In this study, a new diffusion bioreactor was developed to cultivate hidden bacterial communities in their natural environment. The newly developed method was investigated to cultivate microbial communities from the forest soil, and the results were evaluated against traditional culture methods and compared to the results of a pyrosequencing-based molecular survey. The molecular analysis revealed that a diverse bacterial population was present in the soil sample. However, both the newly developed method and the traditional method recovered more than 400 isolates, which belonged to only four phyla: Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. Although these isolates were distributed over only four major phyla, the use of the newly developed technique resulted in the successful cultivation of 35 previously uncultured strains, whereas no such strains were successfully cultivated by the traditional method. Furthermore, the study also found that the recovery of uncultured bacteria and novel isolates was related to sampling season, incubation period, and cultivation media. The use of soil collected in summer, a prolonged incubation period, and low-substrate modified media increased the recovery of uncultured and novel isolates. Overall, the results indicate that the newly designed diffusion bioreactor can mimic the natural environment, which permits the cultivation of previously uncultured bacteria.

This study presents taxonomic description of two novel diesel-degrading, psychrophilic strains: Kopri-42^T and Kopri-43, isolated during screening of oil-degrading psychrotrophs from oil-contaminated Arctic soil. A preliminary 16S rRNA gene sequence and phylogenetic tree analysis indicated that these Arctic strains belonged to the genus Flavobacterium, with the nearest relative being Flavobacterium psychrolimnae LMG 22018^T (98.9% sequence similarity). The pairwise 16S rRNA gene sequence identity between strains Kopri-42^T and Kopri-43 was 99.7%. The DNA-DNA hybridization value between strain Kopri-42^T and Kopri-43 was 88.6 ± 2.1% indicating that Kopri-42^T and Kopri-43 represents two strains of the same genomospecies. The average nucleotide identity and in silico DNA-DNA hybridization values between strain Kopri-42^T and nearest relative F. psychrolimnae LMG 22018^T were 92.4% and 47.9%, respectively. These values support the authenticity of the novel species and confirmed the strain Kopri-42^T belonged to the genus Flavobacterium as a new member. The morphological, physiological, biochemical and chemotaxonomic data also distinguished strain Kopri-42^T from its closest phylogenetic neighbors. Based on the polyphasic data, strains Kopri-42^T and Kopri-43 represents a single novel species of the genus Flavobacterium, for which the name Flavobacterium petrolei sp. nov. is proposed. The type strain is Kopri-42^T (=KEMB 9005-710^T = KACC 19625^T = NBRC 113374^T).

Here, a new medium named as intensive soil extract medium (ISEM) based on new soil extract (NSE) using 80% methanol was used to efficiently isolate previously uncultured bacteria and new taxonomic candidates, which accounted for 49% and 55% of the total isolates examined (n=258), respectively. The new isolates were affiliated with seven phyla (Proteobacteria, Acidobacteria, Firmicutes, Actinobacteria, Verrucomicrobia, Planctomycetes, and Bacteroidetes). The result of chemical analysis showed that NSE included more diverse components of low-molecular-weight organic substances than two conventional soil extracts made using distilled water. Cultivation of previously uncultured bacteria is expected to extend knowledge through the discovery of new phenotypic, physiological and functional properties, and even roles of unknown genes. IMPORTANCE Either metagenomics or single-cell sequencing can detect unknown genes from uncultured microbial strains in environments and may find their significant potential metabolites and roles. However, such gene/genome-based techniques do not allow detailed investigations that possible with cultures. To solve this problem, various approaches for cultivation of uncultured bacteria have been developed, but there are still difficulties in maintaining pure cultures by subculture.