Clostridium Beijerinckii Strain BGS1 Chen Zhang, Tinggang Li and Jianzhong He*

Zhang et al. Biotechnol Biofuels (2018) 11:280 https://doi.org/10.1186/s13068-018-1274-x Biotechnology for Biofuels RESEARCH Open Access Characterization and genome analysis of a butanol–isopropanol‑producing Clostridium beijerinckii strain BGS1 Chen Zhang, Tinggang Li and Jianzhong He* Abstract Background: One of the main challenges of acetone–butanol–ethanol fermentation is to reduce acetone production with high butanol yield. Converting acetone into isopropanol is an alternative pathway to reduce fermentation by-products in the fermentation broth. Here, we aimed to cultivate a wild-type Clostridium strain with high isopropanol and butanol production and reveal its genome information. Results: Clostridium beijerinckii strain BGS1 was found to be capable of producing 10.21 g/L butanol and 3.41 g/L isopropanol, higher than previously known wild-type isopropanol–butanol-producing Clostridium species. Moreover, culture BGS1 exhibited a broad carbon spectrum utilizing diverse sugars such as arabinose, xylose, galactose, cellobiose, and sucrose, with 9.61 g/L butanol and 2.57 g/L isopropanol generated from 60 g/L sucrose and less amount from other sugars. Based on genome analysis, protein-based sequence of strain BGS1 was closer to C. beijerinckii NCIMB 8052, reaching 90.82% similarity, while compared to C. beijerinckii DSM 6423, the similarity was 89.53%. In addition, a unique secondary alcohol dehydrogenase (sAdhE) was revealed in the genome of strain BGS1, which distinguished it from other Clostridium species. Average nucleotide identity analysis identifed strain BGS1 belonging to C. beijerinckii. The transcription profle and enzymatic activity of sAdhE proved its function of converting acetone into isopropanol. Conclusions: Clostridium beijerinckii strain BGS1 is a potential candidate for industrial isopropanol and butanol production. Its genome provides unique information for genetic engineering of isopropanol–butanol-producing microorganisms. Keywords: Isopropanol, Butanol, Clostridium beijerinckii, Genome, Secondary alcohol dehydrogenase Background However, given that both chemicals are mainly produced Isopropanol and butanol are widely used in a variety of from non-renewable petroleum-based materials, micro- industrial applications including solvent applications, bial production of isopropanol and butanol from renew- chemical intermediates and biofuels. When serving as able feedstock becomes more attractive and cost efective a biofuel, butanol can be used as a fuel additive or com- [4]. pletely replace gasoline due to property similarities [1], As a traditional microbial way to produce butanol, while isopropanol can replace methanol for biodiesel acetone–butanol–ethanol (ABE) fermentation by synthesis with reduced biodiesel crystallization tempera- Clostridium species can generate butanol, acetone and ture [2]. Additionally, isopropanol can partially replace ethanol at a ratio of 6:3:1 typically [5]. However, the gasoline so as to increase octane number of the fuels [3]. main by-product, acetone, is not preferred because of its high corrosiveness to engine and low energy density [6]. Although acetone production have been tried to be *Correspondence: [email protected] reduced by knock-out of acetoacetate decarboxylase Department of Civil and Environmental Engineering, National University of Singapore, Block E2‑02‑13, 1 Engineering Drive 3, Singapore 117576, (adc) gene, butanol reduction and acetate accumulation Singapore inevitably occurred with the acetone reduction [7]. In © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhang et al. Biotechnol Biofuels (2018) 11:280 Page 2 of 11 contrast, isopropanol–butanol–ethanol (IBE) fermenta- [14] and further transferred to fresh medium. Among tion by Clostridium species is a more preferred micro- all isolates, one culture designated BGS1 was identi- bial pathway for both renewable butanol and isopropanol fed to be capable of producing 3.88 g/L butanol and production without the undesirable acetone [8]. Com- 1.07 g/L isopropanol with negligible amount of acetone pared to traditional ABE fermentation, IBE fermentation and ethanol. When culture BGS1 was observed under produces a component of isopropanol, an alcohol with light microscopy, the uniform rod-shaped morphology slightly higher energy density than acetone (23.9 MJ/L suggested the purity of the culture. Te sequence of the vs 22.6 MJ/L), and the end-products of IBE mixture can 16S rRNA gene from culture BGS1 showed 99% identity be used directly as a green fuel in spark-ignition (SI) to C. beijerinckii strain TERI-Chilika-02 based on Blast engines, usually with lower pollutant emissions and bet- results. When comparing with 12 previously reported ter engine performance than ABE mixture [9]. bacteria capable of producing butanol or isopropanol, Among all the solvent-producing Clostridium strains, culture BGS1 exhibited closer similarity to strains under a few strains can produce butanol and isopropanol as C. beijerinckii (Fig. 1a). However, phylogenetic tree based major fermentation products, while most isopropanol- on 16S rRNA gene can only identify microorganisms to producing bacteria belong to Clostridium beijerinckii genus level. Based on the genome information, average [10]. In general, most of the isopropanol-producing nucleotide identity (ANI) was applied to identify the spe- strains produce relatively low concentrations of butanol cies of strain BGS1 [15]. ANI of 95% between two bac- and isopropanol, less than 5.5 g/L and 1.5 g/L, respec- terial genomes usually corresponds to 70% DNA–DNA tively, due to the toxicity posed by butanol (> 7.4 g/L) hybridization value, from which it can confrm the two [11]. To produce isopropanol with ABE-producing bacteria belonging to the same species [16]. As shown in Clostridium, an attempt has been made to engineer sec- Fig. 1b, cluster heatmap of ANI analysis clearly depicted ondary alcohol dehydrogenase (sAdhE) gene from C. bei- that Clostridium sp. BGS1 clusters to C. beijerinckii spe- jerinckii NRRL B-593 into non-isopropanol producing cies, of which all ANI values were higher than 96% of C. acetobutylicum [6]. However, the successful conver- similarity but were lower than 80% of ANI values com- sion of acetone into isopropanol also caused reduction of paring with other Clostridium species. Hence, this isolate butanol yield. To date, only one sADH gene from C. bei- is designated C. beijerinckii strain BGS1. jerinckii NRRL B-593 with limited genome information Clostridium beijerinckii strain BGS1 distinguishes itself was identifed, while this wild-type strain produced quite from other known C. beijerinckii strains by its high iso- low titers (< 5 g/L) of butanol and isopropanol [12]. Very propanol production. As a typical C. beijerinckii strain, recently, C. beijerinckii DSM 6423 [13] was also found to NCIMB 8052 mainly produced butanol and ethanol with possess sAdhE gene encoding enzymes for isopropanol acetone as a by-product, but was not able to produce and butanol production but with lower titers (< 10.7 g/L). isopropanol using glucose as a substrate [17]. Terefore, Finding novel wild-type bacteria with higher isopropanol this newly isolated wild-type solvent-producing C. beijer- and butanol production as well as detailed genome infor- inckii BGS1 broadens the pool of isopropanol–butanol- mation is of great interest for isopropanol and butanol producing species [18]. biorefnery. To broaden current knowledge of butanol and isopro- Optimization of fermentation conditions for enhanced panol production, this study aimed to (i) characterize an butanol and isopropanol production isopropanol–butanol-producing microorganism with During the solvent fermentation, pH was a key factor high yield; (ii) analyze the genome of this strain and fur- infuencing fermentation performance [5]. To optimize ther identify functional genes responsible for isopropanol pH, fve batch experiments were conducted at diferent production. pH conditions (5.0, 5.2, 5.5, 5.8, 6.5). pH was adjusted to a defned value by 3 M NaOH in every 12 h. After 3 days Results and discussion of incubation, culture BGS1 generated higher amount of Isolation and characterization of Clostridium sp. strain butanol at pH 5.0 (5.00 g/L), pH 5.2 (4.46 g/L) and pH 5.5 BGS1 (4.11 g/L), and lower amount at pH 5.8 (3.53 g/L) and pH To discover novel isopropanol–butanol-producing 6.5 (1.93 g/L), while similar amounts of isopropanol were microorganisms, soil samples from grass land were used obtained at varied pH conditions. In addition, high pH as inocula for microcosm setup. After three transfers, (6.5) led to signifcantly increased acetic acid (6.36 g/L) four enriched cultures with butanol and isopropanol and butyric acid (6.67 g/L) production in the fermenta- production were selected for further cultivation of indi- tion broth, indicating that it was difcult for strain BGS1 vidual colonies. Eight of well-grown colonies were picked to shift from acidogenesis to solventogenesis

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