Metagenomic and proteomic analysis of bacterial retting community and proteome profile in the degumming process of kenaf bast.

BACKGROUND: Data on the microbial community and functional proteins associated with degumming in kenaf remains scant. Here, we analyzed the microbial communities associated with kenaf (Hibiscus cannabinus) bast fibers during retting to identify potential candidate degumming bacteria. Retting liquids were collected and analyzed at 0 days, 10 days, and 34 days and then evaluated the yield and quality of kenaf fiber at the different retting times. Besides, the microbial communities were characterized using metagenomic and proteomic analysis by LC-MS/MS technology. RESULTS: The data showed that increase in the retting time significantly improves the softness, dispersion, and fiber whiteness of the kenaf fiber. The relative abundance of Acinetobacter increased from 2.88% at the baseline to 6.64% at the 34th retting. On the other hand, some members of Clostridium were reduced from 3% at the baseline to 2% at the 34th retting. Analysis of carbohydrate active enzymes showed constant changes in the utilization of carbohydrates. Besides, benzoquinone reductase, cellobiose dehydrogenase, glucose 1-oxidase, aryl alcohol oxidase and alcohol oxidase were the top five most abundant enzymes in the retting liquids. This present results demonstrated that the expressions of B7GYR8, Q6RYW5 and Q6FFK2 proteins were suppressed in Acinetobacter with the retting time. On the contrary, P05149 was upregulated with the retting time. In Clostridium, P37698, P52040 and P54937 proteins were upregulated with the retting time. CONCLUSION: In addition, bacteria Acinetobacter and Clostridium might be playing important roles in the kenaf degumming process. Similarly, up-regulation of P37698, P52040 and P54937 proteins is an important manifestation and mediates important roles in the degumming process.

Insights on bio-degumming of kenaf bast based on metagenomic and proteomics.

BACKGROUND: Microbes play important roles in kanef-degumming. This study aims at identifying the key candidate microbes and proteins responsible for the degumming of kenaf bast (Hibiscus cannabinus). Kenaf bast was cut into pieces and immersed into microbia fermentation liquid collected from different sites. Fermentation liquid samples were collected at 0, 40, 110 and 150 h and then subjected to the 16S/18S rRNA sequencing analysis and isobaric tag for relative and absolute quantitation (iTRAQ) analysis. The microbial (bacterial and fungal) diversity and the differentially expressed proteins/peptides (DEPs) were identified. RESULTS: With the prolonged degumming time, the weight loss rate increased, the bacterial diversity was decreased. [Weeksellaceae], Enterobacteriaceae and Moraxellaceae were rapidly increased at 0~40 h, and then decreased and were gradually replaced by Bacteroidaceae from 40 h to 150 h. Similarly, Chryseobacterium and Dysgonomonas were gradually increased at 0~110 h and then decreased; Acinetobacter and Lactococcus were increased at 0~40 h, followed by decrease. Bacteroides was the dominant genus at 150 h. Sequencing 18S rRNA-seq showed the gradually decreased Wallemia hederae and increased Codosiga hollandica during degumming. iTRAQ data analysis showed Rds1, and pyruvate kinase I was decreased and increased in the kanef-degumming, respectively. Other DEPs of ferredoxin I, superoxide dismutase and aconitatehydratase were identified to be related to the Glyoxylate and dicarboxylate metabolism (ko00630). CONCLUSIONS: Bacteria including Chryseobacterium, Dysgonomonas, Acinetobacter, Lactococcus and Bacteroidesand fungi like Wallemia hederae and Codosiga hollandica are key candidate microbes for kanef degumming.

Nibribacter flagellatus sp. nov., isolated from rhizosphere of Hibiscus syriacus and emended description of the genus Nibribacter.

A Gram-stain negative, aerobic, motile by flagella, rod-shaped strain (THG-T16T) was isolated from rhizosphere of Hibiscus syriacus. Growth occurred at 10-40 °C (optimum 28-30 °C), at pH 6.0-8.0 (optimum 7.0) and at 0-1.0% NaCl (optimum 0%). Based on 16S rRNA gene sequence analysis, the near phylogenetic neighbours of strain THG-T16T were identified as Nibribacter koreensis KACC 16450T (98.6%), Rufibacter roseus KCTC 42217T (94.7%), Rufibacter immobilis CCTCC AB 2013351T (94.5%) and Rufibacter tibetensis CCTCC AB 208084T (94.4%). The DNA G+C content of strain THG-T16T was determined to be 46.7 mol%. DNA-DNA hybridization values between strain THG-T16T and N. koreensis KACC 16450T, R. roseus KCTC 42217T, R. immobilis CCTCC AB 2013351T, R.tibetensis CCTCC AB 208084T were 33.5 ± 0.5% (31.7 ± 0.7% reciprocal analysis), 28.1 ± 0.2% (25.2 ± 0.2%), 17.1 ± 0.9% (10.2 ± 0.6%) and 8.1 ± 0.3% (5.2 ± 0.1%). The polar lipids were identified as phosphatidylethanolamine, two unidentified aminophospholipids, an unidentified aminolipid and three unidentified lipids. The quinone was identified as MK-7 and the polyamine as sym-homospermidine. The major fatty acids were identified as C16:1 ω5c, C17:1 ω6c, iso-C15:0, summed feature 3 (C16:1 ω7c and/or C16:1 ω6c) and summed feature 4 (iso-C17:1 I and/or anteiso-C17:1 B). On the basis of the phylogenetic analysis, chemotaxonomic data, physiological characteristics, and DNA-DNA hybridization data, strain THG-T16T represents a novel species of the genus Nibribacter, for which the name Nibribacter flagellatus sp. nov. is proposed. The type strain is THG-T16T(= KACC 19188T = CCTCC AB 2016246T).

Two new species of the Fusarium solani species complex isolated from compost and hibiscus (Hibiscus sp.).

Two new species in the Fusarium solani species complex (FSSC) are described and introduced. The new taxa are represented by German isolates CBS 142481 and CBS 142480 collected from commercial yard waste compost and vascular tissue of a wilting branch of hibiscus, respectively. The phylogenetic relationships of the collected strains to one another and within the FSSC were evaluated based on DNA sequences of 6 gene loci. Due to the limited sequence data available for reference strains in GenBank, however, a multi-gene phylogenetic analysis included partial sequences for the internal transcribed spacer region and intervening 5.8S nrRNA gene (ITS), translation elongation factor 1-alpha (tef1) and the RNA polymerase II second largest subunit (rpb2). Morphological and molecular phylogenetic data independently showed that these strains are distinct populations of the FSSC, nested within Clade 3. Thus, we introduce Fusarium stercicola and Fusarium witzenhausenense as novel species in the complex. In addition, 19 plant species of 7 legume genera were evaluated for their potential to host the newly described taxa. Eighteen plant species were successfully colonized, with 6 and 9 of these being symptomatic hosts for F. stercicola and F. witzenhausenense, respectively. As plants of the family Fabaceae are very distant to the originally sourced material from which the new taxa were recovered, our results suggest that F. stercicola and F. witzenhausenense are not host-specific and are ecologically fit to sustain stable populations in variety of habitats.

Sphingomonas rhizophila sp. nov., isolated from rhizosphere of Hibiscus syriacus.

A Gram-stain-negative, aerobic, non-motile, rod-shaped, catalase-positive and oxidase-positive bacteria (THG-T61T), was isolated from rhizosphere of Hibiscus syriacus. Growth occurred at 10-37 °C (optimum 25-30 °C), at pH 5.0-9.0 (optimum 7.0) and in the presence of 0-2.0 % NaCl (optimum without NaCl supplement). Based on 16S rRNA gene sequence analysis, the nearest phylogenetic neighbours of strain THG-T61T were identified as Sphingomonas ginsengisoli KCTC 12630T (97.9 %), Sphingomonas jaspsi DSM 18422T (97.8 %), Sphingomonas astaxanthinifaciens NBRC 102146T (97.4 %), Sphingomonassediminicola KCTC 12629T (97.2 %), 'Sphingomonas swuensis' KCTC 12336 (97.1 %) and Sphingomonas daechungensis KCTC 23718T (96.9 %). The isoprenoid quinone was ubiquinone-10 (Q-10). The major fatty acids were C16 : 0, C17 : 1ω6c, summed feature 4 (iso-C15 : 0 2-OH and/or C16 : 1ω7c) and summed feature 7 (C18 : 1ω7c, C18 : 1ω9t and/or C18 : 1ω12t). The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, sphingoglycolipid, one unidentified lipid, one unidentified phospholipid, one unidentified glycolipid and one unidentified phosphoglycolipid. The polyamine was homospermidine. The DNA G+C content of strain THG-T61T was 65.6 mol%. The DNA-DNA relatedness values between strain THG-T61T and its closest reference strains were less than 49.2 %, which is lower than the threshold value of 70 %. Therefore, strain THG-T61T represents a novel species of the genus Sphingomonas, for which the name Sphingomonas rhizophila sp. nov. is proposed. The type strain is THG-T61T (=KACC 19189T=CCTCC AB 2016245T).

Deinococcus hibisci sp. nov., isolated from rhizosphere of Hibiscus syriacus L. (mugunghwa flower).

A Gram-stain-positive, pink-pigmented, coccus-shaped, strictly aerobic, non-motile bacterium, strain THG-AG1.5T, was isolated from rhizosphere of Hibiscus syriacus L. (Mugunghwa flower) located in Kyung Hee University, Yongin, Gyeonggi, Republic of Korea. The isolated strain grew optimally at 25-30 °C, at pH 6.0-7.5 and in the presence of additional 0-1.5 % (w/v) NaCl. Strain THG-AG1.5T exhibited tolerance to UV radiation (>1500 J m-2) and to gamma radiation (>12 kGy). Based on 16S rRNA gene sequence comparisons, strain THG-AG1.5T was closely related to Deinococcus daejeonensis MJ27T (98.03 %), Deinococcus radiotolerans C1T (97.61 %) and Deinococcus grandis DSM 3963T (97.32 %). The genomic DNA G+C content of strain THG-AG1.5T was 74.8 mol%. The DNA-DNA hybridization values between strain THG-AG1.5T and its closest phylogenetically neighbours were below 63.0 %. The peptidoglycan amino acids were alanine, valine, glutamic acid, glycine, ornithine, lysine and aspartic acid. Strain THG-AG1.5T contained ribose, mannose and glucose as whole-cell-wall sugars and menaquinone-8 (MK-8) as the only isoprenoid quinone. The major component in the polyamine pattern was spermidine. The major polar lipids of strain THG-AG1.5T were a phosphoglycolipid, six unidentified glycolipids and an unidentified aminophospholipid. The major fatty acids were identified as iso-C15 : 0, C15 : 1ω6c, C16 : 0, iso-C17 : 0, C17 : 0, C18 : 0 and summed feature 3. On the basis of our polyphasic taxonomy study, strain THG-AG1.5T represents a novel species within the genus Deinococcus, for which the name Deinococcushibisci sp. nov. is proposed. The type strain is THG-AG1.5T (=KACC 18850T=CCTCC AB 2016078T).

Floricoccus tropicus gen. nov., sp. nov. and Floricoccus penangensis sp. nov. isolated from fresh flowers of durian tree and hibiscus.

Three strains of Gram-staining-positive, coccus-shaped, lactic acid bacteria, designated as HibF3T, HibF2 and HibF5 were isolated from fresh flowers of hibiscus, and a fourth, DF1T, was isolated from fresh flowers of durian tree, in Penang, Malaysia. Taxonomic characterisation was performed by polyphasic analysis. Sequence similarities of the 16S rRNA gene and the housekeeping rpoA and pheS genes of these strains with their closely-related lactococcal and streptococcal relatives were 92-94, 78 and 81 %, respectively. The results of phylogenetic analysis indicated that strains DF1T, HibF2, HibF5 and HibF3T were clustered together but were clearly separated from species of the genera Streptococcus and Lactococcus, indicating that they represent members of a novel genus of the family Streptococcaceae. Calculation of average nucleotide identity (ANI) values between the genomes of DF1T and HibF3T yielded values of 92.50-92.93 %. ANI values below the cut-off value and distinctive chemotaxonomic characteristics supported the hypothesis that these strains represented two novel species. Major cellular fatty acids in DF1T, HibF2 and HibF5 were C18 : 1ω7c and C16 : 0, while C12 : 0 and C14 : 0 were also dominant, in addition to C18 : 1ω7c and C16 : 0, in HibF3T. A novel genus is proposed with the name Floricoccus gen. nov. which consists of two species, Floricoccus tropicus sp. nov as the type species, and Floricoccus penangensis sp. nov. The respective type strains are DF1T (=LMG 29833T=JCM 31733T) and HibF3T (=LMG 29831T=DSM 31735T).

Microbacterium hibisci sp. nov., isolated from rhizosphere of mugunghwa (Hibiscus syriacus L.).

A Gram-stain-positive, aerobic, non-motile, short-rod shaped actinobacterium, designated THG-T2.14T, was isolated from soil sampled from the rhizosphere of mugunghwa. Growth occurred at 10-40 °C (optimum 30 °C), at pH 5.0-10.0 (optimum 7.0) and at 0-7.0 % NaCl (optimum 3.0 %). Based on 16S rRNA gene sequence analysis, the nearest phylogenetic neighbours of strain THG-T2.14T were identified as Microbacterium yannicii DSM 23203T (98.8 %), Microbacterium trichothecenolyticum DSM 8608T (98.8 %), Microbacterium arthrosphaerae DSM 22421T (98.7 %) and Microbacterium jejuense KACC 17124T (98.4 %). The major fatty acids were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, one unidentified lipid, two unidentified phospholipids and two unidentified phosphoglycolipids. The menaquinones were MK-12, and MK-13. The major polyamine was spermidine. The peptidoglycan contained ornithine, alanine, glycine, homoserine and glutamic acid. The diagnostic diamino acid was ornithine. The acyl type of the muramic acid was glycolyl. The whole-cell sugars were rhamnose, ribose, galactose, arabinose, xylose and glucose. The DNA G+C content of strain THG-T2.14T was 71.2 mol%. The DNA-DNA relatedness between strain THG-T2.14T and its closest reference strains were significantly lower than the threshold value of 70 %. On the basis of the phylogenetic analysis, chemotaxonomic data, physiological characteristics and DNA-DNA hybridization data, strain THG-T2.14T represents a novel species of the genus Microbacterium, for which the name Microbacterium hibisci sp. nov. is proposed. The type strain is THG-T2.14T (=KACC 18931T=CCTCC AB 2016180T).

Ramlibacter rhizophilus sp. nov., isolated from rhizosphere soil of national flower Mugunghwa from South Korea.

A Gram-negative, aerobic, non-motile, long rods or coccoid without flagellum strain, designated THG-YS3.2.7T, was isolated from therhizosphere soil of a Mugunghwa flower collected from Kyung Hee University, Yongin, South Korea. Growth occurred at 10-40 °C (optimum 28-37 °C), at pH 6-8 (optimum 7) and at 0-5 % NaCl (optimum 1 %). The predominant ubiquinone was ubiquinone 8 (Q-8). The major cellular fatty acids were C10 : 0, C10 : 0 3OH, C16 : 0, C17 : 0, C17 : 0 cyclo, C18 : 0, C18 : 3ω6c (6,9,12), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and summed feature 8 (C18 : 1 ω7c and/or C18 : 1ω6c). The major polar lipids were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylcholine (PC). The DNA G+C content of strain THG-YS3.2.7T was 69.4 mol%. Based on 16S rRNA gene sequence analysis, the nearest phylogenetic neighbours of strain THG-YS3.2.7T were identified as Ramlibacter henchirensis DSM 14656T (97.92 %), Ramlibacter tataouinensis DSM 14655T (97.90 %), Ramlibacter solisilvae KACC 17567T (97.04 %). DNA-DNA hybridization values between strain THG-YS3.2.7T and R. henchirensis DSM 14656T, R. tataouinensis DSM 14655T, R. solisilvae KACC 17567T were 32.5±1.5, 43.1±1.1, 42.8±1.1 %, respectively. On the basis of the phylogenetic analysis, chemotaxonomic data, physiological characteristics and DNA-DNA hybridization data, strain THG-YS3.2.7T represent a novel species of the genus Ramlibacter, for which the name Ramlibacter rhizophilus sp. nov. is proposed. The type strain is THG-YS3.2.7T (=KCTC 52083T=CCTCC AB 2015357T).

Caulobacter hibisci sp. nov., isolated from rhizosphere of Hibiscus syriacus L. (Mugunghwa flower).

A Gram-stain-negative, smooth, bright yellow-pigmented, aerobic, catalase- and oxidase-positive and rod-shaped bacterial strain was isolated from rhizosphere of Hibiscus syriacus L. (Mugunghwa flower) located in Kyung Hee University, Yongin, Gyeonggi, South Korea. Cells were dimorphic, non-motile or non-stalked, and motile by means of peritrichous flagellum. The strain, named THG-AG3.4T, grew at 15-35 °C, at pH 6.5-9.0 and in the presence of 0-1.5 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain THG-AG3.4T was most closely related to Caulobacter segnis ATCC 21756T (98.64 % similarity), Caulobacter vibrioides CB51T (98.57 %) and Caulobacter henricii ATCC 15253T (97.41 %). The DNA G+C content of strain THG-AG3.4T was 64.0 mol%. In DNA-DNA hybridization, the DNA-DNA relatedness between strain THG-AG3.4T and its closest phylogenetic neighbour was below 55.0 %. The predominant isoprenoid quinone detected in strain THG-AG3.4T was ubiquinone-10 (Q-10). The major polar lipids were found to be an unidentified lipid, two unidentified phosphoglycolipids, five unidentified glycolipids, eight unidentified aminolipids and phosphatidylglycerol. The major fatty acids were C16 : 0, summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). Thus, based on the report of the phenotypic, genotypic and phylogenetic characterization of strain THG-AG3.4T, it has been concluded that the isolate represents a novel species of the genus Caulobacter, for which the name Caulobacter hibisci sp. nov. is proposed. The type strain is THG-AG3.4T (=KACC 18849T=CCTCC AB 2016077T).

Roseomonas hibiscisoli sp. nov., isolated from the rhizosphere of Mugunghwa (Hibiscus syriacus).

A Gram-stain-negative, aerobic, non-motile and coccoid to short-rod-shaped bacterial strain (THG-N2.22T) was isolated from the rhizosphere of Mugunghwa (Hibiscus syriacus). Growth occurred at 20-40 °C (optimum 28 °C), at pH 5-9 (optimum 7) and with 0-4 % (w/v) NaCl (optimum 1 %). Based on 16S rRNA gene sequence analysis, the nearest phylogenetic neighbours of strain THG-N2.22T were identified as Roseomonas rhizosphaerae YW11T (98.5 % similarity), Roseomonas rubra S5T (98.5 %), Roseomonas cervicalis ATCC 49957T (98.2 %), Roseomonas aestuarii JC17T (97.8 %), Roseomonas oryzae JC288T (97.3 %) and Roseomonas ludipueritiae 170/96T (97.3 %); levels of similarity with the type strains of other Roseomonas species were lower than 97.0 %. The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, two unidentified aminolipids, three unidentified phospholipids and three unidentified lipids. The major quinone was ubiquinone-10. The major fatty acids were C16 : 0, C18 : 1 2-OH, C18 : 1ω7c and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c). The DNA G+C content of strain THG-N2.22T was 64.1 mol%. DNA-DNA hybridization values between strain THG-N2.22T and R. rhizosphaerae YW11T, R. rubra S5T, R. cervicalis ATCC 49957T, R. aestuarii JC17T, R. oryzae JC288T and R. ludipueritiae 170/96T were 43.1 % (30.2 %, reciprocal analysis), 39.0 % (24.7 %), 34.4 % (15.2 %), 18.0 % (14.5 %), 14.7 % (9.7 %) and 11.0 % (5.6 %), respectively. On the basis of the phylogenetic analysis, chemotaxonomic data, physiological characteristics and DNA-DNA hybridization data, strain THG-N2.22T represents a novel species of the genus Roseomonas, for which the name Roseomonas hibiscisoli sp. nov. is proposed. The type strain is THG-N2.22T (=KACC 18935T=CCTCC AB 2016176T).

Paracoccus hibiscisoli sp. nov., isolated from the rhizosphere of Mugunghwa (Hibiscus syriacus).

A Gram-reaction-negative, aerobic, non-motile, short-rod-shaped bacterium (THG-T2.31T) was isolated from the rhizosphere of Mugunghwa (Hibiscus syriacus). Growth occurred at 10-35 °C (optimum 28 °C), at pH 5.0-8.0 (optimum pH 7.0) and with 0-4.0 % NaCl (optimum 1.0 %). Based on 16S rRNA gene sequence analysis, the nearest phylogenetic neighbours of strain THG-T2.31T were identified as Paracoccus marcusii DSM 11574T (98.4 %), Paracoccus haeundaensis BC74171T (98.3 %), Paracoccus carotinifaciens E-396T (98.3 %), Paracoccus aestuarii B7T (97.3 %) and Paracoccus seriniphilus MBT-A4T (97.0 %); levels of similarity with the type strains of other species of the genus Paracoccus were lower than 97.0 %. The polar lipids were diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, one unidentified aminolipid and two unidentified phospholipids. The major fatty acids were C16 : 0, C18 : 0, C10 : 0 3-OH, and C18 : 1ω7c. The quinone was ubiquinone-10 (Q-10). The DNA G+C content of strain THG-T2.31T was 69.1 mol%. DNA-DNA hybridization values between strain THG-T2.31T and P. marcusii DSM 11574T, P. haeundaensis BC74171T, P. carotinifaciens E-396T, P. aestuarii B7T and P. seriniphilus MBT-A4T were 38.9 % (34.9 %, reciprocal analysis), 29.1 % (23.5 %), 28.0 % (19.7 %), 18.9 % (9.3) and 13.1 % (6.2 %). On the basis of the phylogenetic analysis, chemotaxonomic data, physiological characteristics and DNA-DNA hybridization data, strain THG-T2.31T represents a novel species of the genus Paracoccus, for which the name Paracoccus hibiscisoli sp. nov. is proposed. The type strain is THG-T2.31T (=KACC 18933T=CCTCC AB 2016182T).

Paracoccus hibisci sp. nov., isolated from the rhizosphere of Hibiscus syriacus L. (Mugunghwa flower).

A Gram-stain-negative, aerobic, short-rod-shaped bacterium, motile by means of one flagellum (THG-T2.8T), was isolated from the rhizosphere of Mugunghwa flower. Growth occurred at 10-37 °C (optimum 28 °C), at pH 6-8 (optimum 7) and with 0-5 % NaCl (optimum 1 %). The major quinone was ubiquinone-10 (Q-10). The major fatty acids were C10 : 0 3-OH, C16 : 0, C18 : 0 and C18 : 1ω7c. The polar lipids were diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine, phosphatidylglycerol, one unidentified aminolipid, two unknown phospholipids, one unknown glycolipid and one unidentified lipid. The DNA G+C content of strain THG-T2.8T was 65.5 mol%. Based on 16S rRNA gene sequence analysis, the nearest phylogenetic neighbours of strain THG-T2.8T were identified as Paracoccus tibetensis Tibet-S9a3T (98.6 %), Paracoccus aestuarii B7T (98.4 %), Paracoccus rhizosphaerae CC-CCM15-8T (98.3 %) and Paracoccus beibuensis JLT1284T (98.2 %). Levels of sequence similarity among strain THG-T2.8T and other species of the genus Paracoccus were lower than 98.0 %. DNA-DNA hybridization values between strain THG-T2.8T and P. tibetensis Tibet-S9A3TT, P. aestuarii B7T, P. rhizosphaerae CC-CCM15-8T and P. beibuensisJLT1284T were 36.5 % (38.8 %, reciprocal analysis), 32.8 % (34.8 %), 31.6 % (33.8 %) and 15.3 % (24.8 %), respectively. On the basis of the phylogenetic analysis, chemotaxonomic data, physiological characteristics and DNA-DNA hybridization data, strain THG-T2.8T represents a novel species of the genus Paracoccus, for which the name Paracoccus hibisci sp. nov. is proposed. The type strain is THG-T2.8T (=KACC 18932T=CCTCC AB 2016181T).

Niabella hibiscisoli sp. nov., isolated from soil of a Rose of Sharon garden.

A Gram-stain-negative, strictly aerobic, non-motile, rod-shaped and yellow-pigmented bacterium, designated strain THG-DN5.5T, was isolated from soil of a Rose of Sharon garden in Daejeon, South Korea. According to 16S rRNA gene sequence comparisons, strain THG-DN5.5T was found to be most closely related to Niabella yanshanensis CCBAU 05354T (97.7 % sequence similarity), Niabella ginsengisoli GR10-1T (97.0 %), 'Niabella terrae' ICM 1-15 (96.0 %), Niabella soli DSM 19437T (95.7 %) and Niabella aquatica RP-2T (95.6 %). The DNA-DNA relatedness between strain THG-DN5.5T and its phylogenetically closest neighbours was below 50.0 %. The DNA G+C content was 43.1 mol%. The major polar lipid of strain THG-DN5.5T was found to be phosphatidylethanolamine. The major fatty acids were identified as C16 : 0, iso-C15 : 0, iso-C15 : 1 G, and iso-C17 : 0 3-OH. MK-7 was the only menaquinone present. These data supported the affiliation of strain THG-DN5.5T to the genus Niabella. Strain THG-DN5.5T was distinguished from related species of the genus Niabellaby physiological and biochemical tests. In conclusion, strain THG-DN5.5T represents a novel species of the genus Niabella, for which the name Niabella hibiscisolisp. nov. is proposed. The type strain is THG-DN5.5T (=KACC 18857T=CCTCC AB 2016086T).

Flavobacterium hibisci sp. nov., isolated from the rhizosphere of Hibiscus syriacus L.

A Gram-stain-negative, smooth, bright-yellow-pigmented, rod-shaped bacterial strain, slightly motile by gliding, catalase- and oxidase-positive and aerobic, but growing weakly under anaerobic conditions, was isolated from the rhizosphere of the flower mugunghwa (Hibiscus syriacus L.) located in Kyung Hee University, Yongin, Gyeonggi, South Korea. The strain named THG-HG1.4T grew at 15-35 °C, pH 6.5-9.0 and in the presence of 0-2.5 % (w/v) NaCl. The phylogenetic analysis based on 16S rRNA gene sequence showed that strain THG-HG1.4T was most closely related to Flavobacterium gyeonganense HME7524T (98.83 %) and Flavobacterium arsenitoxidans S2-3HT (97.28 %). The DNA G+C content of strain THG-HG1.4T was 41.2 mol%. In DNA-DNA hybridization, the DNA-DNA relatedness between strain THG-HG1.4T and its closest phylogenetic neighbour was below 64.1 %. The predominant isoprenoid quinone detected in strain THG-HG1.4T was menaquinone-6 (MK-6). The major polar lipids were found to be phosphatidylethanolamine, three unidentified lipids, two unidentified glycolipids and an unidentified aminolipid. The major fatty acids were identified as iso-C15 : 0, iso-C15 : 0 3-OH, C16 : 0, iso-C17 : 0 3-OH and summed feature 3. Thus, based on the report of the phenotypic, genotypic and phylogenetic characterization of strain THG-HG1.4T, it has been concluded that the novel isolate represents a novel species of the genus Flavobacterium.Flavobacterium hibisci sp. nov. is proposed, with THG-HG1.4T (=KACC 18852T=CCTCC AB 2016178T) as the type strain.

Niastellahibisci sp. nov., isolated from rhizosphere soil of mugunghwa, the Korean national flower.

A Gram-stain-negative, aerobic, non-motile, long rods with no flagellum strain, designated THG-YS3.2.1T, was isolated from rhizosphere soil of mugunghwa, collected from Kyung Hee University, Yongin, South Korea. Growth occurred at 10-40 °C (optimum 28 °C), at pH 6.0-8.0 (optimum pH 7.0) and with 0-1.0 % NaCl (optimum 1.0 %). The predominant menaquinone was menaquinone-7 (MK-7). The major cellular fatty acids were anteiso-C12 : 0, iso-C14 : 0, iso-C15 : 0, anteiso-C15 : 0, iso-C15 : 1 G, anteiso-C15 : 1 A, C15 : 0 2-OH, C16 : 0, iso-C16 : 0, C16 : 0 3-OH, iso-C16 : 0 3-OH, iso-C16 : 1 G, C17 : 0 2-OH, iso-C17 : 0 3-OH, C17 : 1ω6c, C18 : 3ω6c (6, 9, 12), C18 : 0, summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH). The major polar lipids were phosphatidylmethylethanolamine), phosphatidylethanolamine, five unidentified aminolipids and three unidentified lipids. The DNA G+C content of strain THG-YS3.2.1T was 45.3 mol%. Based on 16S rRNA gene sequence analysis, the nearest phylogenetic neighbours of strain THG-YS3.2.1T were Niastella populi KCTC 22560T (98.7 % 16S rRNA gene sequence similarity), Niastella gongjuensis KACC 17399T (96.9 %), Niastella vici KCTC 42474T (96.2 %), Niastella yeongjuensis KACC 11466T (95.5 %) and Niastella koreensis KACC 11465T (95.1 %). DNA-DNA hybridization values between strain THG-YS3.2.1T and N. populi KCTC 22560T, N. gongjuensis KACC 17399T, N.vici KCTC 42474T, N. yeongjuensis KACC 11466T and N. koreensis KACC 11465Twere 55.8±1.0, 39.5±0.5, 35.2±0.1, 17.6±0.3 and 12.5±1.2 %, respectively. On the basis of the phylogenetic analysis, chemotaxonomic data, physiological characteristics and DNA-DNA hybridization data, strain THG-YS3.2.1T represents a novel species of the genus Niastella, for which the name Niastellahibisci sp. nov. is proposed. The type strain is THG-YS3.2.1T (=KCTC 52084T=CCTCC AB 2015356T).

Lysobacter rhizophilus sp. nov., isolated from rhizosphere soil of mugunghwa, the national flower of South Korea.

A Gram-staining-negative, aerobic, motile, rod-shaped, catalase- and oxidase-negative strain with one polar flagellum, designated THG-YS3.6T, was isolated from rhizosphere soil of a mugunghwa flower collected from Kyung Hee University, Yongin, South Korea. Growth occurred at 10-37 °C (optimum 25-30 °C), at pH 6-8 (optimum 7.0) and with 0-2.0 % NaCl (optimum 1 %). The isoprenoid quinone was ubiquinone-8 (Q-8). The major cellular fatty acids were iso-C11 : 0, iso-C11 : 0 3-OH, iso-C15 : 0, iso-C16 : 0, C16 : 1ω7c alcohol, C16 : 0, iso-C17 : 0 and summed feature 9 (iso-C17 : 1ω9c and/or C16 : 0 10-methyl). The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine, one unknown phospholipid, one unknown lipid and three unknown aminolipids. The DNA G+C content of strain THG-YS3.6T was 65.3 mol%. Based on 16S rRNA gene sequence analysis, the nearest phylogenetic neighbours of strain THG-YS3.6T were identified as Lysobacter yangpyeongensis KACC 11407T (98.7 %), Lysobacter oryzae KCTC 22249T (98.0 %), Lysobacter niabensis KACC 11587T (97.6 %) and Lysobacter terrae KACC 17646T (97.1 %). The DNA-DNA relatedness values between strain THG-YS3.6T and L. yangpyeongensis KACC 11407T, L. oryzae KCTC 22249T, L. niabensis KACC 11587T and L. terrae KACC 17646T were 53.8±1.0 %, 12.9±1.2 %, 10.9±0.6 % and 7.0±1.9 %, respectively. On the basis of the phylogenetic analysis, chemotaxonomic data, physiological characteristics and DNA-DNA hybridization data, strain THG-YS3.6T represents a novel species of the genus Lysobacter, for which the name Lysobacter rhizophilus sp. nov. is proposed. The type strain is THG-YS3.6T (=KCTC 52082T=CCTCC AB 2015358T).

Bacillus amyloliquefaciens ssp. plantarum strains as potential protective starter cultures for the production of Bikalga, an alkaline fermented food.

AIMS: To identify and screen dominant Bacillus spp. strains isolated from Bikalga, fermented seeds of Hibiscus sabdariffa for their antimicrobial activities in brain heart infusion (BHI) medium and in a H. sabdariffa seed-based medium. Further, to characterize the antimicrobial substances produced. METHODS AND RESULTS: The strains were identified by gyrB gene sequencing and phenotypic tests as B. amyloliquefaciens ssp. plantarum. Their antimicrobial activity was determined by the agar spot and well assay, being inhibitory to a wide range of Gram-positive and Gram-negative pathogenic bacteria and fungi. Antimicrobial activity against Bacillus cereus was produced in H. sabdariffa seed-based medium. PCR results revealed that the isolates have potential for the lipopeptides iturin, fengycin, surfactin, the polyketides difficidin, macrolactin, bacillaene and the dipeptide bacilysin production. Ultra-high-performance liquid chromatography-time of flight mass spectrometry analysis of antimicrobial substance produced in BHI broth allowed identification of iturin, fengycin and surfactin. CONCLUSIONS: The Bacillus amyloliquefaciens ssp. plantarum exhibited broad-spectrum antifungal and antibacterial properties. They produced several lipopeptide antibiotics and showed good potential for biological control of Bikalga. SIGNIFICANCE AND IMPACT OF THE STUDY: Pathogenic bacteria often occur in spontaneous food fermentations. This is the first report to identify indigenous B. amyloliquefaciens ssp. plantarum strains as potential protective starter cultures for safeguarding Bikalga.

Biodiversity of aerobic endospore-forming bacterial species occurring in Yanyanku and Ikpiru, fermented seeds of Hibiscus sabdariffa used to produce food condiments in Benin.

Yanyanku and Ikpiru made by the fermentation of Malcavene bean (Hibiscus sabdariffa) are used as functional additives for Parkia biglobosa seed fermentations in Benin. A total of 355 aerobic endospore-forming bacteria (AEFB) isolated from Yanyanku and Ikpiru produced in northern and southern Benin were identified using phenotypic and genotypic methods, including GTG5-PCR, M13-PCR, 16S rRNA, gyrA and gyrB gene sequencing. Generally, the same 5-6 species of the genus Bacillus predominated: Bacillus subtilis (17-41% of isolates), Bacillus cereus (8-39%), Bacillus amyloliquefaciens (9-22%), Bacillus licheniformis (3-26%), Bacillus safensis (8-19%) and Bacillus altitudinis (0-19%). Bacillus aryabhattai, Bacillus flexus, and Bacillus circulans (0-2%), and species of the genera Lysinibacillus (0-14%), Paenibacillus (0-13%), Brevibacillus (0-4%), and Aneurinibacillus (0-3%) occurred sporadically. The diarrheal toxin encoding genes cytK-1, cytK-2, hblA, hblC, and hblD were present in 0%, 91% 15%, 34% and 35% of B. cereus isolates, respectively. 9% of them harbored the emetic toxin genetic determinant, cesB. This study is the first to identify the AEFB of Yanyanku and Ikpiru to species level and perform a safety evaluation based on toxin gene detections. We further suggest, that the gyrA gene can be used for differentiating the closely related species Bacillus pumilus and B. safensis.

Investigation of the bacterial retting community of kenaf (Hibiscus cannabinus) under different conditions using next-generation semiconductor sequencing.

The microbial communities associated with kenaf (Hibiscus cannabinus) plant fibers during retting were determined in an effort to identify possible means of accelerating this process for industrial scale-up. Microbial communities were identified by semiconductor sequencing of 16S rRNA gene amplicons from DNA harvested from plant-surface associated samples and analyzed using an Ion Torrent PGM. The communities were sampled after 96 h from each of three different conditions, including amendments with pond water, sterilized pond water, or with a mixture of pectinolytic bacterial isolates. Additionally, plants from two different sources and having different pretreatment conditions were compared. We report that the best retting communities are dominated by members of the order Clostridiales. These bacteria appear to be naturally associated with the plant material, although slight variations between source materials were found. Additionally, heavy inoculations of pectinolytic bacteria established themselves and in addition their presence facilitated the rapid dominance of the original plant-associated Clostridiales. These data suggest that members of the order Clostridiales dominate the community and are most closely associated with efficient and effective retting. The results further suggest that establishment of the community structure is first driven by the switch to anaerobic conditions, and subsequently by possible competition for nitrogen. These findings reveal important bacterial groups involved in fiber retting, and suggest mechanisms for the manipulation of the community and retting efficiency by modifying nutrient availability.