For over two decades researchers have been using Fluorescence in situ hybridization (FISH) and fluorescent ribosomal RNA (rRNA) targeted oligonucleotide probes to identify and study the microbes present in many habitats across the globe. A limitation of this technology is the requirement to first isolate the 16S rRNA gene sequences from a target population prior to FISH probe design and subsequent use. Issues arise when target organisms represent only a small percentage of the overall bacterial population, are incorporated into complex structures such as flocs or biofilms, and/or possess varying amounts of attached growth making physical isolation (e.g. cell sorting via flow cytometry or micromanipulation) difficult. This situation is especially true for the complex microbial communities present in many activated sludge wastewater treatment systems, and the reason why some important organisms remain unidentified beyond their morphological descriptions. Bacteria possessing a filamentous morphotype are of particular concern due to their ability to cause problems (such as bulking) even when their relative abundance remains small.
Here we describe a process where Roche 454 amplicon sequencing data (commonly used for microbial diversity analysis) was used as a basis for ‘identifying’ 16S rRNA sequences of interest, allowing FISH probe design for an organism contributing less than 0.5% of the total (sequenced) microbial population. Application of the Roche 454 and other next generation sequencing services have allowed for more sensitive profiling than clone libraries, when studying the microbial diversity of complex systems. These platforms recover up to 106 individual 16S rRNA sequences per sequencing effort. While this information is currently limited to a 300-500 bp region of the 16S rRNA gene, developments providing approx. 1000 bp are becoming available and in the future coverage of the whole 16S rRNA gene may be possible.