A novel denaturing high performance liquid chromatography (DHPLC)-based technique was developed to rapidly separate and identify signature in pre16S rRNA levels among pure cultures of E.coli and A. calcoaceticus as well as activated sludge samples. The chromatography results showed the distinct differences in retention time of the individual species for pre 16S ssDNA, thus providing a qualitative and quantitative characterization of species in a mixture of pre 16S ssDNA. This study is an expansion of previous results reporting the development of a reverse transcription and primer extension assay, and it is the first to document activated sludge response to toxic shock loadings using DHPLC-based quantification of pre16S rRNA levels. The anticipated outcome is to demonstrate the effectiveness of ribosome genesis as a sensitive indicator of toxic loading.
The activated sludge system is the most popular form of biological sewage treatment. However, shock loads of toxic chemicals represent a significant problem to activated sludge systems because they disrupt microbial metabolism resulting in a deterioration of process performance (Love et al., 2002). Due to the significance of these disruptions on process performance, a number of research projects have been undertaken to develop upset early warning devices (UEWDs) that can detect the presence of toxic chemicals in sewage (Gutierrez et al., 2002; Aulenta et al., 2002). The current study was guided by the hypothesis that sensitive measurements of ribosome genesis can be used as a quantitative indicator of overall response to toxic loads. Ribosome genesis starts with the transcription of rrn operons to produce a polycistronic transcript, which is then processed in two steps by RNases to produce precursor rRNAs and then mature rRNAs. The rRNAs are finally combined with ribosomal proteins to produce a functional ribosome. It is well documented that high levels of pre16S rRNA correspond to the presence of a toxic inhibition in pure cultures as well as activated sludge (Cangelosi et al., 1997; Licht et al., 1999; Oerther et al., 2000; Oerther et al., 2002).
Recently, a novel reverse transcription and primer extension (RT&PE) approach was developed to investigate the secondary processing of precursor 16S rRNA for pure cultures by determining the ratio of pre16S-5’ rRNA to 16S rRNA (Stroot, 2004). This method was then tested successfully with activated sludge samples. The previous study suggested that the novel RT&PE method could be used to determine whether specific microbial populations are inhibited in activated sludge systems under shock loads of toxic chemicals. To expand this method, the current study examines the use of denaturing high performance liquid chromatography (DHPLC) to rapidly separate and identify signature in pre16S rRNA levels among mixed cultures of environmental bacteria. This approach was used to investigate the secondary processing of the 5’ end of precursor 16S rRNA (pre16S-5’ rRNA) for pure cultures exposed to chloramphenicol. This study represents the first of its kind to document activated sludge response to toxic shock loadings using DHPLC-based quantification of pre16S rRNA levels. This study is significant because it provides us with a better understanding of bacterial growth inhibition under toxic shock loading. In addition, the methods reported in this study could be developed into a standard approach for assaying toxic materials in activated sludge simply on the basis of the DHPLC profile obtained.