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Characterization of a Novel Clostridium Sp. Sp17eb1 and Its Application Ability

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Characterization of a Novel Clostridium Sp. Sp17eb1 and Its Application Ability Anaerobe 61 (2020) 102096 Contents lists available at ScienceDirect List of publications for 2019: Khanok RATANAKHANOKCHAI Anaerobe journal homepage: www.elsevier.com/locate/anaerobe 1. Nakazono-Nagaoka, E., Fujikawa, T., Shikata, A., Tachaapaikoon, C., Waeonukul, R., Pason, P., Ratanakhanokchai, K., and Kosugi, A. (2019). Draft genome sequence Anaerobes in the environment data of Clostridium thermocellum PAL5 possessing high cellulose-degradation Characterization of a novel Clostridium sp. SP17eB1 and its application ability. Data in Brief 25: 104274, 1-5. for succinic acid production from hevea wood waste hydrolysate DOI: https://doi.org/10.1016/j.dib.2019.104274. Sukanya Phuengjayaem a, Somboon Tanasupawat b, Siriluk Teeradakorn c, * 2. Sorn, V., Chang, K-L., Phitsuwan, P., Ratanakhanokchai, K., and Dong, C-D. (2019). a Program in Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand Effect of microwave-assisted ionic liquid/acidic ionic liquid pretreatment on the b Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, 10330, Thailand c The Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, Bangkok, 10330, Thailand morphology, structure, and enhanced delignification of rice straw. Bioresource Technology 293: 121929. https://doi.org/10.1016/j.biortech.2019.121929. article info abstract Article history: An anaerobic, gram-positive, rod-shaped bacterium strain SP17eB1, isolated from dog saliva, was taxo- 3. Pason, P., Sermsathanaswadi, J., Waeonukul, R., Tachaapaikoon, C., Baramee, S., Received 16 May 2019 nomically characterized on the basis of phenotypic, chemotaxonomic, and genotypic characteristics. It Received in revised form Ratanakhanokchai, K., and Kosugi, A. (2019). Molecular characterization of was cultured in 4% (w/v) NaCl at a pH range of 5.0e8.0 (optimally at pH 7) and at 30Ce40 C (optimally 19 August 2019 at 37 C). Its major cellular fatty acids are C (36.3%), C cyclo (9.7%), C u9c (13.9%), and C u9c hypothetical scaffolding-like protein S1 in multienzyme complex produced by Accepted 2 September 2019 16:0 17:0 16:1 18:1 (10.7%), and its DNA guanineecytosine content is 40.8 mol%. On the basis of the 16S rRNA gene sequence Available online 4 September 2019 Paenibacillus curdlanolyticus B-6. AMB Express. 9: 171. analysis, it was determined that the strain belonged to the genus Clostridium and was closely related to https://doi.org/10.1186/s13568-019-0896-0. Handling Editor: Dena Lyras C. amygdalinum BR-10T (97.8%), C. saccharolyticum WM1T (97.8%), and C. celleracrescens DSM 5628T (97.7%). This strain showed a low level of DNAeDNA relatedness with the closely related strains, sug- Keywords: gesting that it is a novel species in the genus Clostridium. Recent studies have demonstrated the pro- 4. Phakeenuya, V., Ratanakhanokchai, K., Kosugi, A., and Tachaapaikoon, C. (2020). Clostridium duction of succinic acid using Clostridium strains. Strain SP17eB1 produced 25.1 ± 1.3 and 15.3 ± 1.5 g/L of A novel multifunctional GH9 enzyme from Paenibacillus curdlanolyticus B-6 Succinic acid succinic acid from 40 g/L of glucose and 30 g/L of hevea wood waste hydrolysate (HH), respectively, after Hevea wood waste hydrolysate 24 h. When detoxified HH was used as a substrate, the lag phase was reduced and cell growth was Detoxification exhibiting endo/exo functions of cellulase, mannanase and xylanase activities. enhanced by 7 fold (OD660 0.4e3.0) within 12 h. Detoxification using granular activated carbon may have Granular activated carbon Applied Microbiology and Biotechnology 104: 2079-2096. reduced the levels of furfural and HMF without interfering with the amount of sugars in HH. © 2019 Elsevier Ltd. All rights reserved. DOI: 10.1007/s00253-020-10388-3. 5. Aikawa, S., Thianheng, P., Baramee, S., Ungkulpasvich, U., Tachaapaikoon, C., Waeonukul, R., Pason, P., Ratanakhanokchai, K., and Kosugi, A. (2020). Phenotypic characterization and comparative genome analysis of two strains of thermophilic, 1. Introduction possess all the metabolic pathways for all fermentation products, including the EmbdeneMeyerhofeParnas and pentose phosphate anaerobic, cellulolytic-xylanolytic bacterium Herbivorax saccincola. Enzyme and Succinic acid is an intermediate of the tricarboxylic acid (TCA) pathways, suggesting that they have the ability to produce succinic Microbial Technology 136: 109517. cycle and a fermentative end-product of anaerobic metabolism. acid [9]. Most studies on Clostridium strains focus on a few com- https://doi.org/10.1016/j.enzmictec.2020.109517. Several anaerobic and facultative bacteria that produce succinic mercial products important to the energy industry, such as aceto- acid as a major fermentation product from carbohydrates have neebutanoleethanol and hydrogen gas. Clostridium amygdalinum been isolated from bovine rumen such as Actinobacillus succino- BR10T, which was isolated from an up-flow anaerobic sludge bed genes, Anaerobiospirillum succiniciproducens, Bacteroides fragilis, reactor treating potato starch waste water, can produce benzyl Klebsiella pneumoniae, and Mannheimia succiniciproducens [1,2]. alcohol and energy [10]. Clostridium amygdalinum strain C9, which Non-rumen bacteria such as Corynebacterium crenatum [3], was isolated from oil water mixtures, can use xylose, arabinose, and C. glutamicum [4], and Enterococcus faecalis [5] have been isolated in starch as carbon sources to produce hydrogen. This strain can various anaerobic environments such as domestic sludge, cattle achieve maximum production rates of 40 mmol H2 per g xylan; waste, rice paddy, marine shipworm, and dog mouths [6,7]. Be- 2.2e2.5 mol H2 per gram xylose; and 390 ml H2 per g of starch [11]. sides, A. succiniciproducens has been isolated from the mouth, The first discovery of C. saccharolyticum, which was isolated from a throat, and feces of a beagle dog [8]. Members of the genus Clos- methanogenic cellulose-enriched culture from sewage sludge, tridium are obligately anaerobic, rod-shaped bacteria and are pre- demonstrated the ability of utilizing a wide variety of carbohy- sent in soils and in the intestinal tract of animals and humans. They drates when grown in a complex media. It is capable of producing high yields of ethanol of up to 1.8 mol of ethanol/mole of glucose [12]. Clostridium celerecrescens DSM 5628T from a methanogenic * Corresponding author. culture, which was initiated from a cow manure inoculum and T E-mail address: [email protected] (S. Teeradakorn). C. xylanolyticum DSM 6555 from Pinus patula chip pile, could https://doi.org/10.1016/j.anaerobe.2019.102096 1075-9964/© 2019 Elsevier Ltd. All rights reserved. _______________________________ Reproduced from Anaerobe 61: 102096 (2020). Siriluk Teeradakorn: Participant of the 18th UM, 1990-1991. 328 329 2 S. Phuengjayaem et al. / Anaerobe 61 (2020) 102096 2. Materials and methods Nomenclature 2.1. Isolation, cultivation, and bacterial strains AC Activated carbon HH Hevea wood waste hydrolysate Strain SP17eB1 was isolated from dog saliva in the Suphanburi HPLC High-performance liquid chromatography province, Thailand. The bacteria were first screened by inoculating RCM Reinforced Clostridial medium the sample into enrichment medium and then by transferring the SPSS Statistical Package for the Social Sciences enrichment medium to a screening plate. Positive isolates exhibited TCA Tricarboxylic acid clear zones on the screening plate. The selected isolates were TSA Tryptic Soy Agar analyzed for succinic acid production qualitatively and quantita- TSB Tryptic soy broth tively by thin layer chromatography and high-performance liquid chromatography (HPLC), respectively. The details of the screening process and medium composition are described by Phuengjayaem, Phinkian, Tanasupawat and Teeradakorn [22]. The type strains of reportedly produce hydrogen, ethanol, and organic acids from closely related Clostridium species, including C. amygdalinum DSM cellulosic biomass and transform cinnamic acid to 3- 12857T, C. celerecrescens DSM 5628T, and C. saccharolyticum DSM phenylpropionic acid [13]. However, research on succinic acid 2544T, were obtained from DSMZ (Deutsche Sammlung von Mik- production using Clostridium strains has been neglected, and to the roorganismen und Zellkulturen, Braunschweig, Germany). All best of our knowledge, succinic acid has only been reported in bacterial strains were maintained in tryptic soy agar (TSA) (Difco, C. coccoides [14] and C. thermosuccinogenes [9] to date. Clostridium France) at 37 �C. Anaerobic conditions were maintained using an coccoides can convert glucose (5 g/L) to acetate (3.0 g/L), succinate Anaero Pack (Mitsubishi Gas Chemical, Japan). Cultures were grown (0.57 g/L), and lactate (0.58 g/L) after 24 h, whereas for 24e48 h prior to further study. C. thermosuccinogenes can convert various carbohydrates to succi- nate and acetate as major products [9]. These findings suggest that 2.2. Characterization of the strain SP17eB1 the genus Clostridium is an attractive source of succinic acid producers. 2.2.1. Phenotypic characterization Para rubber (Hevea brasiliensis) is an abundant and important The colonies on TSA plates were characterized on the basis of economic crop in Thailand [15]. Hevea is a softwood tree with a color, shape, margin, optical property, and elevation after growing high cellulose content (53.01%) [16]. Softwoods have higher cellu- under anaerobic conditions at 37 �C for 24e48 h. Cell morphology lose and lignin
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