Microbial community analysis assessed by pyrosequencing of rRNA gene: Community comparisons, organism identification, and its enhancement

There are more than 1030 bacteria on Earth, with their members embedding 3.8 billion years evolutionary history and having evolved to take advantage of virtually every energy-yielding niche hospitable to life. This makes the microbial world extremely diverse, ubiquitous and essential to Earth's habitability. Hence, determining which microbes make up these communities is an initial goal for understanding microbial communities. Recently, pyrosequencing of ribosomal RNA genes has become a popular tool for in-depth analyses of microbial communities. I used pyrosequencing of rRNA's hypervariable V4-region to characterize a wide variety of microbial communities. Soil microbial communities in the tropics are potentially more dynamic than temperate ones due to longer and more favorable temperature, moisture and energy resources from primary productivity. I studied the effect on soil Bacteria of different soil-crop management systems in Eastern Ghana, one of which lost 50% of its stored soil organic carbon (SOC) within 4 years. Canonical correspondence analysis and stepwise multiple regression of the 290,000 V4-rRNA sequences showed that SOC was the most important factor that explained differences in microbial community structure among managements. The data indicate that the use of a pigeon-pea crop (a legume) during the winter season (normally fallow) promotes a higher microbial diversity and sequesters more soil organic carbon, which is important for soil structure, nutrient retention and recycling, and general soil health. I also evaluated analysis methods for 211 rRNA-determined bacterial assemblages, comprising 1.3 million rRNA sequences from seven habitat types. A taxonomy-supervised method, using taxonomy-bins, was advantageous in its ability to compare non-overlapping sequences, and requiring minimal computation capacity compared to the non-taxonomy-supervised (clustering-determined) method. The taxonomy supervised method produced results that were significantly correlated to the clustering method, which is the current standard, and as taxonomy improves should provide even better resolution. Because of the much greater depth and replication provided by pyrosequencing, more robust determination of microbial species distribution, diversity, organism identification, community comparisons and dynamics is possible. As a result of microbes' long history, they harbor considerable genetic diversity and some of their genes likely have more desirable properties that those known. I used stable isotope probing (SIP) with biphenyl as substrate to retrieve novel biphenyl dioxygenase which showed PCB oxidative activity in the 31.8 kb cosmid clone made from the [13C]-DNA. The discrepancy of G+C content near the bphAE genes implies their recent acquisition, possibly by horizontal transfer, and suggests dispersed dioxygenase gene organization in nature. I also used V4-16S rRNA gene pyrosequencing of the [13C]-biphenyl-derived DNA from three PCB-contaminated environmental matrices: rhizosphere, industrial soil, and river sediment to more specifically identify the PCB- and biphenyl-utilizing populations of the three sites. I found little commonality in the abundant members of three sites but new candidate groups that may be involved in PCB degradation.