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Population and metabolic activity dynamics of resin-acid-degrading bacteria as determined by the RNA:DNA ratio Muttray, Annette Friederike

Abstract

This thesis describes the development and application of a new quantitative and species-specific approach to measure the abundance and metabolic activity of selected populations of microorganisms in complex communities. The approach is based on a positive correlation between growth rate and RNA:DNA ratio, which was shown to exist in selected microorganisms able to degrade resin acids from pulp and paper mill effluent. Species included in the study belonged to the genera Pseudomonas, Sphingomonas, Zoogloea and Escherichia. Only the zoogloeal isolate did not exhibit a positive correlation between growth rate and RNA: DNA ratio during exponential growth, and neither were the trends in its RNA:DNA ratio similar to the trends in other species during batch growth. In other organisms, during batch growth, the ratio was low during lag phase, had a brief maximum during exponential growth and declined during decelerating and stationary phase. In no case was the ratio observed to remain high during the entire period of exponential growth. Slot-blot hybridization and competitive RT-PCR/PCR assays, targeting hypervariable regions of the 16S ribosomal RNA sequences, were used to measure the DNA and RNA concentrations in a species-specific manner. The hybridization assay proved to be not sensitive enough and burdened with high background when applied to activated sludge samples. The competitive RT-PCR/PCR assay had a much higher sensitivity (10 target copies per ml of activated sludge) and no background. Sphingomonas sp. DhA-33 and Pseudomonas abietaiphila BKME-9 were examined for their ability to survive and to contribute to the resin acid biodegradation in mixed communities. In both cases, DNA data showed survival of the strains, and the RNA:DNA ratio measured their metabolic activity over time, which varied with changes in resin acid concentration, aeration and pH. The quantitative competitive PCR/RT-PCR method contributes significantly to our ability to estimate growth or turnover rates of populations in complex environments when their population sizes appear to be stable. The method allows for the processing of large representative samples and, in conjunction with other molecular microbiological techniques, will contribute toward our understanding of microbial activities in situ.

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