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

Anaerobe 61 (2020) 102096

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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

3. Pason, P., Sermsathanaswadi, J., Waeonukul, R., Tachaapaikoon, C., Baramee, S., Article history: An anaerobic, gram-positive, rod-shaped bacterium strain SP17eB1, isolated from dog saliva, was taxo- 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.

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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 succinate 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 and lower pentosan content than hardwoods or of the isolate was determined by light microscopy (model Nikon angiosperms [17]. Moreover, hevea wood waste is a by-product YS2eH, Japan) and scanning electron microscopy (model JSM-5410 from the furniture industry and can be used as an alternative LV; JEOL, Japan). For scanning electron microscopy, samples were substrate for succinic acid production. prepared as described by Kudo, Matsushima, Itoh, Sasaki and Pretreatment of the waste is necessary to remove lignin and Suzuki [23]. Gram staining was performed according to standard hemicellulose and reduce cellulose crystallinity; moreover, pre- procedures [24]. Flagella staining was performed as described by treatment also increases the porosity of materials [18]. Diluted-acid Forbes [25]. The effects of temperature (20 �C, 30 �C, 37 �C, 40 �C, pretreatment is a simple and widely used process. However, it is 50 �C, and 60 �C) and pH (5.0, 6.0, 7.0, 8.0, and 9.0) on growth were conducted under severe conditions that produce inhibitors in the determined in tryptic soy broth (TSB; Difco, France) medium. solution. These include furfural and 5-hydroxymethylfurfural Catalase was tested by exposing the cell material to 10% H2O2 [26]. (HMF), which are produced by the degradation of pentoses and The biochemical characteristics were examined on the basis of hexoses, respectively [19]. To use the hydrolysate from waste more arginine hydrolysis, nitrate reduction, starch hydrolysis, and slime efficiently, it must be detoxified. For example, overliming with formation, as described by Tanasupawat et al. [27]. Acid production Ca(OH)2 is used to chemically modify inhibitors in the hydrolysate was tested on the following carbohydrates: L-arabinose, D-cello- to reduce toxicity, but the resulting solution may have high pH and biose, D-glucose, glycerol, D-lactose, D-maltose, D-mannitol, D- temperature [20]. Hodge et al. (2009) have reported that the use of mannose, D-melezitose, D-raffinose, L-rhamnose, salicin, D-sorbitol, fi activated carbon (AC) for the detoxi cation of softwood dilute acid sucrose, D-trehalose, and D-xylose. Gelatin hydrolysis (bovine hydrolysates is convenient, fast, and cost-effective [21]. origin), esculin hydrolysis (with ferric citrate) and urease were In the present study, we isolated Clostridium species from dog analyzed after growth under anaerobic conditions at 37 �C for 48 h saliva, which has not been reported elsewhere to the best of our using the API 20A biochemical kits (Identification system for knowledge. The advantage of using this strain is that it can utilize Anaerobic bacteria; BioMe'rieux; France) [28]. The activities of the several substrates and hydrolyze starch. Clostridium SP17eB1 was following enzymes were tested: alkaline phosphatase, esterase (C characterized on the basis of its phenotypic, chemotaxonomic, and 4), esterase lipase (C 8), lipase (C 14), leucine arylamidase, valine genotypic characteristics and then used to produce succinic acid arylamidase, cystine arylamidase, trypsin, a-chymotrypsin, acid from hevea wood waste hydrolysate (HH). To detoxify HH, a com- phosphatase, naphthol-AS-BI-phosphohydrolase, a-galactosidase, bination of neutralization and AC was studied. To date, a few studies b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N- have reported about succinic acid production using Clostridium sp. acetyl-b-glucosaminidase, a-mannosidase, and a-fucosidase. This strain can utilize abundant agriculture residues as carbon Enzyme activities were determined after anaerobic growth on TSB sources to increase the added value of widely available biomass. at 37 �C for 48 h using the API ZYM systems (System for the Moreover, Clostridium SP17eB1 can produce succinic acid using a research of enzymatic activity; BioMe'rieux; France) [28] according lignocellulosic material as a carbon source, thereby being cost- to the manufacturer's instructions. effective and providing added value to residual agricultural wastes. Therefore, the present study focused on this strain and 2.2.2. Chemotaxonomic characterization taxonomically characterized it on the basis of phenotypic, chemo- Bacterial strains were cultivated in Reinforced Clostridial me- taxonomic, and genotypic characteristics. dium (RCM) broth on a rotary shaker at 200 rpm at 37 �C for 48 h under anaerobic conditions. The guanineecytosine (G C) content þ

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2. Materials and methods of DNA was determined by HPLC [29]. DNAeDNA hybridization of 2.5. Detoxification of hevea wood waste hydrolysate Nomenclature strains was performed as described by Ezaki, Hashimoto and 2.1. Isolation, cultivation, and bacterial strains Yabuuchi [30]. Cellular fatty acids were analyzed using gas chro- To determine the optimal conditions for detoxification, we AC Activated carbon matography according to the Microbial Identification System investigated different ratios of AC:HH (1:2.5, 1:5, and 1:10) and HH Hevea wood waste hydrolysate Strain SP17eB1 was isolated from dog saliva in the Suphanburi [Sherlock Microbial Identification System (MIDI)] [31]. different contact times (0, 5, and 10 min). The reaction mixtures HPLC High-performance liquid chromatography province, Thailand. The bacteria were first screened by inoculating were stirred at 100 rpm for the specified time period at 30 �C. The RCM Reinforced Clostridial medium the sample into enrichment medium and then by transferring the 2.2.3. Genotypic characterization detoxified HH was recovered using vacuum filtration and was then SPSS Statistical Package for the Social Sciences enrichment medium to a screening plate. Positive isolates exhibited Bacterial strains were anaerobically cultivated in RCM broth as centrifuged and filtered through 0.45 mm filter papers before TCA Tricarboxylic acid clear zones on the screening plate. The selected isolates were described above. Bacterial cells were harvested by refrigerated analyzing for total reducing sugars and inhibitors by the DNS TSA Tryptic Soy Agar analyzed for succinic acid production qualitatively and quantita- centrifugation at 12,000 g (model 6500, Kubota, Japan). Wet method [39] and HPLC, respectively. � TSB Tryptic soy broth tively by thin layer chromatography and high-performance liquid biomass was washed with a solution comprising 0.15 M NaCl and chromatography (HPLC), respectively. The details of the screening 0.1 M EDTA (pH 8.0) and transferred to a fresh buffer solution. Egg 2.6. Effect of different types of AC on reducing the levels of inhibitors process and medium composition are described by Phuengjayaem, white lysozyme (Sigma, Germany) was added (10 mg/mL) to digest Phinkian, Tanasupawat and Teeradakorn [22]. The type strains of the bacterial cell wall and was then incubated at 37 �C for 2 h, fol- We investigated the effects of AC powder and granular AC on reportedly produce hydrogen, ethanol, and organic acids from closely related Clostridium species, including C. amygdalinum DSM lowed by the addition of 1% SDS and buffer B (0.15 M Tris-HCl). DNA reducing the levels of the inhibitors in HH. AC powder was mixed cellulosic biomass and transform cinnamic acid to 3- 12857T, C. celerecrescens DSM 5628T, and C. saccharolyticum DSM was precipitated by adding absolute ethanol, followed by succes- with HH at a ratio of 1:5 (w/v) and stirred at 100 rpm at 30 �C for phenylpropionic acid [13]. However, research on succinic acid T 2544 , were obtained from DSMZ (Deutsche Sammlung von Mik- sive washing with 70% ethanol and 95% ethanol. The DNA was 5 min. The detoxified HH was recovered and then analyzed as production using Clostridium strains has been neglected, and to the roorganismen und Zellkulturen, Braunschweig, Germany). All dissolved in 0.1 SSC and then stored at 4 C. Further purification � � described above. best of our knowledge, succinic acid has only been reported in bacterial strains were maintained in tryptic soy agar (TSA) (Difco, was conducted according to the method of Saito and Miura [32]. C. coccoides [14] and C. thermosuccinogenes [9] to date. Clostridium France) at 37 C. Anaerobic conditions were maintained using an The 16S rRNA gene was sequenced using PCR with two primers � 2.7. Effect of substrate concentrations on cell growth and succinic coccoides can convert glucose (5 g/L) to acetate (3.0 g/L), succinate Anaero Pack (Mitsubishi Gas Chemical, Japan). Cultures were grown (20F: 5 -AGTTTGATCCTGGCTC-3 and 1530R: 5 -AAGGAGGTGATC- 0 0 0 acid production (0.57 g/L), and lactate (0.58 g/L) after 24 h, whereas for 24e48 h prior to further study. CAGCC-30). The PCR products were sequenced using Macrogen and C. thermosuccinogenes can convert various carbohydrates to succi- universal primers [33]. The 16S rRNA gene sequences were Different concentrations (20e40 g/L) of HH and detoxified HH nate and acetate as major products [9]. These findings suggest that 2.2. Characterization of the strain SP17eB1 analyzed using the BioEdit software version 7.0.2 (http://www. were compared with those of glucose. The substrates were the genus Clostridium is an attractive source of succinic acid mbio.ncsu.edu/BioEdit/bioedit.html), and BLAST analysis was per- substituted for glucose in the production medium. The cells were producers. 2.2.1. Phenotypic characterization formed using the EzTaxon database [34]. A phylogenetic tree was cultivated under anaerobic conditions at 37 C with shaking at Para rubber (Hevea brasiliensis) is an abundant and important The colonies on TSA plates were characterized on the basis of constructed using the neighbor-joining method and the MEGA � 200 rpm for 48 h. Samples were collected every 12 h and centri- economic crop in Thailand [15]. Hevea is a softwood tree with a color, shape, margin, optical property, and elevation after growing program version 6 [35]. Bootstrap analysis of Felsenstein [36] was fuged at 10,000 rpm for 5 min. The pellet was re-suspended and high cellulose content (53.01%) [16]. Softwoods have higher cellu- under anaerobic conditions at 37 C for 24e48 h. Cell morphology performed with 1000 replications to determine the confidence � then analyzed for cell growth after removing the insoluble MgCO lose and lignin and lower pentosan content than hardwoods or of the isolate was determined by light microscopy (model Nikon values of individual branches in the phylogenetic tree. 3 in the sample by adding 0.2 M HCl [40], while the supernatant was angiosperms [17]. Moreover, hevea wood waste is a by-product YS2eH, Japan) and scanning electron microscopy (model JSM-5410 filtered and then analyzed for residual reducing sugars and organic from the furniture industry and can be used as an alternative LV; JEOL, Japan). For scanning electron microscopy, samples were 2.3. Inoculation and preparation of medium for succinic acid acids. substrate for succinic acid production. prepared as described by Kudo, Matsushima, Itoh, Sasaki and production Pretreatment of the waste is necessary to remove lignin and Suzuki [23]. Gram staining was performed according to standard hemicellulose and reduce cellulose crystallinity; moreover, pre- procedures [24]. Flagella staining was performed as described by The seed culture was anaerobically cultivated in 50 mL of 3% TSB 2.8. Analytical method treatment also increases the porosity of materials [18]. Diluted-acid Forbes [25]. The effects of temperature (20 �C, 30 �C, 37 �C, 40 �C, at 37 �C with shaking at 200 rpm for 24 h. Subsequently, 10% of the After removing MgCO , cell growth was estimated using a pretreatment is a simple and widely used process. However, it is 50 �C, and 60 �C) and pH (5.0, 6.0, 7.0, 8.0, and 9.0) on growth were culture was inoculated into the production medium and then 3 spectrophotometer at 660 nm (UV160, Shimadzu Corporation) as conducted under severe conditions that produce inhibitors in the determined in tryptic soy broth (TSB; Difco, France) medium. cultivated under anaerobic conditions at 37 �C with shaking at described by Phuengjayaem, Phinkian, Tanasupawat and Teer- solution. These include furfural and 5-hydroxymethylfurfural Catalase was tested by exposing the cell material to 10% H2O2 [26]. 200 rpm. Fermentation was conducted in a 250-mL flask with (HMF), which are produced by the degradation of pentoses and The biochemical characteristics were examined on the basis of 50 mL of production medium comprising (g/L) yeast extract, 30; adakorn [22]. The inhibitors in HH such as furfural and HMF were . detected by HPLC (HPLC, Varian Prostar) using the following pa- hexoses, respectively [19]. To use the hydrolysate from waste more arginine hydrolysis, nitrate reduction, starch hydrolysis, and slime urea, 2.0; MgCl26H2O, 2.0; CaCl2, 1.5; MnCl2, 0.07; Na2HPO4, 4.4; fi fi rameters: UV-VIS detector system, set at 285 nm, 12% (v/v) meth- ef ciently, it must be detoxi ed. For example, overliming with formation, as described by Tanasupawat et al. [27]. Acid production NaH2PO4, 3.3; and MgCO3, 30, and the pH was adjusted to 7 [37]. w/v Ca(OH)2 is used to chemically modify inhibitors in the hydrolysate was tested on the following carbohydrates: L-arabinose, D-cello- Membrane-filtered (0.22 mm, Millipore Express, Ireland) biotin anol in 1% ( ) acetic acid as a mobile phase, and a Varian C18 size to reduce toxicity, but the resulting solution may have high pH and 250 4.6 mm column at flow rate 1.0 mL/min. The soluble reducing biose, D-glucose, glycerol, D-lactose, D-maltose, D-mannitol, D- (0.3 mg/L) and thiamin (0.2 mg/L) were also added to the medium. � sugars in the initial substrate (HH) and the residual reducing sugars temperature [20]. Hodge et al. (2009) have reported that the use of mannose, D-melezitose, D-raffinose, L-rhamnose, salicin, D-sorbitol, Glucose was separately sterilized at 115 �C for 20 min and then fi activated carbon (AC) for the detoxi cation of softwood dilute acid sucrose, D-trehalose, and D-xylose. Gelatin hydrolysis (bovine added to the medium at a concentration of 60 g/L. remaining in the culture medium were determined by the DNS hydrolysates is convenient, fast, and cost-effective [21]. origin), esculin hydrolysis (with ferric citrate) and urease were method [39]. Subsequently, monosaccharides such as glucose, galactose, mannose, xylose, and arabinose were analyzed by HPLC, In the present study, we isolated Clostridium species from dog analyzed after growth under anaerobic conditions at 37 �C for 48 h 2.4. Hevea wood waste hydrolysate saliva, which has not been reported elsewhere to the best of our using the API 20A biochemical kits (Identification system for as described by Poonsrisawat, Phuengjayaem, Petsom and Teer- knowledge. The advantage of using this strain is that it can utilize Anaerobic bacteria; BioMe'rieux; France) [28]. The activities of the Hevea wood waste (Hevea brasiliensis) was supplied by Panel adakorn [38]. The organic acids (succinic, lactic, formic, and acetic several substrates and hydrolyze starch. Clostridium SP17eB1 was following enzymes were tested: alkaline phosphatase, esterase (C Plus Company (Ban Khu Nai Sang, Songkhla, Thailand). The major acid) were detected by HPLC, as described by Phuengjayaem and characterized on the basis of its phenotypic, chemotaxonomic, and 4), esterase lipase (C 8), lipase (C 14), leucine arylamidase, valine compositions of hevea wood waste were as follows: cellulose Teeradakorn [41]. genotypic characteristics and then used to produce succinic acid arylamidase, cystine arylamidase, trypsin, a-chymotrypsin, acid 44.87%; hemicellulose14.51%, and lignin 13.12%. Hevea wood waste from hevea wood waste hydrolysate (HH). To detoxify HH, a com- phosphatase, naphthol-AS-BI-phosphohydrolase, a-galactosidase, was chopped and milled in a hammer mill to a size that passed 2.9. Statistical analysis bination of neutralization and AC was studied. To date, a few studies b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N- through a 100-mesh sieve (100 mm) using an in-house saccharifi- have reported about succinic acid production using Clostridium sp. acetyl-b-glucosaminidase, a-mannosidase, and a-fucosidase. cation reactor [38]. The milled hevea wood waste was mixed (10%) All the experiments evaluating the effects of treatment on in- This strain can utilize abundant agriculture residues as carbon Enzyme activities were determined after anaerobic growth on TSB in a solution comprising 3% (v/v)H2SO4 and was then incubated at hibitors, cell growth, and succinic acid production were performed sources to increase the added value of widely available biomass. at 37 �C for 48 h using the API ZYM systems (System for the 170 �C for 30 min. The mixture was neutralized with 40% NaOH, in triplicates and analyzed using ANOVA, followed by Duncan's new Moreover, Clostridium SP17eB1 can produce succinic acid using a research of enzymatic activity; BioMe'rieux; France) [28] according centrifuged, and filtered through a Whatman No. 1 filter paper. The multiple range tests to evaluate the differences among means lignocellulosic material as a carbon source, thereby being cost- to the manufacturer's instructions. pH of the hydrolysate was adjusted to 7 prior to its use in the based on the type of AC, the ratio of AC to HH, and contact time for effective and providing added value to residual agricultural fermentation process. The supernatant was called “hevea wood detoxification. The same tests were used to analyze the effects of wastes. Therefore, the present study focused on this strain and 2.2.2. Chemotaxonomic characterization waste hydrolysate (HH).” HH was evaporated to a Brix value within the type and concentration of substrate and cultivation time on taxonomically characterized it on the basis of phenotypic, chemo- Bacterial strains were cultivated in Reinforced Clostridial me- 25%e30%, then analyzed for monosugars, furfural, and 5- succinic acid production. Statistical analyses were performed using taxonomic, and genotypic characteristics. dium (RCM) broth on a rotary shaker at 200 rpm at 37 �C for 48 h hydroxymethyl furfural by HPLC, and stored at 4 �C for later use the Statistical Package for the Social Sciences program (SPSS) under anaerobic conditions. The guanineecytosine (G C) content as a substrate for succinic acid production [38]. version 15. þ

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[43]. All the strains are negative for catalase test. Only strain SP17eB1 is positive for arginine hydrolysis. Both the strains SP17eB1 and C. amygdalinum DSM 12857T can hydrolyze starch. All the strains showed positive reaction for acid phosphatase, alkaline phosphatase, esterase (C 4), a-glucosidase, and naphthol-AS-BI- phosphohydrolase but negative for cystine arylamidase, a-fucosidase, and a-mannosidase. The differential characteristics are described in Table 1. The G C content of strain SP17eB1 was 40.8 mol%, which is þ characteristic of the species within the genus Clostridium (Table 1). The main cellular fatty acid components of SP17eB1 are C12:0,C14:0, C16:0,C17:0 cyclo, C18:0,C19:0 cyclo u8c,C16:1 u9c, and C18:1 u9c, which comprise 3.2%, 8.2%, 36.3%, 9.7%, 2.2%, 3.4%, 13.9%, and 10.7% of the total fatty acid components, respectively. All the type strains showed the same major fatty acid profiles as follows for C12:0,C14:0, C16:0,C18:0, and C18:1 u9c as well as the same minor fatty acid component in all species, including C15:0 anteiso, C17:0,C17:0 anteiso, C15:1 u8c, and iso-C16:0. Summed feature 1 (iso-C15:1 H/C13:0 3OH or Fig. 1. Scanning electron micrograph of Clostridium SP17eB1 (Bar, 1.0 mm) which iso- C13:0 3OH/iso-C15:1 H) and summed feature 7 (C19:1 u7c/C19:1 u6c or lated from dog saliva in the Suphanburi province, Thailand, and cultivated on TSA plate C19:1 u6c/ u7c/19cy) comprised less than 2% of the total fatty acids. at 37 �C under the anaerobic condition. The major fatty acid profile of strain SP17eB1 is closely related to that of C. amygdalinum DSM 12857T. However, differences in the relative amounts of particular fatty acids were observed; for 3. Results and discussion example, C17:0 cyclo (9.7%) and C16:1 u9c (13.9%) were 10-fold higher in SP17eB1 than in C. amygdalinum DSM 12857T. 3.1. Identification Conversely, C17:0 2OH summed in features 3 and 8 were not detected in the strain SP17eB1 but were present at high levels in Strain SP17eB1 is a rod-shaped (0.3e0.7 2.9e3.5 mm) bacte- � C. amygdalinum DSM 12857T (9.7% and 14.7%, respectively). The rium with terminal oval endospores (Fig. 1); its colonies are cream- results of the fatty acid profile places SP17eB1 and the three type colored, convex, and opaque with entire margins and 1e2 mm in strains within the genus Clostridium. In addition, on the basis of the diameter. Its cells are motile by peritrichous flagella. This strain is BLAST analysis of the 16S rRNA gene sequence, strain SP17eB1 positive for aesculin hydrolysis, nitrate reduction, urease, and it (1398 bp) was found to be closely related to C. amygdalinum BR-10T produces acid from L-arabinose, D-cellobiose, D-glucose, D-lactose, (97.8%), C. saccharolyticum WM1T (97.8%), and C. celleracrescens D-maltose, D-mannitol, D-mannose, salicin, D-sucrose, D-trehalose, T T DSM 5628 (97.7%). The phylogenetic tree based on 16S rRNA gene and D-xylose, similar to C. amygdalinum DSM 12857 [10], sequences using the neighbor-joining method (Fig. 2) showed that C. celerecrescens DSM 5628T [42], and C. saccharolyticum DSM 2544T

Table 1 Characteristics of strain Clostridium SP17eB1 and closely related Clostridium species.

Characteristics 1234

Cell form Rods Rods Rods Rods Starch hydrolysis ee þþ Indole formation e e þ þ Gelatin hydrolysis eee þ pHa 5.0e8.0 (7.0) 6.5e8.0 (7.0) 6.0e8.8 (7.5) 7e8 (7.0) b Temperature (�C) 30e40 (37) 20e60 (45) 17e43 (37) 30e37 (37) Utilization of: Glycerol ee þ þ D-Melezitose e þþ þ D-Rafﬁnose e e þ þ L-Rhamnose e þþþ D-Sorbitol e þ þþ API ZYM: N-acetyl-b-Glucosaminidase e (w) (w) e þ þ a-Chymotrypsin e (w) e þ þ Esterase lipase (C 8) (w) ee þþ þ a-Galactosidase eee þþ b-Galactosidase eee þþ b-Glucosidase ee þþ þþ b-Glucuronidase e ee þ Leucine arylamidase e (w) (w) þþ þ þ Lipase (C 14) ee (w) e þ Trypsin (w) e (w) þþ þ DNA G C content (mol%) 40.8 32 45 43.9 þ Strains: 1, Clostridium SP17eB1; 2, C. amygdalinum DSM 12857T; 3, C. saccharolyticum DSM 2544T; 4, C. celerecrescens DSM 5628T All data are obtained from this study. , Positive reaction; w, weak reaction; , negative reaction. þ � a pH expressed as: minimumemaximum (pH optimum). b , temperature expressed as: minimumemaximum (optimum temperature).

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[43]. All the strains are negative for catalase test. Only strain SP17eB1 is positive for arginine hydrolysis. Both the strains SP17eB1 and C. amygdalinum DSM 12857T can hydrolyze starch. All the strains showed positive reaction for acid phosphatase, alkaline phosphatase, esterase (C 4), a-glucosidase, and naphthol-AS-BI- phosphohydrolase but negative for cystine arylamidase, a-fucosidase, and a-mannosidase. The differential characteristics are described in Table 1. The G C content of strain SP17eB1 was 40.8 mol%, which is þ characteristic of the species within the genus Clostridium (Table 1). The main cellular fatty acid components of SP17eB1 are C12:0,C14:0, C16:0,C17:0 cyclo, C18:0,C19:0 cyclo u8c,C16:1 u9c, and C18:1 u9c, which comprise 3.2%, 8.2%, 36.3%, 9.7%, 2.2%, 3.4%, 13.9%, and 10.7% of the total fatty acid components, respectively. All the type strains showed the same major fatty acid profiles as follows for C12:0,C14:0, C16:0,C18:0, and C18:1 u9c as well as the same minor fatty acid component in all species, including C15:0 anteiso, C17:0,C17:0 anteiso, C15:1 u8c, and iso-C16:0. Summed feature 1 (iso-C15:1 H/C13:0 3OH or Fig. 1. Scanning electron micrograph of Clostridium SP17eB1 (Bar, 1.0 mm) which iso- C13:0 3OH/iso-C15:1 H) and summed feature 7 (C19:1 u7c/C19:1 u6c or lated from dog saliva in the Suphanburi province, Thailand, and cultivated on TSA plate C19:1 u6c/ u7c/19cy) comprised less than 2% of the total fatty acids. at 37 �C under the anaerobic condition. The major fatty acid profile of strain SP17eB1 is closely related to that of C. amygdalinum DSM 12857T. However, differences in the relative amounts of particular fatty acids were observed; for 3. Results and discussion example, C17:0 cyclo (9.7%) and C16:1 u9c (13.9%) were 10-fold higher in SP17eB1 than in C. amygdalinum DSM 12857T. 3.1. Identification Conversely, C17:0 2OH summed in features 3 and 8 were not detected in the strain SP17eB1 but were present at high levels in Strain SP17eB1 is a rod-shaped (0.3e0.7 2.9e3.5 mm) bacte- � C. amygdalinum DSM 12857T (9.7% and 14.7%, respectively). The rium with terminal oval endospores (Fig. 1); its colonies are cream- results of the fatty acid profile places SP17eB1 and the three type colored, convex, and opaque with entire margins and 1e2 mm in strains within the genus Clostridium. In addition, on the basis of the Fig. 2. Neighbor-joining phylogenetic tree on the basis of16S rRNA gene sequences. Bootstrap values are expressed as percentages of 1000 replications. diameter. Its cells are motile by peritrichous flagella. This strain is BLAST analysis of the 16S rRNA gene sequence, strain SP17eB1 positive for aesculin hydrolysis, nitrate reduction, urease, and it (1398 bp) was found to be closely related to C. amygdalinum BR-10T produces acid from L-arabinose, D-cellobiose, D-glucose, D-lactose, (97.8%), C. saccharolyticum WM1T (97.8%), and C. celleracrescens strain SP17eB1 formed a tight cluster with the type strains of the SP17eB1 is a distinct species within the genus Clostridium. Even- D-maltose, D-mannitol, D-mannose, salicin, D-sucrose, D-trehalose, T e e T DSM 5628 (97.7%). The phylogenetic tree based on 16S rRNA gene genus Clostridium. The DNA of strain SP17 B1 is 36.1%, 40.7%, and tually, strain SP17 B1 may be considered a novel species on the and D-xylose, similar to C. amygdalinum DSM 12857 [10], T T T sequences using the neighbor-joining method (Fig. 2) showed that 35.8% related to those of C. amygdalinum DSM 12857 , basis of the phenotypic, chemotaxonomic, and genotypic charac- C. celerecrescens DSM 5628 [42], and C. saccharolyticum DSM 2544 C. saccharolyticum DSM 2544T, and C. celerecrescens DSM 5628T, teristics described above. Furthermore, to the best of our knowl- respectively. These values are lower than the 70% cut-off value for edge, Clostridium SP17eB1 has not been reported elsewhere. The assigning strains to the same species [44], indicating that strain distinct characteristic of this strain on the basis of phenotypic study Table 1 Characteristics of strain Clostridium SP17eB1 and closely related Clostridium species.

Characteristics 1234

Cell form Rods Rods Rods Rods Starch hydrolysis ee þþ Indole formation e e þ þ Gelatin hydrolysis eee þ pHa 5.0e8.0 (7.0) 6.5e8.0 (7.0) 6.0e8.8 (7.5) 7e8 (7.0) b Temperature (�C) 30e40 (37) 20e60 (45) 17e43 (37) 30e37 (37) Utilization of: Glycerol ee þ þ D-Melezitose e þþ þ D-Rafﬁnose e e þ þ L-Rhamnose e þþþ D-Sorbitol e þ þþ API ZYM: N-acetyl-b-Glucosaminidase e (w) (w) e þ þ a-Chymotrypsin e (w) e þ þ Esterase lipase (C 8) (w) ee þþ þ a-Galactosidase eee þþ b-Galactosidase eee þþ b-Glucosidase ee þþ þþ b-Glucuronidase e ee þ Leucine arylamidase e (w) (w) þþ þ þ Lipase (C 14) ee (w) e þ Trypsin (w) e (w) þþ þ DNA G C content (mol%) 40.8 32 45 43.9 þ Strains: 1, Clostridium SP17eB1; 2, C. amygdalinum DSM 12857T; 3, C. saccharolyticum DSM 2544T; 4, C. celerecrescens DSM 5628T All data are obtained from this study. , Positive reaction; w, weak reaction; , negative reaction. þ � a pH expressed as: minimumemaximum (pH optimum). Fig. 3. Detoxiﬁcation of hevea wood waste hydrolysate using granular and powder AC with different AC:HH ratios (1:2.5, 1:5, and 1:10) and different contact times ranging from 0 to b , temperature expressed as: minimumemaximum (optimum temperature). 10 min. The reaction mixtures were stirred at 100 rpm at 30 C. All results are mean of triplicates with error bar as standard deviation.

332 333 6 S. Phuengjayaem et al. / Anaerobe 61 (2020) 102096 is that it can utilize several substrates and hydrolyze starch.

3.2. Detoxiﬁcation of hevea wood waste hydrolysate

The detoxification of hevea wood waste hydrolysate was analyzed using granular and powder AC with different ratios of AC:HH and contact times ranging from 0 to 10 min. The results showed that the adsorption process occurred rapidly with all type of AC. The removal of furfural and HMF was appropriate within 5 min but not distinct when the contact time exceeded 5 min. Therefore, in the subsequent analysis, the contact time of 5 min was used for detoxification (Fig. 3AeD). The results of comparing the ratios of granular AC:HH at 1:2.5, 1:5, and 1:10 for a contact time of 5 min indicated that the removal of furfural was determined to be 51.6%, 79.1%, and 100% at granular AC:HH ratios of 1 2.5, 1:5, and 1:10, respectively (Fig. 3A). The removal of HMF was determined to be 62.2%, 91.8%, and 99.6% at granular AC:HH ratios of 1: 2.5, 1:5, and 1:10, respectively (Fig. 3B). On comparing the detoxification using powder AC or granular AC at same ratio and condition, the removal of furfural was determined to be 25.6%, 86.1%, and 98.2% at powder AC:HH ratios of 1:2.5, 1:5 and 1:10, respectively (Fig. 3C). The removal of HMF was determined to be 62.5%, 95.2%, and 100% at powder AC:HH ratios of 1:2.5, 1:5 and 1:10, respectively (Fig. 3D). Results from both type of AC showed the same phenomenon of detoxification process and suggested that AC:HH ratios of 1:5 and 1:10 are more effective in removing the inhibitors than AC: HH ratio of 1:2.5. For economic concerns, the AC:HH ratio of 1: 5 was considered suitable for detoxification process.

3.3. Effects of different types of AC on reducing the level of inhibitors

Saccharified HH was detoxified by AC at an AC:HH ratio of 1:5. This ratio is determined to be optimum for reducing the levels of inhibitors such as furfural and HMF. We also optimized the contact time for detoxification. Results from above study (section 3.2) have showed that contact times greater than 5 min did not affect the concentrations of furfural and HMF. Then, we used statistical analysis (Duncan's new multiple range tests) to evaluate the effects of contact times of 0e5 min on detoxification. The results showed that the contact time of 5 min almost completely reduced the levels of inhibitors in HH. Granular AC, when used to detoxify HH, can remove furfural and HMF of 87.3% and 94.9%, respectively, whereas powder AC can remove furfural and HMF of 100% and 99.9%, respectively, as shown in Fig. 4A and B. Fig. 4. Effect of different types of activated carbon used to reduce the levels of inhibitors. The reaction mixture comprised a granular AC:HH ratio of 1:5, stirred at On comparing the effects of granular and powder AC on 100 rpm at 30 C. All results are mean of triplicates with error bar as standard detoxification, the results showed that granular AC is more effective deviation. in reducing the level of inhibitors because granular AC did not affect the monosugar concentrations in the HH (27.7% removal) (Fig. 4C). This result is supported by the findings of Gonzales, Hong, Park, have previously reported that the optimum conditions for detoxi- Kumar and Kim [45], who studied the mechanism of HMF fying sugarcane bagasse hydrolysate are 1.0% AC for 30 min at 60 C, adsorption on granular AC in algae hydrolysates. They reported that with shaking at 100 rpm and at pH 2.5. In both these studies the adsorption of inhibitors into granular AC prevented the in- [46,47], although low amounts of AC were used, the contact time hibitors from interfering with the sugar concentration in hydroly- used was longer than that used in our present study, thus sug- sates. The optimum condition for HH detoxification was the gesting that these factors (amount of AC and contact time), as well granular AC:HH ratio of 1:5 (20% w/v), with stirring at 100 rpm for as the composition of lignocellulose, affect the detoxification pro- 5 min at 30 C. Under these conditions (5 min of contact time), cess. Thus, optimized conditions may also differ depending on the furfural (79 mg/L) and HMF (13 mg/L) were completely removed. type of lignocellulosic material used. This contact time was effective in reducing the amount of by- Furthermore, to reduce the toxification of inhibitors in the products in HH while not affecting the monosugar concentrations present study, we used the adsorption technique, which is conve- (Fig. 4). Other research has reported that the optimum conditions nient and cost-effective. AC was used as a sorbent due to its high for detoxifying sago trunk hydrolysate are 2.5% AC and a contact sorption capacity, which depends on the total surface area of the time of 60 min; these conditions enabled a 58%-reduction of AC. furfural levels [46]. However, Marton, Felipe, Silva and Pessoa [47]

334 335 6 S. Phuengjayaem et al. / Anaerobe 61 (2020) 102096 S. Phuengjayaem et al. / Anaerobe 61 (2020) 102096 7 is that it can utilize several substrates and hydrolyze starch. Table 2 Effect of substrate concentrations on cell growth and succinic acid production of Clostridium SP17eB1 after 24 h.

3.2. Detoxiﬁcation of hevea wood waste hydrolysate Substrate Cultivation time (h) Cell growth (OD660) Reducing sugars (g/L) Succinic acid (g/L)

Substrate concentrations (g/L) The detoxification of hevea wood waste hydrolysate was 20A 30C 40B 20 30 40 20D 30F 40E analyzed using granular and powder AC with different ratios of AC:HH and contact times ranging from 0 to 10 min. The results Glucose 0 0.1 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 18.7 ± 3.2 27.1 ± 0.6 35.9 ± 3.5 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 3 showed that the adsorption process occurred rapidly with all type (control) 12 0.2 ± 0.1 0.4 ± 0.0 0.3 ± 0.1 4.7 ± 0.6 5.9 ± 1.6 5.1 ± 0.7 10.2 ± 0.5 9.1 ± 0.5 9.9 ± 0.5 24 2.3 ± 0.0 4.7 ± 0.2 4.1 ± 0.1 1.4 ± 0.1 1.3 ± 0.1 2.0 ± 0.2 12.9 ± 0.6 18.5 ± 0.9 25.1 ± 1.3 of AC. The removal of furfural and HMF was appropriate within 36 2.9 ± 0.2 7.2 ± 0.1 4.4 ± 0.0 0.2 ± 0.1 0.2 ± 0.0 0.3 ± 0.0 14.5 ± 0.7 18.6 ± 0.9 23.4 ± 1.2 5 min but not distinct when the contact time exceeded 5 min. 48 3.0 ± 0.1 4.9 ± 0.1 6.9 ± 0.1 0.1 ± 0.0 0.1 ± 0.0 0.5 ± 0.1 13.7 ± 0.7 19.1 ± 1.0 24.4 ± 1.2 Therefore, in the subsequent analysis, the contact time of 5 min was HH1 0 0.1 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 19.9 ± 1.2 27.9 ± 0.6 37.6 ± 3.2 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 used for detoxification (Fig. 3AeD). 12 0.1 ± 0.0 0.4 ± 0.0 0.0 ± 0.0 3.7 ± 0.3 4.5 ± 3.4 10.6 ± 1.6 8.1 ± 0.8 12.3 ± 1.2 8.3 ± 0.8 24 0.1 ± 0.0 0.4 ± 0.0 0.2 ± 0.0 3.2 ± 0.7 6.2 ± 0.4 10.2 ± 1.4 7.6 ± 0.8 15.3 ± 1.5 8.6 ± 0.9 The results of comparing the ratios of granular AC:HH at 1:2.5, 36 0.0 ± 0.0 0.0 ± 0.0 0.1 ± 0.0 3.7 ± 0.4 6.6 ± 0.1 8.9 ± 0.3 7.5 ± 0.8 10.5 ± 1.1 9.6 ± 1.0 1:5, and 1:10 for a contact time of 5 min indicated that the removal 48 0.0 ± 0.0 0.0 ± 0.0 0.1 ± 0.0 3.7 ± 0.1 3.4 ± 0.1 7.4 ± 0.6 4.6 ± 0.5 4.8 ± 0.5 5.8 ± 0.6 of furfural was determined to be 51.6%, 79.1%, and 100% at granular Detoxified HH2 0 0.1 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 13.8 ± 0.3 25.6 ± 1.0 36.4 ± 4.4 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 AC:HH ratios of 1 2.5, 1:5, and 1:10, respectively (Fig. 3A). The 12 1.9 ± 0.1 3.0 ± 0.3 0.9 ± 0.0 1.8 ± 0.1 5.6 ± 0.1 28.4 ± 1.1 7.3 ± 0.0 9.3 ± 0.1 7.5 ± 0.1 removal of HMF was determined to be 62.2%, 91.8%, and 99.6% at 24 1.0 ± 0.1 2.0 ± 0.2 1.5 ± 0.0 1.3 ± 0.4 4.7 ± 0.3 8.3 ± 0.5 6.5 ± 0.0 11.0 ± 0.1 8.9 ± 0.1 36 2.0 ± 1.6 1.7 ± 0.5 2.1 ± 0.1 2.2 ± 0.1 6.1 ± 0.2 2.1 ± 0.3 7.1 ± 0.0 10.5 ± 0.1 7.8 ± 0.1 granular AC:HH ratios of 1: 2.5, 1:5, and 1:10, respectively (Fig. 3B). 48 1.7 ± 0.0 3.3 ± 0.1 3.8 ± 0.1 2.4 ± 0.3 12.6 ± 1.0 17.1 ± 1.0 4.0 ± 0.0 5.6 ± 0.0 4.5 ± 0.0 On comparing the detoxification using powder AC or granular AC at same ratio and condition, the removal of furfural was determined to be 25.6%, 86.1%, and 98.2% at powder AC:HH ratios of 1:2.5, 1:5 and 1:10, respectively (Fig. 3C). The removal of HMF was deter- 3.4. Effect of substrate concentrations on cell growth and succinic acid production in strain SP17eB1. Using the detoxified HH as a mined to be 62.5%, 95.2%, and 100% at powder AC:HH ratios of acid production substrate reduced the lag phase of growth and produced succinic 1:2.5, 1:5 and 1:10, respectively (Fig. 3D). Results from both type of acid within 12 h (Table 2). Statistical analysis using Duncan's new fi AC showed the same phenomenon of detoxi cation process and Strain SP17eB1 is an obligately bacterium that can grow well multiple range tests at a P value of 0.05 differentiated 5 groups of suggested that AC:HH ratios of 1:5 and 1:10 are more effective in under anaerobic conditions and survive under limited aerated succinic acid production from detoxified HH on the basis of culti- removing the inhibitors than AC: HH ratio of 1:2.5. For economic conditions. When Clostridium SP17eB1 was cultured under anaer- vation time. The best yield of succinic acid production (8.8 g/L) was concerns, the AC:HH ratio of 1: 5 was considered suitable for obic conditions for 24 h, it produced a maximum amount of suc- obtained from the 24 h group (Supplementary data). Although fi detoxi cation process. cinic acid of 25.1 g/L from 40 g/L of glucose, as well as small detoxified HH did not enhance succinic acid production, it pro- amounts of formic (9.8 g/L) and acetic acids (4.2 g/L) (Table 2). moted a higher level of cell growth (Table 2). Because succinic acid 3.3. Effects of different types of AC on reducing the level of Lower glucose concentrations of 20 and 30 g/L produced maximum is an intermediate in the TCA cycle as well as a fermentative end- inhibitors succinic acid levels of 12.9 and 18.5 g/L, respectively (Table 2). product of anaerobic metabolism [6], its synthesis is growth- Similarly, glucose was completely consumed in 24 h (Table 2), but a associated. Therefore, further studies are warranted to investigate Saccharified HH was detoxified by AC at an AC:HH ratio of 1:5. maximum cell growth (OD660) of 2.9, 7.2, and 4.4 were obtained the relationship between cell growth and succinic acid production This ratio is determined to be optimum for reducing the levels of from 20, 30, and 40 g/L of glucose, respectively, after 36 h of culti- in strain SP17eB1. inhibitors such as furfural and HMF. We also optimized the contact vation (Supplementary data). Increasing the cultivation time from Zhang, Xia, Lu, and Tu [49] have previously reported that in fi time for detoxi cation. Results from above study (section 3.2) have 24 to 48 h produced no significantly different effects on acid pro- poplar prehydrolysates, overliming efficiently reduced the amounts showed that contact times greater than 5 min did not affect the duction (Supplementary data). In the present study, Clostridium of 2,5-furandicarboxyaldehyde, 5-ethylfuran-2-carbaldehyde, and concentrations of furfural and HMF. Then, we used statistical SP17eB1 produced a succinic acid yield of 25.1 g product/40 g 2,5-hexanedione, whereas AC effectively reduces the amounts of analysis (Duncan's new multiple range tests) to evaluate the effects glucose (62.3%). Gokarn et al. (1997) [14] have reported that phenolic acids. The combination of overliming and AC results in fi of contact times of 0e5 min on detoxi cation. The results showed C. coccoides not only produced a succinic acid yield of 0.57 g outstanding fermentation capability and butanol yields of up to that the contact time of 5 min almost completely reduced the levels product/5 g glucose (11.40%) but also produced acetate and lactate 0.22 g/g substrate in C. saccharobutylicum [49]. In addition, Chen, of inhibitors in HH. Granular AC, when used to detoxify HH, can yields of 3.0 and 0.58 g/L, respectively, after 24 h. In the present Jiang, Wei, Yao, and Wu [50] have reported that the combination of remove furfural and HMF of 87.3% and 94.9%, respectively, whereas study, with HH as a carbon source at 20, 30, and 40 g/L, maximum CaCO3 neutralization and AC treatment is effective in removing powder AC can remove furfural and HMF of 100% and 99.9%, levels of succinic acid of 7.6, 15.3 g/L, and 8.6, respectively, were inhibitors, particularly furfural (91.9% removed; from 206.1 g/L to Fig. 4. Effect of different types of activated carbon used to reduce the levels of in- respectively, as shown in Fig. 4A and B. fi hibitors. The reaction mixture comprised a granular AC:HH ratio of 1:5, stirred at achieved (Table 2). The highest succinic acid yield of 15.3 g product/ 24 mg/L of furfural), in acid hydrolysates of corn ber. This resulted On comparing the effects of granular and powder AC on 100 rpm at 30 C. All results are mean of triplicates with error bar as standard 30 g HH (51%) was obtained from 30 g/L of HH. However, an even in a maximum succinic acid production of 35.4 g/L, which corre- fi detoxi cation, the results showed that granular AC is more effective deviation. higher yield of reducing sugars in 40 g/L of HH resulted in a sponded to a yield of 72.5% by A. succinogenes NJ113 after 36 h of in reducing the level of inhibitors because granular AC did not affect reduction of succinic acid production (Table 2). In this case, the very cultivation. the monosugar concentrations in the HH (27.7% removal) (Fig. 4C). high concentration of HH was accompanied by high levels of by- Although the efficiency of producing succinic acid is less with fi have previously reported that the optimum conditions for detoxi- This result is supported by the ndings of Gonzales, Hong, Park, products, such as furfural and HMF. Wang and coworkers [48] strain SP17eB1 than with A. succinogenes, relatively little research fying sugarcane bagasse hydrolysate are 1.0% AC for 30 min at 60 C, Kumar and Kim [45], who studied the mechanism of HMF have reported that furfurals and HMF affect cell growth and suc- has been conducted on succinic acid production in Clostridium. At with shaking at 100 rpm and at pH 2.5. In both these studies adsorption on granular AC in algae hydrolysates. They reported that cinic acid production using engineered Escherichia coli. Both cell present, the discovery of a Clostridium strain that can produce [46,47], although low amounts of AC were used, the contact time the adsorption of inhibitors into granular AC prevented the in- growth and succinic acid production were severely inhibited by succinic acid is an attractive novelty. Clostridium SP17eB1 can used was longer than that used in our present study, thus sug- hibitors from interfering with the sugar concentration in hydroly- furfural and HMF when their concentrations were higher than produce succinic acid from glucose and lignocellulosic material fi gesting that these factors (amount of AC and contact time), as well sates. The optimum condition for HH detoxi cation was the 0.8 mg/L. Cell growth was completely inhibited when the concen- waste; moreover, it can utilize starch. Further studies must apply as the composition of lignocellulose, affect the detoxification pro- granular AC:HH ratio of 1:5 (20% w/v), with stirring at 100 rpm for tration of furfural was higher than 6.4 g/L or when the concentra- genome analysis to discover other novel species. Moreover, the cess. Thus, optimized conditions may also differ depending on the 5 min at 30 C. Under these conditions (5 min of contact time), tion of HMF was higher than 12.8 g/L. The maximum concentration relationship between the cell growth and succinic acid production type of lignocellulosic material used. furfural (79 mg/L) and HMF (13 mg/L) were completely removed. of inhibitors tolerated was 3.2 g/L; at this concentration, furfural in strain SP17eB1 should be investigated in more detail. Furthermore, to reduce the toxification of inhibitors in the This contact time was effective in reducing the amount of by- decreased cell growth by 77.8%, succinic acid production decreased present study, we used the adsorption technique, which is conve- products in HH while not affecting the monosugar concentrations by 36.1%, and HMF decreased the cell growth and succinic acid nient and cost-effective. AC was used as a sorbent due to its high (Fig. 4). Other research has reported that the optimum conditions production by 13.6% and 18.3%, respectively [48]. Therefore, the 4. Conclusions sorption capacity, which depends on the total surface area of the for detoxifying sago trunk hydrolysate are 2.5% AC and a contact detoxification process (combined neutralization and granular AC) AC. time of 60 min; these conditions enabled a 58%-reduction of was necessary to remove the by-products in HH to improve succinic Clostridium SP17eB1 is an obligate anaerobic bacterium furfural levels [46]. However, Marton, Felipe, Silva and Pessoa [47] belonging to the genus Clostridium. It has the ability to produce

334 335 8 S. Phuengjayaem et al. / Anaerobe 61 (2020) 102096 succinic acid at levels of 25.1 g/L and 11.0 g/L from 40 g/L glucose Can. J. Microbiol. 29 (1983) 342e347. and 30 g/L detoxified HH, respectively. The fermentation efficiency, [13] M. Chamkha, J.L. Garcia, M. Labat, Metabolism of cinnamic acids by some Clostridiales and emendation of the descriptions of Clostridium aerotolerans, as well as cell growth and sugar utilization, was improved when Clostridium celerecrescens and Clostridium xylanolyticum, Int. J. Syst. Evol. detoxified HH was used as a substrate. The mechanisms involved in Microbiol. 51 (2001) 2105e2111. reducing the levels of inhibitors by AC and the resulting enhanced [14] R.R. Gokarn, M.A. Eiteman, J. Sridhar, Production of succinate by anaerobic microorganisms, Acs. Sym. Ser. 666 (1997) 237e263. succinic acid production are currently under investigation. [15] J. Riyaphan, T. Phumichai, T. Neimsuwan, S. Witayakran, K. Sungsing, R. Kaveeta, C. Phumichaia, Variability in chemical and mechanical properties Conflicts of interest of Para rubber (Hevea brasiliensis) trees, Sci. Asia 41 (2015) 251e258. [16] A.M.K. Alhasan, D. Kuang, A.B. Mohammad, R.R. Sharma-Shivappa, Combined effect of nitric acid and sodium hydroxide pretreatments on enzymatic The authors declare that there are no conflicts of interest. saccharification of rubber wood (Heavea brasiliensis), Int. J. Chem. Technol. 2 (2010) 12e20. [17] R. RM, Handbook of Wood Chemistry and Wood Composites. New York, 2005. Acknowledgments [18] Y. Sun, J. Cheng, Hydrolysis of lignocellulosic materials for ethanol production: a review, Bioresour. Technol. 83 (2002) 1e11. This work was supported by Chulalongkorn University Grant, [19] D. Chiaramonti, M. Prussi, S. Ferrero, L. Oriani, P. Ottonello, P. Torre, F. Cherchi, Review of pretreatment processes for lignocellulosic ethanol production, and 2017 (grant number GB-B_60_108_61_05) and 2018 (grant number development of an innovative method, Biomass Bioenergy 46 (2012) 25e35. GB-B_61_095_61_01), Thailand. The authors would like to thank [20] A. Martinez, M.E. Rodriguez, M.L. Wells, S.W. York, J.F. Preston, L.O. Ingram, fi Enago (www.enago.com) for the English language review. Detoxi cation of dilute acid hydrolysates of lignocellulose with lime, Bio- technol. Prog. 17 (2001) 287e293. [21] D.B. Hodge, C. Andersson, K.A. Berglund, U. Rova, Detoxification requirements Appendix A. Supplementary data for bioconversion of softwood dilute acid hydrolyzates to succinic acid, Enzym. Microb. Technol. 44 (2009) 309e316. [22] S. Phuengjayaem, N. Phinkian, S. Tanasupawat, S. Teeradakorn, Diversity and Supplementary data to this article can be found online at succinic acid production of lactic acid bacteria isolated from animals, soils and https://doi.org/10.1016/j.anaerobe.2019.102096. tree barks, Res. J. Microbiol. 12 (2017) 177e186. The results are means of triplicates ± standard deviation. The [23] T. Kudo, K. Matsushima, T. Itoh, J. Sasaki, K. Suzuki, Description of four new species of the genus Kineosporia: Kineosporia succinea sp. nov., Kineosporia strain was cultivated under the anaerobic condition at 37 �C, with rhizophila sp. nov., Kineosporia mikuniensis sp. nov. and Kineosporia rhamnosa shaking at 200 rpm for 48 h. The clustering result of glucose con- sp. nov., isolated from plant samples, and amended description of the genus centrations on cell growth defined groups A, B, and C. The clus- Kineosporia, Int. J. Syst. Bacteriol. 48 Pt 4 (1998) 1245e1255. [24] G.J. Hucker, H.J. Conn, Method of gram staining, Tech. Bull. N. Y. St. Agric. Exp. tering result of glucose concentrations on succinic acid production Sta. 93 (1923) 3e37. defined groups D, E, and F. The clustering result of the different [25] L. Forbes, Rapid flagella stain, J. Clin. Microbiol. 13 (1981) 807e809. substrates on cell growth and succinic acid production defined [26] M. Gagnon, W.M. Hunting, W.B. Esselen, New method for catalase determi- e groups 1, 2, and 3. The grouping results were obtained by multiple nation, Anal. Chem. 31 (1959) 144 146. [27] S. Tanasupawat, N. Chamroensaksri, T. Kudo, T. Itoh, Identification of comparisons using a statistical program by Duncan's new multiple moderately halophilic bacteria from Thai fermented fish ( pla-ra ) and pro- range tests. posal of Virgibacillus siamensis sp. nov, J. Gen. Appl. Microbiol. 56 (2010) 369e379. [28] M. Hosny, S. Benamar, G. Durand, N. Armstrong, C. Michelle, F. Cadoret, B. La References Scola, N. Cassir, Description of Clostridium phoceensis sp. nov., a new species within the genus Clostridium, New Microbes New Infect 14 (2016) 85e92. [1] C. Thakker, Z. Burhanpurwala, G. Rastogi, Y. Shouche, D. Ranade, Isolation and [29] M. Peyret, J. Freney, H. Meugnier, J. Fleurette, Determination of G C content þ characterization of a new osmotolerant, non-virulent Klebsiella pneumoniae of DNA using high-performance liquid chromatography for the identification strain SAP for biosynthesis of succinic acid, Indian J. Exp. Biol. 44 (2006) of staphylococci and micrococci, Res. Microbiol. 140 (1989) 467e475. 142e150. [30] T. Ezaki, Y. Hashimoto, E. Yabuuchi, Fluorometric deoxyribonucleic acid- [2] J.J. Beauprez, M. De Mey, W.K. Soetaert, Microbial succinic acid production: deoxyribonucleic acid hybridization in microdilution wells as an alternative natural versus metabolic engineered producers, Process Biochem. 45 (2010) to membrane filter hybridization in which radioisotopes are used to deter- 1103e1114. mine genetic relatedness among bacterial strains, Int. J. Syst. Bacteriol. 39 [3] X.J. Chen, S.T. Jiang, Z. Zheng, L.J. Pan, S.Z. Luo, Effects of culture redox po- (1989) 224e229. tential on succinic acid production by Corynebacterium crenatum under [31] M. Athalye, W.C. Noble, D.E. Minnikin, Analysis of cellular fatty acids by gas anaerobic conditions, Process Biochem. 47 (2012) 1250e1255. chromatography as a tool in the identification of medically important [4] M. Inui, S. Murakami, S. Okino, H. Kawaguchi, A.A. Vertes, H. Yukawa, Meta- coryneform bacteria, J. Appl. Bacteriol. 58 (1985) 507e512. bolic analysis of Corynebacterium glutamicum during lactate and succinate [32] H. Saito, K.I. Miura, Preparation of transforming deoxyribonucleic acid by productions under oxygen deprivation conditions, J. Mol. Microbiol. Bio- phenol treatment, Biochim. Biophys. Acta 72 (1963) 619e629. technol. 7 (2004) 182e196. [33] D.J. Lane, 16S/23S rRNA sequencing, in: E.S.M. Goodfellow (Ed.), In Nucleic [5] H.W. Ryu, K.H. Kang, J.S. Yun, Bioconversion of fumarate to succinate using Acid Techniques in Bacterial Systematics, Wiley, Chichester, 1991, glycerol as a carbon source, Appl. Biochem. Biotechnol. 77e79 (1999), pp. 115e148. 511e220. [34] O.S. Kim, Y.J. Cho, K. Lee, S.H. Yoon, M. Kim, H. Na, S.C. Park, Y.S. Jeon, J.H. Lee, [6] H. Song, S.Y. Lee, Production of succinic acid by bacterial fermentation, Enzym. H. Yi, S. Won, Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence Microb. Technol. 39 (2006) 352e361. database with phylotypes that represent uncultured species, Int. J. Syst. Evol. [7] J.B. McKinlay, J.G. Zeikus, C. Vieille, Insights into Actinobacillus succinogenes Microbiol. 62 (2012) 716e721. fermentative metabolism in a chemically defined growth medium, Appl. En- [35] K. Tamura, G. Stecher, D. Peterson, A. Filipski, S. Kumar, MEGA6: molecular viron. Microbiol. 71 (2005) 6651e6656. evolutionary genetics analysis version 6.0, Mol. Biol. Evol. 30 (2013) [8] N.P. Nghiem, B.H. Davison, B.E. Suttle, G.R. Richardson, Production of succinic 2725e2729. acid by Anaerobiospirillum succiniciproducens, Appl. Biochem. Biotechnol. [36] J. Felsenstein, Confidence limits on phylogenies: an approach using the 63e65 (1997) 565e576. bootstrap, Evolution 39 (1985) 783e791. [9] J.G. Koendjbiharie, K. Wiersma, R. van Kranenburg, Investigating the central [37] L.Y. Liang, R.M. Liu, J.F. Ma, K.Q. Chen, M. Jiang, P. Wei, Increased production of metabolism of Clostridium thermosuccinogenes, Appl. Environ. Microbiol. 84 succinic acid in Escherichia coli by overexpression of malate dehydrogenase, (2018) e00363e18. Biotechnol. Lett. 33 (2011) 2439e2444. [10] S.N. Parshina, R. Kleerebezem, J.L.S. Sanz, G. Lettinga, A.N. Nozhevnikova, [38] A. Poonsrisawat, S. Phuengjayaem, A. Petsom, S. Teeradakorn, Conversion of N.A. Kostrikina, A.M. Lysenko, A.J. Stams, Soehngenia saccharolytica gen. nov., sweet sorghum straw to sugars by dilute acid saccharification, Sugar Technol. sp nov and Clostridium amygdalinum sp nov., two novel anaerobic, 15 (2013) 322e327. benzaldehyde-converting bacteria, Int. J. Syst. Evol. Microbiol. 53 (2003) [39] G.L. Miller, Use of dinitrosalicylic acid reagent for determination of reducing 1791e1799. sugar, Anal. Chem. 31 (1959) 426e428. [11] H.S. Jayasinghearachchi, S. Singh, P.M. Sarma, A. Aginihotri, B. Lal, Fermenta- [40] P. Zheng, J.J. Dong, Z.H. Sun, Y. Ni, L. Fang, Fermentative production of succinic tive hydrogen production by new marine Clostridium amygdalinum strain C9 acid from straw hydrolysate by Actinobacillus succinogenes, Bioresour. Tech- isolated from offshore crude oil pipeline, Int. J. Hydrogen Energy 35 (2010) nol. 100 (2009) 2425e2429. 6665e6673. [41] S. Phuengjayaem, S. Teeradakorn, Producing succinic acid with Actinobacillus [12] W.D. Murray, A.W. Khan, Ethanol production by a newly isolated anaerobe, succinogenes: optimizing the composition of the medium using Plackett- Clostridium saccharolyticum: effects of culture medium and growth conditions, Burman Design, CMU. J. Nat. Sci. 15 (2016) 253e264.

336 337 8 S. Phuengjayaem et al. / Anaerobe 61 (2020) 102096 S. Phuengjayaem et al. / Anaerobe 61 (2020) 102096 9 succinic acid at levels of 25.1 g/L and 11.0 g/L from 40 g/L glucose Can. J. Microbiol. 29 (1983) 342e347. [42] M. Palop, S. Valles, F. Pinaga, A. Flors, Isolation and characterization of an Detoxification of sago trunk hydrolysate using activated charcoal for xylitol and 30 g/L detoxified HH, respectively. The fermentation efficiency, [13] M. Chamkha, J.L. Garcia, M. Labat, Metabolism of cinnamic acids by some anaerobic, cellulolytic bacterium, Clostridium celerecrescens sp. nov, Int. J. Syst. production, Procedia Food Sci. 1 (2011) 908e913. Clostridiales and emendation of the descriptions of Clostridium aerotolerans, Bacteriol. 39 (1989) 68e71. [47] J.M. Marton, M.G.A. Felipe, J.B.A.E. Silva, A. Pessoa, Evaluation of the activated as well as cell growth and sugar utilization, was improved when Clostridium celerecrescens and Clostridium xylanolyticum, Int. J. Syst. Evol. [43] W.D. Murray, A.W. Khan, L. Van Den Berg, Clostridium saccharolyticum sp. nov., charcoals and adsorption conditions used in the treatment of sugarcane detoxified HH was used as a substrate. The mechanisms involved in Microbiol. 51 (2001) 2105e2111. a saccharolytic species from sewage sludge, Int. J. Syst. Bacteriol. 32 (1982) bagasse hydrolysate for xylitol production, Braz. J. Chem. Eng. 23 (2006) 9e21. reducing the levels of inhibitors by AC and the resulting enhanced [14] R.R. Gokarn, M.A. Eiteman, J. Sridhar, Production of succinate by anaerobic 132e135. [48] D. Wang, H. Wang, J. Wang, N. Wang, J. Zhang, J. Xing, [Effects of furfural and microorganisms, Acs. Sym. Ser. 666 (1997) 237e263. [44] L.G. Wayne, D.J. Brenner, R.R. Colwell, P.A.D. Grimont, O. Kandler, 5-hydroxymethylfurfural on succinic acid production by Escherichia coli], succinic acid production are currently under investigation. [15] J. Riyaphan, T. Phumichai, T. Neimsuwan, S. Witayakran, K. Sungsing, M.I. Krichevsky, L.H. Moore, W.E.C. Moore, R. Murray, E.S.M.P. Stackebrandt, Sheng Wu Gong Cheng Xue Bao 29 (2013) 1463e1472. R. Kaveeta, C. Phumichaia, Variability in chemical and mechanical properties M.P. Starr, Report of the ad hoc committee on reconciliation of approaches to [49] Y. Zhang, C.L. Xia, M.M. Lu, M.B. Tu, Effect of overliming and activated carbon fi Conflicts of interest of Para rubber (Hevea brasiliensis) trees, Sci. Asia 41 (2015) 251e258. bacterial systematics, Int. J. Syst. Bacteriol. 37 (1987) 463e464. detoxi cation on inhibitors removal and butanol fermentation of poplar [16] A.M.K. Alhasan, D. Kuang, A.B. Mohammad, R.R. Sharma-Shivappa, Combined [45] R.R. Gonzales, Y. Hong, J.H. Park, G. Kumar, S.H. Kim, Kinetics and equilibria of prehydrolysates, Biotechnol. Biofuels 11 (2018), 178. effect of nitric acid and sodium hydroxide pretreatments on enzymatic 5-hydroxymethylfurfural (5-HMF) sequestration from algal hydrolyzate using [50] K.Q. Chen, M. Jiang, P. Wei, J.M. Yao, H. Wu, Succinic acid production from acid The authors declare that there are no conflicts of interest. saccharification of rubber wood (Heavea brasiliensis), Int. J. Chem. Technol. 2 granular activated carbon, J. Chem. Technol. Biotechnol. 91 (2016) hydrolysate of corn fiber by Actinobacillus succinogenes, Appl. Biochem. Bio- (2010) 12e20. 1157e1163. technol. 160 (2010) 477e485. [17] R. RM, Handbook of Wood Chemistry and Wood Composites. New York, 2005. [46] S.M. Mustapa Kamal, N.L. Mohamad, A.G. Liew Abdullah, N. Abdullah, Acknowledgments [18] Y. Sun, J. Cheng, Hydrolysis of lignocellulosic materials for ethanol production: a review, Bioresour. Technol. 83 (2002) 1e11. This work was supported by Chulalongkorn University Grant, [19] D. Chiaramonti, M. Prussi, S. Ferrero, L. Oriani, P. Ottonello, P. Torre, F. Cherchi, Review of pretreatment processes for lignocellulosic ethanol production, and 2017 (grant number GB-B_60_108_61_05) and 2018 (grant number development of an innovative method, Biomass Bioenergy 46 (2012) 25e35. GB-B_61_095_61_01), Thailand. The authors would like to thank [20] A. Martinez, M.E. Rodriguez, M.L. Wells, S.W. York, J.F. Preston, L.O. Ingram, fi Enago (www.enago.com) for the English language review. Detoxi cation of dilute acid hydrolysates of lignocellulose with lime, Bio- technol. Prog. 17 (2001) 287e293. [21] D.B. Hodge, C. Andersson, K.A. Berglund, U. Rova, Detoxification requirements Appendix A. Supplementary data for bioconversion of softwood dilute acid hydrolyzates to succinic acid, Enzym. Microb. Technol. 44 (2009) 309e316. [22] S. Phuengjayaem, N. Phinkian, S. Tanasupawat, S. Teeradakorn, Diversity and Supplementary data to this article can be found online at succinic acid production of lactic acid bacteria isolated from animals, soils and https://doi.org/10.1016/j.anaerobe.2019.102096. tree barks, Res. J. Microbiol. 12 (2017) 177e186. The results are means of triplicates ± standard deviation. The [23] T. Kudo, K. Matsushima, T. Itoh, J. Sasaki, K. Suzuki, Description of four new species of the genus Kineosporia: Kineosporia succinea sp. nov., Kineosporia strain was cultivated under the anaerobic condition at 37 �C, with rhizophila sp. nov., Kineosporia mikuniensis sp. nov. and Kineosporia rhamnosa shaking at 200 rpm for 48 h. The clustering result of glucose con- sp. nov., isolated from plant samples, and amended description of the genus centrations on cell growth defined groups A, B, and C. The clus- Kineosporia, Int. J. Syst. Bacteriol. 48 Pt 4 (1998) 1245e1255. [24] G.J. Hucker, H.J. Conn, Method of gram staining, Tech. Bull. N. Y. St. Agric. Exp. tering result of glucose concentrations on succinic acid production Sta. 93 (1923) 3e37. defined groups D, E, and F. The clustering result of the different [25] L. Forbes, Rapid flagella stain, J. Clin. Microbiol. 13 (1981) 807e809. substrates on cell growth and succinic acid production defined [26] M. Gagnon, W.M. Hunting, W.B. Esselen, New method for catalase determi- e groups 1, 2, and 3. The grouping results were obtained by multiple nation, Anal. Chem. 31 (1959) 144 146. [27] S. Tanasupawat, N. Chamroensaksri, T. Kudo, T. Itoh, Identification of comparisons using a statistical program by Duncan's new multiple moderately halophilic bacteria from Thai fermented fish ( pla-ra ) and pro- range tests. posal of Virgibacillus siamensis sp. nov, J. Gen. Appl. Microbiol. 56 (2010) 369e379. [28] M. Hosny, S. Benamar, G. Durand, N. Armstrong, C. Michelle, F. Cadoret, B. La References Scola, N. Cassir, Description of Clostridium phoceensis sp. nov., a new species within the genus Clostridium, New Microbes New Infect 14 (2016) 85e92. [1] C. Thakker, Z. Burhanpurwala, G. Rastogi, Y. Shouche, D. Ranade, Isolation and [29] M. Peyret, J. Freney, H. Meugnier, J. Fleurette, Determination of G C content þ characterization of a new osmotolerant, non-virulent Klebsiella pneumoniae of DNA using high-performance liquid chromatography for the identification strain SAP for biosynthesis of succinic acid, Indian J. Exp. Biol. 44 (2006) of staphylococci and micrococci, Res. Microbiol. 140 (1989) 467e475. 142e150. [30] T. Ezaki, Y. Hashimoto, E. Yabuuchi, Fluorometric deoxyribonucleic acid- [2] J.J. Beauprez, M. De Mey, W.K. Soetaert, Microbial succinic acid production: deoxyribonucleic acid hybridization in microdilution wells as an alternative natural versus metabolic engineered producers, Process Biochem. 45 (2010) to membrane filter hybridization in which radioisotopes are used to deter- 1103e1114. mine genetic relatedness among bacterial strains, Int. J. Syst. Bacteriol. 39 [3] X.J. Chen, S.T. Jiang, Z. Zheng, L.J. Pan, S.Z. Luo, Effects of culture redox po- (1989) 224e229. tential on succinic acid production by Corynebacterium crenatum under [31] M. Athalye, W.C. Noble, D.E. Minnikin, Analysis of cellular fatty acids by gas anaerobic conditions, Process Biochem. 47 (2012) 1250e1255. chromatography as a tool in the identification of medically important [4] M. Inui, S. Murakami, S. Okino, H. Kawaguchi, A.A. Vertes, H. Yukawa, Meta- coryneform bacteria, J. Appl. Bacteriol. 58 (1985) 507e512. bolic analysis of Corynebacterium glutamicum during lactate and succinate [32] H. Saito, K.I. Miura, Preparation of transforming deoxyribonucleic acid by productions under oxygen deprivation conditions, J. Mol. Microbiol. Bio- phenol treatment, Biochim. Biophys. Acta 72 (1963) 619e629. technol. 7 (2004) 182e196. [33] D.J. Lane, 16S/23S rRNA sequencing, in: E.S.M. Goodfellow (Ed.), In Nucleic [5] H.W. Ryu, K.H. Kang, J.S. Yun, Bioconversion of fumarate to succinate using Acid Techniques in Bacterial Systematics, Wiley, Chichester, 1991, glycerol as a carbon source, Appl. Biochem. Biotechnol. 77e79 (1999), pp. 115e148. 511e220. [34] O.S. Kim, Y.J. Cho, K. Lee, S.H. Yoon, M. Kim, H. Na, S.C. Park, Y.S. Jeon, J.H. Lee, [6] H. Song, S.Y. Lee, Production of succinic acid by bacterial fermentation, Enzym. H. Yi, S. Won, Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence Microb. Technol. 39 (2006) 352e361. database with phylotypes that represent uncultured species, Int. J. Syst. Evol. [7] J.B. McKinlay, J.G. Zeikus, C. Vieille, Insights into Actinobacillus succinogenes Microbiol. 62 (2012) 716e721. fermentative metabolism in a chemically defined growth medium, Appl. En- [35] K. Tamura, G. Stecher, D. Peterson, A. Filipski, S. Kumar, MEGA6: molecular viron. Microbiol. 71 (2005) 6651e6656. evolutionary genetics analysis version 6.0, Mol. Biol. Evol. 30 (2013) [8] N.P. Nghiem, B.H. Davison, B.E. Suttle, G.R. Richardson, Production of succinic 2725e2729. acid by Anaerobiospirillum succiniciproducens, Appl. Biochem. Biotechnol. [36] J. Felsenstein, Confidence limits on phylogenies: an approach using the 63e65 (1997) 565e576. bootstrap, Evolution 39 (1985) 783e791. [9] J.G. Koendjbiharie, K. Wiersma, R. van Kranenburg, Investigating the central [37] L.Y. Liang, R.M. Liu, J.F. Ma, K.Q. Chen, M. Jiang, P. Wei, Increased production of metabolism of Clostridium thermosuccinogenes, Appl. Environ. Microbiol. 84 succinic acid in Escherichia coli by overexpression of malate dehydrogenase, (2018) e00363e18. Biotechnol. Lett. 33 (2011) 2439e2444. [10] S.N. Parshina, R. Kleerebezem, J.L.S. Sanz, G. Lettinga, A.N. Nozhevnikova, [38] A. Poonsrisawat, S. Phuengjayaem, A. Petsom, S. Teeradakorn, Conversion of N.A. Kostrikina, A.M. Lysenko, A.J. Stams, Soehngenia saccharolytica gen. nov., sweet sorghum straw to sugars by dilute acid saccharification, Sugar Technol. sp nov and Clostridium amygdalinum sp nov., two novel anaerobic, 15 (2013) 322e327. benzaldehyde-converting bacteria, Int. J. Syst. Evol. Microbiol. 53 (2003) [39] G.L. Miller, Use of dinitrosalicylic acid reagent for determination of reducing 1791e1799. sugar, Anal. Chem. 31 (1959) 426e428. [11] H.S. Jayasinghearachchi, S. Singh, P.M. Sarma, A. Aginihotri, B. Lal, Fermenta- [40] P. Zheng, J.J. Dong, Z.H. Sun, Y. Ni, L. Fang, Fermentative production of succinic tive hydrogen production by new marine Clostridium amygdalinum strain C9 acid from straw hydrolysate by Actinobacillus succinogenes, Bioresour. Tech- isolated from offshore crude oil pipeline, Int. J. Hydrogen Energy 35 (2010) nol. 100 (2009) 2425e2429. 6665e6673. [41] S. Phuengjayaem, S. Teeradakorn, Producing succinic acid with Actinobacillus [12] W.D. Murray, A.W. Khan, Ethanol production by a newly isolated anaerobe, succinogenes: optimizing the composition of the medium using Plackett- Clostridium saccharolyticum: effects of culture medium and growth conditions, Burman Design, CMU. J. Nat. Sci. 15 (2016) 253e264.

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