Final Report: 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 are major players in the nutrient cycles. This study identified the endogenous microbial community present in the Canacadea Creek watershed 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 objectives of this project were 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.

Summary/Accomplishments (Outputs/Outcomes):

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

Two student employees were hired during summer 2002, and we made significant progress in developing our protocols both in the laboratory and in the field. We 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 then were collected, processed, and used to optimize carbohydrate utilization studies, using the Biolog system.

Genomic fingerprinting using denaturing gradient gel electrophoresis (DGGE) 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 polymerase chain reactions (PCRs). PCR reaction conditions then were 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 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 we 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 2: Testing and Sample Collection (May 2003 - August 2003)

Genomic DNA was isolated from bulked soil samples as per our previously determined protocols. Extensive analysis and troubleshooting of our protocols suggested that although 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, although optimal for use as a template in DGGE analysis, would not be acceptable for T-RFLP analysis. It was possible, however, to re-optimize the genomic preparation protocols to allow the larger DNA products, and hence T-RFLP analysis. In addition to 16s rDNA amplification, we also used the T-RFLP protocol to analyze denitrifier communities, by focusing on amplification of nirS, nirK, and nos genes.

We 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.

Phase 3: Data Analysis (August 2003 -May 2004)

Although the initial project completion date was August 2003, work on this project is not yet complete and we are waiting for all data to be final before we make any final conclusions. Our 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 laboratories without the budget to afford more expensive sequencing/acrylamide-based analysis.

This study has not yet resulted in any publications or presentations, but I anticipate at least three papers and one to two poster presentations will result from this work. The first research-based paper will present the results analyzing the denitrification populations in different regions of the watershed, and the second research-based paper will examine and present genomic and metabolic fingerprints in relation to sulfate and nitrate inputs. Our modified method of agarose-based gel separation and visualization of fluorescent end-labeled DNA fragments for T-RFLP will be adapted for classroom use in BIO 221 Microbial Ecology this spring, assessed as an exercise, and prepared for publication in an education-based journal (e.g., Microbiology Education.) Finally, I will be submitting a poster for presentation at the American Society for Microbiology’s General Meeting in May 2004.

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

Relevant Websites: Exit Exit

Progress and Final Reports:

Original Abstract
  • 2002

  • Main Center Abstract and Reports:

    R830420    Center for Environmental and Energy Research (CEER)

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R828737C001 Environmental Impact of Fuel Cell Power Generation Systems
    R828737C002 Regional Economic and Material Flows
    R828737C003 Visualizing Growth and Sustainability of Water Resources
    R828737C004 Vibratory Residual Stress Relief and Modifications in Metals to Conserve Resources and Prevent Pollution
    R828737C005 Detecting and Quantifying the Evolution of Hazardous Air Pollutants Produced During High Temperature Manufacturing: A Focus on Batching of Nitrate Containing Glasses
    R828737C006 Sulfate and Nitrate Dynamics in the Canacadea Watershed
    R828737C007 Variations in Subsurface Denitrifying and Sulfate-Reducing Microbial Populations as a Result of Acid Precipitation
    R828737C008 Recycling Glass-Reinforced Thermoset Polymer Composite Materials
    R828737C009 Correlating Clay Mineralogy with Performance: Reducing Manufacturing Waste Through Improved Understanding
    R830420C001 Accelerated Hydrogen Diffusion Through Glass Microspheres: An Enabling Technology for a Hydrogen Economy
    R830420C002 Utilization of Paper Mill Waste in Ceramic Products
    R830420C003 Development of Passive Humidity-Control Materials
    R830420C004 Microarray System for Contaminated Water Analysis
    R830420C005 Material and Environmental Sustainability in Ceramic Processing
    R830420C006 Interaction of Sealing Glasses with Metallic Interconnects in Solid Oxide and Polymer Fuel Cells
    R830420C007 Preparation of Ceramic Glaze Waste for Recycling using Froth Flotation
    R830420C008 Elimination of Lead from Ceramic Glazes by Refractive Index Tailoring
    R830420C010 Nanostructured C6B: A Novel Boron Rich Carbon for H2 Storage