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.

Using a newly developed culture method for not yet cultured soil bacteria, three Gram-stain-negative, aerobic, non-spore-forming, motile, and rod-shaped bacteria (strain designated J18T, J11T and J9T) were isolated from forest soil at Kyonggi University, South Korea. Isolates were subjected to a taxonomic study by using a polyphasic approach. According to a phylogenetic tree based on 16S rRNA gene sequences, strains J18T, J11T and J9T belonged to the genus Massilia and clustered with Massilia haematophila CCUG 38318T (similarity range: 97.6~98.0 %). The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol, and the genomic DNA G+C contents of strains J18T, J11T and J9T were 63.4, 68.7 and 64.5 mol%, respectively. The major polyamines were putrescine and 2-hydroxyputescine, which were detected in all three strains. DNA-DNA between the three tested strains and the reference strains much lower than 70 %, the recommended threshold value for the delineation of genomic species. The predominant respiratory quinine was ubiquinone-8 (Q-8) and the major cellular fatty acids were Summed feature 3 (C16 : 1ω6c/C16 : 1ω7c) and C16 : 0. On the basis of phenotypic and genotypic data and DNA-DNA hybridization results, the three isolates are considered to represent three novel species of the genus Massilia, for which the names Massilia solisilvae sp. nov. for type strain J18T (=KEMB 9005-366T=JCM 31607T), Massilia terrae sp. nov. for type strain J11T (=KEMB 9005-360T=JCM 31606T) and Massilia agilis sp. nov. for type strain J9T (=KEMB 9005-359T=JCM 31605T) are proposed.

A Gram-stain-negative, aerobic, motile with a single polar flagellum, non-spore-forming and rodshaped bacterial strain named T33T was isolated from forest soil collected at Kyonggi University, South Korea. The strain was catalase-and oxidase-positive, colonies grew on R2A agar at 32 C. The genus Massilia was first proposed with the type species Massilia timonae, for a strain isolated from the blood of an immunocompromised patient with cerebellar lesions (La Scola et al., 1998). Since then, other strains representing species of the genus Massilia have been isolated from air (Weon et al., 2008(Weon et al., , 2009(Weon et al., , 2010Orthov a et al. 2015), blood (K€ ampfer et al., 2011, 2012), ice (Shen et al., 2013(Shen et al., , 2015, water (Gallego et al., 2006) and soil samples (Zhang et al., 2006;Zul et al., 2008;Wang et al., 2012;Du et al., 2012;Kong et al., 2013;Luo et al., 2013;Kim, 2014;Rodríguez-Díaz et al., 2014;Singh et al., 2015;Feng et al., 2016; EmbarcaderoJim enez et al., 2016). Species of the genus Massilia show typical characteristics; they are aerobic, Gram-stain-negative, motile, non-spore-forming and rod-shaped cells. Summed feature 3 (comprising C 16 : 1 !7c and/or iso-C 15 : 0 2-OH) and C 16 : 0 are the major fatty acids, Q-8 is the predominant isoprenoid quinone, and the DNA G+C content is in the range of 62.2-68.9 mol%. Phosphatidylglycerol, phosphatidylethanolamine and diphosphatidylglycerol are the major polar lipids.In the present study, we attempted to isolate microorganisms from forest soil at Kyonggi University, located in Suwon region, South Korea. Moreover, a novel species was characterized by a polyphasic taxonomic approach, including phylogenetic analysis based on 16S rRNA gene sequences and chemotaxonomic and physiological properties. Soil samples were collected and dried at room temperature. Pebbles and plant debris were removed by sieving the samples through a 2 mm mesh sieve. In this study, a modified culture method using transwell plates (Corning) (Van Pham & Kim, 2012) was utilized with following steps: Approximately 3 g of soil was added into each transwell plate, 3 ml of R2A medium (MB-R2230, MB Cell: proteose peptone, 0.5 g; yeast extract, 0.5 g; acid digest of casein, 0.5 g; glucose, 0.5 g; soluble starch, 0.5 g; dipotassium phosphate, 0.3 g; magnesium sulphate, 0.024 g; and sodium pyruvate, 0.3 g). The pH was adjusted to 7.2±0.2 and 100 µl of a soil suspension (1 g of soil in 9 ml of distilled water, thoroughly stirred and settled) was added to each transwell insert. The transwell plate was incubated in a shaking incubator at 120 rpm and 28 C for 2 weeks. The culture was serially diluted on an R2A agar plate and incubated for 1 week at 28 C. We reviewed 16S rRNA gene sequences from 12 isolates and identified one isolate that had <99 % sequence identity with valid reference sequences and thus possibly represented a novel species. The novel strain T33 T was then cultured routinely on R2A agar at 28 C and maintained as glycerol suspensions (20 %, w/v) at À70C.

No abstract

Two novel strains, J77-1 and J76-1, were isolated from farmland soil and were taxonomically characterized by a polyphasic approach. Both strains were yellow, Gram-stain-negative, strictly aerobic, non-motile and rod-shaped bacteria. These strains were non-sporulating, catalase-positive and oxidase-negative. J77-1 and J76-1 were able to grow at 15-40 °C, pH 5.0-10.0, and 0-1.0 % (w/v) NaCl concentration. Phylogenetic analyses revealed that both strains formed a distinct separate lineage within the family Cytophagaceae of the phylum Bacteroidetes. J77-1 and J76-1 showed low 16S rRNA gene sequence similarity to the most closely related type strain Dyadobacter koreensis WPCB159 (85.09 %) and exhibited less than 85.0 % sequence similarity with other members of the family Cytophagaceae. The pairwise sequence similarity between strains J77-1 and J76-1 was observed to be 99.86 %. In both strains, the only respiratory quinone was menaquinone-7 (MK-7); the major polar lipid was phosphatidylethanolamine; and the major fatty acids were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C16 : 0, iso-C15 : 0, iso-C17 : 0 3-OH C16 : 1ω5c, and C16 : 0 3-OH. The genomic DNA G+C content values of J77-1 and J76-1 were 50.1 and 50.9 mol%, respectively. On the basis of the results of phenotypic, genotypic and phylogenetic analysis, J77-1 represents a novel species of a novel genus, for which the name Ravibacter arvi gen. nov., sp. nov. is proposed, within the family Cytophagaceae. The type strain of Ravibacter arvi is J77-1 (=KEMB 9005-548=KACC 19172=JCM 31920), and J76-1 is an additional strain.