Detection of bacteriophage infection using absorbance, bioluminescence, and fluorescence tests

Date of Award

2011

Degree Name

M.S. in Civil Engineering

Department

Department of Civil and Environmental Engineering and Engineering Mechanics

Advisor/Chair

Advisor: Denise Taylor

Abstract

The activated sludge treatment process is a common method employed to treat wastewater. Normal operation of this process results in a floc-forming bacterial mixture, which settles rapidly. However, filamentous bacteria can cause sludge bulking, which interferes with the compaction and settling of flocs. A common method to control sludge bulking is adding a chemical such as chlorine to the activated sludge basin, which kills not only the problematic bacteria, but also the essential floc-forming bacteria. Bacteriophages (phages) are viruses that only infect bacteria. It is hypothesized that phages of filamentous bacteria can be added to the activated sludge basin to control sludge bulking, rather than a chemical. Due to the unique morphology of filamentous bacteria, traditional methods such as the plate method do not work well to detect phage infection. The purpose of this thesis was to detect infection of bacteria by phages using absorbance, bioluminescence, and fluorescence broth tests. E. coli and T2 phage was first used to establish a model of the bacteria-phage relationship using these tests. All three broth methods show evidence of phage infection in T2 phage and E. coli mixtures. Following this, phages were isolated from activated sludge systems and were applied to E. coli and S. natans, an example of filamentous bacteria found in activated sludge bulking problems. Their growth patterns were observed using the above mentioned tests. E. coli showed obvious infection patterns, but S. natans test sets were highly variable and phage infection patterns could not be distinguished. For the absorbance test and fluorescence test, ratios of bacteria to phage that clearly showed phage infection were 1000:1 and 100:1. Low concentrations of bacteria (i.e. 10⁵ cfu/mL) are recommended for use when preparing samples because even if the titer of phage from an environmental sample is higher than expected, phage infection patterns will still be detected. The bioluminescence test showed infection patterns for all ratios of bacteria to phage. The concentration of bacteria used to prepare these ratios did not affect these patterns. The bioluminescence test is recommended to detect infection of bacteria by phages from activated sludge samples for these reasons.

Keywords

Bacteriophages Industrial applications, Sludge bulking Biological control, Sewage Purification Activated sludge process Biological control, Bioluminescence, Fluorescence

Rights Statement

Copyright © 2011, author

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