DESIGNING PRIMERS TO TEST FOR DENITRIFICATION BIOMARKERS IN SEPTIC SYSTEM LEACH FIELDS
Septic sewer systems are an increasingly popular method for wastewater treatment in domestic households across the United States. Investigating their greenhouse gas emissions and the microbial communities controlling the cycling of the two most common greenhouse gases, nitrous oxide (N2O) and methane (CH4), provides important information on their potential atmospheric impacts and helps streamline the use of molecular biology techniques to correlate microbial activity to net greenhouse gas fluxes. Over the course of a summer, gas flux measurements and soil samples from the leachfields and control plots of nine different septic systems in central New York were analyzed. While preliminary gas flux measurements and quantitative polymerase chain reaction (qPCR) data suggests that soil depth is a greater predictor of denitrifier activity than the presence of a septic leachfield, more investigation on the potential quantities, roles, and types of denitrifiers must be conducted. Thus degenerate forward and reverse primers targeting the biomarker genes of nosZ and norB as well as their appropriate protocols were designed, validated, and optimized using an array of biomolecular techniques such as PCR, gel electrophoresis, qPCR, and high-throughput sequencing. Long amplicon forward and reverse primers and their design protocols were also created, validated, and optimized in order to amplify the limited quantity of genetic material extracted from the soil samples without depleting original sources. Standard curves of these degenerate primers are being created and optimized for further in-depth analysis of the exact abundance of selected denitrifiers in the soil samples. Both the degenerate and long amplicon primers will be additionally used to help determine the microbial community composition at the septic system leachfields involved in greenhouse gas cycling. The results from this study will help inform future mitigation methods for potential greenhouse gas emissions from septic system leachfields.
Attribution 4.0 International
dissertation or thesis
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