Variations in Subsurface Denitrifying and Sulfate-Reducing Microbial Populations as a Result of Acid Precipitation

EPA Grant Number: R828737C007
Subproject: this is subproject number 007 , established and managed by the Center Director under grant R830420
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Center for Environmental and Energy Research (CEER)
Center Director: Earl, David A.
Title: Variations in Subsurface Denitrifying and Sulfate-Reducing Microbial Populations as a Result of Acid Precipitation
Investigators: Cardinale, Jean
Institution: Alfred University
EPA Project Officer: Hahn, Intaek
Project Period: September 1, 2001 through August 31, 2003
RFA: Targeted Research Center (2000) Recipients Lists
Research Category: Hazardous Waste/Remediation , Targeted Research


Sulfur oxide and nitrogen oxide deposition as a result of acid precipitation has long-term detrimental effects on ecosystems. Although much attention has been paid to the cycling of nutrients within a defined system, little attention has been paid to the microbiota that inhabit these systems, and that are major players in the nutrient cycles. This study will identify the endogenous microbial community present in the Canacadea Creek watershed in order to determine denitrification and sulfate reduction potentials of the community, and to assess the flux in community structure in response to pollutant stress.

The specific aims of this project are to (1) determine the denitrifying capacity of the subsurface; (2) determine if there is a sulfate-reducing capacity in the subsurface; (3) determine if there are differences in the spatial and temporal distributions of denitrifying and sulfate-reducing microbial populations within the Canacadea Creek Watershed; and (4) correlate all community and reduction potential data obtained for input of sulfate and nitrate in the watershed.


Phase (1) Initial Testing and Assay Development: October 2001-April 2003

The investigators have made significant progress in developing protocols both in the lab and in the field. They developed and optimized sample handling, collection, and processing protocols, as well as DNA extraction and carbohydrate utilization analysis studies. Nine sampling sites were chosen, representing three levels of development, and mapped using global positioning satellite (GPS) technology.

Bulked site soil samples were collected and used as a test run of sample processing protocols and carbohydrate utilization studies. Reliable soil samples were then collected, processed, and used to optimize carbohydrate utilization studies, using the Biolog system.

Genomic fingerprinting using denaturing gradient gel electrophoresis initially suggested that it would be a promising technique for analysis of total bacterial community and species diversity. We optimized DNA extraction via a bead beater method and obtained good template for use in PCR reactions. PCR reaction conditions were then optimized to amplify the 16s rDNA gene. Initial DGGE gels showed faint fuzzy bands, suggesting little variety of bacterial species, or improper gradient formation in the gels. As several subsequent gels repeated the pattern of non-discrete bands, we decided to isolate genomic DNA from standard laboratory strains of bacteria to generate a DGGE DNA standard, which would also in turn help to optimize gradient gel preparation and formation.

The DNA standards along with commercial DNA standards were used in a series of attempts to optimize denaturing gradient gel preparation, but investigators were unable to reproduce acceptable gradient gels. We decided to attempt an alternate protocol for evaluating species diversity and richness using terminal restriction fragment length polymorphism (T-RFLP). Enhanced available public domain software for searching rRNA databases and expansion of ribosomal sequence databases suggested that T-RFLP would allow the same level of analysis that we had expected from the denaturing gradient gel electrophoresis, in addition to allowing for species identification.

Phase Two: Testing and Sample Collection (May 2003-August 2003) and Phase Three: Data Analysis (August 2003-May 2004)

Genomic DNA was isolated from bulked soil samples as per our previously determined protocols. Extensive analysis and troubleshooting of our protocols suggested that while our DNA preparation had been optimal for amplification of 1 kB DNA fragments, there was sufficient shearing as to prevent amplification of a slightly larger 1.5 kB product. What this suggested was that all previously prepared genomic DNA, while optimal for use as template in DGGE analysis, would not be acceptable for T-RFLP analysis. However, it was possible to re-optimize the genomic prep protocols to allow the larger DNA products, and hence T-RFLP analysis. In addition to 16s rDNA amplification, we will also be using the T-RFLP protocol to analyze denitrifier communities, by focusing on amplification of nirS, nirK, and nos genes.

They were able to obtain smaller (amplifiable) PCR products using modified primer sets. We used this smaller fluorescently labeled PCR product to successfully show that we could visualize end labeled DNA using an available imaging system and agarose gels, rather than sequencer imaging systems and acrylamide gels.

Expected Results:

While the initial project completion date was August 2003, work on this project is not yet complete and investigators are waiting for all data to be final before we make any final conclusions. Their success using the agarose based gels for the analysis of fluorescently-end labeled PCR and restriction products suggests a new method that may be of interest to smaller labs without the budget to afford more expensive sequencing/acrylamide based analysis.

Relevant Websites: Exit Exit

Supplemental Keywords:

denitrification, genomic fingerprints, metabolic fingerprints, agarose-based gel separation, terminal restriction fragment length polymorphism, T-RFLP, fluorescently-end labeled PCR, Genomic DNA, 16s rDNA amplification, denaturing gradient gel electrophoresis, DNA extraction, carbohydrate utilization analysis, Canacadea Watershed., RFA, Scientific Discipline, Water, Water & Watershed, Environmental Chemistry, Environmental Monitoring, Ecology and Ecosystems, Watersheds, atmospheric processes, dentrification, acid precipitation, geochemical map, microbial community, aquatic ecosystems, ecological indicators, ecosystem stress, watershed assessment

Progress and Final Reports:

  • 2002
  • Final Report

  • Main Center Abstract and Reports:

    R830420    Center for Environmental and Energy Research (CEER)

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