Grantee Research Project Results
2002 Progress Report: Adaptation of Subsurface Microbial Biofilm Communities in Response to Chemical Stressors
EPA Grant Number: R828770C006Subproject: this is subproject number 006 , established and managed by the Center Director under grant R828770
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: Center for Air, Climate, and Energy Solutions
Center Director: Robinson, Allen
Title: Adaptation of Subsurface Microbial Biofilm Communities in Response to Chemical Stressors
Investigators: Bishop, Paul , Stevens, Am , Love, Nancy
Current Investigators: Love, Nancy , Stevens, Am
Institution: University of Cincinnati , Virginia Tech
Current Institution: Virginia Tech , University of Cincinnati
EPA Project Officer: Aja, Hayley
Project Period: October 1, 2001 through September 30, 2003
Project Period Covered by this Report: October 1, 2001 through September 30, 2002
Project Amount: Refer to main center abstract for funding details.
RFA: Hazardous Substance Research Centers - HSRC (2001) Recipients Lists
Research Category: Hazardous Waste/Remediation , Land and Waste Management
Objective:
A thorough understanding of how microbiological subsurface communities adapt to the changing concentrations of chemical stressors is necessary for the effective restoration of contaminated subsurface environments to function properly. We hypothesize that both catabolism and microbial stress responses significantly affect how subsurface biofilm communities adapt to dynamic changes in the concentration and type of chemical stressor, and that the relative contributions of these processes are distinguishable. We are focusing on the study of a specific stress response, the glutathione-gated potassium efflux (GGKE) system, which is activated in response to electrophilic chemical stressors (see Figure 1). A matrix of environmentally relevant chemical stressors (pentachlorophenol [PCP], cadmium, and benzene) have been selected for this study, allowing the relative roles of catabolism versus the GGKE system on biofilm community adaptation during restoration of contaminated sites to be determined.
Specially designed soil columns will be constructed and operated aerobically by exposing the columns to slowly increasing, stabilizing, and decreasing concentrations of a given contaminant over an 18-month period. The selected contaminants cover a range of properties; two are electrophilic (PCP and Cd) and two are capable of being catabolized (PCP and benzene). Changes in the structure of the soil column microbial communities over space and time will be determined microscopically based on biochemical variations and microbial community shifts, using confocal scanning laser microscopy to visualize physical heterogeneities. Changes in function will be monitored with microelectrodes that target key constituents, including dissolved oxygen (DO), oxidation-reduction potential (ORP), pH, and K+, and by monitoring contaminant fate in pore water samples. Fluorescent in situ hybridization (FISH) also will be used to track catabolically active regions of the biofilm, which will be compared to regions that are actively engaging the GGKE system, as determined biochemically and with the K+ microelectrode.
Progress Summary:
This research project has been underway for approximately 9 months. Graduate students involved with the project have compiled extensive literature. Several iterations of soil column reactors were made and tested to find one that will give the desired performance. This also allowed them to learn the necessary techniques to run these systems over extended periods, as well as the analytical procedures required (e.g., gas chromatography [GC], plate counting, carbohydrates, proteins, lipid phosphate, etc.). The graduate research assistants also have developed and refined the necessary techniques for making DO, pH, and ORP microelectrodes (tip diameters of 3-15 µm); they soon will begin work on constructing a potassium microelectrode. A benzene-degrading enrichment culture has been obtained and is being used to inoculate the soil columns. A modified sacrificial flow cell design has been developed to enable sampling of sufficient soil biomass to conduct the desired analyses without disturbing the main column. Protocols for extracting DNA from biofilm communities and analyzing diversity using temperature gradient gel electrophoresis (TGGE) have been established. Use of reverse transcription polymerase chain reaction (RT-PCR) and terminal restriction fragment length polymorphism (TRFLP) are being investigated as complimentary methods to TGGE.
Research Apparatus Construction. The University of Cincinnati (UC) group designed a complete soil column system for use in this work, and both the UC and the Virginia Polytechnic Institute and State University (VT) groups have fabricated four column systems (see Figure 1). Two additional columns are under construction in the event any of the others break. The columns are 43 cm long and have an inside diameter of 3.8 cm. The reactors are filled to a 32 cm depth with sand and are operated in an upflow manner with a 5.0 m/d pore water velocity. The column systems at both sites are identical, allowing comparative research results to be obtained.
Figure 1. Soil Column Setup
The original soil columns from a previous research project (slightly larger than the ones that are now being used) were run for several months at UC to establish baseline conditions and to eradicate any difficulties with the experimental design. We established procedures to run the column with various operational and maintenance schedules. This included plans for preparing influent solutions, collecting and analyzing samples, and cleaning and replacing the equipment. The influent solutions in our preliminary studies included mineral salts, sodium acetate, and PCP, one of the chemical stressors in question. The sodium acetate acted as a supplemental carbon source. In our current experiments, we are using the same mineral salt recipe; however, we are not using sodium acetate as the supplemental carbon source. Instead, we are using a special feed solution, biogenic organic feed solution (BOFS), developed at VT, that provides all the essential nutrients from proteins, sugars, and organic acids. The synthetic growth medium contains a diluted form of M9 as the basal salt solution, which is supplemented with a range of protein extracts, carbohydrates, and organic acids. The final concentration of organic carbon constituents is 20 mg/L as chemical oxygen demand (COD).
VT developed a culture of microorganisms capable of biodegrading benzene. Soil cultures were obtained by mixing the Ap, B, and Bt horizons of a Frederick soil with soil extract (obtained from the same soil) for 2 weeks. Bacteria from this mixture were cultured, using 1.0 X M9 Media with 1,000 mg/L BOFS as COD, and transferred serially for 2 months prior to the benzene degradation experiment. To perform benzene degradation experiments, media containing 0.1 X M9 salts with 20 mg/L BOFS as COD and approximately 3.5 mg/L benzene were prepared the evening prior to use. Hydrogen peroxide (H2O2) and catalase (excess amounts according to stoichiometry) were added prior to use to increase DO to approximately 19 mg/L O2. We used a pressurized bottle system to distribute the solution to individual preinoculated U.S. Environmental Protection Agency (EPA) vials. Each EPA vial was inoculated with 25 µL of soil bacteria, and filled with the high DO, benzene, and BOFS-containing medium. Samples were prepared in triplicate. We used two types of controls: a benzene control and dissolved oxygen/benzene controls (one for every time point). Benzene concentrations were determined by our standard benzene analytical protocol, as described in the May 2002 report. A similar experiment was conducted for PCP, except that aeration was provided by using an air-permeable sponge top on the flasks containing the inoculum; the sponge also was used for abiotic controls. Additionally, a set of flasks was inoculated with biomass from the local Blacksburg/VT wastewater treatment facility to enhance the chance of locating a PCP degrading community.
Results from the benzene biodegradation experiments are shown in Figures 1 and 2. Figure 2 shows that benzene was lost from the sacrificial flasks, starting after 49 hours, and was completely consumed. Differences in DO profiles in the benzene/DO controls relative to the experimental flasks show that the DO uptake rate increased simultaneously as benzene loss was observed, strongly suggesting that biodegradation was occurring. Results from the PCP exposed cultures are provided in Figure 3. There is variation over time, because of some issues with changed operational conditions in the GC, which have been rectified. However, when the experimental and control flasks are compared, we cannot conclude that PCP degradation is occurring in this culture. Consequently, the inoculum used to seed the column soil communities will need to be supplemented with another source known to aerobically degrade PCP.
The UC reactors have been started on BOFS using the benzene-degrading soil enrichment culture developed at VT and supplemented with mixed liquor collected from a local wastewater treatment facility that is exposed to a broad range of industrial inputs. After 1 month of culturing, the sand media will be removed from the columns, mixed to ensure homogeneity, and divided for use by the two groups. The VT portion will be shipped overnight to VT for use in their columns. In this way, we can be assured that both groups will be starting with the same initial culture.
Figure 2. Benzene Degradation Experiment With Soil Enrichment Culture. Upper chart shows benzene concentration profiles over time. Lower chart shows dissolved oxygen concentrations in sacrificed benzene/DO controls as well as experimental flasks.
Figure 3. PCP Biodegradation Experiment With Either a Soil Enrichment Culture or Biomass From the Blacksburg/VT Wastewater Treatment Plant. Variation in controls and inoculated cultures over time are an effect of analytical difficulties. Comparisons need to be made between controls and inoculated flasks.
Analytical Techniques. VT has developed protocols for analysis of benzene, PCP, and cadmium within the synthetic feed matrix. The UC team has adapted these procedures to their analytical equipment. The benzene protocol involves hexane extraction in a ratio of 7 parts sample to 1 part hexane. The extracted hexane layer is aspirated and analyzed using GC and flame ionization detection. The GC injection and analysis conditions have been optimized. For PCP analysis, samples are derivatized by acetylation and extracted hexane, and 2,4,6-trichlorophenol serves as an internal standard. Extracted samples are analyzed by GC with electron capture detection. Finally, cadmium is analyzed from nitric acid stabilized samples using atomic absorption spectroscopy.
The UC research assistants produced and calibrated several microelectrodes. The DO microelectrode is a solid-state electrode made from a low melting point alloy and has a gold-plated tip. This microelectrode amperometrically measures DO from zero concentration to saturation. The ORP microelectrode is a solid-state electrode made from a platinum wire. The pH microelectrode is a liquid-membrane, ion-selective electrode used for potentiometric measurement from pH 5.5-12. They have successfully fabricated several DO and ORP microelectrodes that are now stored for future use in the soil biofilm analysis. They also have practiced making pH microelectrodes, but these are only functional for a few days, so they cannot be prefabricated and stored. They now are researching methods for construction of a liquid ion exchange potassium microelectrode.
Future Activities:
In the coming year, we will be operating all reactors at both universities in parallel to study the specific stress response caused by the GGKE system, which is activated in response to electrophilic chemical stressors. The soil columns will be operated aerobically by exposing the columns to slowly increasing, stabilizing, and decreasing concentrations of a given contaminant over an 18-month period. The selected contaminants cover a range of properties; two are electrophilic (PCP and Cd) and two are capable of being catabolized (PCP and benzene). Changes in the structure of the soil column microbial communities over space and time will be determined based on biochemical variations, microbial community shifts, and microscopically, using confocal scanning laser microscopy to visualize physical heterogeneities. Changes in function will be monitored with microelectrodes targeting key constituents, including DO, ORP, pH, and K+, and by monitoring contaminant fate in pore water samples. FISH also will be used to track catabolically active regions of the biofilm, which will be compared to regions that are actively engaging the GGKE system, as determined biochemically and with the K+ microelectrode.
Journal Articles:
No journal articles submitted with this report: View all 4 publications for this subprojectSupplemental Keywords:
pentachlorophenol, PCP, cadmium, benzene, soil, glutathione-gated potassium efflux, GGKE system, fluorescent in situ hybridization, FISH, microelectrodes., RFA, Scientific Discipline, Toxics, Waste, Water, Chemical Engineering, Contaminated Sediments, Environmental Chemistry, Environmental Microbiology, Hazardous Waste, Bioremediation, Molecular Biology/Genetics, Hazardous, 33/50, Environmental Engineering, microbiology, biofilm, microbial biofilm, microbial degradation, genetics, catabolic biodegradation, cadmium & cadmium compounds, PCP, bioavailability, biodegradation, laser scanner microscopy, contaminated sediment, microbes, contaminated soil, contaminants in soil, fluorescent in situ hybridization, bioremediation of soils, biochemistry, toxic chemicals, phytoremediation, cadmiumRelevant Websites:
http://bridge.ecn.purdue.edu/~mhsrc/ Exit
Progress and Final Reports:
Original AbstractMain Center Abstract and Reports:
R828770 Center for Air, Climate, and Energy Solutions Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R828770C001 Technical Outreach Services for Communities
R828770C002 Technical Outreach Services for Native American Communities
R828770C003 Sustainable Remediation
R828770C004 Incorporating Natural Attenuation Into Design and Management
Strategies For Contaminated Sites
R828770C005 Metals Removal by Constructed Wetlands
R828770C006 Adaptation of Subsurface Microbial Biofilm Communities in Response to Chemical Stressors
R828770C007 Dewatering, Remediation, and Evaluation of Dredged Sediments
R828770C008 Interaction of Various Plant Species with Microbial PCB-Degraders
in Contaminated Soils
R828770C009 Microbial Indicators of Bioremediation Potential and Success
The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.
Project Research Results
Main Center: R828770
108 publications for this center
14 journal articles for this center