Grantee Research Project Results

2002 Progress Report: Microbial Indicators of Bioremediation Potential and Success

EPA Grant Number: R828770C009
Subproject: this is subproject number 009 , 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: Duke University Center for Environmental Implications of NanoTechnology
Center Director: Wiesner, Mark R.
Title: Microbial Indicators of Bioremediation Potential and Success
Investigators: Banks, M. Katherine , Alleman, Jim
Institution: Purdue University
EPA Project Officer: Aja, Hayley
Project Period: October 1, 2001 through September 30, 2002
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:

The objectives of this research project are to: (1) evaluate innovative biodiversity assessment techniques to predict microbial community changes in contaminated soil; and (2) assess whether any of the proposed techniques provide results that correlate positively or negatively to contaminant removal during phytoremediation.

Progress Summary:

Several molecular biological techniques can be applied to meet this objective (e.g., denaturing gradient gel electrophoresis [DGGE], phospholipid-fatty acid [PLFA], thermal-GGE [TGGE]); however, after a thorough literature review and two visits to internationally known microbiology laboratories this summer, we decided that terminal-restriction fragment length polymorphism (T-RFLP) is the most appropriate technique available to meet our project objectives.

T-RFLP is a technique that uses polymerase chain reaction (PCR), in which one of the primers is fluorescently labeled. After amplification, the PCR product is digested with one or more restriction enzymes, generating fragments with different lengths (depending on the DNA sequence of the bacteria analyzed and the specificity of the enzyme). T-RFLP is a molecular approach that can assess subtle genetic differences between strains as well as provide insight into the structure and function of microbial communities. The technique has both high sensitivity and throughput; it is an ideal tool for comparative analyses.

Well-characterized techniques such as DGGE and TGGE separate amplification products by sequence-dependent helix denaturation and the accompanying change in electrophoretic mobility. Another approach, single-strand conformation polymorphism (SSCP), takes advantage of sequence-dependent conformational differences between reannealed single-stranded products, which also results in electrophoretic mobility changes. The T-RFLP was developed most recently and has three clear advantages. First, direct reference to the sequence database is possible. Second, the nucleic acid sequencing technology has considerably greater resolution than the electrophoretic systems of either DGGE or SSCP. Third, the T-RFLP gel analysis is instantaneous and the output is digital.

DGGE, TGGE, and SSCP allow for the analysis of multiple samples, but the community fingerprints generated do not directly translate into taxonomic information. For example, T-RFLP and DGGE analyses have been compared for complex marine bacterial communities. Both methods were optimized for accurate determination of operational taxonomic units from the samples, but using T-RFLP resulted in a more sensitive data set. Amplified ribosomal DNA restriction analysis (ARDRA) is another community analysis technique that provides a representation of the microbial community through restriction analysis of clones in a rDNA library. Although ARDRA was effective for identifying phylogenetic groups in a highly diverse community, it is prohibitively expensive and time consuming for this research project, because of the construction of clones and the identification of environmental clones by sequence analysis. In conclusion, T-RFLP provides a sensitive and rapid technique for assessing amplification-produced diversity within a community as well as comparative distribution across communities. We propose to focus on this molecular technique for the remainder of this research project. We anticipate correlating results from the T-RFLP with other microbial assessments such as most probable number (MPN) of contaminant degraders and plate counts.

Future Activities:

After the technique development phase of this research project is complete (anticipated January 2003), we will begin sample collection. Limitations of the technique, as a result of change in environmental conditions, will be noted and discussed in detail in progress reports. The potential use of this technique as a site assessment and/or screening tool also will be discussed in depth in the final report.

Supplemental Keywords:

bioavailability, biochemistry, biodegradation, bioremediation, contaminant, contaminated sediment, contaminated soil, contaminated soils, degradation, denaturing gradient gel electrophoresis, genetics, microbes, microbial degradation., RFA, Scientific Discipline, Waste, Water, Hazardous, Environmental Chemistry, Contaminated Sediments, Microbiology, Hazardous Waste, Environmental Microbiology, Molecular Biology/Genetics, Bioremediation, bioavailability, bioremediation of soils, microbial degradation, contaminated sediment, phytoremediation, biochemistry, contaminated soil, biodegradation, denaturing gradient gel electrophoresis, genetics, microbes, degradation, contaminants in soil

Relevant Websites:

http://bridge.ecn.purdue.edu/~mhsrc/ Exit

Progress and Final Reports:

Original Abstract

Final

Main Center Abstract and Reports:

R828770    Duke University Center for Environmental Implications of NanoTechnology

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