Identification of microbial populations in activated sludge by 16S rDNA analysis and fluorescent in situ hybridization

Author

Prakash Koderi Narayana-Karanth

Date of Award

12-1997

Degree Type

Thesis

Degree Name

Master of Science

Major

Life Sciences

Major Professor

Gary S. Sayler

Committee Members

Jeffery M. Becker, Robert N. Moore

Abstract

Total DNA was extracted from three different samples of the activated sludge taken from the wastewater system at Eastman Chemical Company, Kingsport, TN. 16S rDNA methodologies were used to characterize the microbial populations of the activated sludge and the 16S rDNA (1500 bp) was amplified by polymerase chain reaction (PCR), and subsequently cloned into the TA cloning vector. The clones were sequenced and the sequences were analyzed by comparing them with the published sequences in the Ribosomal Database Project (RDP) maintained by the Department of Microbiology, University of Illinois, Urbana-Champaigne, IL. Phylogenetic trees based on distance matrix methods were constructed from the aligned sequences. Some of the sequences were similar to the reference sequences from known organisms, but none of the sequences were identical. Comparative sequence analysis of clones from different months indicated a shift in the microbial populations of the activated sludge. Bacteria representing the α, β, γ, and σ subclasses of the Proteobacteria, as well as members of the Cytophagales/Flavobacter/Flexibacter group, and Gram-positive organisms with low (G-t-C) content were found in all three samples analyzed (January, April and November 1995). Organisms belonging to the α subclass of the Proteobacteria were dominant in April and November and organisms belonging to the β subclass of the Proteobacteria were dominant in January. Microbial diversity in activated sludge was also demonstrated by restriction fragment length polymorphism (RFLP) analysis. Sequences similar to Hyphomicrobium spp., belonging to the a subclass of the Proteobacteria, were present as 10 of the 64 sequences analyzed. Based on comparative sequence analysis, a fluorescein-labeled oligonucleotide probe complementary to the conserved region of the rRNA of Hyphomicrobium spp. was designed. The probe was designed with 1 degeneracy to encompass Hyphomicrobium spp. in the RDP and the observed library sequences. Sequencing of a Hyphomicrobium spp. isolate (Hyphomicrobium,/em> M3) from the activated sludge indicated that the library sequences and the isolate were identical in the probe target region, whereas the RDP sequences differed by 1 nucleotide. Whole-cell hybridizations of target organism and reference bacteria with one, two, four and eight mismatches in the target region were used to determine the optimal hybridization conditions for the probe. The efficacy of the probe was demonstrated by in situ hybridization of activated sludge samples. In general, the effectiveness of the use of 16S rDNA libraries for identification of important microorganisms in activated sludge was demonstrated. This was confirmed by fluorescent in situ hybridization with a probe designed based on the library sequences. Independent confirmation was provided by visible, transmission and scanning electron micrographs of the sludge. Transmission and scanning electron micrographs of the sludge indicated the presence of budding bacteria resembling Hyphomicrobium spp. in the Bergey’s Manual of Determinative Bacteriology (1994).