Semester

Spring

Date of Graduation

2022

Document Type

Thesis

Degree Type

MS

College

Statler College of Engineering and Mineral Resources

Department

Civil and Environmental Engineering

Committee Chair

Emily Garner

Committee Co-Chair

John Quaranta

Committee Member

John Quaranta

Committee Member

Lian-Shin Lin

Abstract

Escherichia coli, a fecal coliform, is an essential component of water quality monitoring, entering waterways through sewage deposits, agricultural land and livestock, and wildlife. Various fast and user-friendly methods for quantifying E. coli make it the principal indicator bacteria for fecal contamination. The most common methods for E. coli quantification are culture-based and involve cultivating samples onto nutrient agar and counting the visible colonies. However, E. coli can enter a viable but non-culturable (VBNC) state in stressful environments. Bacteria in a VBNC state are still alive but reduce key metabolic activities that typically encourage growth and division, allowing them to remain undetected using standard culture-based methods. VBNC E. coli have demonstrated the ability to both remain pathogenic and resuscitate when the stressor is removed. The main objective of this study is to investigate whether E. coli can be induced into a VBNC state in mining-influenced waters and streams that have been treated for acid mine drainage. Additionally, this study aims to determine whether culture, substrate coliform test, and molecular methods are well suited for detecting E. coli and fecal coliforms in this environment.

Samples from the Decker’s Creek and West Run watersheds were tested for pH and the common acid mine drainage metals, aluminum, iron, calcium, magnesium, and manganese, were determined as well as potassium, phosphorus, and sodium. E. coli was first dosed into different dilutions of an acid mine drainage sample to compare quantification results between culture and microscopy staining methods with increasing environmental stress. E. coli was then dosed into 20 different stream samples to compare culture and microscopy methods with the total number of uidA gene copies across different stream environments. Additionally, 36 stream samples were tested for E. coli using standard quantification methods and compared with the number of gene copies.

This study found that the percentage of living cells detected by microscopy staining was not as influenced by increasing environmental stress as standard culture methods were. Across varying water quality, the percentage of viable cells quantified through culturing was small compared to the percentage of live cells determined by microscopy staining. Additionally, standard filtration-based culture methods may be more sensitive to physicochemical water quality. These results indicated the presence of VBNC cells in mining-influenced waters, which is causing inaccuracies in standard quantification methods.

Embargo Reason

Publication Pending

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