Bioprospecting of facultatively oligotrophic bacteria from non-rhizospheric soils

Conventional cultivation method relies on the use of nutrient-rich medium, which might limit the exploration of oligotrophic bacteria from soil ecosystems. In the present study attempts were made to isolate oligotrophic bacteria from non-rhizospheric samples collected from mountain roadside, beach, bay and wetland using 1000-fold diluted nutrient agar. Phylogenetic analysis and biogeographic studies of these isolates were performed based on their 16S rDNA sequences. Besides, tests for the utilization of methanol or agar as their carbon source (methylotrophic or agarolytic), light as their energy source (photoheterotrophic), fixation of nitrogen or solubilization of tricalcium phosphate were conducted. A total of 74 isolates with facultatively oligotrophic behavior were obtained, since they can proliferate on both 1000-fold diluted and conventional nutrient agar. 16S rDNA sequence analyses assigned them mainly to class Actinobacteria and α-Proteobacteria. All these isolates belonged to 24 genera which encompassed 35 species, demonstrating that these facultatively oligotrophic bacteria occupied a wide range of bacterial lineages. Fifty-seven out of 74 isolates formed colonies on methanol-containing agar, while only 21 showed better growth in methanol-containing medium. Nine isolates were verified to utilize agar instead of methanol for growth. A total of 4 isolates showed photoheterotrophic trait and the pufLM gene was successfully PCR amplified and sequenced. The methylotrophic or photoheterotrophic behaviors might provide advantages for them to inhabit oligotrophic environments. Members affiliated with many genera were first demonstrated to fix nitrogen or solubilize phosphate, and isolates with profound activities have potentials to be developed as bioinoculants used to promote plant growth. In addition, the use of diluted nutrient agar helped to explore several unrecognized species from non-rhizospheric soils. All these isolates may provide opportunities for studying their oligotrophic growth, metabolism and interaction with plants in a near future.