Soil Diffusion System Enriches the Growth of Diverse and Previously Uncultivated Bacterial Taxa

Abstract

Recent studies have demonstrated that culturing microorganisms in association with their native habitat promoted the growth of diverse and previously uncultivable bacteria. We developed a soil diffusion system (SDS) where soil microorganisms were inoculated and allowed to grow on regenerated cellulose filters (RCF) that were tightly coupled with the soil habitat. The objective of the study was to assess the influence of the native habitat and the biotic (microbe–microbe) and abiotic soil effects on the selective growth of microbial communities. Regenerated cellulose filters were used as support for the growing microbial community. A polycarbonate membrane (0.003-μm pore size) was inserted between the RCF and the soil to prevent the movement of bacteria and larger organisms. Three different SDS treatments were used to differentiate biotic and abiotic growth effects along with a control-SDS treatment and a traditional culturing medium. The treatments were: (i) inoculated RCF on unsterilized soil (BioticRCF), (ii) inoculated RCF on autoclave-sterilized soil (Abiotic-HNRCF), (iii) inoculated RCF without soil but amended with sterilized soil extracts (Abiotic-LNRCF), and (iv) sterilized cellulose–Congo red agar medium (CCRA). A fifth uninoculated RCF (UninocRCF) treatment on unsterilized soil was included as control to check for contamination from the soil, and all the treatments were replicated thrice. Following 20 d of incubation, the developing communities from all treatments were characterized using 16S rRNA gene clone libraries. Our results showed that nutrient levels had a small effect on the growing communities, and as hypothesized, the community growing in association with the unsterilized soil (BioticRCF) was the richest and most unique among all treatments. Previously uncultured members of the phyla Proteobacteria, Bacteroidetes, and few members of Verrucomicrobia, Planctomycetes, and OP10 were detected on the BioticRCF treatment. Members of the phylum Firmicutes were the dominating bacteria in abiotic treatments, followed by the members of phylum Planctomycetes. The CCRA medium supported the growth of a less diverse community, with ∼91% of the sequences closely related to isolates of the phyla Proteobacteria, Bacteroidetes, and Firmicutes. The presence or absence of a living microbial community appears to have a significant impact on the richness and structure of bacterial community growth and development.