Isolation of previously uncultivable bacteria from a nickel contaminated soil using a diffusion-chamber-based approach

A cultivable portion of the bacterial community exhibits important metabolic activities in metal-contaminated soils. We aimed to isolate, identify and characterise hardly cultivable and previously uncultured bacteria (with similarity < 97% to closest relatives) from Ni-contaminated soil in Slovakia by a diffusion-chamber-based approach. Screened bacteria were either directly grown on minimal solid media or passaged through four generations of diffusion chamber. The phylogeny was reconstructed using partial sequences of 16S rDNA genes from separated isolates inoculum and PCR-DGGE analysis. A total of 260 isolates obtained from both, diffusion-chamber-based approach and traditional cultivation techniques, represented 108 species belonging to 6 bacterial phyla: Actinobacteria, Bacteroidetes, Firmicutes, α-, β- and γ-Proteobacteria, and one yet unclassified species. Up to 51 discrete bands from PCR-DGGE analysis, representing different taxa were assigned to same phyla. 29.6% of the isolates and 56.9% of the bands were found as previously uncultivable bacteria, and 8.5% of the isolates and 19.6% of the bands were found with similarity < 97% to their closest relatives. The phylogeny was also reconstructed with partial sequences of selected heavy-metal resistance genes. In 14 of previously uncultured β- or γ-Proteobacteria nccA-like gene products were identified. Majority of them were assigned to a system of transmembrane metal pumps. All these isolates were resistant to Ni, Co, Zn, Cu and Cd ions. The comparison of diffusion-chamber approach with traditional cultivation revealed that diffusion chamber cultivation produced more isolates of a real phylogenetic novelty and led to in vitro cultivation of poorly cultivable species.