A novel isolate of Clostridium butyricum for efficient butyric acid production by xylose fermentation

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 growth rate (μ _m ) and the specific butyric acid yield were 0.1466 h⁻¹ 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.

Introduction

Butyric acid is not only an important feedstock in chemical, food, cosmetic, and pharmaceutical industries, but also the precursor for biobutanol production via bioconversion (Zigová and Šturdík 2000; Zhu and Yang 2004). Currently, butyric acid is mainly produced through petrochemical synthesis such as butyraldehyde oxidation (Zhang et al. 2009a). However, with the increasing demand and consumption of the nonrenewable fossil fuel, chemical synthesis seems unsustainable and unfavorable. As an alternative method, microbial fermentation of renewable biomass can be used to produce bioenergy (Forrest et al. 2010; Junghare et al. 2012; Pagliano et al. 2017) and biochemicals (Saratale et al. 2016; Liu et al. 2017; Ventorino et al. 2017) has been extensively investigated, through different microbial processes. To valorize biomass, waste materials derived from agriculture, food processing factories, and municipal organic waste can be used to produce bio-based production, and there is a growing interest in bioconversion of lignocellulosic biomass (the most abundant renewable source of carbohydrates) for butyric acid production (Zhang et al. 2009a; Jiang et al. 2010; Baroi et al. 2015; Fu et al. 2017; Pagliano et al. 2017).

Normally, pretreatment to removal lignin and enzymatic saccharification of cellulose and hemicellulose are required previous to fermentation (Saratale et al. 2016; Ventorino et al. 2016). As one of the main component of lignocellulose with a mass ratio of 35–45%, hydrolysate xylose is difficult to be utilized by fermentative bacteria, resulting in a low specific yield of bioenergy and biochemicals (Menon and Rao 2012; Jönsson et al. 2013). Numerous bacterial species belonging to the genera of Butyrivibrio, Clostridium, Butyribacterium, Eubacterium, Fusobacterium, Megasphera, and Sarcina have been described as butyric acid producers (Starr et al. 1981; Zigová and Šturdík 2000; Rogers et al. 2006), but the reported pure bacterial cultures are not satisfactory in xylose fermentation for butyric acid production with a specific yield less than 0.33 g/g xylose (Zhu and Yang 2004; Khamtib and Reungsang 2012; An et al. 2014). Though mutants of the pure cultures can enhance the fermentative butyric acid production from xylose (Liu and Yang 2006; Baroi et al. 2015), search for novel isolates fermenting xylose more efficiently is essential and represents one of the main approach to the cost reduction of fermentative butyric acid production from lignocellulose (Ren et al. 2008; Junghare et al. 2012).

To develop the microbial resources for fermentative butyric acid production from xylose, a novel strain was isolated in the present research. After identified by physiological-biochemical and 16S rDNA gene analyses, performance of the novel strain in batch fermentation was evaluated with various temperature, initial pH and xylose concentration for the maximal butyric acid production. The kinetic characteristics of the isolate in xylose fermentation process were further investigated under optimal condition.

Materials and methods

Bacterial isolation resource, media, and procedure

A microflora stored in the laboratory (State Key Laboratory of Urban Water Resource and Environment, China) was used as the bacterial isolation resource. The microflora was a cellulose-degrading and butyrate-producing microbial community which was derived from a mixture of cattle manure, pig manure compost, soil and rotten wood (Ai et al. 2014). Before bacteria isolation, the microflora was enriched at 37 °C 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 0.41 g, Na₂HPO₄ 1.06 g, MgCl₂·6H₂O 0.1 g, (NH₄)₂SO₄ 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 solution, 0.2 mL vitamin solution and 1 mL ferric salt solution (Angelidaki and Sanders 2004). The pH of the basic medium was about 8.5. The procedure for bacteria isolation was as follows: (1) aliquot 0.5 mL of the enriched culture was aseptically moved to solid medium (with 1.5% (w/v) agar in the basic medium) and incubated for 48 h at 37 °C, (2) single colonies on the solid medium were selected and separately mixed into 10 mL deoxidized sterile normal saline, (3) 0.5 mL of the bacterial suspension was inoculated on fresh solid medium and incubated for 48 h at 37 °C, and (4) operation (2) and (3) were repeated for several times until pure cultures of isolates were obtained. The purity of the isolates was checked with optical microscope (BX51, OLYMPUS) and scanning electron microscopy (S-3400N, Hitachi). Based on the fermentative butyric acid production from xylose, the isolates with better productivity were selected for further investigation.

Physiological-biochemical and phylogenetic analysis of the isolates

The physiological-biochemical characteristics of the isolates were checked by API 20A system (Biomerieux company, French) (Park et al. 2015). Total genomic DNA was separately extracted from the isolates with Genomic DNA extraction kit (HuaShun, ShangHai). Each of the 16S rDNA gene was amplified using the PCR primers: 27f (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492r (5′-GGTTACCTTGTTACGACTT-3′), and sequenced by Sangon Biotech Company (Shanghai, China). Each 50 μL PCR mixture contained 5 μL of 10× Ex Taq buffer, 4 μL 2 mM dNTP mixture, 1 μl 20 μM forward and reverse primers, 0.5 U Ex Taq DNA polymerase (Takara, Dalian, China), 38 μL sterile distilled 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, 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 available in the NCBI database using the algorithm of Basic Local Alignment Search Tool (BLAST). Closely related sequence was retrieved from the database and aligned. Similarity analysis was performed using the program CLUSTAL_X. Phylogenetic tree was constructed from the evolutionary distance matrix calculated through the neighbor-joining method by the software MEGA 5. Confidence in the tree topology was evaluated by re-sampling 1000 bootstrap trees.

Preparation of bacteria suspension and batch fermentation

The isolate single colonies on solid medium were collected and inoculated in fresh liquid basic medium including 1% xylose and incubated at 37 °C for 48 h and then the cultured mixture with a cell density (dry weight) of about 0.77 g/L served as the inoculum. Performance of the isolate in butyric acid production from xylose was evaluated by batch fermentation under various of temperature (20 °C–47 °C), pH (5.0–10.0) and xylose concentration (6–20 g/L), separately. The batch fermentations were all performed in 20-mL anaerobic tubes (ϕ1.5 cm × 18 cm for each), incubated in a ventilated incubator (HZQ-C, Beijing Donglian Har Instrument Manufacture Co., Ltd., China) with a temperature controlling precision of 0.5 °C. Each of the tubes was loaded with 9.5-mL sterile medium and 0.5 mL inoculum of the isolate.

For the temperature tests, six fermentations were conducted 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/L. As for the pH tests, a series of fermentations were performed at initial pH ranged from 5.0 to 10.0 with an interval of 0.5 (adjusted by 4 M HCl and 4 M NaOH), also incubated at 37 °C for 72 h. The initial xylose concentration in the fermentations with various pH was the same 10 g/L. For the xylose concentration tests, a concentration range from 6 to 20 g/L with an interval of 2 g/L was set up and all of the batch fermentations were conducted at 37 °C and pH 9.0 for 72 h.

Performance of the isolate in xylose fermentation was further investigated under the identified optimum condition: 37 °C, initial pH 9.0 and 8 g/L xylose. During the 72-h fermentation, xylose, biomass (cell dry weight, CDW), volatile fatty acids (VFAs), hydrogen and pH were all detected every 4 h.

Description of kinetic model

The kinetic analysis of cell growth and butyric acid production of the isolate was established by Logistic model (Weiss and Ollis 1980) and Leudeking-Piret model (Luedeking and Piret 2000), respectively. Logistic model was used to simulate the kinetics of cell growth in monosaccharide fermentation and described as following Eq. (1):

$$ x=\frac{x_0{\mathrm{e}}^{\mu_mt}}{1-\frac{x_0}{x_m}\left(1-{\mathrm{e}}^{\mu_mt}\right)} $$

(1)

where x was the biomass of the isolate (g/L), x ₀ was the initial biomass (g/L), x _m was the maximum biomass (g/L), μ_m was the maximum specific growth rate of the isolate (h⁻¹). After rearrangement, the Eq. (1) was also expressed as following Eq. (2):

$$ \mathit{\ln}\frac{x}{x_m-x}={\mu}_mt-\mathit{\ln}\left(\frac{x_m}{x_0}-1\right) $$

(2)

The value of x _m could be obtained from experimental data. Both μ _m and $\mathit{\ln}\left(\frac{x_m}{x_0}-1\right)$ were estimated by linear regression of Eq. (2).

Butyrate production (P) of the isolate could be simulated by Leudeking-Piret model as following Eq. (3):

$$ P=\alpha \left(x-{x}_0\right) $$

(3)

where α was the ratio of butyrate production rate (g/(L∙h)) to cell growth rate (g/(L∙h)), i.e. the specific production efficiency of butyric acid by xylose fermentation of the isolate (g/g CDW).

Analytical methods

Cell density was determined by monitoring the optical density (OD) at 600 nm using a spectrophotometer (UV 2300, ShangHai TianMei). CDW was measured by constant weight method, and the relationship of cell density with CDW was calculated by d = 0.4852 OD_600nm (R² = 0.98). Xylose concentration was measured by 3,5-dinitrosalicylic acid colorimetry (DNS) method at 540 nm using the spectrophotometer (Silva et al. 2015).

The biogas produced in each of the anaerobic tubes was measured by releasing the gas pressure using 50-mL glass syringes to equilibrate the room pressure. Hydrogen in the biogas was detected by the gas chromatography (SP-6800A, Shandong Lunan Instrument Factory, China) equipped with a thermal conductivity detector (TCD) and a 2-m stainless column packed with Porapak Q (60/80 mesh, Lanzhou ZhongKeKaiDi Chemical New-tech Co., Ltd., China). 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 were taken out with an air tight syringe and injected into the gas chromatography. The operational temperature of the injection port, oven, and detector was set to 50, 50, and 80 °C, respectively. The hydrogen yield in each fermentation was calculated by Eq. (4)

$$ \mathrm{HY}=\frac{\left(V+v\right)}{22.4}\times c\% $$

(4)

where HY was the hydrogen yield by the end of the fermentation (mmol/L), V was the biogas released from the anaerobic tube (mL), v was the head space of the anaerobic tube (10 mL), and c was the hydrogen percentage in the released biogas (%).

Liquid samples from each fermentation were centrifuged at 13,000 rpm for 3 min and then 1 mL of the supernatant was collected and acidified with 0.1 mL 25% phosphoric acid. The acidified supernatant was then used for VFAs analysis. VFAs were analyzed by another gas chromatography (SP-6800A, Shandong Lunan Instrument Factory, China) equipped with the flame ionization detector and a FFAP capillary column (30 m × 0.32 mm, SHIMADZU Japan). The operational temperature of the injection port, column oven and detector were 210, 180, and 210 °C, respectively. Nitrogen was applied as the carrier gas, with a 0.5 MPa column head pressure. The injection volume of the acidified supernatant was 1 μL.

All of the experiments were performed in triplicate. Experimental results were presented as the mean ± standard deviation of the three parallel measurements. Statistical analyses were performed by one-way ANOVA, followed by Dunnett’s t tests. The difference was considered to be statistically significant when p value was less than 0.05. Statistical analyses were performed using SPSS software Ver.20, and plotting using Origin software Ver.8.5.

Results and discussion

Identification of the isolated bacterium

According to the fermentative butyric acid production from xylose, 15 strains were isolated from the microflora obtained by Ai et al. (2014). Among the 15 pure cultures was Strain B10 with the maximal butyric acid yield. Besides butyric acid, acetic acid and hydrogen were also produced as byproducts during the xylose fermentation. Observation via optical microscope (Fig. 1a) and scanning electron microscope (Fig. 1b) showed that Strain B10 was a 0.6–1.2 × 3.0–7.0 μm, Gram-positive, non-flagellum, straight or rod bacterium occurred singly or in pair.

Fig. 1

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

Physiological characteristics of Strain B10 were inspected by API 20A system (Park et al. 2015). The results (Table 1) showed that Strain B10 could utilize a variety of carbohydrates for growth except melezitose, rhamnose, raffinose, and glycerol. Strain B10 could neither produce indole, urease and contact enzyme, nor hydrolyze gelatin and nitrate. The physiological characteristics above suggested that Strain B10 was a bacterium of Clostridium.

Table 1 Physiological characteristics of the isolate Strain B10

According to the phylogenetic analysis of 16S rDNA gene sequence (GeneBank accession No. KY937197), a phylogenetic tree for Strain B10 rooted with C. butyricum was constructed. As shown in Fig. 2, Strain B10 had a similarity of 97% with C. butyricum Strain VPI 3266. However, the isolate was unable to grow on glycerol which could be utilized by Strain VPI 3266 to produce 1,3-propanediol (González-Pajuelo et al. 2004). Both phenotypical analysis and 16S rDNA gene analysis indicated the isolate was a novel strain belonging to C. butyricum and designed as C. butyricum B10.

Fig. 2

Phylogenetic position of the isolate Strain B10

Performance of C. butyricum B10 in fermentative butyric acid production from xylose

Effect of temperature on the performance of C. butyricum B10

Temperature is a critical factor in butyric acid fermentation by affecting the cell growth and enzymatic reaction (Da et al. 2012). To enhance the butyric acid production of Strain B10, its performance in xylose fermentation was investigated at 20, 25, 32, 37, 42, and 47 °C, respectively, with 10 g/L xylose and initial pH of 8.5 in the broth. As shown in Table 2, both VFAs production and cell growth were remarkably affected by fermentation temperature. The maximum butyric acid yield (BY) of about 2.55 g/L, with a selectivity (PB, percentage of butyric acid in the total VFAs) of about 72.44%, was obtained at 37 °C, as well as the maximum biomass (about 0.97 g/L) and the specific yield of butyric acid by consumed xylose (SBY, about 0.35 g/g xylose). The BY increased 2.57-fold with the temperature increasing from 20 to 37 °C, but decreased 11.59 folds when the temperature was further increased from 37 to 47 °C. Though there was no significant difference in BY, the butyric acid productivity (BP) of about 0.058 g/(L·h) at 37 °C was observably higher than that of 0.044 g/(L·h) at 32 °C, indicating 37 °C as the optimum temperature for fermentative butyric acid production from xylose. The present results were in accordance with previous studies in which the optimum temperature of 35–37 °C was identified for Clostridium sp. (Hahnke et al. 2014; Yin and Wang 2017).

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

It was interesting to find that the PB decreased from 88.39 to 72.44% following the temperature increasing from 20 to 37 °C, though the BY increased from 0.99 to 2.55 g/L. As known, the increased temperature within a certain range will promote growth and metabolism of the inhabitants (Da et al. 2012; Junghare et al. 2012). Furthermore, more energy would be needed by bacteria when cultured at a higher temperature, and acetic acid formation would produce more energy than that of butyric acid formation (Verhaart et al. 2010; Junghare et al. 2012). Thus, the metabolic alteration of Strain B10 resulted in the increase of VFAs production and decrease of acetate/butyrate ratio following the culturing temperature increased from 20 to 37 °C (Table 2). However, pH in the fermentation processes of Strain B10 was also found a decrease with an increase of following the increasing temperature (Table 2). It was essential to understand what effect of the pH variation had produced on the metabolic alteration of Strain B10.

Effect of initial pH on the performance of C. butyricum B10

As illustrated in Table 2, accumulation of acetic and butyric acids in xylose fermentation process increased following the temperature increasing from 20 °C to 37 °C, resulting in a decrease of pH from 5.85 to 4.58. Khanal et al. (2004) reported that a pH decrease would cause a metabolic alteration of bacteria and resulted in a variation of acetate/butyrate ratio. The changed acetate/butyrate ratio should be attributed to environmental changes such as pH, partial pressure of hydrogen and the accumulation of intermediate products. Angenent et al. (2004) indicated that a hydrogen partial pressure more than 60 Pa was more favorable for the production of butyric acid but acetic acid. However, the hydrogen partial pressure in the xylose fermentation processes of Strain B10 at 20, 25, 32, and 37 °C was counted to be about 56, 77, 157, and 157 kPa, respectively, indicating the hydrogen partial pressure was not the major factors affecting the PB. Zhu and Yang (2004) had investigated the effect of pH on butyric acid fermentation of xylose by C. tyrobutyricum ATCC 25755 and indicated that (1) the clostridia would produce more acetate and lactate at pH lower than 5.7 and 92) activity of phosphotransacetylase as the key enzyme controlling acetate formation was higher in the cells at pH 5.0 than that at pH 6.3. Therefore, pH was identified as the dominant factor affecting the PB, which should be further researched to enhance the BY in the xylose fermentation of Strain B10.

To enhance the BY in the xylose fermentation of Strain B10, the performance of the strain in batch fermentation was evaluated at various initial pHs with a constant temperature of 37 °C. The results (Table 3) revealed that the growth and butyric acid production of Strain B10 were pH dependent. The xylose uptake, biomass, HY, VFAs, SBY, and PB all increased following the initial pH increasing from 5.0 and to 9.0, but sharply decreased when the initial pH was further increased to 9.5 and 10.0. The BY was promoted by 11.73-fold with the initial pH increased from 5.0 to 9.0. Though the BYs, as well as SBYs and PBs, were not different significantly 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 pH 9.0 were obviously higher than that of the other pHs. As known, a higher pH means more alkalinity to neutralize the produced acetic and butyric acids during the batch fermentation, which was conducive to better growth and metabolism of acidogenic bacteria (Jo et al. 2008). With the maximal xylose uptake of about 5.25 g/L and BY of about 2.58 g/L, the initial pH 9.0 was suggested as the optimal for the batch fermentation of Strain B10 to produce butyric acid from xylose. The present results were accorded with that of Junghare and coworkers who also found that pH 9.0 was the optimal for the growth and fermentation of C. butyricum TM-9A in batch experiment (Junghare et al. 2012).

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

Effect of initial xylose concentration on the performance of C. butyricum B10

Concentration of xylose as substrate has been reported to affect cellular respiration and cell growth of fermentative bacteria, resulting in variation in performance of butyric acid fermentation (Khamtib and Reungsang 2012). Zhang et al. (2009b) investigated the effect of glucose concentration on the butyric acid production of C. thermobutyricum ATCC 49875 and a maximum yield of 12.05 g/L was obtained only with 34.9 g/L glucose in the broth. Song et al. (2010) reported that the favorable glucose for the maximum specific cell growth (0.426 h⁻¹) and butyric acid yield (62.48 g/L) of C. tyrobutyricum were obviously different (20 g/L and 120 g/L, respectively). When the glucose in the broth was as high as 150 g/L, the lag phase was found to be remarkbly extended to about 20 h. To optimize the xylose concentration for Strain B10 to produce butyrate, a series of xylose concentration (6~20 g/L) was evaluated.

As shown in Table 4, when xylose concentration in broth was increased from 6.00 to 8.00 g/L, the xylose uptake, HY, BY and PB increased from about 6.00 g/L, 58.69 mmol/L, 1.97 g/L and 70.12% to 7.52 g/L, 75.02 mmol/L, 2.52 g/L and 75.22%, respectively, without obvious difference in biomass, acetic acid yield (AY) and SBY. With more VFAs accumulated, the final pH decreased from 4.99 to 4.69 following the increased xylose concentration. When the xylose concentration was further increased from 8 to 10 and then to 20 g/L, no obvious difference in xylose uptake, biomass, final pH, HY, BY and SBY was observed. However, the AY increased obviously from about 0.83 to 0.90 g/L as the xylose concentration was increased from 8 to 20 g/L, resulting the PB decreased from about 75.22 to 73.76%. The results indicated that the decreased pH (1) was more favorable to the production of acetic acid but butyric acid (Zhu and Yang 2004) (2) had made the fermentation tend to be terminated (Wu and Yang 2003). Wu and Yang (2003) also found that acidogenic fermentation of bacteria would be terminated by low pH condition. More researches indicated that the lower pH would inhibit bacterial cell growth and metabolism by disrupting the transmembrane pH gradient, reducing ATP level and activities of acid-forming enzymes (Zigová and Šturdík 2000; Zhu and Yang 2003).

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

Kinetic analysis

To understand the kinetic characteristics of Strain B10 in xylose fermentation, batch fermentation was conducted again under the identified optimal 37 °C, initial pH 9.0 and 8 g/L xylose. The results (Fig. 3) showed that both biomass and BY found a logarithmic phase from the 8th to the 44th hour after a lag phase (0~8 h). By the end of the logarithmic phase at the 44th hour, the biomass and BY reached about 0.82 and 2.51 g/L, respectively, with a xylose absorptivity of about 97.02%.

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

As shown in Fig. 3, the growth of Strain B10 was well met with the Logistic model (Weiss and Ollis 1980), while BY was met well with the Leudeking-Piret model (Luedeking and Piret 2000), both of which were validated by an R² of 0.988 (Table 5). The results illustrated in Table 5 indicated the simulated x ₀ and μ_m by linear regression of Eq. (2) was 0.0165 g/L and 0.1466 h⁻¹, respectively. It was found that the simulated x ₀ (0.0165 g/L) was much lower than that of the experimental value (0.082 g/L), suggesting many cells in the inoculum was inactive and the improvement of inoculation methods would be helpful to optimizing the fermentation process. The generation time of Strain B10 under optimum condition was calculated to be 2.61 h based on the μ _m of 0.1466 h⁻¹. According to Ledueking-Piret model (Eq. (3)), α of Strain B10 reached 3.6274 g/g CDW.

Table 5 Kinetic parameters of C. butyricum B10 in xylose fermentation process

The fermentation efficiency of C. butyricum B10 compared with other clostridia

Though thermophilic fermentation is more efficient than mesophilic fermentation for hydrogen and VFAs production from various saccharides (Verhaart et al. 2010), fermentation 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 fermentation by species of Clostridium under mesophilic condition were collected with the results illustrated in Table 6. With the same mode of bath fermentation without pH control, the highest BY 3.79 g/L came to C. butyricum CGS5 with a PB of 42% (Lo et al. 2008). Though the BY 2.58 g/L of Strain B10 was lower than that of Strain CGS5, its PB reached 75.00% that was much higher than that of Strain CGS5. C. butyricum CGS2 (Lo et al. 2008), TM-9A (Junghare et al. 2012) and INET1 (Yin and Wang 2017) all performed not well in fermentative butyric acid production when compared their BY and PB with Strain CGS5 and Strain B10. The results indicated C. butyricum B10 a good performance in xylose utilization and a potential application to fermentative butyric acid production from lignocellulose. Obviously, batch fermentation (Fu et al. 2017) and fed batch fermentation (Liu and Yang 2006; Jiang et al. 2010; Baroi et al. 2015) with a constant pH were more feasible for effective butyric acid production than batch fermentation without pH control during fermentation process. The results suggested that fed batch fermentation with pH control should be further investigated to enhance the butyric acid production efficiency of C. butyricum B10.

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

Conclusion

The isolate Strain B10 was identified as a novel bacterium of C. butyricum, with acetic and butyric acids as the main liquid products. The optimal temperature and initial pH in batch fermentation was 37 °C and 9.0, respectively. Loaded with 8 g/L xylose in the broth, a butyric acid yield and specific butyric acid yield as high as 2.58 g/L and 0.36 g/g xylose, respectively, was obtained along with 73.86 mmol/L hydrogen collected. The cell growth rate and the specific butyric acid yield of the strain in xylose fermentation reached 0.1466 h⁻¹ and 3.6274 g/g CDW, respectively, indicating a good application potential in butyric acid production by lignocellulose fermentation.