Annals of Microbiology (2018) 68:321–330 https://doi.org/10.1007/s13213-018-1340-4 ORIGINAL ARTICLE A novel isolate of Clostridium butyricum for efficient butyric acid production by xylose fermentation Xin Wang1,2 & Jianzheng Li2 & Xue Chi 2 & Yafei Zhang2 & Han Yan2 & Yu Jin1 & Juanjuan Qu1 Received: 1 September 2017 /Accepted: 26 April 2018 /Published online: 9 May 2018 # Springer-Verlag GmbH Germany, part of Springer Nature and the University of Milan 2018 Abstract Bacterial fermentation of lignocellulose has been regarded as a sustainable approach to butyric acid production. However, the yield of butyric acid is hindered by the conversion efficiency of hydrolysate xylose. A mesophilic alkaline-tolerant strain designated as Clostridium butyricum B10 was isolated by xylose fermentation with acetic and butyric acids as the principal liquid products. To enhance butyric acid production, performance of the strain in batch fermentation was evaluated with various temperatures (20–47 °C), initial pH (5.0–10.0), and xylose concentration (6–20 g/L). The results showed that the optimal temperature, initial pH, and xylose concentration for butyric acid production were 37 °C, 9.0, and 8.00 g/L, respectively. Under the optimal condition, the yield and specific yield of butyric acid reached about 2.58 g/L and 0.36 g/g xylose, respectively, with 75.00% butyric acid in the total volatile fatty acids. As renewable energy, hydrogen was also collected from the xylose fermentation with a yield of about 73.86 mmol/L. The kinetics of growth and product formation indicated that the maximal cell −1 growth rate (μm) and the specific butyric acid yield were 0.1466 h and 3.6274 g/g cell (dry weight), respectively. The better performance in xylose fermentation showed C. butyricum B10 a potential application in efficient butyric acid production from lignocellulose. Keywords Lignocellulose . Xylose . Fermentation . Butyric acid . Clostridium butyricum Introduction bioenergy (Forrest et al. 2010; Junghare et al. 2012; Pagliano et al. 2017) and biochemicals (Saratale et al. 2016; Butyric acid is not only an important feedstock in chemical, Liu et al. 2017; Ventorino et al. 2017) has been extensively food, cosmetic, and pharmaceutical industries, but also the investigated, through different microbial processes. To valo- precursor for biobutanol production via bioconversion rize biomass, waste materials derived from agriculture, food (Zigová and Šturdík 2000; Zhu and Yang 2004). Currently, processing factories, and municipal organic waste can be used butyric acid is mainly produced through petrochemical syn- to produce bio-based production, and there is a growing inter- thesis such as butyraldehyde oxidation (Zhang et al. 2009a). est in bioconversion of lignocellulosic biomass (the most However, with the increasing demand and consumption of the abundant renewable source of carbohydrates) for butyric acid nonrenewable fossil fuel, chemical synthesis seems unsustain- production (Zhang et al. 2009a; Jiang et al. 2010; Baroi et al. able and unfavorable. As an alternative method, microbial 2015;Fuetal.2017; Pagliano et al. 2017). fermentation of renewable biomass can be used to produce Normally, pretreatment to removal lignin and enzymatic saccharification of cellulose and hemicellulose are required previous to fermentation (Saratale et al. 2016;Ventorino * Jianzheng Li et al. 2016). As one of the main component of lignocellulose [email protected] with a mass ratio of 35–45%, hydrolysate xylose is difficult to * Juanjuan Qu be utilized by fermentative bacteria, resulting in a low specific [email protected] yield of bioenergy and biochemicals (Menon and Rao 2012; 1 College of Resources and Environment, Northeast Agricultural Jönsson et al. 2013). Numerous bacterial species belonging to University, Harbin, China the genera of Butyrivibrio, Clostridium, Butyribacterium, 2 State Key Laboratory of Urban Water Resource and Environment, Eubacterium, Fusobacterium, Megasphera,andSarcina have Harbin Institute of Technology, Harbin, China been described as butyric acid producers (Starr et al. 1981; 322 Ann Microbiol (2018) 68:321–330 Zigová and Šturdík 2000; Rogers et al. 2006), but the reported Based on the fermentative butyric acid production from xy- pure bacterial cultures are not satisfactory in xylose fermenta- lose, the isolates with better productivity were selected for tion for butyric acid production with a specific yield less than further investigation. 0.33 g/g xylose (Zhu and Yang 2004;Khamtiband Reungsang 2012;Anetal.2014). Though mutants of the pure Physiological-biochemical and phylogenetic analysis cultures can enhance the fermentative butyric acid production of the isolates from xylose (Liu and Yang 2006; Baroi et al. 2015), search for novel isolates fermenting xylose more efficiently is essential The physiological-biochemical characteristics of the isolates and represents one of the main approach to the cost reduction were checked by API 20A system (Biomerieux company, of fermentative butyric acid production from lignocellulose French) (Park et al. 2015). Total genomic DNAwas separately (Ren et al. 2008;Junghareetal.2012). extracted from the isolates with Genomic DNA extraction kit To develop the microbial resources for fermentative butyric (HuaShun, ShangHai). Each of the 16S rDNA gene was am- acid production from xylose, a novel strain was isolated in the plified using the PCR primers: 27f (5′-AGAGTTTGATCCTG present research. After identified by physiological-biochemical GCTCAG-3′) and 1492r (5′-GGTTACCTTGTTAC and 16S rDNA gene analyses, performance of the novel strain in GACTT-3′), and sequenced by Sangon Biotech Company batch fermentation was evaluated with various temperature, ini- (Shanghai, China). Each 50 μL PCR mixture contained tial pH and xylose concentration for the maximal butyric acid 5 μL of 10× Ex Taq buffer, 4 μL 2 mM dNTP mixture, 1 μl production. The kinetic characteristics of the isolate in xylose 20 μM forward and reverse primers, 0.5 U Ex Taq DNA fermentation process were further investigated under optimal polymerase (Takara, Dalian, China), 38 μL sterile distilled condition. water, and 0.5 μL of the DNA extract. Cycling conditions were a 5-min hot start at 94 °C; 20 cycles of 30s at 92 °C, Materials and methods 2 min at 48 °C, and 1.5 min at 72 °C; and a final 5-min extension step at 72 °C. The 16S rDNA gene sequence each of the isolates was compared with other reference sequences Bacterial isolation resource, media, and procedure available in the NCBI database using the algorithm of Basic Local Alignment Search Tool (BLAST). Closely related se- A microflora stored in the laboratory (State Key Laboratory of quence was retrieved from the database and aligned. Urban Water Resource and Environment, China) was used as Similarity analysis was performed using the program the bacterial isolation resource. The microflora was a CLUSTAL_X. Phylogenetic tree was constructed from the cellulose-degrading and butyrate-producing microbial com- evolutionary distance matrix calculated through the munity which was derived from a mixture of cattle manure, neighbor-joining method by the software MEGA 5. pig manure compost, soil and rotten wood (Ai et al. 2014). Confidence in the tree topology was evaluated by Before bacteria isolation, the microflora was enriched at 37 °C re-sampling 1000 bootstrap trees. for 48 h, with 1% xylose (w/v) as the sole carbon source in basic medium. The basic medium was composed of (1/L): NaHCO3 2.5 g, yeast extract 0.1 g, cysteine 0.5 g, KH2PO4 Preparation of bacteria suspension and batch 0.41 g, Na2HPO4 1.06 g, MgCl2·6H2O0.1g,(NH4)2SO4 fermentation 0.3 g, CaCl2·2H2O 0.11 g, FeCl2·4H2O 0.0045 g, EDTA· Na2 0.00165 g, 1 mL acid trace solution, 1 mL alkaline trace The isolate single colonies on solid medium were collect- solution, 0.2 mL vitamin solution and 1 mL ferric salt solution ed and inoculated in fresh liquid basic medium including (Angelidaki and Sanders 2004). The pH of the basic medium 1% xylose and incubated at 37 °C for 48 h and then the was about 8.5. The procedure for bacteria isolation was as cultured mixture with a cell density (dry weight) of about follows: (1) aliquot 0.5 mL of the enriched culture was asep- 0.77 g/L served as the inoculum. Performance of the iso- tically moved to solid medium (with 1.5% (w/v)agarinthe late in butyric acid production from xylose was evaluated basic medium) and incubated for 48 h at 37 °C, (2) single by batch fermentation under various of temperature colonies on the solid medium were selected and separately (20 °C–47 °C), pH (5.0–10.0) and xylose concentration mixed into 10 mL deoxidized sterile normal saline, (3) (6–20 g/L), separately. The batch fermentations were all 0.5 mL of the bacterial suspension was inoculated on fresh performed in 20-mL anaerobic tubes (ϕ1.5 cm × 18 cm solid medium and incubated for 48 h at 37 °C, and (4) oper- for each), incubated in a ventilated incubator (HZQ-C, ation (2) and (3) were repeated for several times until pure Beijing Donglian Har Instrument Manufacture Co., Ltd., cultures of isolates were obtained. The purity of the isolates China) with a temperature controlling precision of 0.5 °C. was checked with optical microscope (BX51, OLYMPUS) Each of the tubes was loaded with 9.5-mL sterile medium and scanning electron microscopy (S-3400N, Hitachi). and 0.5 mL inoculum of the isolate. Ann Microbiol (2018) 68:321–330 323 For the temperature tests, six fermentations were conducted Analytical methods for 72 h at 20, 25, 32, 37, 42, and 47 °C, respectively, with the same initial pH of 8.5 and initial xylose concentration of 10 g/ Cell density was determined by monitoring the optical L. As for the pH tests, a series of fermentations were per- density (OD) at 600 nm using a spectrophotometer (UV formed at initial pH ranged from 5.0 to 10.0 with an interval 2300, ShangHai TianMei).