Characterization of dilution enrichment cultures obtained from size-fractionated soil bacteria by BIOLOG® community-level physiological profiles and restriction analysis of 16S rRNA genes

Dilution cultures have advantages for studying small-celled oligotrophic bacteria because culture selection favors the most abundant, rather than the most nutrient-tolerant, organisms. In the present study, dilution cultures were employed to enrich small-celled (SC; <0.45 μm) and large-celled (LC; >0.45 μm) bacteria from A and B horizons of cultivated and forested Guernsey silt–loam soil. BIOLOG® GN microtitre plates were used to assess functional diversity and were combined with restriction digest of PCR-amplified 16S rRNA genes to evaluate changes in the bacterial community among flasks of increasing dilution. SC obtained from more highly diluted enrichment cultures of A horizon soils produced community-level physiological profiles (CLPP) distinct both from all enrichments of LC and from SC obtained in less dilute enrichments. Principal-component analysis (PCA) based on adjusted average well color development (AWCD) separated A horizon samples by cell size and inoculum dilution at both study sites, with SC from the most dilute enrichments clearly separated from LC and all other SC samples. At any given inoculum dilution, SC from A horizon soil utilized a greater number of substrates than SC from the B horizon. PCA separated A and B horizon SC samples by soil horizon and inoculum dilution. The least-dilute B horizon enrichment generally grouped with A horizon samples, while more dilute B horizon samples were distinct from the A horizon at both sites. Restriction digestion with Sau 3AI and Rsa I enzymes demonstrated that the dilution culture enriched distinct bacterial sub-populations from a single soil sample. Dilution-enrichment resulted in a larger number of distinct RFLP patterns in the cultivated site compared with the forested site.