Grantee Research Project Results

2005 Progress Report: Development and Characterization of Microbial Inocula for High-Performance Passive Treatment of Acid Mine Drainage

EPA Grant Number: R829515C009
Subproject: this is subproject number 009 , established and managed by the Center Director under grant R829515
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: Center for Comprehensive, optimaL, and Effective Abatement of Nutrients
Center Director: Arabi, Mazdak
Title: Development and Characterization of Microbial Inocula for High-Performance Passive Treatment of Acid Mine Drainage
Investigators: Reardon, Kenneth F. , Pruden, Amy
Institution: Colorado State University
EPA Project Officer: Aja, Hayley
Project Period: October 1, 2003 through September 30, 2006
Project Period Covered by this Report: October 1, 2004 through September 30, 2005
RFA: Hazardous Substance Research Centers - HSRC (2001) Recipients Lists
Research Category: Hazardous Waste/Remediation , Land and Waste Management

Objective:

The overall objective of this research project is to investigate the role of inocula in the performance of sulfate-reducing permeable reactive zones (SR-PRZs). Although our past work on project R829515C003 focused on the role of the organic matter used, this research project focuses on the role of the microbes. We currently are applying the molecular tools that were developed in project R829515C003 to characterizing and comparing inocula. The specific objective of this research project is to compare inocula with respect to three parameters: (1) startup time; (2) sulfate reduction rate; and (3) activity retention time.

Progress Summary:

Pilot Column Research

The purpose of this research was to provide proof-of-concept that the source of inoculum can impact the overall column performance. Three pairs of duplicate columns were operated in parallel differing only in the source of the inoculum: (1) dairy manure; (2) acclimated column material (from another Center project); and (3) no inoculum. The columns were fed simulated mine drainage water containing 10 mg/L zinc, 5 mg/L cadmium, and 20 mg/L iron(II) at a pH of 5.5 and were operated for 5 months. This research demonstrated that the sulfate removal rate, the metal removal rate, and the extent of pH neutralization were all superior in the columns inoculated with the acclimated column material. A manuscript on this work currently is in preparation to be submitted for publication.

Batch Research

The purpose of the batch research was to screen further and test promising inocula that will provide the basis for development of optimized inocula for SR-PRZs. Five inocula were compared: (1) dairy manure; (2) acclimated column material; (3) anaerobic digester sludge; (4) Luttrell SR-PRZ (Montana); and, (5) Peerless Jenny SR-PRZ (Montana). Also, bottles without inocula were included in the research, and the effect of agitating the bottles was tested. Total gas production was monitored weekly, and the headspace composition of bottles was analyzed at the end of the research for carbon dioxide, methane, and hydrogen sulfide by gas chromatography with a thermal conductivity detector. In addition, the structure of the microbial community was monitored using denaturing gradient gel electrophoresis (DGGE), quantitative polymerase chain reaction (Q-PCR) to quantify sulfate-reducers, and cloning and restriction fragment length polymorphism (RFLP) to determine the initial diversity of the starting material. Sulfate, metals, and pH also were monitored by sacrificing bottles with time. The results of this research confirmed that inoculum plays a role in performance. In particular, the Luttrell SR-PRZ inoculum was superior with respect to its overall gas production and start-up time, whereas the Peerless Jenny inoculum and the dairy manure inocula were slow to start up, but eventually reached sulfate-reducing rates equivalent to or greater than the Luttrell inoculum. The acclimated column material performed as poorly as the blank inoculum, which was likely due to the low inoculum density and significant exposure of the inoculum to oxygen prior to the experiment. Molecular analyses of the research correlated well to the observed performance: for example, sulfate reducers were detectable during times of active sulfate-reduction, and a notable degradation of community structure was correlated in some cases to loss of sulfate-reduction. A manuscript is being prepared to summarize the results of this research.

Microbial Community Analysis

We are applying the various molecular tools developed under another Center project to characterize the inocula and monitor the behavior of the microbial communities during start up, during active sulfate reduction periods, and during decline of sulfate reduction. We then will use these tools as guides for optimizing inocula for site-specific SR-PRZ concerns. We have used DGGE to monitor and compare columns and batch reactors and have been able to identify several functional groups (cellulose degraders, fermenters, and sulfate-reducers). Cloning of 16S rRNA genes followed by RFLP screening also is being performed on select samples and provides an assessment of the overall diversity of the inoculum. Real-time Q-PCR is being used as a complementary method to specifically quantify two different groups of sulfate-reducers: Desulfovibrio and Desulfobacterium. Finally, cloning and sequencing of the apsA gene specific to sulfate-reducers provides an overview of the kinds of sulfate-reducers present in the samples. This is necessary because, based on our previous work, the concentrations of sulfate-reducers is relatively low (<1% of the total community) and thus difficult to detect by DGGE or 16S gene cloning.

Field Research

To transition our work to applicability in the field, we have begun working with several field sites, including: Luttrell, Montana; Peerless Jenny King, Montana; Leviathan, California; Elizabeth, Vermont; and Golinsky, California. Access to sites and samples from sites has been coordinated through Jim Gusek of Golder Associates, Robb Amick of EQ Management, Inc., and David Reisman of the U.S. Environmental Protection Agency (EPA). In June and August 2005, the investigators traveled to Luttrell and Peerless Jenny King for comprehensive sample collection for microbial community analyses. Smaller sample sets from other sites have been provided directly by Golder and/or EQ. Our primary goal first is to characterize the microbial communities active at these sites and to correlate community composition to overall performance in the field. It is expected that by the end of December 2005, we will have completed a comprehensive evaluation of the microbial communities present in the field reactors, including: cloning and 16S gene RFLP, cloning and sequencing of apsA genes, and Q-PCR of Desulfovibrio and Desulfobacterium. Initial results indicate that the microbial populations at Luttrell are significantly different than those at Peerless Jenny. Furthermore, within Luttrell, the microbial community is relatively uniform, whereas at Peerless Jenny there is significant heterogeneity with distance and with depth. In particular, there is evidence that microaerophillic conditions in the reactor have had a substantial influence on the microbial community. We expect to have a manuscript prepared for submission by January 2006, that summarizes the results of this initial research. The next step following this work will be to begin to conduct inoculum experiments at field-scale.

Supplemental Keywords:

waste treatment, acid mine drainage, acid mine runoff, aquatic ecosystems, biodegradation, contaminant, transport, contaminated waste sites, microbial degradation, permeable reactive barrier, sediment transport, sulfate reducing bacterium, suspended sediment,, RFA, Scientific Discipline, Industry Sectors, TREATMENT/CONTROL, INTERNATIONAL COOPERATION, Waste, Hazardous, Remediation, Ecological Risk Assessment, Waste Treatment, Contaminated Sediments, Hazardous Waste, Treatment Technologies, Geology, Mining - NAIC 21, Bioremediation, Ecology and Ecosystems, Environmental Engineering, contaminant transport, heavy metals, stream ecosystems, metal removal, microbial degradation, mining impacted watershed, mining, acid mine drainage, treatment, suspended sediment, permeable reactive barrier, sulfate reducing bacterium, natural organic matter, mining wastes, risk assessment, redox, remediation technologies, acid mine runoff, biodegradation, groundwater, sediment transport, field monitoring, aquatic ecosystems, contaminated waste sites, runoff

Relevant Websites:

http://www.engr.colostate.edu/hsrc/ Exit

Progress and Final Reports:

Original Abstract

2004 Progress Report

Final

Main Center Abstract and Reports:

R829515    Center for Comprehensive, optimaL, and Effective Abatement of Nutrients

Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R829515C001 Redox Transformations, Complexation and Soil/Sediment Interactions of Inorganic Forms of As and Se in Aquatic Environments: Effects of Natural Organic Matter
R829515C002 Fate and Transport of Metals and Sediment in Surface Water
R829515C003 Metal Removal Capabilities of Passive Bioreactor Systems: Effects of Organic Matter and Microbial Population Dynamics
R829515C004 Evaluating Recovery of Stream Ecosystems from Mining Pollution: Integrating Biochemical, Population, Community and Ecosystem Indicators
R829515C005 Rocky Mountain Regional Hazardous Substance Research Center Training and Technology Transfer Program
R829515C006 Technical Outreach Services for Communities and Technical Assistance to Brownfields
R829515C007 Evaluation of Hydrologic Models for Alternative Covers at Mine Waste Sites
R829515C008 Microbial Reduction of Uranium in Mine Leachate by Fermentative and Iron-Reducing Bacteria
R829515C009 Development and Characterization of Microbial Inocula for High-Performance Passive Treatment of Acid Mine Drainage
R829515C010 Reactive Transport Modeling of Metal Removal From Anaerobic Biozones
R829515C011 Assessment of Electrokinetic Injection of Amendments for Remediation of Acid Mine Drainage
R829515C012 Metal Toxicity Thresholds for Important Reclamation Plant Species of the Rocky Mountains
R829515C013 An Improved Method for Establishing Water Quality Criteria for Mining Impacted Streams