Characterizing the Cultivable Soil Bacteria Growing on Cellulose Media Using Traditional and Novel Methods.

The cultivation of soil microorganisms using traditional methods recovers only a small fraction (<1%) of the total soil microbial diversity. Part of the reason for this low recovery may be due to incompatible cultivation conditions and their failure to mimic the native microbial habitat.  The objective of our experiment was to grow aerobic cellulose degrading bacteria in close association with the soil using a new technique and to compare it to a traditional method that is commonly used for the cultivation of cellulose degrading bacteria. In the new method, bacteria were inoculated on duplicate 0.2um regenerated cellulose filters (RCF) which were used as both the carbon source and physical support for microbial growth. A 0.03 um polycarbonate membrane was first seated on a mound of moist soil and then topped with the inoculated RCF. Traditional plates consist of duplicate Congo red agar (CRA) plates containing cellulose. Following 10 days incubation, DNA was extracted from the cultures and 16S rDNA clone libraries were developed to characterize the bacterial communities. A total of 41 OTU’s were derived from approximately 100 sequenced clones. The results of LIBSHUFF, show that the communities in the two methods were significantly different (<0.0019) from one another. Moreover, the bacterial communities of two methods have completely dissimilar members (operational taxonomic units defined at D=0.03). Both the Shannon and evenness indices for the communities were higher when grown on RCF than on CRA. While we have not yet confirmed the cellulolytic character of all clones, the presence of more unique sequences on the filter papers and their close association with predominately uncultured members in the RDP database suggests that the soil environment encouraged the growth of bacteria that were not detected on traditional plates.