Engineered bioreactors for wastewater and water treatment contain complex communities of microorganisms. Often the relationships between organisms influence how systems can be designed and operated. As a result, understanding of microbial ecology is critical for modern environmental biotechnology. In fact, this is one instance in which ecologists and engineers often ask the same questions:
- What factors make a community more and less stable to perturbations?
- What is the role of diversity in the functional stability of a microbial community?
- Can communities be designed or managed to enhance functional stability?
In order to address such questions, we need to understand the nature of the organisms present within these communities. Until recently, this was nearly impossible because tools for the assessment of microbial community structure were lacking. Over the past decade, however, a number of exciting molecular techniques have emerged for the analysis of community structure.
One method of community analysis that we employ was first described by Liu et al. This technique involves analysis of terminal restriction fragments of the 16S rRNA gene (sometimes called analysis of the Terminal Restriction Fragment Length Polmorphisms, T-RFLP). In this method, 16S genes within a microbial community are amplified using the polymerase chain reaction (PCR) and a fluorescently labeled primer is incorporated in the amplified products. By cutting the products with restriction enzymes and sizing the resulting fragments, we are able to identify major "ribotypes" within the community.
There are several limitations to the above approach: primers used in the PCR step may fail to amplify all 16S rRNA genes, the amplification step may result in biased amplification of some genes more than others, and, in fact, the number of copies of the 16S genes in a cell varies, depending on the organism. To overcome these problems, we are currently testing the feasibility of a bead-based method for gene capture and use of rpo C gene as a target for community structure analysis in oligonucleotide arrays.
Funding for this research is provided by an NIH Traineeship in Biotechnology awarded to Laurel Crosby
Crosby, L. D., and C. S. Criddle, 2003. Understanding systematic error in microbial community analysis techniques as a result of ribosomal RNA (rrn) operon copy number. BioTechniques 34, 790-803. (Click here for a PDF copy).