A Report of 37 Unrecorded Anaerobic Bacterial Species Isolated from the Geum River in South Korea

Journal of Species Research 9(2):105-116, 2020

A report of 37 unrecorded anaerobic bacterial species isolated from the Geum River in South Korea

Changsu Lee, Joon Yong Kim, Yeon Bee Kim, Juseok Kim, Seung Woo Ahn, Hye Seon Song and Seong Woon Roh*

Microbiology and Functionality Research Group, World Institute of Kimchi, Gwangju 61755, Republic of Korea

A total of 37 anaerobic bacteria strains within the classes Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Bacteroidia, Flavobacteriia, Bacilli, Clostridia, and Fusobacteriia were isolated from freshwater and sediment of the Geum River in Korea. The unreported species were related with Rhizobium and Oleomonas of the class Alphaproteobacteria; Acidovorax, Pseudogulbenkiania, and Aromatoleum of the class Betaproteobacteria; Tolumonas, Aeromonas, Cronobacter, Lonsdalea, and Phytobacter of the class Gammaproteobacteria; Bacteroides, Dysgonomonas, Macellibacteroides, and Parabacteroides of the class Bacteroidia; Flavobacterium of the class Flavobacteriia; Bacillus and Paenibacillus of the class Bacilli; Clostridium, Clostridioides, Paraclostridium, Romboutsia, Sporacetigenium, and Terrisporobacter of the class Clostridia; and Cetobacterium and Ilyobacter of the class Fusobacteriia. A total of 37 strains, with >98.7% 16S rRNA gene sequence similarity with validly published bacterial species, but not reported in Korea, were determined to be unrecorded anaerobic bacterial species in Korea. Keywords: ‌16S rRNA, anaerobic bacteria, bacterial diversity, taxonomy, unrecorded species

Introduction lated culture in Korea. In the present study, we attempted to isolate anaerobic Since the Nagoya Protocol and the Convention on Bi- microorganisms from freshwater and sediment in the ological Diversity, securing and managing of biological Geum River of Korea. Here, 37 unreported species be- resources have become more important (Buck & Ham- longing to the classes Alphaproteobacteria, Betaproteo ilton, 2011). In order to be competitive with national bi- bacteria, Gammaproteobacteria, Bacteroidia, Flavobac ological resources, various biological resources should teriia, Bacilli, Clostridia, and Fusobacteriia are reported be secured. However, to date, systematic research on and described. domestic freshwater biological resources is insufficient. Freshwater basins are expected to have high diversity due to their diverse environmental conditions. According to Materials and Methods the Korean Society of Ecology, reported in 1994, it is estimated that 100,000 native species inhabit Korea, while Freshwater and sediment samples were collected from 52,628 species have been reported (National species list urban streams and wetland of the Geum River watershed of Korea, 2019); so there is an urgent need to study un- in 2019. A total of 37 anaerobic bacteria were isolated reported species. In particular, prokaryotes are a resource using various agar plates made of Deutsche Sammlung of biological industry and have the highest industrial val- von Mikroorganismen und Zellkulturen (DSMZ) medi- ue, but reported species are less than 1% of the estimated um No. 311c, DSMZ medium No. 320, DSMZ medium species. Most aerobic prokaryotes are being investigated No. 1451, and reinforced clostridial medium. These agar among reported species. Although anaerobic prokaryotic plates were incubated at 15-25℃ under anaerobic con- microorganisms derived from freshwater environment are dition (BD GasPak EZ Anaerobe Pouch System) for 3-7 actively being studied globally due to their high novelty days. Isolated bacterial strains were purified by serial di- as biological resources, there are few cases of purely iso- lution spreading and the pure cells were preserved in 20% 106 JOURNAL OF SPECIES RESEARCH Vol. 9, No. 2

(v/v) glycerol suspension containing 10% skimmed milk um aurantibutyricum, Acidovorax wautersii, Bacteroides at -80℃ and as lyophilized ampoules. luti, Flavobacterium tyrosinilyticum (Du & Yi, 2016), Colony morphology of the strains was observed by eye Lonsdalea britannica (Brady et al., 2012), Macellibacte or a magnifying glass after the cells were cultivated to roides fermentans (Jabari et al., 2012), Paraclostridium their stationary phase on agar plates. Cellular morphology benzoelyticum (Tushar et al., 2015), Tolumonas auensis and cell size were examined by field emission transmis- (Chertkov et al., 2011), Aeromonas rivipollensis (Marti sion electron microscopy (JEM 2100F; Jeol). Growth in & Balcázar, 2015), Cronobacter dublinensis subsp. lau the presence of oxygen was tested by aerobic incubation sannensis (Grim et al., 2013), Cetobacterium somerae for seven days. Gram staining was performed using a (Finegold et al., 2003), Aromatoleum toluolicum (Krieger Gram-staining kit (BD). Biochemical characteristics were et al., 1999), Parabacteroides chartae (Tan et al., 2012), evaluated by using API 20NE (bioMérieux) according to Dysgonomonas oryzarvi (Kodama et al., 2012), Clostrid the manufacturer’s instructions. ium amazonense (O’Neal et al., 2015), Clostridium chro Chromosomal DNA extraction, PCR amplification, miireducens (Inglett et al., 2011), Ilyobacter delafieldii, and 16S rRNA gene sequencing were performed using Phytobacter diazotrophicus (Zhang et al., 2008), and Ba standard procedures as described elsewhere (Kim et al., cillus endoradicis (Zhang et al., 2012). 2019). For the determination of 16S rRNA gene sequenc- Fourteen strains were isolated from freshwater and the es, primers 27F, 337F, 518R, 785F, and 1492R were used. others were isolated from sediment. Based on 16S rRNA Based on full 16S rRNA gene sequences, the closely re- gene sequences, phylogenetic position of 37 unrecorded lated type species were obtained using the EzBioCloud strains is shown in Fig. 2. Detailed physiological and server (Yoon et al., 2017). 16S rRNA gene sequences morphological characteristics of the 37 unrecorded bac- were aligned with the most closely related strains using terial strains determined in present study are given in the Clustal W (Thompson et al., 1994). The phylogenetic following strain descriptions. trees were constructed using neighbor-joining (Saitou & Nei, 1987), maximum likelihood (Felsenstein, 1981), and Description of Clostridioides mangenotii CBA7501 maximum parsimony methods (Fitch, 1971) in MEGA7 Cells are obligate anaerobic, Gram-stain-positive, non- (Kumar et al., 2016) with bootstrap values based on 1,000 pigmented, and cocci-shaped. Colonies are circular, con- randomly generated trees. vex, and entire after incubation for four days on DSMZ medium No. 311c at 25℃. Positive for esculin hydrolysis and gelatinase. Negative for nitrate reduction, indole pro- Results and Discussion duction, glucose fermentation, arginine dihydrolase, urease, and β-galactosidase activity. Does not utilize d-glu- The designation of strains, ID, similarity, and source cose, l-arabinose, d-mannose, d-mannitol, N-acetyl-glu- of isolation are described at Table 1. Thirty-seven strains cosamine, potassium gluconate, d-maltose, capric acid, were distributed into eight classes: two strains in Al adipic acid, malic acid, trisodium citrate, and phenylacetic phaproteobacteria, four strains in Bacilli, four strains acid. Strain CBA7501 (=NNIBR2019644BA4) was iso- in Bacteroidia, three strains in Betaproteobacteria, 16 lated from a sediment sample, Jinan-gun, Jeollabuk-do, strains in Clostridia, one strain in the Flavobacteriia, two Korea. strains in Fusobacteriia, and five strains in Gammapro teobacteria. Unrecorded anaerobic bacterial strains in Description of Clostridium botulinum CBA7502 the eight classes were identified as following species in the order of strain ID (Fig. 1): Clostridioides mangenotii, Cells are obligate anaerobic, Gram-stain-positive, non- Clostridium botulinum, Clostridium lundense (Cirne et pigmented, and rod-shaped. Colonies are irregular, raised, al., 2006), Pseudogulbenkiania subflava, Rhizobium alvei and entire after incubation for four days on DSMZ me- (Sheu et al., 2015), Sporacetigenium mesophilum (Chen dium No. 311c at 25℃. Positive for esculin hydrolysis. et al., 2006), Terrisporobacter glycolicus (Collins et al., Negative for nitrate reduction, indole production, glucose 1994), Clostridium huakuii (Ruan et al., 2014), Oleo fermentation, arginine dihydrolase, urease, gelatinase, monas sagaranensis, Clostridium algidicarnis, Clostridi and β-galactosidase activity. Does not utilize d-glucose, um intestinale (Collins et al., 1994), Paenibacillus sonchi l-arabinose, d-mannose, d-mannitol, N-acetyl-glucos- (Hong et al., 2009), Paenibacillus riograndensis, Clos amine, potassium gluconate, d-maltose, capric acid, adip- tridium sartagoforme (Stackebrandt et al., 1999), Clos ic acid, malic acid, trisodium citrate, and phenylacetic tridium gasigenes (Broda et al., 2000), Romboutsia sed acid. Strain CBA7502 (=NNIBR2019644BA41) was iso- imentorum (Wang et al., 2015), Bacillus benzoevorans, lated from a sediment sample, Jangsu-gun, Jeollabuk-do, Clostridium senegalense (Mishra et al., 2012), Clostridi Korea. May 2020 Lee et al. A report of 37 unrecorded anaerobic bacterial species in Korea 107

source Isolation F reshwater S ediment S ediment F reshwater F reshwater F reshwater S ediment S ediment S ediment S ediment Sediment S ediment F reshwater S ediment S ediment S ediment F reshwater S ediment S ediment F reshwater S ediment Sediment Sediment Freshwater Sediment Sediment Sediment Freshwater Sediment Sediment Sediment Sediment F reshwater F reshwater F reshwater F reshwater F reshwater (%) 99.72 99.39 99.02 99.93 99.35 99.30 99.44 99.93 99.02 99.02 99.86 99.51 99.21 99.85 99.49 99.86 98.96 99.51 100.00 99.93 98.74 100.00 99.66 99.38 99.72 99.45 99.79 99.24 99.39 99.66 99.64 99.57 99.79 99.44 99.71 99.55 99.66 Gammaproteobac Similarity , and AJKY01000076 FR775967 CP001616 FR733681 AJ438155 KR232271 X76750 AY682207 KF443808 LBBT01000182 FR733662 CP001056 AY858804 KC967412 jgi.1107655 X76740 Y18175 AF092548 JF824801 LZYW01000149 KP281434 AY228334 AF129465 FXAG01000045 jgi.1068022 JN029805 HQ020488 AB547446 jgi.1107761 DQ358736 LN831776 D78311 GU434676 D45202 DQ821583 HE649224 JF311442 Accession number Fusobacteriia , LMG 23824 P2G1 DSM 9187 DSM 5704 WAL 14325 WAL THGDN8.8 DSM 1288 ZLJ115 LAM201 JC272 DSM 1289 Eklund 17B DSM 17049 LAM1030 DSM 15099 DSM 6191 DSM 1292 DSM 12272 JC122 DSM 793 NE08V GCAF-1 T DSM 22618 DSM 27981 NS31-3 LIND7H Dy73 DSM 26991 X19-5 SBR5 DSM 5391 CCBAU 05776 HD-1 LS 8 TNR-22 LMG 26267 Most closely related species Closest type strain Flavobacteriia , number Accession Clostridia MN646995 MN646994 MN646993 MN647002 MN646996 MN646989 MN646972 MN646971 MN646982 MN646992 MN646967 MN960557 MN646968 MN646974 MN646976 MN646977 MN646980 MN646981 MN646984 MN646985 MN647000 MN647001 MN646997 MN646969 MN646986 MN646998 MN646991 MN646999 MN646987 MN646978 MN646979 MN646983 MN971626 MN646975 MN647003 MN646970 MN646990 , Betaproteobacteria NNIBR2019644BA32 NNIBR2019644BA31 NNIBR2019644BA30 NNIBR2019644BA39 NNIBR2019644BA33 NNIBR2019644BA26 NNIBR2019644BA9 NNIBR2019644BA8 NNIBR2019644BA19 NNIBR2019644BA29 NNIBR2019644BA4 NNIBR2019644BA41 NNIBR2019644BA5 NNIBR2019644BA11 NNIBR2019644BA13 NNIBR2019644BA14 NNIBR2019644BA17 NNIBR2019644BA18 NNIBR2019644BA21 NNIBR2019644BA22 NNIBR2019644BA37 NNIBR2019644BA38 NNIBR2019644BA34 NNIBR2019644BA6 NNIBR2019644BA23 NNIBR2019644BA35 NNIBR2019644BA28 NNIBR2019644BA36 NNIBR2019644BA24 NNIBR2019644BA15 NNIBR2019644BA16 NNIBR2019644BA20 NNIBR2019644BA42 NNIBR2019644BA12 NNIBR2019644BA40 NNIBR2019644BA7 NNIBR2019644BA27 NNIBR ID , CBA7532 CBA7531 CBA7530 CBA7539 CBA7533 CBA7526 CBA7508 CBA7507 CBA7519 CBA7529 CBA7501 CBA7502 CBA7503 CBA7510 CBA7512 CBA7513 CBA7517 CBA7518 CBA7521 CBA7522 CBA7537 CBA7538 CBA7534 CBA7505 CBA7523 CBA7535 CBA7528 CBA7536 CBA7524 CBA7514 CBA7516 CBA7520 CBA7541 CBA7511 CBA7540 CBA7506 CBA7527 Strain ID Bacteroidia . , subsp Bacilli , lausannensis dublinensis rivipollensis auensis delafieldii somerae tyrosinilyticum glycolicus mesophilum sedimentorum benzoelyticum mangenotii botulinum lundense huakuii algidicarnis intestinale sartagoforme gasigenes senegalense aurantibutyricum amazonense chromiireducens toluolicum subflava wautersii chartae fermentans oryzarvi luti sonchi riograndensis benzoevorans endoradicis sagaranensis diazotrophicus alvei britannica Species Alphaproteobacteria Cronobacter Aeromonas Tolumonas Ilyobacter Cetobacterium Flavobacterium Terrisporobacter Sporacetigenium Romboutsia Paraclostridium Clostridioides Clostridium Aromatoleum Pseudogulbenkiania Acidovorax Parabacteroides Macellibacteroides Dysgonomonas Bacteroides Paenibacillus Bacillus Oleomonas Phytobacter Rhizobium Lonsdalea Genus Enterobacteriaceae Aeromonadaceae Fusobacteriaceae Flavobacteriaceae Peptostreptococcaceae Clostridiaceae Rhodocyclaceae Chromobacteriaceae Comamonadaceae Tannerellaceae Porphyromonadaceae Dysgonamonadaceae Bacteroidaceae Paenibacillaceae Bacillaceae Acetobacteraceae unclassified Rhizobiaceae Pectobacteriaceae Family Enterobacterales Aeromonadales Fusobacteriales Flavobacteriales Clostridiales Rhodocyclales Neisseriales Burkholderiales Bacteroidales Bacillales Rhodospirillales Rhizobiales Order Summary of strains isolated belonging to the classes the to belonging isolated of strains Summary and their taxonomic affiliations. Gammaproteobacteria Fusobacteriia Flavobacteriia Clostridia Betaproteobacteria Bacteroidia Bacilli teria Alphaproteobacteria Table 1. Table Class 108 JOURNAL OF SPECIES RESEARCH Vol. 9, No. 2

1 2 3 4 5 6

7 8 9 10 11 12

13 14 15 16 17 18

19 20 21 22 23 24

25 26 27 28 29 30

31 32 33 34 35 36

Fig. 1. Transmission electron micrographs of cells. Strains: 1, CBA7501; 2, CBA7502; 3, CBA7503; 4, CBA7505; 5, CBA7506; 6, CBA7507; 7, CBA7508; 8, CBA7510; 9, CBA7511; 10, CBA7512; 11, CBA7513; 12, CBA7514; 13, CBA7516; 14, CBA7517; 15, CBA7518; 16, CBA7519; 17, CBA7520; 18, CBA7521; 19, CBA7522; 20, CBA7523; 21, CBA7524; 22, CBA7526; 23, CBA7527; 24, CBA7528; 25, CBA7529; 26, CBA7530; 27, CBA7531; 28, CBA7532; 29, CBA7533; 30, CBA7534; 31, CBA7535; 32, CBA7536; 33, CBA7537; 34, CBA7538; 35, CBA7539; 36, CBA7540; 37, CBA7541.

Description of Clostridium lundense CBA7503 pigmented, and rod-shaped. Colonies are circular, convex, and entire after incubation for four days on DSMZ medi- Cells are obligate anaerobic, Gram-stain-positive, non- um No. 311c at 25℃. Positive for glucose fermentation May 2020 Lee et al. A report of 37 unrecorded anaerobic bacterial species in Korea 109

Fig. 2. Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showing the relationship between the strains isolated in this study. Bootstrap values (expressed as percentages of 1000 replications) of above 70% are shown at branch points. Filled circles and empty circles indicate nodes recovered by all three or two algorithms (neighbor-joining, maximum likelihood, and maximum parsimony), respec- tively. Halolamina sediminis halo-7T (CVUA01000001) was used as an outgroup. Bar, 0.05 substitutions per nucleotide position. 110 JOURNAL OF SPECIES RESEARCH Vol. 9, No. 2 and esculin hydrolysis. Negative for nitrate reduction, in- lated from a sediment sample, Buyeo-gun, Chungcheong- dole production, arginine dihydrolase, urease, gelatinase, nam-do, Korea. and β-galactosidase activity. Does not utilize d-glucose, l-arabinose, d-mannose, d-mannitol, N-acetyl-glucos- Description of Terrisporobacter glycolicus CBA7508 amine, potassium gluconate, d-maltose, capric acid, adip- Cells are obligate anaerobic, Gram-stain-positive, non- ic acid, malic acid, trisodium citrate, and phenylacetic pigmented, and rod-shaped. Colonies are circular, convex, acid. Strain CBA7503 (=NNIBR2019644BA5) was iso- and entire after incubation for three days on DSMZ me- lated from a sediment sample, Jinan-gun, Jeollabuk-do, dium No. 311c at 25℃. Positive for esculin hydrolysis. Korea. Negative for nitrate reduction, indole production, glucose Description of Pseudogulbenkiania subflava CBA7505 fermentation, arginine dihydrolase, urease, gelatinase, and β-galactosidase activity. Utilize d-glucose. Does not Cells are facultative anaerobic, Gram-stain-negative, utilize l-arabinose, d-mannose, d-mannitol, N-acetyl-glu- non-pigmented, and rod-shaped. Colonies are circular, cosamine, potassium gluconate, d-maltose, capric acid, convex, and entire after incubation for three days on adipic acid, malic acid, trisodium citrate, and phenylacetic DSMZ medium No. 311c at 25℃. Positive for esculin acid. Strain CBA7508 (=NNIBR2019644BA9) was iso- hydrolysis. Negative for nitrate reduction, indole pro- lated from a freshwater sample, Jinan-gun, Jeollabuk-do, duction, glucose fermentation, arginine dihydrolase, Korea. urease, gelatinase, and β-galactosidase activity. Utilizes d-glucose, d-maltose, and potassium gluconate. Does not Description of Clostridium huakuii CBA7510 utilize l-arabinose, d-mannose, d-mannitol, N-acetyl-glucosamine, capric acid, adipic acid, malic acid, trisodium Cells are obligate anaerobic, Gram-stain-positive, non- citrate, and phenylacetic acid. Strain CBA7505 (=NNI- pigmented, and rod-shaped. Colonies are circular, convex, BR2019644BA6) was isolated from a sediment sample, and entire after incubation for four days on DSMZ medi- Dong-gu, Daejeon, Korea. um No. 311c at 25℃. Positive for esculin hydrolysis and β-galactosidase activity. Negative for nitrate reduction, Description of Rhizobium alvei CBA7506 indole production, glucose fermentation, arginine dihydrolase, urease, and gelatinase. Does not utilize d-glu- Cells are facultative anaerobic, Gram-stain-negative, cose, l-arabinose, d-mannose, d-mannitol, N-acetyl-glu- non-pigmented, and rod-shaped. Colonies are circular, cosamine, potassium gluconate, d-maltose, capric acid, convex, and curled after incubation for three days on adipic acid, malic acid, trisodium citrate, and phenylacetic DSMZ medium No. 311c at 25℃. Positive for esculin acid. Strain CBA7510 ( =NNIBR2019644BA11) was hydrolysis and β-galactosidase activity. Negative for isolated from a freshwater sample, Dong-gu, Daejeon, nitrate reduction, indole production, glucose fermenta- Korea. tion, arginine dihydrolase, urease, and gelatinase activity. Does not utilize d-glucose, l-arabinose, d-mannose, Description of Oleomonas sagaranensis CBA7511 d-mannitol, N-acetyl-glucosamine, potassium gluconate, d-maltose, capric acid, adipic acid, malic acid, trisodium Cells are facultative anaerobic, Gram-stain-negative, citrate, and phenylacetic acid. Strain CBA7506 (=NNI- non-pigmented, and rod-shaped. Colonies are circular, BR2019644BA7) was isolated from a freshwater sample, convex, and entire after incubation for three days on Buyeo-gun, Chungcheongnam-do, Korea. DSMZ medium No. 320 at 25℃. Positive for esculin hydrolysis. Negative for nitrate reduction, indole produc- Description of Sporacetigenium mesophilum tion, glucose fermentation, arginine dihydrolase, urease, CBA7507 gelatinase, and β-galactosidase activity. Does not utilize d-glucose, l-arabinose, d-mannose, d-mannitol, N-ace- Cells are obligate anaerobic, Gram-stain-positive, non- tyl-glucosamine, potassium gluconate, d-maltose, capric pigmented, and rod-shaped. Colonies are circular, con- acid, adipic acid, malic acid, trisodium citrate, and pheny- vex, and entire after incubation for three days on DSMZ lacetic acid. Strain CBA7511 (=NNIBR2019644BA12) medium No. 311c at 25℃. Positive for esculin hydro- was isolated from a freshwater sample, Gunsan-si, Jeolla- lysis. Negative for nitrate reduction, indole production, buk-do, Korea. glucose fermentation, arginine dihydrolase, urease, gel atinase, and β-galactosidase activity. Does not utilize d- Description of Clostridium algidicarnis CBA7512 glucose, l-arabinose, d-mannose, d-mannitol, N-acetyl- glucosamine, potassium gluconate, d-maltose, capric acid, Cells are facultative anaerobic, Gram-stain-positive, adipic acid, malic acid, trisodium citrate, and phenylacetic non-pigmented, and rod-shaped. Colonies are puncti- acid. Strain CBA7507 (=NNIBR2019644BA8) was iso- form, convex, and lobate after incubation for four days May 2020 Lee et al. A report of 37 unrecorded anaerobic bacterial species in Korea 111 on DSMZ medium No. 311c at 25℃. Positive for esculin citrate, and phenylacetic acid. Strain CBA7516 (=NNI- hydrolysis. Negative for nitrate reduction, indole produc- BR2019644BA16) was isolated from a sediment sample, tion, glucose fermentation, arginine dihydrolase, urease, Dong-gu, Daejeon, Korea. gelatinase, and β-galactosidase activity. Does not utilize d-glucose, l-arabinose, d-mannose, d-mannitol, N-ace- Description of Clostridium sartagoforme CBA7517 tyl-glucosamine, potassium gluconate, d-maltose, capric Cells are obligate anaerobic, Gram-stain-positive, non- acid, adipic acid, malic acid, trisodium citrate, and pheny- pigmented, and rod-shaped. Colonies are circular, con- lacetic acid. Strain CBA7512 (=NNIBR2019644BA13) vex, and entire after incubation for four days on DSMZ was isolated from a sediment sample, Dong-gu, Daejeon, medium No. 1451 at 15℃. Positive for glucose fermen- Korea. tation and esculin hydrolysis. Negative for nitrate re- Description of Clostridium intestinale CBA7513 duction, indole production, arginine dihydrolase, urease, gelatinase, and β-galactosidase activity. Utilizes d-glu- Cells are obligate anaerobic, Gram-stain-positive, non- cose, d-mannitol, N-acetyl-glucosamine, and d-maltose. pigmented, and rod-shaped. Colonies are circular, convex, Does not utilize l-arabinose, d-mannose, potassium glu- and entire after incubation for four days on DSMZ medi- conate, capric acid, adipic acid, malic acid, trisodium um No. 1451 at 15℃. Positive for esculin hydrolysis and citrate, and phenylacetic acid. Strain CBA7517 (=NNI- β-galactosidase activity. Negative for nitrate reduction, BR2019644BA17) was isolated from a sediment sample, indole production, glucose fermentation, arginine dihy- Buyeo-gun, Chungcheongnam-do, Korea. drolase, urease, and gelatinase. Utilizes d-glucose, d-mannose, d-mannitol, N-acetyl-glucosamine, and malic acid. Description of Clostridium gasigenes CBA7518 Does not utilize l-arabinose, potassium gluconate, d-maltose, capric acid, adipic acid, trisodium citrate, and pheny- Cells are obligate anaerobic, Gram-stain-positive, non- lacetic acid. Strain CBA7513 (=NNIBR2019644BA14) pigmented, and rod-shaped. Colonies are circular, convex, was isolated from a sediment sample, Jangsu-gun, Jeolla- and entire after incubation for four days on DSMZ medi- buk-do, Korea. um No. 320 at 15℃. Negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, Description of Paenibacillus sonchi CBA7514 urease, esculin hydrolysis, gelatinase, and β-galactosidase activity. Utilize d-mannitol. Does not utilize d-glucose, Cells are facultative anaerobic, Gram-stain-positive, l-arabinose, d-mannose, N-acetyl-glucosamine, potassium non-pigmented, and rod-shaped. Colonies are circular, gluconate, d-maltose, capric acid, adipic acid, malic acid, umbonate, and entire after incubation for three days on trisodium citrate, and phenylacetic acid. Strain CBA7518 DSMZ medium No. 1451 at 15℃. Positive for glucose (=NNIBR2019644BA18) was isolated from a freshwater fermentation and esculin hydrolysis. Negative for ni- sample, Buyeo-gun, Chungcheongnam-do, Korea. trate reduction, indole production, arginine dihydrolase, urease, gelatinase, and β-galactosidase activity. Utilizes Description of Romboutsia sedimentorum CBA7519 d-mannitol, potassium gluconate, d-maltose, and adipic acid. Does not utilize d-glucose, l-arabinose, d-mannose, Cells are obligate anaerobic, Gram-stain-positive, non- N-acetyl-glucosamine, capric acid, malic acid, trisodium pigmented, and rod-shaped. Colonies are irregular, con- citrate, and phenylacetic acid. Strain CBA7514 (=NNI- vex, and undulate after incubation for three days on BR2019644BA15) was isolated from a sediment sample, DSMZ medium No. 1451 at 25℃. Positive for glucose Dong-gu, Daejeon, Korea. fermentation. Negative for nitrate reduction, indole production, arginine dihydrolase, urease, esculin hydrolysis, Description of Paenibacillus riograndensis CBA7516 gelatinase, and β-galactosidase activity. Does not utilize d-glucose, l-arabinose, d-mannose, d-mannitol, N-ace- Cells are facultative anaerobic, Gram-stain-positive, tyl-glucosamine, potassium gluconate, d-maltose, capric non-pigmented, and rod-shaped. Colonies are circular, acid, adipic acid, malic acid, trisodium citrate, and pheny- convex, and entire after incubation for three days on lacetic acid. Strain CBA7519 (=NNIBR2019644BA19) DSMZ medium No. 1451 at 15℃. Positive for glucose was isolated from a sediment sample, Jangsu-gun, Jeolla- fermentation, esculin hydrolysis, and β-galactosidase ac- buk-do, Korea. tivity. Negative for nitrate reduction, indole production, arginine dihydrolase, urease, and gelatinase. Utilizes Description of Bacillus benzoevorans CBA7520 l-arabinose, N-acetyl-glucosamine, potassium gluconate, and d-maltose. Does not utilize d-glucose, d-mannose, Cells are facultative anaerobic, Gram-stain-positive, d-mannitol, capric acid, adipic acid, malic acid, trisodium non-pigmented, and cocci-shaped. Colonies are circular, 112 JOURNAL OF SPECIES RESEARCH Vol. 9, No. 2

umbonate, and entire after incubation for three days on utilize d-glucose, l-arabinose, d-mannose, N-acetyl-glu- DSMZ medium No. 1451 at 25℃. Negative for nitrate re- cosamine, potassium gluconate, d-maltose, capric acid, duction, indole production, glucose fermentation, arginine adipic acid, and phenylacetic acid. Strain CBA7523 dihydrolase, urease, esculin hydrolysis, gelatinase, and (=NNIBR2019644BA23) was isolated from a freshwa- β-galactosidase activity. Utilizes l-arabinose, d-mannitol, ter sample, Dong-gu, Daejeon, Korea. and adipic acid. Does not utilize d-glucose, d-mannose, N-acetyl-glucosamine, potassium gluconate, d-maltose, Description of Bacteroides luti CBA7524 capric acid, malic acid, trisodium citrate, and pheny- Cells are obligate anaerobic, Gram-stain-negative, non- lacetic acid. Strain CBA7520 (=NNIBR2019644BA20) pigmented, and rod-shaped. Colonies are circular, con- was isolated from a sediment sample, Buyeo-gun, Chung vex, and entire after incubation for seven days on DSMZ cheongnam-do, Korea. medium No. 311c at 25℃. Positive for β-galactosidase activity. Negative for nitrate reduction, indole produc- Description of Clostridium senegalense CBA7521 tion, glucose fermentation, arginine dihydrolase, urease, Cells are obligate anaerobic, Gram-stain-positive, non- esculin hydrolysis, and gelatinase. Does not utilize d-glu- pigmented, and cocci-shaped. Colonies are irregular, cose, l-arabinose, d-mannose, d-mannitol, N-acetyl-glu- convex, and undulate after incubation for four days on cosamine, potassium gluconate, d-maltose, capric acid, reinforced clostridial medium at 25℃. Positive for glu- adipic acid, malic acid, trisodium citrate, and phenylacetic cose fermentation and gelatinase. Negative for nitrate re- acid. Strain CBA7524 ( =NNIBR2019644BA24) was duction, indole production, arginine dihydrolase, urease, isolated from a freshwater sample, Jangsu-gun, Jeolla- esculin hydrolysis, and β-galactosidase activity. Does not buk-do, Korea. utilize d-glucose, l-arabinose, d-mannose, d-mannitol, N-acetyl-glucosamine, potassium gluconate, d-maltose, Description of Flavobacterium tyrosinilyticum capric acid, adipic acid, malic acid, trisodium citrate, and CBA7526 phenylacetic acid. Strain CBA7521 (=NNIBR2019644 Cells are facultative anaerobic, Gram-stain-negative, BA21) was isolated from a sediment sample, Buyeo-gun, non-pigmented, and rod-shaped. Colonies are circular, Chungcheongnam-do, Korea. convex, and entire after incubation for three days on DSMZ medium No. 320 at 25℃. Positive for glucose Description of Clostridium aurantibutyricum fermentation, esculin hydrolysis, and β-galactosidase CBA7522 activity. Negative for nitrate reduction, indole produc- Cells are obligate anaerobic, Gram-stain-positive, non- tion, arginine dihydrolase, urease, and gelatinase. Uti- pigmented, and rod-shaped. Colonies are circular, con- lize capric acid. Does not utilize d-glucose, l-arabinose, vex, and entire after incubation for four days on DSMZ d-mannose, d-mannitol, N-acetyl-glucosamine, potassium medium No. 311c at 25℃. Positive for glucose fer- gluconate, d-maltose, adipic acid, malic acid, trisodium mentation, esculin hydrolysis, gelatinase, and β-galac- citrate, and phenylacetic acid. Strain CBA7526 (=NNI- tosidase activity. Negative for nitrate reduction, indole BR2019644BA26) was isolated from a freshwater sam- production, arginine dihydrolase, and urease. Utilizes ple, Jangsu-gun, Jeollabuk-do, Korea. d-glucose and d-mannose. Does not utilize l-arabinose, d-mannitol, N-acetyl-glucosamine, potassium gluconate, Description of Lonsdalea britannica CBA7527 d-maltose, capric acid, adipic acid, malic acid, trisodium Cells are facultative anaerobic, Gram-stain-negative, citrate, and phenylacetic acid. Strain CBA7522 (=NNI- non-pigmented, and rod-shaped. Colonies are circular, BR2019644BA22) was isolated from a sediment sample, convex, and entire after incubation for three days on Jinan-gun, Jeollabuk-do, Korea. DSMZ medium No. 311c at 25℃. Positive for glucose fermentation and esculin hydrolysis. Negative for ni- Description of Acidovorax wautersii CBA7523 trate reduction, indole production, arginine dihydrolase, Cells are facultative anaerobic, Gram-stain-negative, urease, gelatinase, and β-galactosidase activity. Utilize non-pigmented, and short rod-shaped. Colonies are cir- capric acid. Does not utilize d-glucose, l-arabinose, cular, convex, and entire after incubation for three days d-mannose, d-mannitol, N-acetyl-glucosamine, potassium on DSMZ medium No. 320 at 25℃. Positive for nitrate gluconate, d-maltose, adipic acid, malic acid, trisodium reduction and glucose fermentation. Negative for indole citrate, and phenylacetic acid. Strain CBA7527 (=NNI- production, arginine dihydrolase, urease, esculin hydro- BR2019644BA27) was isolated from a freshwater sam- lysis, gelatinase, and β-galactosidase activity. Utilizes ple, Dong-gu, Daejeon, Korea. d-mannitol, malic acid, and trisodium citrate. Does not May 2020 Lee et al. A report of 37 unrecorded anaerobic bacterial species in Korea 113

Description of Macellibacteroides fermentans fermentation. Negative for nitrate reduction, indole pro- CBA7528 duction, arginine dihydrolase, urease, esculin hydrolysis, gelatinase, and β-galactosidase activity. Does not Cells are facultative anaerobic, Gram-stain-positive, utilize d-glucose, l-arabinose, d-mannose, d-mannitol, non-pigmented, and rod-shaped. Colonies are circular, N-acetyl-glucosamine, potassium gluconate, d-malt- umbonate, and entire after incubation for three days on ose, capric acid, adipic acid, malic acid, trisodium ci- DSMZ medium No. 1451 at 25℃. Positive for nitrate re- trate, and phenylacetic acid. Strain CBA7531 ( =NNI- duction, glucose fermentation, esculin hydrolysis, gelati- BR2019644BA31) was isolated from a freshwater sam- nase, and β-galactosidase activity. Negative for indole ple, Dong-gu, Daejeon, Korea. production, arginine dihydrolase, and urease. Does not utilize d-glucose, l-arabinose, d-mannose, d-mannitol, Description of Cronobacter dublinensis subsp. N-acetyl-glucosamine, potassium gluconate, d-malt- lausannensis CBA7532 ose, capric acid, adipic acid, malic acid, trisodium citrate, and phenylacetic acid. Strain CBA7528 ( =NNI- Cells are facultative anaerobic, Gram-stain-negative, BR2019644BA28) was isolated from a sediment sample, non-pigmented, and rod-shaped. Colonies are circular, Jangsu-gun, Jeollabuk-do, Korea. pulvinate, and entire after incubation for three days on DSMZ medium No. 311c at 25℃. Positive for nitrate Description of Paraclostridium benzoelyticum reduction, indole production, glucose fermentation, ar- CBA7529 ginine dihydrolase, esculin hydrolysis, gelatinase, and β-galactosidase activity. Negative for urease. Utilizes po- Cells are obligate anaerobic, Gram-stain-positive, non- tassium gluconate, malic acid, and trisodium citrate. Does pigmented, and rod-shaped. Colonies are irregular, flat, not utilize d-glucose, l-arabinose, d-mannose, d-manni- and curled after incubation for three days on reinforced tol, N-acetyl-glucosamine, d-maltose, capric acid, adipic clostridial medium at 25℃. Positive for glucose fermen- acid, and phenylacetic acid. Strain CBA7532 ( =NNI- tation. Negative for nitrate reduction, indole production, BR2019644BA32) was isolated from a freshwater sam- arginine dihydrolase, urease, esculin hydrolysis, gelati- ple, Dong-gu, Daejeon, Korea. nase, and β-galactosidase activity. Does not utilize d-glucose, l-arabinose, d-mannose, d-mannitol, N-acetyl-glu- Description of Cetobacterium somerae CBA7533 cosamine, potassium gluconate, d-maltose, capric acid, adipic acid, malic acid, trisodium citrate, and phenylacetic Cells are facultative anaerobic, Gram-stain-negative, acid. Strain CBA7529 (=NNIBR2019644BA29) was iso- non-pigmented, and cocci-shaped. Colonies are circu- lated from a sediment sample, Jangsu-gun, Jeollabuk-do, lar, convex, and entire after incubation for three days on Korea. DSMZ medium No. 320 at 25℃. Positive for glucose fermentation, esculin hydrolysis, and β-galactosidase activity. Negative for nitrate reduction, indole produc- Description of Tolumonas auensis CBA7530 tion, arginine dihydrolase, urease, and gelatinase. Uti- Cells are facultative anaerobic, Gram-stain-negative, lize capric acid. Does not utilize d-glucose, l-arabinose, non-pigmented, and rod-shaped. Colonies are circular, d-mannose, d-mannitol, N-acetyl-glucosamine, potas- convex, and entire after incubation for three days on sium gluconate, d-maltose, adipic acid, malic acid, tri- DSMZ medium No. 1451 at 25℃. Negative for nitrate sodium citrate, and phenylacetic acid. Strain CBA7533 reduction, indole production, glucose fermentation, argi- (=NNIBR2019644BA33) was isolated from a freshwa- nine dihydrolase, urease, esculin hydrolysis, gelatinase, ter sample, Dong-gu, Daejeon, Korea. and β-galactosidase activity. Does not utilize d-glucose, l-arabinose, d-mannose, d-mannitol, N-acetyl-glucos- Description of Aromatoleum toluolicum CBA7534 amine, potassium gluconate, d-maltose, capric acid, adipic Cells are facultative anaerobic, Gram-stain-negative, acid, malic acid, trisodium citrate, and phenylacetic acid. non-pigmented, and rod-shaped. Colonies are circular, Strain CBA7530 (=NNIBR2019644BA30) was isolated convex, and entire after incubation for three days on from a sediment sample, Dong-gu, Daejeon, Korea. DSMZ medium No. 320 at 25℃. Positive for nitrate reduction and glucose fermentation. Negative for indole Description of Aeromonas rivipollensis CBA7531 production, arginine dihydrolase, urease, esculin hydro- Cells are facultative anaerobic, Gram-stain-negative, lysis, gelatinase, and β-galactosidase activity. Does not non-pigmented, and rod-shaped. Colonies are circular, utilize d-glucose, l-arabinose, d-mannose, d-mannitol, convex, and entire after incubation for three days on N-acetyl-glucosamine, potassium gluconate, d-maltose, DSMZ medium No. 311c at 25℃. Positive for glucose capric acid, adipic acid, malic acid, trisodium citrate, 114 JOURNAL OF SPECIES RESEARCH Vol. 9, No. 2 and phenylacetic acid. Strain CBA7534 (=NNIBR2019 pigmented, and rod-shaped. Colonies are circular, convex, 644BA34) was isolated from a sediment sample, Dong- and entire after incubation for four days on DSMZ medi- gu, Daejeon, Korea. um No. 311c at 25℃. Positive for glucose fermentation. Negative for nitrate reduction, indole production, argi- Description of Parabacteroides chartae CBA7535 nine dihydrolase, urease, esculin hydrolysis, gelatinase, and β-galactosidase activity. Does not utilize d-glucose, Cells are facultative anaerobic, Gram-stain-negative, l-arabinose, d-mannose, d-mannitol, N-acetyl-glucos- non-pigmented, and rod-shaped. Colonies are circu- amine, potassium gluconate, d-maltose, capric acid, adip- lar, convex, and entire after incubation for three days ic acid, malic acid, trisodium citrate, and phenylacetic on reinforced clostridial medium at 25℃. Positive for acid. Strain CBA7538 (=NNIBR2019644BA38) was iso- glucose fermentation, esculin hydrolysis, and β-galac- lated from a sediment sample, Jangsu-gun, Jeollabuk-do, tosidase activity. Negative for nitrate reduction, indole Korea. production, arginine dihydrolase, urease, and gelatinase. Does not utilize d-glucose, l-arabinose, d-mannose, Description of Ilyobacter delafieldii CBA7539 d-mannitol, N-acetyl-glucosamine, potassium gluconate, d-maltose, capric acid, adipic acid, malic acid, trisodium Cells are obligate anaerobic, Gram-stain-positive, non- citrate, and phenylacetic acid. Strain CBA7535 (=NNI- pigmented, and rod-shaped. Colonies are circular, convex, BR2019644BA35) was isolated from a sediment sample, and entire after incubation for three days on DSMZ medi- Jangsu-gun, Jeollabuk-do, Korea. um No. 1451 at 25℃. Negative for nitrate reduction, indole production, glucose fermentation, arginine dihydro- Description of Dysgonomonas oryzarvi CBA7536 lase, urease, esculin hydrolysis, gelatinase, and β-galactosidase activity. Does not utilize d-glucose, l-arabinose, Cells are obligate anaerobic, Gram-stain-negative, non- d-mannose, d-mannitol, N-acetyl-glucosamine, potassium pigmented, and rod-shaped. Colonies are circular, convex, gluconate, d-maltose, capric acid, adipic acid, malic acid, and entire after incubation for three days on DSMZ medi- trisodium citrate, and phenylacetic acid. Strain CBA7539 um No. 311c at 25℃. Positive for glucose fermentation, (=NNIBR2019644BA39) was isolated from a sediment esculin hydrolysis, and β-galactosidase activity. Negative sample, Dong-gu, Daejeon, Korea. for nitrate reduction, indole production, arginine dihydrolase, urease, and gelatinase. Does not utilize d-glucose, Description of Phytobacter diazotrophicus CBA7540 l-arabinose, d-mannose, d-mannitol, N-acetyl-glucosamine, potassium gluconate, d-maltose, capric acid, adip- Cells are facultative anaerobic, Gram-stain-negative, ic acid, malic acid, trisodium citrate, and phenylacetic non-pigmented, and short rod-shaped. Colonies are cir- acid. Strain CBA7536 (=NNIBR2019644BA36) was iso- cular, convex, and entire after incubation for three days lated from a sediment sample, Jangsu-gun, Jeollabuk-do, on DSMZ medium No. 1451 at 25℃. Positive for ni- Korea. trate reduction, indole production, glucose fermentation, esculin hydrolysis, gelatinase, and β-galactosidase ac- Description of Clostridium amazonense CBA7537 tivity. Negative for arginine dihydrolase and urease. Uti- lizes d-mannitol, potassium gluconate, malic acid, and Cells are obligate anaerobic, Gram-stain-positive, non- trisodium citrate. Does not utilize d-glucose, l-arabinose, pigmented, and cocci-shaped. Colonies are irregular, d-mannose, N-acetyl-glucosamine, d-maltose, capric convex, and lobate after incubation for four days on re- acid, adipic acid, and phenylacetic acid. Strain CBA7540 inforced clostridial medium at 25℃. Positive for β-ga- (=NNIBR2019644BA40) was isolated from a freshwater lactosidase activity. Negative for nitrate reduction, indole sample, Dong-gu, Daejeon, Korea. production, glucose fermentation, arginine dihydrolase, urease, esculin hydrolysis, and gelatinase. Utilize capric Description of Bacillus endoradicis CBA7541 acid. Does not utilize d-glucose, l-arabinose, d-man- Cells are facultative anaerobic, Gram-stain-positive, nose, d-mannitol, N-acetyl-glucosamine, potassium non-pigmented, and rod-shaped. Colonies are circular, gluconate, d-maltose, adipic acid, malic acid, trisodium convex, and entire after incubation for three days on citrate, and phenylacetic acid. Strain CBA7537 (=NNI- DSMZ medium No. 320 at 25℃. Positive for glucose fer- BR2019644BA37) was isolated from a freshwater sam- mentation and esculin hydrolysis. Negative for nitrate re- ple, Dong-gu, Daejeon, Korea. duction, indole production, arginine dihydrolase, urease, gelatinase, and β-galactosidase activity. Utilizes d-glu- Description of Clostridium chromiireducens CBA7538 cose, l-arabinose, d-mannose, N-acetyl-glucosamine, Cells are obligate anaerobic, Gram-stain-positive, non- and d-maltose. Does not utilize d-mannitol, potassium May 2020 Lee et al. A report of 37 unrecorded anaerobic bacterial species in Korea 115 gluconate, capric acid, adipic acid, malic acid, trisodium Collins, M., P. Lawson, A. Willems, J. Cordoba, J. Fernan- citrate, and phenylacetic acid. Strain CBA7541 (=NNI- dez-Garayzabal, P. Garcia, J. Cai, H. Hippe and J. Farrow. BR2019644BA42) was isolated from a sediment sample, 1994. The phylogeny of the genus Clostridium: proposal Jangsu-gun, Jeollabuk-do, Korea. of five new genera and eleven new species combinations. International Journal of Systematic and Evolutionary Mi- crobiology 44(4):812-826. Acknowledgements Du, J. and T.-H. Yi. 2016. Flavobacterium tyrosinilyticum sp. nov., isolated from the rhizosphere of wild strawberry. 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