Dieter M. TourloussePh.D. Candidate 2006-contd. |
Assessment of Diversity and Dynamics of Complex Microbial CommunitiesMyresearch aims at understanding the microbial diversity and population dynamics of microbial communities harboring tens to hundreds or even thousands distinct bacterial and archaeal species. Studies on such complex microbial communities are being made possible by recent advances in high throughput molecular tools. These techniques now allow researchers to depict microbial communities with an unprecedented resolution due to the ability to analyze a large sample number in great depth. We are implementing two independent but complementary molecular techniques, high throughput quantitative real time PCR (Q-PCR) and DNA sequencing, to investigate various microbial communities present in different niches, including bioreactors, sewer systems, and pant roots. Both techniques use advanced instruments available at Michigan State University.
Figure 1. 16S rRNA based community analysis of anaerobic microbial communities In one project, I (with my other colleagues) am investigating the microbial diversity and population dynamics of anaerobic bioreactor communities in response to stress. This work elaborates on previous studies by Hashsham et al. (2000) and Fernandez et al. (2000) who characterized the communities in part by clone library sequencing and ARDRA. It is anticipated that using the ‘better eyes’ provided by high throughput Q-PCR and DNA sequencing will provide a more in-depth picture of the communities and their dynamics after substrate shock load. First, high throughput Q-PCR using the BioTroveTM platform (www.BioTrove.com) is being applied to monitor the population dynamics of the bioreactor communities after perturbation. Our research group previously validated this technology for the simultaneous detection of 20 microbial pathogens in environmental matrices (Stedtfeld et al., 2006; Stedtfeld et al., in preparation). A set of 56 primer pairs targeting the 16S rRNA gene was developed targeting numerous bacteria and archaea at various taxonomic levels (Hashmi et al., 2007). Bacterial primers target over 25 fatty acid oxidizing synthrophic microorganisms, including Smithella propionica, Pelotomaculum spp., and Syntrophobacter spp. Archaeal primers target all known lineages of methanogens, including Methanosarcinaceae and Methanosaetaceae. With the BioTroveTM platform we are able to simultaneously analyze 3×48 reactor samples in a single instrument run in a matter of hours, thereby providing a very comprehensive depiction of the population dynamics. Second, sequencing of the 16S rRNA genes in the bioreactor community is being performed using the Genome Sequencer 20TM (www.454.com). We are adopting this technology using an approach described in the work by Herndl and co-workers (Sogin et al., 2006). A new set of universal archaeal and bacterial universal primers was designed targeting selected hypervariable regions of the 16S rRNA gene. Using the Genome Sequencer 20TM system we are able to analyze up to 10 samples simultaneously yielding 2 Mbp of sequence information per sample (or over 100,000 sequence reads per sample). This ability yields information on rare microorganisms which are in general not detected using less high throughput techniques. Also, sequence information gathered from 16S rRNA gene sequencing will be evaluated to design a second set of PCR primers. Using this approach of re-iterative primer design a detailed view of the population dynamics will be obtained. Both molecular techniques are also being applied to other microbial niches and communities, including:
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