Jaisoo Kim

Cultivation of unculturable soil bacteria.

Despite the abundance of bacterial species in soil, more than 99% of these species cannot be cultured by traditional techniques. In addition, the less than 1% of bacteria that can be cultured are not representative of the total phylogenetic diversity. Hence, identifying novel species and their new functions is still an important task for all microbiologists. Cultivating techniques have played an important role in identifying new species but are still low-throughput processes. This review discusses the issues surrounding cultivation, including achievements, limitations, challenges, and future directions.

Arvibacter flaviflagrans gen. nov., sp. nov., isolated from forest soil.

During a study of the bacterial community in forest soil of Kyonggi University, Suwon, South Korea, a glistening yellow-pigmented, Gram-stain-negative, aerobic, motile and rod-shaped bacterium, designated strain C-1-16T, was isolated. This strain was non-sporulating, catalase-negative and oxidase-positive. It was able to grow at 20-42 °C, at pH 6.0-9.0 and with 0-0.5 % (w/v) NaCl. This strain was taxonomically characterized by a polyphasic approach. Based on the results of 16S rRNA gene sequence analysis, strain C-1-16T formed a lineage within the family Chitinophagaceae of the phylum Bacteroidetes and closely related to the genera Filimonas(93.53 % sequence similarity), Sediminibacterium(92.52-90.75 %), Lacibacter(91.99-91.19 %) and Parasegetibacter(91.88-91.78 %). Flexirubin-type pigment was present for strain C-1-16T. The only respiratory quinone was menaquinone-7 (MK-7) and the major polar lipid was phosphatidylethanolamine. The predominant fatty acids of strain C-1-16T were iso-C15 : 0, iso-C17 : 0 3-OH, iso-C15 : 1 G, summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c) and C16 : 0. The genomic DNA G+C content of this novel strain was 45.5 mol%. On the basis of phenotypic, genotypic and phylogenetic analysis, strain C-1-16T represents a novel species of a new genus in the family Chitinophagaceae, for which the name Arvibacter flaviflagrans gen. nov., sp. nov. is proposed. The type strain of Arvibacter flaviflagrans is C-1-16T (=KEMB 900-374T=KACC 18717T=JCM 31293T).

Rhabdobacter roseus gen. nov., sp. nov., isolated from soil.

An aerobic, Gram-stain-negative, oxidase- and catalase-positive, non-motile, non-spore-forming, rod-shaped, pink-pigmented bacterium, designated strain R49T, was isolated from soil. Flexirubin-type pigments were absent. Phylogenetic analysis based on its 16S rRNA gene sequence revealed that strain R49T formed a lineage within the family Cytophagaceae of the phylum Bacteroidetes that was distinct from the most closely related genera Dyadobacter (91.98-93.85 % sequence similarity), Persicitalea (88.69 %) and Runella (84.79-85.81 %). The major isoprenoid quinone was menaquinone-7 (MK-7) and the major polar lipid was phosphatidylethanolamine. The major cellular fatty acids were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), iso-C15 : 0, C16 : 1ω5c, C16 : 0 and iso-C17 : 0 3-OH. The DNA G+C content of strain R49T was 53.9 mol%. On the basis of phenotypic, genotypic and phylogenetic analysis, strain R49T represents a novel species of a new genus in the family Cytophagaceae, for which the name Rhabdobacter roseus gen. nov., sp. nov. is proposed. The type strain of Rhabdobacter roseus is R49T ( = KEMB 9005-318T = KACC 18395T = JCM 30685T).

Microvirga soli sp. nov., an alphaproteobacterium isolated from soil

An aerobic, Gram-stain-negative, catalase-positive and oxidase-negative, non-motile, non-spore-forming, rod-shaped, pink-pigmented bacterium designated strain R491T was isolated from soil. Flexirubin-type pigments were absent. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain R491T formed a lineage within the family Methylobacteriaceae of the phylum Proteobacteria that was distinct from various species of the genus Microvirga, including Microvirgaaerilata 5420S-16T (97.83 % 16S rRNA gene sequence similarity), Microvirga zambiensis WSM3693T (97.76 %), Microvirga flocculans ATCC BAA-817T (97.41 %), and Microvirga lupini Lut6T, Microvirga subterranea DSM 14364T, Microvirga vignae BR3299T, and Microvirga guangxiensis 25BT (96.99 %). The major polar lipids of strain R491T were phosphatidylcholine, phosphatidylglycerol and phosphatidylethanolamine. The major cellular fatty acids were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c; 71.2 %), C16 : 0 (12.0 %), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c; 4.7 %), and C18 : 0 (4.2 %). The DNA G+C content of strain R491T was 61.8 mol%. DNA-DNA hybridization values between strain R491T and other members of the genus Microvirga ranged from 27 to 57 %. On the basis of phenotypic, genotypic and phylogenetic analyses, strain R491T represents a novel species of the genus Microvirga, for which the name Microvirga soli sp. nov. is proposed. The type strain is R491T (=KEMB 9005-408T=KACC 18969T=NBRC 112417T).

Sphingomonas naphthae sp. nov., isolated from oil-contaminated soil

During the study of hydrocarbon-degrading bacteria in the oil-contaminated soil of Gunsan, North Jeolla Province, South Korea, a yellow, Gram-stain-negative, aerobic, motile, rod-shaped bacterium, designated strain DKC-5-1T, was isolated. This strain was non-sporulating, catalase-negative and oxidase-positive. It was able to grow at 10-33 °C, pH 6.0-8.5 and at an NaCl concentration of 0-1.5 % (w/v). This strain was characterized taxonomically using a polyphasic approach. Based on 16S rRNA gene sequence analysis, strain DKC-5-1T belongs to the genus Sphingomonas and is closely related to Sphingomonas laterariae LNB2T (96.65 % sequence similarity), Sphingomonas haloaromaticamans A175T (96.63 % sequence similarity), Sphingomonas histidinilytica UM2T (96.63 % sequence similarity), and Sphingomonas wittichii RW1T (96.43 % sequence similarity). The only respiratory quinone was ubiquinone-10 and the major polyamine was homospermidine. The polar lipid profile revealed the presence of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, sphingoglycolipid and phosphatidyldimethylethanolamine. The predominant fatty acids of strain DKC-5-1T were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), C16 : 0, C14 : 0 2-OH, summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C17 : 1ω6c and C14 : 0. The genomic DNA G+C content of this novel strain was 65.9 mol%. Morphological, physiological, chemotaxonomic and phylogenetic analyses clearly distinguished this strain from its closest phylogenetic neighbours. Thus, strain DKC-5-1T represents a novel species of the genus Sphingomonas, for which the name Sphingomonasnaphthae sp. nov. is proposed. The type strain is DKC-5-1T (=KEMB 9005-380T=KACC 18716T=JCM 31294T).

Pedobacter humicola sp. nov., a member of the genus Pedobacter isolated from soil.

An aerobic, Gram-stain-negative, oxidase-negative, catalase-positive, non-motile, non-spore-forming, rod-shaped, light pink-pigmented bacterium, designated strain R135T, was isolated from soil in Hwaseong, South Korea. Flexirubin-type pigments were absent. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain R135T formed a lineage within the family Sphingobacteriaceae of the phylum Bacteroidetes. It was distinct from various species of the genus Pedobacter, including P. terrae DS-57T (98.13 % sequence similarity), P. alluvionis NWER-II11T (97.76 %), P. suwonensis 15-52T (97.71 %), P. kyungheensis KACC 16221T (97.37 %), P. roseus CG-GP80T (97.24 %), P. soli 15-51T (97.23 %) and P. sandarakinus DS-27T (97.09 %). The major isoprenoid quinone was menaquinone-7 (MK-7), and the major polar lipid was phosphatidylethanolamine. The major cellular fatty acids were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), iso-C15 : 0, iso-C17 : 0 3-OH and C16 : 0. The DNA G+C content of strain R135T was 40.4 mol%. Levels of DNA-DNA hybridization similarities between strain R135Tand other members of the genus Pedobacter ranged from 25 % to 43 %. On the basis of phenotypic, genotypic and phylogenetic analysis, strain R135T represents a novel species of the genus Pedobacter, for which the name Pedobacter humicola sp. nov. is proposed. The type strain is R135T (=KEMB 9005-332T=KACC 18452T=JCM 31010T).

Massilia kyonggiensis sp. nov., isolated from forest soil in Korea

A Gram-negative, short, rod-shaped bacterium, TSA1T, was isolated from forest soil collected at Kyonggi University, South Korea. Assessment of 16S rRNA gene sequence similarity indicated that the strain is related to Massilia niastensis 5516S-1T (98.3%), M. haematophila CCUG 38318T (97.9%), M. aerilata 5516S-11T (97.9%), M. tieshanensis TS3T (97.6%), and M. varians CCUG 3529T (97.1%). Colonies grown on Reasoner’s 2A agar at 30°C for 2 days were transparent, white, round, smooth, and glossy. The cells grew at 10–42°C (optimum: 25–37°C) and pH 5–9 (optimum: 5–9) and in 0–2% NaCl (optimum: 0–1%). TSA1T was able to grow on trypticase soy and nutrient agar, but not on Luria-Bertani or MacConkey agar. The strain was catalase- and oxidasepositive and able to degrade starch and casein, but not carboxymethyl cellulose. The predominant quinone of TSA1T was Q-8, the major fatty acids were summed feature 3 and C16:0, and the DNA G+C content was 66.7 mol%. Given these findings, we propose that this strain is a novel species of the genus Massilia. We suggest the name Massilia kyonggiensis sp. nov. (type strain, KACC 17471T =KEMB 9005-031T =JCM 19189T).

Simple surface foam application enhances bioremediation of oil-contaminated soil in cold conditions

Landfarming of oil-contaminated soil is ineffective at low temperatures, because the number and activity of micro-organisms declines. This study presents a simple and versatile technique for bioremediation of diesel-contaminated soil, which involves spraying foam on the soil surface without additional works such as tilling, or supply of water and air. Surfactant foam containing psychrophilic oil-degrading microbes and nutrients was sprayed twice daily over diesel-contaminated soil at 6 °C. Removal efficiencies in total petroleum hydrocarbon (TPH) at 30 days were 46.3% for landfarming and 73.7% for foam-spraying. The first-order kinetic biodegradation rates for landfarming and foam-spraying were calculated as 0.019 d(-1) and 0.044 d(-1), respectively. Foam acted as an insulating medium, keeping the soil 2 °C warmer than ambient air. Sprayed foam was slowly converted to aqueous solution within 10-12h and infiltrated the soil, providing microbes, nutrients, water, and air for bioaugmentation. Furthermore, surfactant present in the aqueous solution accelerated the dissolution of oil from the soil, resulting in readily biodegradable aqueous form. Significant reductions in hydrocarbon concentration were simultaneously observed in both semi-volatile and non-volatile fractions. As the initial soil TPH concentration increased, the TPH removal rate of the foam-spraying method also increased.

Chryseobacterium nepalense sp. nov., isolated from oil-contaminated soil

During the study of hydrocarbon-degrading bacteria in the oil-contaminated soil of Biratnagar, Morang, Nepal, a yellow-coloured, Gram-staining-negative, aerobic, non-motile, and rod-shaped bacterium, designated strain C-5-3T, was isolated. This strain was characterized taxonomically by a polyphasic approach. Based on the 16S rRNA gene sequence analysis, strain C-5-3T belonged to the genus Chryseobacterium and was closely related to Chryseobacterium profundimaris DY46T (98.19 % sequence similarity), Chryseobacterium takakiae AG1-2T (98.15 % sequence similarity), Chryseobacterium taiwanense BCRC 17412T (98.14 % sequence similarity), Chryseobacterium camelliae THG C4-1T (97.73 % sequence similarity) and Chryseobacterium hispalense DSM 25574T (97.60 % sequence similarity). The predominant respiratory quinone was menaquinone-6, and phosphatidylethanolamine was the major polar lipid. The predominant fatty acids of strain C-5-3T were iso-C15 : 0, summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), iso-C17 : 0 3-OH and summed feature 9 (iso- C17 : 1ω9c and/or C16 : 0 10-methyl). The genomic DNA G+C content of this novel strain was 38.6 mol%. The DNA-DNA relatedness between strain C-5-3T and Chryseobacterium profundimaris JCM 19801T, C. takakiae DSM 26898T, C. taiwanense KACC 13400T, C. camelliae KACC 16985T and C. hispalense DSM 25574T was 53.3, 42.7, 47.3, 33.0 and 28.0 %, respectively. The morphological, physiological, chemotaxonomic and phylogenetic analyses clearly distinguished this strain from its closest phylogenetic neighbours. Thus, strain C-5-3T represents a novel species of the genus Chryseobacterium, for which the name Chryseobacterium nepalense sp. nov. is proposed. The type strain is C-5-3T (=KEMB 9005-411T=KACC 18907T=JCM 31469T).

Flavobacterium fulvum sp. nov., Flavobacterium pedocola sp. nov. and Flavobacterium humicola sp. nov., three new members of the family Flavobacteriaceae, isolated from soil.

Four Gram-stain-negative, non-endospore-forming, non-motile strains were found in soil, South Korea. Based on their 16S rRNA gene sequences, strains UCM-R15T and UCM-R21 are most closely related to Flavobacterium enshiense DK69T (97.4-97.5 %, pairwise similarity) while strains UCM-R36T and UCM-46T are most closely related to Flavobacterium suncheonense GH29-5T (97.5 % and 98.3 %, respectively), with all four strains sharing less than 97 % pairwise similarity to the type strain of any other species of the genus Flavobacterium. None of the four strains can reduce/digest nitrate or urea. The only menaquinone detected was MK-6 and the major fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH, iso-C15 : 1 G and summed feature 9 in all the type strains. Phosphatidylethanolamine was found in three strains as the major polar lipid, phosphatidylserine was found in both strains UCM-R15T and UCM-R36T, but not UCM-46T, and phosphatidylmonomethylethanolamine only occurred in strain UCM-R15T. The genomic DNA G+C content values of strains UCM-R15T, UCM-R21, UCM-R36T and UCM-46T were 35.3-39.0  mol%. Taking into account their physiological and biochemical characteristics, we suggest that three of the strains are novel members of the genus Flavobacterium. We propose the names Flavobacterium fulvum sp. nov. for type strain UCM-R15T (=KACC 18666T=NBRC 111764T), and strain UCM-R21 as an additional strain Flavobacterium pedocola sp. nov. for type strain UCM-R36T (=KACC 18668T=NBRC 111765T), and Flavobacterium humicola sp. nov. for type strain UCM-46T (=KACC 18575T=NBRC 111657T).