Journal114 of Species Research 7(2):114-122, 2018JOURNAL OF SPECIES RESEARCH Vol. 7, No. 2 A report of 22 unrecorded bacterial species in Korea, isolated from Namhangang Chaeyun Baek1 and Hana Yi1,2,* 1Department of Public Health Sciences, Graduate School, Korea University, Seoul 02841, Republic of Korea 2School of Biosystem and Biomedical Science, Korea University, Seoul 02841, Republic of Korea *Correspondent: [email protected] As part of a larger study of indigenous prokaryotic species diversity in South Korea, various samples from Namhangang were subjected to analyses. Fresh water, underwater sediment, and moss-inhabiting aerobic and anaerobic bacteria were isolated. 22 of the isolates were identified as unrecorded bacterial species in Korea that had ≥98.7% 16S rRNA gene sequence similarity with published species. The aerobic strains isolated were Kurthia gibsonii and Massilia plicata. Also identified were four facultative anaerobic strains: Bacillus hisashii, Enterococcus rotai, Paenibacillus vini, and Pediococcus pentosaceus. 16 strictly anaerobic strains were identified as Bacteroides xylanolyticus, Carnobacterium maltaromaticum, Clostridium argentinense, Clostridium beijerinckii, Clostridium butyricum, Clostridium cavendishii, Clostridium diolis, Clostridium frigidicarnis, Clostridium perfringens, Clostridium saccharoperbutylacetonicum, Clostridium sphenoides, Clostridium subterminale, Cutibacterium acnes, Paraclostridium bifermentans, Prevotella paludivivens, and Romboutsia lituseburensis. Based on the examination of morphological, cultural, physiological, and biochemical properties of the isolates, descriptive information of these previously unrecorded species is provided here. Keywords: anaerobes, Namhangang, unrecorded species Ⓒ 2018 National Institute of Biological Resources DOI:10.12651/JSR.2018.7.2.114 INTRODUCTION and Jungnyeongcheon, are freshwater rivers with 10.19 km2, 69.11 km2, 150.5 km2, and 130.19 km2 areas, respec- While molecular methods have supplanted traditional tively. The Jodaeneub Marshy Land is downstream of microbiological culture-based tools as the favored method Sinnaecheon and is a wetland that developed as a result of identification of microorganisms, isolation of cultivable of the accumulation of sandy loam along shoals. Sainam bacterial strains is still useful in helping to understand the Valley is a low land area with streams between cliffs. The physiological and functional properties of bacteria (Stew- six sampling sites are evenly distributed across the Nam- art, 2012). Toward this end, the Korean government is hangang. directing projects to examine and gather unrecorded bac- Here, we report 22 newly isolated bacterial strains be- terial species in Korea as a part of a larger catalogue of longing to the phylum Actinobacteria, Bacteroidetes, indigenous genetic resources. Through this effort, a num- Firmicutes, and Proteobacteria, all of which are new re- ber of unrecorded bacterial species have been discovered cords for South Korea. and registered in the national resource database of Korea (NIBR, 2017). In this study, we aimed to investigate the diversity of MATERIALS AND METHODS cultivable anaerobic bacteria from freshwater samples, which have been relatively under-studied in previous Fresh water, underwater sediment, and moss were col- projects. Six locations in the Namhangang (Namhang lected between May 16 and Jun 27, 2017. Sampling sites River) tributary area were selected as sampling targets: were Sinnaecheon (N37°27ʹ42.21ʺ; E127°33ʹ16.71ʺ), Sinnaecheon, Hanpocheon, Jodaeneub Marshy Land, Yo- Hanpocheon (N37°4ʹ44.13ʺ; E127°50ʹ14.83ʺ), Jodaeneub docheon, Sainam Valley, and Jungnyeongcheon. The four Marshy Land (N37°5ʹ56.85ʺ; E127°49ʹ15.91ʺ), Yodo- rivers, namely Sinnaecheon, Hanpocheon, Yodocheon, cheon (N36°59ʹ33.79ʺ; E127°45ʹ32.67ʺ), Sainam Valley May 2018 Baek and Yi. Unrecorded bacterial species from Namhangang 115 (N36°53ʹ32.65ʺ; E128°20ʹ29.58ʺ), and Jungnyeongcheon identified as unrecorded bacterial species in the Republic (N36°11ʹ34.30ʺ; E128°21ʹ4.49ʺ). of Korea. The similarity-based identification was further To isolate aerobic bacteria, the samples were inoculated supported by phylogenetic trees (Fig. 1). Each isolate and incubated on Reasoner’s 2A (R2A; BD) agar medi- formed a well-supported monophyletic clade with the um at 25°C for 1-7 days. To isolate anaerobic bacteria, type strain of identified bacterial species, confirming the Anaerobe Basal Agar (ABA; Oxoid) was used and incu- proper assignment of the isolate to the species with pub- bated at 30°C for 1-7 days under anaerobic gas condition lished names. The tree topology of the maximum-like- (80% nitrogen, 10% carbon dioxide, and 10% hydrogen) lihood method was consistent with that of the neigh- using anaerobic workstation (Whitley A35, Don Whit- bor-joining tree. The strain information and identification ley Scientific). Pure cultures of bacterial isolates were results are summarized in Table 1. maintained using R2A or ABA. The optimum growth The unrecorded species belonged to the class Actino- temperature was determined by incubating the isolates bacteria (1 strain) of the phylum Actinobacteria, the class on corresponding agar medium for up to 1 week. For Betaproteobacteria (1 strain) of the phylum Proteobacte- long-term preservation, the isolates were stored using ria, the class Bacteroidia (2 strains) of the phylum Bacte- 20% glycerol suspension at -80°C and lyophilized am- roidetes, the classes Bacilli (6 strains), and Clostridia (12 poules. The designation of strains, source of isolation, strains) of the phylum Firmicutes. At generic and family culture media, and incubation conditions are summa- level, those strains belonged to 13 different genera in 11 rized in Table 1. families: Bacillus of Bacillaceae, Carnobacterium of PCR amplification and gene sequencing of 16S rRNA Carnobacteriaceae, Clostridium and Bacteroides of Clos- gene was done using universal primers (27F, 5ʹ-AGA tridiaceae and Lachnospiraceae, Enterococcus of Entero- GTTTGATCMTGGCTCAG-3ʹ and 1492R, 5ʹ-TACG coccaceae, Pediococcus of Lactobacillaceae, Massilia GYTACCTTGTTACGACTT-3ʹ) as described previous- of Oxalobacteraceae, Paenibacillus of Paenibacillaceae, ly (Shin et al., 2016). The sequences were identified by Paraclostridium and Romboutsia of Peptostreptococcace- comparing them to the type strain sequence database ae, Kurthia of Planococcaceae, Prevotella of Prevotella- hosted by the EzBioCloud server (Yoon et al., 2017). ceae, and Cutibacterium of Propionibacteriaceae. Based on pairwise sequence similarity, an isolate show- The cells of isolates were Gram-reaction-negative or ing 98.7% or higher similarity to a type strain with a positive, rods or cocci, flagellated or non-flagellated bac- published name, but whose presence has not been re- teria. Colonial colors were white or yellow. None of the ported in Korea, was identified as an unrecorded species. isolates produced diffusible pigment on R2A or ABA. For phylogenetic analyses, the 16S rRNA gene se- Two of the isolates were strict aerobes, four were facul- quences were aligned using EzEditor program (Jeon et tative anaerobes, and 16 were strict anaerobes. Aerobic al., 2014). Phylogenetic trees were inferred using the strains were isolated from moss, and anaerobic strains neighbor-joining (NJ) and maximum-likelihood (ML) al- were isolated from fresh water, underwater sediment, gorithms implemented in MEGA v. 7.0 program (Kumar and moss. All the isolates exhibited specific physiolog- et al., 2016). The Jukes-Cantor model and general time ical characteristics and enzymatic properties. The de- reversible model were used for calculating evolutionary tailed feature of carbon source utilization, glucose fer- distances of NJ and ML trees, respectively. Bootstrap mentation, degradation of high molecular weight com- analysis with 1,000 re-samplings was used to evaluate pounds, and presence of metabolic enzymes are given in the trees. the strain descriptions below. Colonial morphology was observed using cells of 2- to From the results of sequence similarities and phyloge- 3-days old on R2A or ABA agar plates. Cellular morphol- netic trees, we identified 22 strains from the Namhangang ogy and cell size were examined by transmission electron samples, including Bacillus hisashii, Bacteroides xy- microscopy (JEM-3010, Jeol). Gram staining was per- lanolyticus, Carnobacterium maltaromaticum, Clostrid- formed using a Gram-Staining kit (Sigma-Aldrich). Phys- ium argentinense, Clostridium beijerinckii, Clostridium iological properties were examined by using API 20NE butyricum, Clostridium cavendishii, Clostridium diolis, for aerobic strains or API 20A galleries for anaerobic Clostridium frigidicarnis, Clostridium perfringens, Clos- strains (bioMérieux). Oxygen requirement for growth was tridium saccharoperbutylacetonicum, Clostridium sphe- determined by incubating the inoculated R2A or ABA noides, Clostridium subterminale, Cutibacterium acnes, agar plates at both aerobic and anaerobic conditions. Enterococcus rotai, Kurthia gibsonii, Massilia plicata, Paenibacillus vini, Paraclostridium bifermentans, Pedi- ococcus pentosaceus, Prevotella paludivivens, and Rom- RESULTS AND DISCUSSION boutsia lituseburensis. These 22 bacterial species have 22 isolates, which had at least 98.7% sequence similar- been previously reported in other locations, but are new ity with previously recognized bacterial type strains, were reports for Korea. 116 Table 1. Taxonomic affiliations and summary of unrecorded species