed PCB-biodegrading organisms in PCB-contaminated site soils.
Objective of Task 3: Design, construct and test the Bioaugmented Tilled Soil Reactor (BTSR) for sequential anaerobic-aerobic remediation of a PCB contaminated soil.
Progress Summary:Rationale: PCBs remain a problematic environmental pollutant to remediate. This has produced a remediation strategy of sequential anaerobic-aerobic treatment. The barriers to application were the slow rate of anaerobic PCB dechlorination which usually yielded incompletely dechlorinated PCBs, the inability of wild type microbes to actually grow on important PCBs in nature and the means to couple these processes in a cost-effective in situ remediation scheme. Previously we discovered, isolated, and characterized aromatic ring dechlorinases that metabolize the products of PCB metabolism in bacteria. We have also demonstrated the proof of concept, namely that we can use these genes to construct a genetically modified organism that grows on certain important PCB congeners. This project is focused on developing and testing field-ready technologies that would allow employing the recombinant bacteria in the two-phase anaerobic-aerobic PCB bioremediation scheme.
Approach: Two types of soils will be used in the HU and MSU studies. These will include (1) soils from contaminated sites, and (2) artificially spiked soil. The question of obtaining sufficient quantities of PCB contaminated soil has always been a big problem for both teams. Since MSU already has some experience using Picatinny soil and has a couple of gallons to spare, the HU team will establish a BTSR using Picatinny soil as well. However, MSU does not possess, and is not likely to receive, sufficient soil to conduct the entire series of lab-scale simulations of potential field demonstrations. Thus, the other soils to be used will be from another site as well as an artificially contaminated soil. From a research perspective, this will enable researchers to create a uniformly and homogeneously contaminated soil matrix for use in all studies, facilitating consistent comparisons of biodegradation research results.
The MSU research is testing several details of the process that need to be optimized at the flask scale. These include testing the effect of Fe ion (residual from anaerobic phase treatment) on efficacy of aerobic treatment, applicability of use of vermiculite for delivery of microbial inocula, optimum ratios of mixed inoculate strains, and verifying the extent of growth and plasmid stability. These experiments should help define critical parameters and protocol for use of genetically modified organisms for PCB remediation on a larger scale.
The laboratory simulation of an in situ field bioremediation process is being conducted at HU as follows: The box is first filled to 2/3 depth with an uncontaminated sandy soil mixture. This will facilitate drainage and aeration. Next, additional soil is contaminated with PCBs (or site soil is used) and this soil is layered on top of the uncontaminated drainage soil. Next, the aerator is inserted into the soil and nitrogen is pumped through it to strip oxygen from the soil. Finally, the contaminated soil layer is tilled with a "rake" (1) while a starch solution is mixed into this layer to ensure the rapid development of completely anoxic conditions. The lid of the BTSR box is then sealed closed using a gasket liner. Reduced anaerobic medium (RAMM) is periodically pumped through the liquid nutrient line onto the soil surface to keep the soil matrix moist. This sealed box is then left undisturbed and unmixed for four months to maximize the anaerobic dechlorination processes that will be ongoing. At the end of the four-month period, the box will be opened and sampled in triplicate. It will be important to make sure that samples are withdrawn from several locations to determine the homogeneity of the ongoing processes. Samples will be analyzed at HU for PCB congener levels and samples will also be sent to MSU for analysis of byproducts of PCB congener bio-transformations.
After four months of anaerobic conditions, the fluid circuits to the reactor will be opened and nutrient media to support the GEMs will be pumped through the bed of soil. The media will be oxygenated and spiked with a cosubstrate that will induce the aerobic biodegradation of PCB congeners by the GEMs. Thus, the aerobic cometabolic biotransformative phase of the cycling anaerobic-aerobic process will be simulated. The aerobic phase will be permitted to operate for 1 month. At the end of the month, samples, again spatially distributed, will be withdrawn and analyzed. (2)
1. The raking
approach is common to landfarming techniques since it uses conventionally
available soil rakes to perform the surface mixing. The soil will NOT be
completely mixed since this is not the approach being proposed and mixing of
large quantities of soil in the field would both defeat an in-situ strategy, and
be prohibitively costly.
2. The BTSR is designed with a lid that can seal the
box reactor so that head-space conditions including oxygen level can be
controlled. The lid is designed for easy removal and replacement, tightened with
bolts and wing-nuts that seal over a gasket so that the BTSR can be sealed for
the anaerobic phase. After the contaminated soil layer has been tilled and the
starch mixed in and BTSR container sealed, the BTSR is then connected to a
reservoir and a pump that permits media to be sprayed onto the tilled soil
surface to maintain soil moisture. The reservoir has a stopper port with a gas
inlet that permits the oxygenation of media if required.
Current Status: The sites apparently available for securing samples are Latex and the US Navy Yard, Washington DC. We are arranging for 55-gallon drums of contaminated soils/sediments for testing. Shipping is expected in the next month or two. Since we are dealing with the US Army Corps of Engineers and the US EPA, we may expect a "glitch" in obtaining either or both of the samples. Two possible sites provide a safety factor of sorts.
The experimental design is complete and is being used to guide the reactor-testing phase. The prototype reactor is ready and testing has already occurred. The materials are in hand for all the necessary reactors for the testing phase.
We have designed recombinant strain Burkholderia sp. LB400 that now possesses two ortho-dehalogenating primary oxygenases (bph and ohb) in addition to pre-existing chlorocatechol ortho-cleavage pathway. We showed that this organism is capable of an efficient dechlorination and growth on a wide range of ortho- and ortho+para-chlorinated PCB congeners. Particularly, it grew on high concentrations (of at least up to 5 mM) of defined Mixes M and C which represent PCB patterns typically observed in anaerobically dechlorinated PCB-contaminated sediments. This efficient degradation appears to further improve when mixed inoculum that consists of LB44(ohb) and Rhodococcus sp. RHA1(fcb) is used (the latter strain is capable of growth on para-CB(A)).
Survival of one of our recombinant organisms, Rhodococcus sp. strain RHA1(fcb)RifR was evaluated in microcosm experiments with soil from a non-contaminated area at the Picatinny Arsenal to which we added the target congeners. The indigenous microbial population present in the soil samples was evaluated using staining with 5-(4,6-dichlorotriazine-2-yl) aminofluoroscein (DTAF) followed by epifluorescence microscopy. The non-sterile soil (moisture content adjusted to 30%) was then amended with 100 ppm 4-CB and inoculated with the recombinant organism at an inoculation density of 10^4 cells/g of soil. Survival of the recombinant was evaluated at 1-week intervals for up to 60 d of incubation by measuring colony-forming units (CFU) on rifampicin-containing LB-plates. We observed an order of magnitude increase in the CFU with a peak after 10 days of inoculation and the 4-CB removal (96%), followed by a decrease in the number of CFU. Using specific PCR-primers, we found that in every sampling, all 50 randomly chosen RifR colonies retained the fcb genes. Achieving growth and PCB removal in non-sterile soil is a major step towards the practical goal.
Experiments are in progress studying the effect of FeSO4, FeS and combination FeSO4+FeS on growth and degradative activity of recombinant strains LB400(ohb) and RHA1(fcb). Our preliminary data indicated that at concentrations likely to be used during stimulated anaerobic PCB dechlorination, FeSO4 and FeS do not affect survivability and PCB degradation activity of the recombinant strains.
We are also conducting microcosm experiments to work out protocol for microbial inocula delivery. Our results indicated that use of carrier vermiculite greatly improved survivability of LB400(ohb) in 2-CB contaminated non-sterile Picatinny Arsenal soil. Effect of vermiculite on survivability of RHA1(fcb) is currently being studied.
Client/Users -Technology Transfer and Outreach Plan: We are moving the use of bioremediation technologies for field use in cleaning up PCB contaminated sites closer to full scale application; We have demonstrated that cometabolic biotransformation of various PCB congeners is supported by cosubstrates such as biphenyl, naphthalene and terpenic compounds and that the two organisms that we obtained from MSU, Comomonas testosteroni and Rhodococcus erythropolis can biotransform various PCB congeners, ranging from di- to hexa.; We have demonstrated this technology using the named organisms in aqueous, slurry and soil systems; We have also demonstrated that a cycling anaerobic/aerobic process can result in the reduction and biotransformation of higher chlorinated PCB congeners, which dechlorinate under anaerobic conditions when the contaminated media is amended with anaerobic sediments and fed Reduced Anaerobic Mineral Media (RAMM) in a re-circulating loop through a packed soil bed reactor.
All of our efforts have so far been at the laboratory scale, in vitro, where all the aforementioned technologies and processes have been successfully demonstrated. We are in the process of establishing a bench-scale field simulation using genetically modified organisms from Jim Tiedje's (MSU) laboratory and contaminated site soils.
It is important that practicing environmental engineers be made aware of this technology and the progress being made. Through the course of the project from 1995 to 1999, various technical presentations have been made at professional meetings and research papers have been published both as part of proceedings of conferences and peer-reviewed journals. In addition to the professional environmental remediation research and development community, local communities that are affected by PCB contaminated sites should be made aware of the potential of these technologies to clean up their immediate environment. The consulting engineering community and the technical community responsible for applying technologies for remediation are not typically reviewing academic peer reviewed research journals. They are more likely to review and be impacted by information exchange in such publications as Pollution Engineering, Remediation, Groundwater, etc.. A list of the most prominent ones will be developed and the staff of the Project will be encouraged to submit publications to these. The trade literature is more and more important as we move into the applications phase.
We will be using all the standard mechanisms of information dissemination, including professional presentations and journal publications. In addition, publications will be submitted to trade journals that are more focused on the applied remediation community as mentioned above, and through Internet web sites that promote reporting on applications technology such as the EPA NCERQA sites.
Journal Articles:No journal articles submitted with this report: View all 6 publications for this subproject
Supplemental Keywords:Water, INTERNATIONAL COOPERATION, TREATMENT/CONTROL, Scientific Discipline, Waste, RFA, Chemical Engineering, Hazardous Waste, Environmental Engineering, Environmental Chemistry, Contaminated Sediments, Hazardous, Ecology and Ecosystems, Treatment Technologies, Bioremediation, bioavailability, biodegradation, environmentally acceptable endpoints, bioacummulation, fate and transport , membrane processes, bioaccumulation, groundwater, kinetic studies, contaminated sediment, hazardous organic compounds, alternative endpoints, contaminant transport, in-situ bioremediation, contaminants in soil, contaminated soils, contaminated soil, bioremediation of soils, contaminated groundwater, groundwater remediation, PCB, genetically engineered microorganisms, sequestration
Progress and Final Reports:
Original Abstract
2000 Progress Report
Main Center Abstract and Reports:
R825540 HSRC (1989) - Great Lakes/Mid Atlantic HSRC
Subprojects under this Center:
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R825540C001 Development and Verification of A Molecular Modeling Approach for Predicting the Sequestration and Bioavailability/Biotoxicity Reduction of Organic Contaminants by Soils and Sediments
R825540C002 Molecular Modeling of Hydrophobic Organic Contaminants Uptake and Sequestration by Soil Organic Matter
R825540C003 The Use of Microfiltration and Ultrafiltration Membranes for the Separation, Recovery, and Reuse of Surfactant/Contaminant Solutions
R825540C004 A Contained Simulation of Field Application of Genetically Engineered Microorganisms (Gems) for the Bioremediation of PCB Contaminated Soils