Graduation Year

2009

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Biology (Integrative Biology)

Major Professor

Valerie J. Harwood, Ph.D.

Co-Major Professor

Daniel V. Lim, Ph.D.

Committee Member

Kathleen Scott, Ph.D.

Committee Member

Diane TeStrake, Ph.D.

Committee Member

Degeng Wang, Ph.D.

Keywords

community structure, DGGE, methanogens, BOX-PCR, VRE.

Abstract

Degradation of the several million tons of solid waste produced in the U.S. annually is microbially mediated, yet little is known about the structure of prokaryotic communities actively involved in the waste degradation process. In the first study, leachates generated during degradation of municipal solid waste (MSW) in the presence (co-disposal) or absence of biosolids were analyzed using laboratory-scale bioreactors over an eight-month period. Archaeal and bacterial community structures were investigated by denaturing gradient gel electrophoresis (DGGE) targeting 16S rRNA genes.

Regardless of waste composition, microbial communities in bioreactor leachates exhibited high diversity and temporal trends. Methanogen sequences from a co-disposal bioreactor were predominantly affiliated with the orders

Methanosarcinales and Methanomicrobiales. Effect of moisture content on indicator organism (IO) survival

during waste degradation was studied using culture-based methods. Fecal coliform and

Enterococcus concentrations in leachate decreased below detection limits within fifty days of bioreactor operation during the hydrated phase. IOs could be recovered from the bioreactor leachate even after a prolonged dry period. This study advances the basic understanding of changes in the microbial community during solid waste decomposition.

The purpose of the second study was to compare the ability of BOX-PCR to determine genetic relatedness with that of the "gold standard" method, 16S rRNA gene sequencing. BOX-PCR typing could clearly differentiate the strains within different

Enterococcus species but closely related genera were not as distinguishable. In contrast, 16S rRNA gene sequencing clearly differentiates between closely related genera but cannot distinguish between different strains of Enterococcus species. This study adds to our knowledge of genetic relationships of enterococci portrayed by two separate molecular methods.

The incidence of vancomycin resistant enterococci (VRE) in environmental matrices, residential and hospital wastewater was also investigated. Low-level VRE (

vanC genotype) were isolated from environmental matrices and residential wastewater. VRE isolates from hospital wastewater were identified as E. faecium and demonstrated resistance to ampicillin, ciprofloxacin and vancomycin (vanA genotype), but sensitivity to chloramphenicol and rifampin. Although no high-level VRE were isolated from surface waters, the high proportion of low-level VRE in environmental matrices is a cause for concern from the public health perspective.

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