Jeong Myeong Kim

Metagenomic Analysis of Kimchi, a Traditional Korean Fermented Food

ABSTRACT Kimchi, a traditional food in the Korean culture, is made from vegetables by fermentation. In this study, metagenomic approaches were used to monitor changes in bacterial populations, metabolic potential, and overall genetic features of the microbial community during the 29-day fermentation process. Metagenomic DNA was extracted from kimchi samples obtained periodically and was sequenced using a 454 GS FLX Titanium system, which yielded a total of 701,556 reads, with an average read length of 438 bp. Phylogenetic analysis based on 16S rRNA genes from the metagenome indicated that the kimchi microbiome was dominated by members of three genera: Leuconostoc, Lactobacillus, and Weissella. Assignment of metagenomic sequences to SEED categories of the Metagenome Rapid Annotation using Subsystem Technology (MG-RAST) server revealed a genetic profile characteristic of heterotrophic lactic acid fermentation of carbohydrates, which was supported by the detection of mannitol, lactate, acetate, and ethanol as fermentation products. When the metagenomic reads were mapped onto the database of completed genomes, the Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 and Lactobacillus sakei subsp. sakei 23K genomes were highly represented. These same two genera were confirmed to be important in kimchi fermentation when the majority of kimchi metagenomic sequences showed very high identity to Leuconostoc mesenteroides and Lactobacillus genes. Besides microbial genome sequences, a surprisingly large number of phage DNA sequences were identified from the cellular fractions, possibly indicating that a high proportion of cells were infected by bacteriophages during fermentation. Overall, these results provide insights into the kimchi microbial community and also shed light on fermentation processes carried out broadly by complex microbial communities.

Influence of Soil Components on the Biodegradation of Benzene, Toluene, Ethylbenzene, and o-, m-, and p-Xylenes by the Newly Isolated Bacterium Pseudoxanthomonas spadix BD-a59

ABSTRACT A bacterium designated strain BD-a59, able to degrade all six benzene, toluene, ethylbenzene, and o-, m-, and p-xylene (BTEX) compounds, was isolated by plating gasoline-contaminated sediment from a gasoline station in Geoje, Republic of Korea, without enrichment, on minimal salts basal (MSB) agar containing 0.01% yeast extract, with BTEX as the sole carbon and energy source. Taxonomic analyses showed that the isolate belonged to Pseudoxanthomonas spadix, and until now, the genus Pseudoxanthomonas has not included any known BTEX degraders. The BTEX biodegradation rate was very low in MSB broth, but adding a small amount of yeast extract greatly enhanced the biodegradation. Interestingly, degradation occurred very quickly in slurry systems amended with sterile soil solids but not with aqueous soil extract. Moreover, if soil was combusted first to remove organic matter, the enhancement effect on BTEX biodegradation was lost, indicating that some components of insoluble organic compounds are nutritionally beneficial for BTEX degradation. Reverse transcriptase PCR-based analysis of field-fixed mRNA revealed expression of the tmoA gene, whose sequence was closely related to that carried by strain BD-a59. This study suggests that strain BD-a59 has the potential to assist in BTEX biodegradation at contaminated sites.

Alteromonas as a key agent of polycyclic aromatic hydrocarbon biodegradation in crude oil-contaminated coastal sediment.

Following the 2007 oil spill in South Korean tidal flats, we sought to identify microbial players influencing the environmental fate of released polycyclic aromatic hydrocarbons (PAHs). Two years of monitoring showed that PAH concentrations in sediments declined substantially. Enrichment cultures were established using seawater and modified minimal media containing naphthalene as sole carbon source. The enriched microbial community was characterized by 16S rRNA-based DGGE profiling; sequencing selected bands indicated Alteromonas (among others) were active. Alteromonas sp. SN2 was isolated and was able to degrade naphthalene, phenanthrene, anthracene, and pyrene in laboratory-incubated microcosm assays. PCR-based analysis of DNA extracted from the sediments revealed naphthalene dioxygenase (NDO) genes of only two bacterial groups: Alteromonas and Cycloclasticus, having gentisate and catechol metabolic pathways, respectively. However, reverse transcriptase PCR-based analysis of field-fixed mRNA revealed in situ expression of only the Alteromonas-associated NDO genes; in laboratory microcosms these NDO genes were markedly induced by naphthalene addition. Analysis by GC/MS showed that naphthalene in tidal-flat samples was metabolized predominantly via the gentisate pathway; this signature metabolite was detected (0.04 μM) in contaminated field sediment. A quantitative PCR-based two-year data set monitoring Alteromonas-specific 16S rRNA genes and NDO transcripts in sea-tidal flat field samples showed that the abundance of bacteria related to strain SN2 during the winter season was 20-fold higher than in the summer season. Based on the above data, we conclude that strain SN2 and its relatives are site natives--key players in PAH degradation and adapted to winter conditions in these contaminated sea-tidal-flat sediments.

Analysis of the fine-scale population structure of "candidatus accumulibacter phosphatis" in enhanced biological phosphorus removal sludge, using fluorescence in situ hybridization and flow cytometric sorting

ABSTRACT To investigate the fine-scale diversity of the polyphosphate-accumulating organisms (PAO) “Candidatus Accumulibacter phosphatis” (henceforth referred to as “Ca. Accumulibacter”), two laboratory-scale sequencing batch reactors (SBRs) for enhanced biological phosphorus removal (EBPR) were operated with sodium acetate as the sole carbon source. During SBR operations, activated sludge always contained morphologically different “Ca. Accumulibacter” strains showing typical EBPR performances, as confirmed by the combined technique of fluorescence in situ hybridization (FISH) and microautoradiography (MAR). Fragments of “Ca. Accumulibacter” 16S rRNA genes were retrieved from the sludge. Phylogenetic analyses together with sequences from the GenBank database showed that “Ca. Accumulibacter” 16S rRNA genes of the EBPR sludge were clearly differentiated into four “Ca. Accumulibacter” clades, Acc-SG1, Acc-SG2, Acc-SG3, and Acc-SG4. The specific FISH probes Acc444, Acc184, Acc72, and Acc119 targeting these clades and some helpers and competitors were designed by using the ARB program. Microbial characterization by FISH analysis using specific FISH probes also clearly indicated the presence of different “Ca. Accumulibacter” cell morphotypes. Especially, members of Acc-SG3, targeted by probe Acc72, were coccobacillus-shaped cells with a size of approximately 2 to 3 μm, while members of Acc-SG1, Acc-SG2, and Acc-SG4, targeted by Acc444, Acc184, and Acc119, respectively, were coccus-shaped cells approximately 1 μm in size. Subsequently, cells targeted by each FISH probe were sorted by use of a flow cytometer, and their polyphosphate kinase 1 (ppk1) gene homologs were amplified by using a ppk1-specific PCR primer set for “Ca. Accumulibacter.” The phylogenetic tree based on sequences of the ppk1 gene homologs was basically congruent with that of the 16S rRNA genes, but members of Acc-SG3 with a distinct morphology comprised two different ppk1 genes. These results suggest that “Ca. Accumulibacter” strains may be diverse physiologically and ecologically and represent distinct populations with genetically determined adaptations in EBPR systems.

Characterization of the Denitrification-Associated Phosphorus Uptake Properties of “Candidatus Accumulibacter phosphatis” Clades in Sludge Subjected to Enhanced Biological Phosphorus Removal

ABSTRACT To characterize the denitrifying phosphorus (P) uptake properties of “Candidatus Accumulibacter phosphatis,” a sequencing batch reactor (SBR) was operated with acetate. The SBR operation was gradually acclimated from anaerobic-oxic (AO) to anaerobic-anoxic-oxic (A2O) conditions by stepwise increases of nitrate concentration and the anoxic time. The communities of “Ca. Accumulibacter” and associated bacteria at the initial (AO) and final (A2O) stages were compared using 16S rRNA and polyphosphate kinase genes and using fluorescence in situ hybridization (FISH). The acclimation process led to a clear shift in the relative abundances of recognized “Ca. Accumulibacter” subpopulations from clades IIA > IA > IIF to clades IIC > IA > IIF, as well as to increases in the abundance of other associated bacteria (Dechloromonas [from 1.2% to 19.2%] and “Candidatus Competibacter phosphatis” [from 16.4% to 20.0%]), while the overall “Ca. Accumulibacter” abundance decreased (from 55.1% to 29.2%). A series of batch experiments combined with FISH/microautoradiography (MAR) analyses was performed to characterize the denitrifying P uptake properties of the “Ca. Accumulibacter” clades. In FISH/MAR experiments using slightly diluted sludge (∼0.5 g/liter), all “Ca. Accumulibacter” clades successfully took up phosphorus in the presence of nitrate. However, the “Ca. Accumulibacter” clades showed no P uptake in the presence of nitrate when the sludge was highly diluted (∼0.005 g/liter); under these conditions, reduction of nitrate to nitrite did not occur, whereas P uptake by “Ca. Accumulibacter” clades occurred when nitrite was added. These results suggest that the “Ca. Accumulibacter” cells lack nitrate reduction capabilities and that P uptake by “Ca. Accumulibacter” is dependent upon nitrite generated by associated nitrate-reducing bacteria such as Dechloromonas and “Ca. Competibacter.”

Henriciella litoralis sp. nov., isolated from a tidal flat, transfer of Maribaculum marinum Lai et al. 2009 to the genus Henriciella as Henriciella aquimarina nom. nov. and emended description of the genus Henriciella.

A Gram-staining-negative, strictly aerobic bacterium, designated strain SD10(T), was isolated from a tidal flat of the Yellow Sea, South Korea. Cells were non-spore-forming rods that showed catalase- and oxidase-positive reactions. Growth of strain SD10(T) was observed at 15-40 °C (optimum, 25-30 °C), at pH 6.0-9.0 (optimum, pH 6.5-8.5) and in the presence of 1-10 % (w/v) NaCl. Strain SD10(T) contained ubiquinone-10 (Q-10) as a major isoprenoid quinone and C(18 : 1)ω7c (39.3 %), C(16 : 0) (20.2 %), C(17 : 0) (8.9 %) and C(17 : 1)ω6c (8.1 %) as major fatty acids. The cellular polar lipids were identified as phosphatidylglycerol, monoglycosyldiglyceride, glucuronopyranosyldiglyceride and two unidentified glycolipids. The G+C content of the genomic DNA was 55.2 mol%. Based on 16S rRNA gene sequence similarities, the strain was most closely related to Henriciella marina Iso4(T) and Maribaculum marinum P38(T), with similarities of 97.8 and 97.0 %, respectively. The DNA-DNA relatedness between strain SD10(T) and H. marina Iso4(T) was 12.0±3.2 %. A phylogenetic analysis based on 16S rRNA gene sequences showed that M. marinum P38(T) and H. marina Iso4(T) formed a monophyletic cluster and that their 16S rRNA gene sequence similarity was 98.1 %. DNA-DNA hybridization between H. marina Iso4(T) and M. marinum LMG 24711(T) was 22.9±2.7 %, indicating that the two strains belong to separate species. On the basis of chemotaxonomic data and molecular properties, we propose that strain SD10(T) represents a novel species of the genus Henriciella, for which the name Henriciella litoralis sp. nov. is proposed. The type strain is SD10(T) ( = KACC 13700(T)  = DSM 22014(T)). In addition, we propose to transfer Maribaculum marinum Lai et al. 2009 to the genus Henriciella as Henriciella aquimarina nom. nov. (type strain P38(T)  = CCTCC AB 208227(T)  = LMG 24711(T)  = MCCC 1A01086(T)), and we present an emended description of the genus Henriciella.

Comparative Genomics Reveals Adaptation by Alteromonas sp. SN2 to Marine Tidal-Flat Conditions: Cold Tolerance and Aromatic Hydrocarbon Metabolism

Alteromonas species are globally distributed copiotrophic bacteria in marine habitats. Among these, sea-tidal flats are distinctive: undergoing seasonal temperature and oxygen-tension changes, plus periodic exposure to petroleum hydrocarbons. Strain SN2 of the genus Alteromonas was isolated from hydrocarbon-contaminated sea-tidal flat sediment and has been shown to metabolize aromatic hydrocarbons there. Strain SN2s genomic features were analyzed bioinformatically and compared to those of Alteromonas macleodii ecotypes: AltDE and ATCC 27126. Strain SN2s genome differs from that of the other two strains in: size, average nucleotide identity value, tRNA genes, noncoding RNAs, dioxygenase gene content, signal transduction genes, and the degree to which genes collected during the Global Ocean Sampling project are represented. Patterns in genetic characteristics (e.g., GC content, GC skew, Karlin signature, CRISPR gene homology) indicate that strain SN2s genome architecture has been altered via horizontal gene transfer (HGT). Experiments proved that strain SN2 was far more cold tolerant, especially at 5°C, than the other two strains. Consistent with the HGT hypothesis, a total of 15 genomic islands in strain SN2 likely confer ecological fitness traits (especially membrane transport, aromatic hydrocarbon metabolism, and fatty acid biosynthesis) specific to the adaptation of strain SN2 to its seasonally cold sea-tidal flat habitat.

Mucilaginibacter oryzae sp. nov., isolated from soil of a rice paddy.

A Gram-negative-staining, non-spore-forming bacterium devoid of flagella, designated strain B9(T), was isolated from rice paddy soil associated with the roots of Oryza sativa collected from Jinju, South Korea. Cells were straight rods, were catalase- and oxidase-positive and were able to hydrolyse pectin, xylan and laminarin. Growth of strain B9(T) was observed between 15 and 35 degrees C (optimum 25-30 degrees C) and between pH 5.0 and 8.0 (optimum pH 6.5-7.5). Strain B9(T) contained menaquinone-7 (MK-7) as a major isoprenoid quinone and summed feature 3 (C(16 : 1)omega7c and/or iso-C(15 : 0) 2-OH), iso-C(15 : 0) and C(16 : 0) as major fatty acids. The G+C content of the genomic DNA was 44.4 mol%. Comparative 16S rRNA gene sequence analysis showed that strain B9(T) belonged to the genus Mucilaginibacter, a member of the family Sphingobacteriaceae, and was most closely related to Mucilaginibacter kameinonensis SCK(T) (95.9 % sequence similarity). On the basis of chemotaxonomic data and molecular properties, strain B9(T) represents a novel species of the genus Mucilaginibacter, for which the name Mucilaginibacter oryzae sp. nov. is proposed. The type strain is B9(T) (=KACC 12816(T) =DSM 19975(T)).

Isolation and Characterization of a New Benzene, Toluene, and Ethylbenzene Degrading Bacterium, Acinetobacter sp. B113

A bacterium designated strain B113, able to degrade benzene, toluene, and ethylbenzene compounds (BTE), was isolated from gasoline-contaminated sediment at a gas station in Geoje, Korea. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolate belonged to the genus Acinetobacter. The biodegradation rates of benzene, toluene, and ethylbenzene were relatively low in MSB broth, but the addition of yeast extract had a substantial impact on the biodegradation of BTE compounds, which suggested that yeast extract might provide a factor that was necessary for its growth or BTE biodegradation activity. However, interestingly, the biodegradation of BTE compounds occurred very quickly in slurry systems amended with sterile soil. Moreover, if soil was combusted first to remove organic matters, the enhancement effect on BTE biodegradation was lost, indicating that some insoluble organic compounds were probably beneficial for BTE degradation in contaminated sediment. This study suggests that strain B113 may play an important role for biodegradation of BTE in the contaminated site.

Litorimonas taeanensis gen. nov., sp. nov., isolated from a sandy beach

A heterotrophic, gram-negative, prosthecate bacterium, designated strain G5(T), was isolated from a sandy beach of Taean in South Korea. Cells of strain G5(T) were aerobic, catalase- and oxidase-positive, straight to slightly curved motile rods with a single flagellum and formed yellow-orange colonies on agar. Growth occurred at 15-40 °C (optimum 25-30 °C) and pH 6-9 (optimum pH 7-8). The major cellular fatty acids were C(18 : 1)ω7c, C(17 : 0), C(16 : 0), 11-methyl C(18 : 1)ω7c, C(17 : 1)ω8c and C(17 : 1)ω6c. The polar lipid pattern indicated the presence of phosphatidylglycerol, monoglycosyldiglyceride, glucuronopyranosyldiglyceride and two unidentified glycolipids. The G+C content of the genomic DNA was 63.6 mol% and the major quinone was Q-10. Comparative 16S rRNA gene sequence analysis showed that strain G5(T) belonged to the branch containing the genera Hellea, Robiginitomaculum and Hypomonas within the family Hyphomonadaceae. Within this group, strain G5(T) was most closely related to Hellea balneolensis 26III/A02/215(T) with 95.8 % 16S rRNA gene sequence similarity. Based on its phylogenetic position and its phenotypic, chemotaxonomic and molecular properties, strain G5(T) represents a novel species of a novel genus of the family Hyphomonadaceae, for which the name Litorimonas taeanensis gen. nov., sp. nov. is proposed. The type strain is G5(T) ( = KACC 13701(T)  = DSM 22008(T)).