Annals of Microbiology (2018) 68:321–330 https://doi.org/10.1007/s13213-018-1340-4

ORIGINAL ARTICLE

A novel isolate of butyricum for efficient production by xylose

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 and 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 , 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 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 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 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). CDW was measured by con- of 0.5 (adjusted by 4 M HCl and 4 M NaOH), also incubated stant weight method, and the relationship of cell density 2 at 37 °C for 72 h. The initial xylose concentration in the with CDW was calculated by d = 0.4852 OD600nm (R = fermentations with various pH was the same 10 g/L. For the 0.98). Xylose concentration was measured by 3,5- xylose concentration tests, a concentration range from 6 to dinitrosalicylic acid colorimetry (DNS) method at 20 g/L with an interval of 2 g/L was set up and all of the batch 540 nm using the spectrophotometer (Silva et al. 2015). fermentations were conducted at 37 °C and pH 9.0 for 72 h. The biogas produced in each of the anaerobic tubes was Performance of the isolate in xylose fermentation was fur- measured by releasing the gas pressure using 50-mL glass ther investigated under the identified optimum condition: 37 °C, syringes to equilibrate the room pressure. Hydrogen in the initial pH 9.0 and 8 g/L xylose. During the 72-h fermenta- biogas was detected by the gas chromatography (SP-6800A, tion, xylose, biomass (cell dry weight, CDW), volatile fatty Shandong Lunan Instrument Factory, China) equipped with a acids (VFAs), hydrogen and pH were all detected every 4 h. thermal conductivity detector (TCD) and a 2-m stainless col- umn packed with Porapak Q (60/80 mesh, Lanzhou ZhongKeKaiDi Chemical New-tech Co., Ltd., China). Description of kinetic model Nitrogen was applied as the carrier gas with a 0.5 MPa column head pressure. 0.5 mL biogas in the head space of each tube The kinetic analysis of cell growth and butyric acid production were taken out with an air tight syringe and injected into the of the isolate was established by Logistic model (Weiss and gas chromatography. The operational temperature of the injec- Ollis 1980) and Leudeking-Piret model (Luedeking and Piret tion port, oven, and detector was set to 50, 50, and 80 °C, 2000), respectively. Logistic model was used to simulate the respectively. The hydrogen yield in each fermentation was kinetics of cell growth in monosaccharide fermentation and calculated by Eq. (4) described as following Eq. (1): ðÞþ ¼ V v  % ð Þ μ t HY c 4 x0ⅇ m 22:4 x ¼ ð1Þ x0 μ − ðÞ−ⅇ mt 1 1 where HY was the hydrogen yield by the end of the fermen- xm tation (mmol/L), V was the biogas released from the anaerobic where x was the biomass of the isolate (g/L), x0 was the initial tube (mL), v was the head space of the anaerobic tube biomass (g/L), xm was the maximum biomass (g/L), μm was (10 mL), and c was the hydrogen percentage in the released − the maximum specific growth rate of the isolate (h 1). After biogas (%). rearrangement, the Eq. (1) was also expressed as following Liquid samples from each fermentation were centrifuged Eq. (2): at 13,000 rpm for 3 min and then 1 mL of the supernatant  was collected and acidified with 0.1 mL 25% phosphoric x x ¼ μ − m − ð Þ acid. The acidified supernatant was then used for VFAs ln − mt ln 1 2 xm x x0 analysis. VFAs were analyzed by another gas chromatogra- phy (SP-6800A, Shandong Lunan Instrument Factory, The value of xm could be obtained from experimental data.  China) equipped with the flame ionization detector and a xm Both μm and ln −1 were estimated by linear regression of x0 FFAP capillary column (30 m × 0.32 mm, SHIMADZU Eq. (2). Japan). The operational temperature of the injection port, Butyrate production (P) of the isolate could be simulated column oven and detector were 210, 180, and 210 °C, re- by Leudeking-Piret model as following Eq. (3): spectively. Nitrogen was applied as the carrier gas, with a 0.5 MPa column head pressure. The injection volume of the ¼ αðÞ− ðÞ P x x0 3 acidified supernatant was 1 μL. where α was the ratio of butyrate production rate (g/(L∙h)) to All of the experiments were performed in triplicate. cell growth rate (g/(L∙h)), i.e. the specific production efficien- Experimental results were presented as the mean ± standard cy of butyric acid by xylose fermentation of the isolate (g/g deviation of the three parallel measurements. Statistical anal- CDW). yses were performed by one-way ANOVA, followed by Dunnett’s t tests. The difference was considered to be statisti- cally significant when p value was less than 0.05. Statistical 324 Ann Microbiol (2018) 68:321–330

Fig. 1 Microscopic observation of the isolate Strain B10. a Optical microscope (× 100). b Scanning transmission electron microscope (× 5000)

analyses were performed using SPSS software Ver.20, and Performance of C. butyricum B10 in fermentative plotting using Origin software Ver.8.5. butyric acid production from xylose

Effect of temperature on the performance of C. butyricum B10 Results and discussion Temperature is a critical factor in butyric acid fermentation by Identification of the isolated bacterium affecting the cell growth and enzymatic reaction (Da et al. 2012). To enhance the butyric acid production of Strain B10, According to the fermentative butyric acid production from its performance in xylose fermentation was investigated at 20, xylose, 15 strains were isolated from the microflora obtained 25, 32, 37, 42, and 47 °C, respectively, with 10 g/L xylose and by Ai et al. (2014). Among the 15 pure cultures was Strain initial pH of 8.5 in the broth. As shown in Table 2,bothVFAs B10 with the maximal butyric acid yield. Besides butyric acid, production and cell growth were remarkably affected by fer- acetic acid and hydrogen were also produced as byproducts mentation temperature. The maximum butyric acid yield (BY) during the xylose fermentation. Observation via optical mi- of about 2.55 g/L, with a selectivity (PB, percentage of butyric croscope (Fig. 1a) and scanning electron microscope (Fig. 1b) acid in the total VFAs) of about 72.44%, was obtained at showed that Strain B10 was a 0.6–1.2 × 3.0–7.0 μm, Gram- 37 °C, as well as the maximum biomass (about 0.97 g/L) positive, non-, straight or rod bacterium occurred and the specific yield of butyric acid by consumed xylose singly or in pair. (SBY, about 0.35 g/g xylose). The BY increased 2.57-fold Physiological characteristics of Strain B10 were inspected with the temperature increasing from 20 to 37 °C, but de- by API 20A system (Park et al. 2015). The results (Table 1) creased 11.59 folds when the temperature was further in- showed that Strain B10 could utilize a variety of carbohy- creased from 37 to 47 °C. Though there was no significant drates for growth except melezitose, rhamnose, raffinose, difference in BY, the butyric acid productivity (BP) of about and glycerol. Strain B10 could neither produce indole, urease 0.058 g/(L·h) at 37 °C was observably higher than that of and contact enzyme, nor hydrolyze gelatin and nitrate. The 0.044 g/(L·h) at 32 °C, indicating 37 °C as the optimum tem- physiological characteristics above suggested that Strain B10 perature for fermentative butyric acid production from xylose. was a bacterium of Clostridium. The present results were in accordance with previous studies According to the phylogenetic analysis of 16S rDNA gene in which the optimum temperature of 35–37 °C was identified sequence (GeneBank accession No. KY937197), a phyloge- for Clostridium sp. (Hahnke et al. 2014; Yin and Wang 2017). netic tree for Strain B10 rooted with C. butyricum was con- It was interesting to find that the PB decreased from 88.39 structed. As shown in Fig. 2, Strain B10 had a similarity of to 72.44% following the temperature increasing from 20 to 97% with C. butyricum Strain VPI 3266. However, the isolate 37 °C, though the BY increased from 0.99 to 2.55 g/L. As was unable to grow on glycerol which could be utilized by known, the increased temperature within a certain range will Strain VPI 3266 to produce 1,3-propanediol promote growth and metabolism of the inhabitants (Da et al. (González-Pajuelo et al. 2004). Both phenotypical analysis 2012;Junghareetal.2012). Furthermore, more energy would and 16S rDNA gene analysis indicated the isolate was a novel be needed by bacteria when cultured at a higher temperature, strain belonging to C. butyricum and designed as C. butyricum and acetic acid formation would produce more energy than B10. that of butyric acid formation (Verhaart et al. 2010;Junghare Ann Microbiol (2018) 68:321–330 325

Table 1 Physiological characteristics of the isolate Strain Project Result Project result Project Result Project Result B10 Indole – Mannose + Sorbitol – Glucose + Urease – Glycerin – Salicin + Galactose + Gelatin – Cellobiose + Melezitose – + Catalase – Raffinose + Trehalose + Arabinose + Nitrate – Rhamnose – Esculin + Maltose + Sucrose + Lactose + Mannitol + Ribose + Xylose + et al. 2012). Thus, the metabolic alteration of Strain B10 re- acid but acetic acid. However, the hydrogen partial pressure in sulted in the increase of VFAs production and decrease of the xylose fermentation processes of Strain B10 at 20, 25, 32, acetate/butyrate ratio following the culturing temperature in- and 37 °C was counted to be about 56, 77, 157, and 157 kPa, creased from 20 to 37 °C (Table 2). However, pH in the fer- respectively, indicating the hydrogen partial pressure was not mentation processes of Strain B10 was also found a decrease the major factors affecting the PB. Zhu and Yang (2004)had with an increase of following the increasing temperature investigated the effect of pH on butyric acid fermentation of (Table 2). It was essential to understand what effect of the xylose by C. tyrobutyricum ATCC 25755 and indicated that pH variation had produced on the metabolic alteration of (1) the would produce more acetate and lactate at pH Strain B10. lower than 5.7 and 92) activity of phosphotransacetylase as the key enzyme controlling acetate formation was higher in Effect of initial pH on the performance of C. butyricum B10 the cells at pH 5.0 than that at pH 6.3. Therefore, pH was identified as the dominant factor affecting the PB, which As illustrated in Table 2, accumulation of acetic and butyric should be further researched to enhance the BY in the xylose acids in xylose fermentation process increased following the fermentation of Strain B10. temperature increasing from 20 °C to 37 °C, resulting in a To enhance the BY in the xylose fermentation of Strain decrease of pH from 5.85 to 4.58. Khanal et al. (2004)report- B10, the performance of the strain in batch fermentation was ed that a pH decrease would cause a metabolic alteration of evaluated at various initial pHs with a constant temperature of bacteria and resulted in a variation of acetate/butyrate ratio. 37 °C. The results (Table 3) revealed that the growth and The changed acetate/butyrate ratio should be attributed to en- butyric acid production of Strain B10 were pH dependent. vironmental changes such as pH, partial pressure of hydrogen The xylose uptake, biomass, HY, VFAs, SBY, and PB all and the accumulation of intermediate products. Angenent increased following the initial pH increasing from 5.0 and to et al. (2004) indicated that a hydrogen partial pressure more 9.0, but sharply decreased when the initial pH was further than 60 Pa was more favorable for the production of butyric increased to 9.5 and 10.0. The BY was promoted by 11.73-

Fig. 2 Phylogenetic position of the isolate Strain B10 326

Table 2 Effect of temperature on performance of Strain B10 in xylose fermentation

Temp. Xylose uptake (g/L) Biomass (g/L) Final pH HY HPP AY BY BP SBY PB

20 °C 3.21 ± 0.18Ca 0.27 ± 0.06D 5.85 ± 0.24C 22.41 ± 1.60D 56 ± 0.66C 0.13 ± 0.02E 0.99 ± 0.06D 0.014 ± 0.01D 0.31 ± 0.02AB 88.39 ± 0.02A 25 °C 5.77 ± 0.25B 0.42 ± 0.12C 5.25 ± 0.13D 47.83 ± 1.92C 77 ± 0.98B 0.59 ± 0.03C 1.65 ± 0.04C 0.023 ± 0.02C 0.29 ± 0.01B 73.66 ± 0.03B 32 °C 6.84 ± 0.25A 0.62 ± 0.05B 4.61 ± 0.25E 69.70 ± 1.34B 157.5 ± 2.44A 0.88 ± 0.02B 2.35 ± 0.13B 0.044 ± 0.02B 0.34 ± 0.02A 72.76 ± 0.03C 37 °C 7.30 ± 0.33A 0.83 ± 0.09A 4.58 ± 0.11E 72.83 ± 3.96A 157.5 ± 3.23A 0.97 ± 0.06A 2.55 ± 0.13A 0.058 ± 0.02A 0.35 ± 0.02A 72.44 ± 0.02D 42 °C 2.99 ± 0.31C 0.10 ± 0.06E 7.72 ± 0.25B 20.34 ± 3.12D 1.2 ± 0.02D 0.35 ± 0.03D 0.80 ± 0.13D 0.011 ± 0.02E 0.27 ± 0.04B 69.57 ± 0.02E 47 °C 1.32 ± 0.35E 0.03 ± 0.00D 8.65 ± 0.24A 6.24 ± 1.60E 0.2 ± 0.00E 0.15 ± 0.01E 0.22 ± 0.01E 0.0003 ± 0.00F 0.17 ± 0.01E 59.46 ± 0.02F

HY, hydrogen yield (mmol/L); HPP, hydrogen partial pressure (kPa); AY, acetic acid yield (g/L); BY, butyric acid yield (g/L); BP, butyric acid productivity g/(L·h); SBY, the specific yield of butyric acid by consumed xylose (g/g); PB, percentage of butyric acid in total VFAs (%) a Means with standard deviation in the same column followed by the same letter do not differ significantly at p = 0.05, according to Duncan’s multiple range test

Table 3 Effect of initial pH on performance of Strain B10 in xylose fermentation

Initial pH Xylose uptake (g/L) Biomass (g/L) Final pH HY AY BY SBY PB

5.0 0.48 ± 0.11Ia 0.03 ± 0.00D 4.51 ± 0.02E 5.15 ± 0.78G 0.12 ± 0.03C 0.22 ± 0.03C 0.46 ± 0.01A 64.70 ± 0.02D 5.5 0.71 ± 0.01H 0.05 ± 0.01C 4.55 ± 0.11E 8.64 ± 1.35F 0.17 ± 0.02C 0.28 ± 0.01C 0.39 ± 0.01 dB 62.22 ± 0.03DE 6.0 0.89 ± 0.10G 0.06 ± 0.01C 4.82 ± 0.14C 9.35 ± 1.10F 0.15 ± 0.05C 0.26 ± 0.08C 0.29 ± 0.09C 63.41 ± 0.04E 6.5 1.69 ± 0.12F 0.09 ± 0.00C 4.66 ± 0.22B 12.17 ± 1.57E 0.10 ± 0.01C 0.24 ± 0.02C 0.14 ± 0.01D 70.59 ± 0.02C 7.0 5.22 ± 0.12E 0.51 ± 0.08B 4.71 ± 0.07CD 59.05 ± 2.75C 0.84 ± 0.05AB 2.07 ± 0.08A 0.40 ± 0.02AB 71.13 ± 0.02C 7.5 5.14 ± 0.03E 0.66 ± 0.01B 4.71 ± 0.01CD 61.88 ± 2.91B 0.82 ± 0.05AB 2.07 ± 0.33A 0.40 ± 0.06AB 71.63 ± 0.04 BC

8.0 5.87 ± 0.09C 0.71 ± 0.00B 4.79 ± 0.04C 62.67 ± 1.25B 0.86 ± 0.02A 2.39 ± 0.21A 0.41 ± 0.04AB 73.54 ± 0.03AB 68:321 (2018) Microbiol Ann 8.5 6.85 ± 0.11B 0.73 ± 0.00B 4.72 ± 0.08C 68.91 ± 1.70A 0.86 ± 0.03A 2.55 ± 0.22A 0.37 ± 0.03B 74.78 ± 0.02AB 9.0 7.25 ± 0.06A 0.80 ± 0.04A 4.64 ± 0.01CD 69.70 ± 2.49A 0.86 ± 0.02A 2.58 ± 0.06A 0.36 ± 0.01B 75.00 ± 0.05A 9.5 5.64 ± 0.07D 0.71 ± 0.12A 5.55 ± 0.19B 44.28 ± 0.87D 0.66 ± 0.03B 1.56 ± 0.08B 0.28 ± 0.01C 72.27 ± 0.02 BC 10.0 0.92 ± 0.13G 0.04 ± 0.00D 9.76 ± 0.10A 0 ± 0.00I 0.05 ± 0.01C 0.12 ± 0.01C 0.13 ± 0.01D 70.59 ± 0.03C

HY, hydrogen yield (mmol/L); AY, acetic acid yield (g/L); BY, butyric acid yield (g/L); SBY, the specific yield of butyric acid by consumed xylose (g/g); PB, percentage of butyric acid in total VFAs (%) a – Means with standard deviation in the same column followed by the same letter do not differ significantly at p = 0.05, according to Duncan’s multiple range test 330 Ann Microbiol (2018) 68:321–330 327

Table 4 Effect of xylose concentration on performance of Strain B10 in xylose fermentation

Initial xylose Xylose Biomass Final pH HY AY BY SBY PB (g/L) uptake(g/L) (g/L)

6.00 6.00 ± 0.11Ba 0.67 ± 0.13A 4.99 ± 0.02A 58.69 ± 1.37B 0.80 ± 0.03B 1.97 ± 0.17B 0.33 ± 0.- 70.12 ± 0.- 03A 02D 8.00 7.52 ± 0.12A 0.78 ± 0.08A 0.83 ± 0.02B 4.69 ± 0.02 - 75.02 ± 0.- 2.52 ± 0.- 0.34 ± 0.- 75.22 ± 0.- BC 84A 08A 01A 03A 10.00 7.56 ± 0.06A 0.78 ± 0.09A 4.72 ± 0.06B 74.93 ± 0.05B 73.86 ± 1.- 0.84 ± 0.05- 2.51 ± 0.- 0.33 ± 0.- 37A AB 23A 03A 20.00 7.64 ± 0.11A 0.78 ± 0.12A 4.63 ± 0.01C 0.90 ± 0.02A 73.76 ± 0.02C 75.35 ± 2.- 2.53 ± 0.- 0.33 ± 0.- 37A 22A 03A

HY, hydrogen yield (mmol/L); AY, acetic acid yield (g/L); BY, butyric acid yield (g/L); SBY, the specific yield of butyric acid by consumed xylose (g/g); PB, percentage of butyric acid in total VFAs (%) a Means with standard deviation in the same column followed by the same letter do not differ significantly at p = 0.05, according to Duncan’s multiple range test fold with the initial pH increased from 5.0 to 9.0. Though the Effect of initial xylose concentration on the performance BYs, as well as SBYs and PBs, were not different significant- of C. butyricum B10 ly with the pH ranged from 7.0 to 9.0, both of the xylose uptake (about 7.25 g/L) and biomass (about 0.80 g/L) at Concentration of xylose as substrate has been reported to af- pH 9.0 were obviously higher than that of the other pHs. As fect cellular respiration and cell growth of fermentative bacte- known, a higher pH means more alkalinity to neutralize the ria, resulting in variation in performance of butyric acid fer- produced acetic and butyric acids during the batch fermenta- mentation (Khamtib and Reungsang 2012). Zhang et al. tion, which was conducive to better growth and metabolism of (2009b) investigated the effect of glucose concentration on acidogenic bacteria (Jo et al. 2008). With the maximal xylose the butyric acid production of C. thermobutyricum ATCC uptake of about 5.25 g/L and BY of about 2.58 g/L, the initial 49875 and a maximum yield of 12.05 g/L was obtained only pH 9.0 was suggested as the optimal for the batch fermenta- with 34.9 g/L glucose in the broth. Song et al. (2010)reported tion of Strain B10 to produce butyric acid from xylose. The that the favorable glucose for the maximum specific cell present results were accorded with that of Junghare and co- growth (0.426 h−1) and butyric acid yield (62.48 g/L) of workers who also found that pH 9.0 was the optimal for the C. tyrobutyricum were obviously different (20 g/L and growth and fermentation of C. butyricum TM-9A in batch 120 g/L, respectively). When the glucose in the broth was as experiment (Junghare et al. 2012). high as 150 g/L, the lag phase was found to be remarkbly

Fig. 3 Performance of C. butyricum B10 in xylose fermentation at 37 °C with initial pH 9.0 and 8.00 g/L xylose in broth 328 Ann Microbiol (2018) 68:321–330

Table 5 Kinetic parameters of C. butyricum B10 in xylose parameter Estimated from Estimated value Deviation Unit fermentation process x0 Intercept of Eq. (2) 0.0165 0.988 g/L

xm Experimental data 0.819 g/L −1 μm Slope of Eq. (2) 0.1466 0.988 h α Slope of Eq. (3) 3.6274 0.9743 g butyric acid/ g cell dry weight

extended to about 20 h. To optimize the xylose concentration Kinetic analysis for Strain B10 to produce butyrate, a series of xylose concen- tration (6~20 g/L) was evaluated. To understand the kinetic characteristics of Strain B10 in As shown in Table 4, when xylose concentration in broth xylose fermentation, batch fermentation was conducted was increased from 6.00 to 8.00 g/L, the xylose uptake, HY, again under the identified optimal 37 °C, initial pH 9.0 BY and PB increased from about 6.00 g/L, 58.69 mmol/L, and 8 g/L xylose. The results (Fig. 3) showed that both 1.97 g/L and 70.12% to 7.52 g/L, 75.02 mmol/L, 2.52 g/L biomass and BY found a logarithmic phase from the 8th and 75.22%, respectively, without obvious difference in bio- to the 44th hour after a lag phase (0~8 h). By the end of mass, acetic acid yield (AY) and SBY. With more VFAs ac- the logarithmic phase at the 44th hour, the biomass and cumulated, the final pH decreased from 4.99 to 4.69 following BY reached about 0.82 and 2.51 g/L, respectively, with a the increased xylose concentration. When the xylose concen- xylose absorptivity of about 97.02%. tration was further increased from 8 to 10 and then to 20 g/L, As shown in Fig. 3, the growth of Strain B10 was well met no obvious difference in xylose uptake, biomass, final pH, with the Logistic model (Weiss and Ollis 1980), while BY was HY, BY and SBY was observed. However, the AY increased met well with the Leudeking-Piret model (Luedeking and obviously from about 0.83 to 0.90 g/L as the xylose concen- Piret 2000), both of which were validated by an R2 of 0.988 tration was increased from 8 to 20 g/L, resulting the PB de- (Table 5). The results illustrated in Table 5 indicated the sim- creased from about 75.22 to 73.76%. The results indicated that ulated x0 and μm by linear regression of Eq. (2) was 0.0165 g/ the decreased pH (1) was more favorable to the production of L and 0.1466 h−1, respectively. It was found that the simulated acetic acid but butyric acid (Zhu and Yang 2004) (2) had made x0 (0.0165 g/L) was much lower than that of the experimental the fermentation tend to be terminated (Wu and Yang 2003). value (0.082 g/L), suggesting many cells in the inoculum was Wu and Yang (2003) also found that acidogenic fermentation inactive and the improvement of inoculation methods would of bacteria would be terminated by low pH condition. More be helpful to optimizing the fermentation process. The gener- researches indicated that the lower pH would inhibit bacterial ation time of Strain B10 under optimum condition was calcu- −1 cell growth and metabolism by disrupting the transmembrane lated to be 2.61 h based on the μm of 0.1466 h .Accordingto pH gradient, reducing ATPlevel and activities of acid-forming Ledueking-Piret model (Eq. (3)), α of Strain B10 reached enzymes (Zigová and Šturdík 2000; Zhu and Yang 2003). 3.6274 g/g CDW.

Table 6 Fermentative butyric acid production from xylose by Strain B10 and other clostridia

Strain Fermentation mode Temp. pH BY SBY PB reference

C. butyricum INET1 Batch 35 7.0 0.03 UA 50 (Yin and Wang 2017) C. butyricum TM-9A Batch 37 8.5 0.92 UA 69.13 (Junghare et al. 2012) C. butyricum CGS2 Batch 37 7.5 2.52 UA 30 (Lo et al. 2008) C. butyricum CGS5 Batch 37 7.5 3.79 UA 42 ATCC 25755 mutant Ct-pTBA Batch (pH controlled) 37 6.0 42.60 0.36 UA (Fu et al. 2017) C. tyrobutyricum DSMZ 2637 Fed batch (pH controlled) 37 7.0 20.00 0.33 80.00 (Baroi et al. 2015) C. tyrobutyricum ATCC 25755 Fed batch (pH controlled) 37 6.0 19.42 0.33 85.44 (Liu and Yang 2006) ATCC 25755 mutant PPTA-Em Fed-batch (pH controlled) 37 6.0 37.22 0.38 89.88 Fed batch with cells immobilized (pH controlled) 37 6.0 51.60 0.45 UA C. tyrobutyricum ATCC 25755 Fed batch with cells immobilized (pH controlled) 37 6.0 10.10 0.40 82.85 (Jiang et al. 2010) C. butyricum B10 Batch 37 9.0 2.58 0.36 75.00 This study

BY (g/L), butyric acid titer; SBY (g/g), butyric acid yield by the consumed xylose; PB (%), percentage of butyric acid in total VFAs; UN, unavailable Ann Microbiol (2018) 68:321–330 329

The fermentation efficiency of C. butyricum B10 Compliance with ethical standards compared with other clostridia Conflict of interest The authors declare that they have no conflict of Though thermophilic fermentation is more efficient than interest. mesophilic fermentation for hydrogen and VFAs production from various saccharides (Verhaart et al. 2010), fermentation References of mesophiles has attracted an increasing interest because of the less energy consumption. To evaluate the fermentation efficiency of C. butyricum B10, previous researches on xylose Ai B, Li J, Chi X, Meng J, Liu C, Shi E (2014) Butyric acid fermentation of sodium hydroxide pretreated rice straw with undefined mixed fermentation by species of Clostridium under mesophilic con- culture. J Microbiol Biotechnol 24(5):629–638 dition were collected with the results illustrated in Table 6. An D, Li Q, Wang X, Yang H, Guo L (2014) Characterization on hydro- With the same mode of bath fermentation without pH control, gen production performance of a newly isolated Clostridium the highest BY 3.79 g/L came to C. butyricum CGS5 with a beijerinckii YA001 using xylose. Int J Hydrogen Energ 39(35): 19928–19936 PB of 42% (Lo et al. 2008). Though the BY 2.58 g/L of Strain Angelidaki I, Sanders W (2004) Assessment of the anaerobic biodegrad- B10waslowerthanthatofStrainCGS5,itsPBreached ability of macropollutants. Rev Environ Sci Bio 3(2):117–129 75.00% that was much higher than that of Strain CGS5. Angenent LT, Karim K, AlDahhan MH, Wrenn BA, C. butyricum CGS2 (Lo et al. 2008), TM-9A (Junghare DomíguezEspinosam R (2004) Production of bioenergy and bio- et al. 2012) and INET1 (Yin and Wang 2017) all performed chemicals from industrial and agricultural wastewater. Trends Biotechnol 22(9):477 not well in fermentative butyric acid production when com- Baroi GN, Baumann I, Westermann P,Gavala HN, Schnürer A, Verstraete pared their BY and PB with Strain CGS5 and Strain B10. The W (2015) Butyric acid fermentation from pretreated and hydrolysed results indicated C. butyricum B10 a good performance in wheat straw by an adapted Clostridium tyrobutyricum strain. Microb xylose utilization and a potential application to fermentative Biotechnol 8(5):874 butyric acid production from lignocellulose. 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The optimal temperature and initial pH in batch Jiang L, Wang J, Liang S, Wang X, Cen P, Xu Z (2010) Production of fermentation was 37 °C and 9.0, respectively. Loaded with butyric acid from glucose and xylose with immobilized cells of 8 g/L xylose in the broth, a butyric acid yield and specific Clostridium tyrobutyricum in a fibrous-bed bioreactor. Appl Biochem Biotechnol 160(2):350 butyric acid yield as high as 2.58 g/L and 0.36 g/g xylose, Jo JH, Lee DS, Park JM (2008) The effects of pH on carbon material and respectively, was obtained along with 73.86 mmol/L hydro- energy balances in hydrogen-producing Clostridium tyrobutyricum gen collected. The cell growth rate and the specific butyric JM1. Bioresour Technol 99(17):8485–8491 acid yield of the strain in xylose fermentation reached Jönsson LJ, Alriksson B, Nilvebrant NO (2013) Bioconversion of ligno- – 0.1466 h−1 and 3.6274 g/g CDW, respectively, indicating a cellulose: inhibitors and detoxification. Biotechnol Biofuels 6(1):1 10 good application potential in butyric acid production by lig- Junghare M, Subudhi S, Lal B (2012) Improvement of hydrogen produc- nocellulose fermentation. tion under decreased partial pressure by newly isolated alkaline tol- erant anaerobe, Clostridium butyricum TM-9A: optimization of pro- Funding information This work was supported financially by National cess parameters. Int J Hydrog Energy 37(4):3160–3168 Natural Science Foundation of China (Grant No. 51478141) and the State Khamtib S, Reungsang A (2012) Biohydrogen production from xylose Key Laboratory of Urban Water Resource and Environment (Harbin by Thermoanaerobacterium thermosaccharolyticum KKU19 isolat- Institute of Technology) (Grant No. 2016DX06). ed from hot spring sediment. Int J Hydrogen Energy 37(17):12219– 12228 330 Ann Microbiol (2018) 68:321–330

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