A NEW MOLECULAR RESPIROMETRY METHOD FOR IDENTIFYING BACTERIA AND DETERMINING THEIR GROWTH STATES IN MIXED CULTURES

By identifying and monitoring the growth of desired bacteria, environmental engineers and scientists may be able to determine the optimal operational conditions for the proliferation of bacteria in biological wastewater treatment systems. Determining the growth state of specific bacteria in mixed cultures is possible by using fluorescence in situ hybridizations with probes that target two predominant molecules in ribosome genesis, precursor 16S rRNA and 16S rRNA. However, this approach is laborious, since it requires a unique precursor 16S rRNA probe for each specific bacteria of interest. We report the development of a novel molecular respirometry method that allows for identification of bacteria in a mixed culture and determination of their respective growth states. This method couples reverse transcription and primer extension (RT&PE) and targets the 0338 site common to all bacteria in the precursor 16S rRNA and 16S rRNA. We used agarose gel electrophoresis analysis to demonstrate that our RT&PE method produces ssDNA from rRNA. In addition, RT&PE products were derived from cultures of Acinetobacter calcoaceticusT for three defined growth conditions. Differences in the abundance of the RT&PE product derived from precursor 16S rRNA corresponds to previously reported FISH results. Slab gel analysis was used to determine the size of the RT&PE products for A. calcoaceticusT, E. coliT, and a mixture of both. Differentiation of both bacteria is possible due to length heterogeneity of RT&PE products. With the RT&PE method described, it will be possible to identify and monitor the bacteria that are responding to changes in their environment. For activated sludge systems, we believe that the RT&PE method will be a powerful method for investigating the impact of operational parameters on the growth of specific bacteria.