The feasibility of utilizing corn straws to produce butanol via fermentation with Clostridium acetobutylicum was evaluated. The supernatant of enzymatically hydrolyzed supernatant of steam-exploded corn straws was used as the raw material. A bacterial strain was selected from Clostridium acetobutylicum zzu-02 and Clostridium beijerinckii zzu-01, which was capable of fermenting the enzymatically hydrolyzed supernatant of steam-exploded corn straw to produce butanol with high yield. The optimal fermentation conditions for the selected strain with enzymatically hydrolyzed supernatant of steam-exploded corn straw were also investigated and they were determined as follows: sugar concentrations in enzymatically hydrolyzed solution of steam exploded corn straws, 57.5 g/L; initial pH, 6.3; the amount of added CaCO3, 5g/L; the bacterial inoculation concentration to enzymatically hydrolyzed solution, 6%; fermentation temperature, 37 oC, the amounts of the added nutritional elements, i.e. yeast extract, CH3COONH4, KH2PO4, and C6H6N2O, 0.8, 6.0, 0.5, and 0.25 g/L, respectively. Under these conditions, the butanol yield reached 9.88 g/L. Based on the butanol metabolism pathways, supplementation of a small amount of C6H6N2O was found to effectively increase the yield of butanol production.
Utilization of Steam-Exploded Corn Straw to Produce Biofuel Butanol via Fermentation with a Newly Selected Strain of Clostridium acetobutylicum
Xia Zhang,a,* Xiaohang Feng,b Hui Zhang,c and Yichang Wei a
The feasibility of utilizing corn straws to produce butanol viafermentation with Clostridium acetobutylicum was evaluated. The supernatant of enzymatically hydrolyzed supernatant of steam-exploded corn straws was used as the raw material. A bacterial strain was selected from Clostridium acetobutylicum zzu-02 and Clostridium beijerinckii zzu-01, which was capable of fermenting the enzymatically hydrolyzed supernatant of steam-exploded corn straw to produce butanol with high yield. The optimal fermentation conditions for the selected strain with enzymatically hydrolyzed supernatant of steam-exploded corn straw were also investigated and they were determined as follows: sugar concentrations in enzymatically hydrolyzed solution of steam exploded corn straws, 57.5 g/L; initial pH, 6.3; the amount of added CaCO3, 5g/L; the bacterial inoculation concentration to enzymatically hydrolyzed solution, 6%; fermentation temperature, 37 oC, the amounts of the added nutritional elements, i.e. yeast extract, CH3COONH4, KH2PO4, and C6H6N2O, 0.8, 6.0, 0.5, and 0.25 g/L, respectively. Under these conditions, the butanol yield reached 9.88 g/L. Based on the butanol metabolism pathways, supplementation of a small amount of C6H6N2O was found to effectively increase the yield of butanol production.
Keywords: Steam-exploded corn straw; Biofuel; Enzymatically hydrolyzed supernatant; Butanol; Clostridium acetobutylicum; Clostridium beijerinckii; Fermentation
Contact information: a: Institute of Environmental & Municipal Engineering, North China University of Water Resources Electric Power, Zhengzhou, 450011, China; b: School of Foreign Languages, Peking University, Beijing, 100871, China; c: School of Management, Henan University of Chinese Medicine, Zhengzhou, 450046, China; *Corresponding authors: email@example.com
Butanol is not only an important organic solvent and a raw material for chemical industry (Guo et al. 2012), but it also is a new type of promising biofuel ( Pyrgakis et al. 2016; Al-Shorgani et al. 2018; Cimino et al. 2018; Diaz and Tost 2018; Oh et al. 2018). Traditionally, acetone-butanol fermentation is a large-scale fermentation process using corn starch as the raw material (Luo et al.2017; Faraji et al. 2018). With the gradual exhaustion of fossil fuel resources, and the potential shortage of food and oil as well as the emergence of problems caused by unfavorable environmental factors such as the increasing greenhouse effects, utilization of agricultural wastes as the raw materials to produce chemical raw materials and the energy-generating substances has attracted more and more attention ( Zheng et al. 2017; Michaels et al. 2018).
Currently, utilization of the fermentation of biomass such as lignocellulose etc. to generate butanol is becoming a hot research topic (Ezeji et al. 2007; Qureshi et al. 2010; Farmanbordar et al.2018; Maiti et al. 2018; Perez-Bibbins et al. 2018). A number of factors such as the types of fermentation bacteria, raw materials, and the ways of pretreatment of raw materials, as well as the fermentation conditions all have significant impacts on the yield of solvent production. Dilute acid treatment of the ethanol extracted biodegradable fraction of municipal solid waste at 140 °C for 60 min resulted in a liquor containing 23 g/L glucose and 41 g/L soluble starch, which was fermented to the highest ABE (acetone-butanol-ethanol) concentration of 17 g/L with productivity of 0.24 g/L/h (Farmanbordar et al. 2018). Parameter optimization (time, pH, and substrate concentration) for acid-catalysed brewery industry liquid waste hydrolysate utilization using central composite model technique produced 307.9 g/kg glucose. Also, 10.62 g/L of ABE was produced by subsequent clostridial fermentation of the substrate (Maiti et al. 2018). The butanol concentration of the fermentation with hydrolytic solution of ‘sugarcorn’ by Clostridium beijerinckii was 8.3 g/L in 257 h (Chen et al. 2017).
The steam-explosion method is a way by which the biomass raw materials are at first steamed by the treatment with high temperature and high pressure for a certain period of time, and that is followed by a sudden decompression. By this way, the chemical decomposition of components of the raw materials, mechanical fracture, and structural recombination can be achieved (Boren et al. 2018). This study aimed to test the feasibility of utilizing corn straw to produce butanol via bacterial fermentation. By using the enzymatically hydrolyzed supernatant of steam-exploded corn straw (Zhang et al. 2017) as the raw material for fermentation, we selected a bacterial strain capable of generating butanol with higher yield and optimized its fermentation conditions. We also investigated the effects of supplement of a small amount of C6H6N2O on the yield of butanol production. This study could provide the experimental basis for increasing the yield of butanol production through proper utilization of the agricultural wastes.
The strain of Clostridium acetobutylicum zzu-02 was cultured and optimized by The Biochemical Industry Laboratory of College of Chemical Industry and Energy Resources, Zhengzhou University (Henan, China). The bacterial spores were cultured with 5% (w/w) corn mash in ratio of corn starch: deionized water of 1:20 and stored at 4 oC. Clostridium beijerinckii zzu-01 was conserved by The Biochemical Industry Laboratory of College of Chemical Industry and Energy Resources, Zhengzhou University. The bacterial spores were cultured in culture medium and stored at 4 oC.
Corn straw samples were collected from the experimental field (50 m×80 m) located at the Suburb of Nanyang City at Henan Province, China. Water steam was channeled to a sealed explosion tank containing naturally air-dried, 1 to 3 mm pieces of corn straw. The pressure within the tank was rapidly increased to 1.5 MPa and then maintained for 5 min. The pressure was suddenly decompressed. The steam-exploded corn straw made in this way were saved for subsequent use. The steam exploded corn straw contained 32.3% cellulose and 16.0% hemicellulose.
Formulation of culture mediums
Culture Medium for Clostridium acetobutylicum zzu-02: the commercially available corn starch obtained from the local market was directly autoclaved under 0.1 MPa at 121 oC for 15 min and dissolved in deionized water to make 5% (w/w) of corn mash with pH 6.3.
Culture Medium for Clostridium beijerinckii zzu-01: this medium contained 500 mL of tryptone pancreatic digest of casein, 500 mL of beef infusion broth, 5.0 g sodium chloride, 5.0 g glucose, 1 g agarose, 0.5 g sodium thioglycollate, and 10 g meat scraps and with pH 7.2 to 7.4.
Fermentation Medium: This medium containing 0.8 g yeast extract, 0.5 g KH2PO4, 6 g CH3COONH4, 0.25 g C6H6N2O, per litter was brought up to 1L with enzymatically hydrolyzed supernatant of steam exploded corn straws at pH 6.3.
Preparation of seed liquids
Clostrisdium acetobutylicum zzu-02 conserved in the freezer was taken out and inoculated into corn mash culture medium in a proportion of 6% (v/v), i.e. 6 mL of Clostrisdium acetobutylicum zzu-02 spore solution was inoculated into 94 mL of corn mash culture medium, and cultured anaerobically at a constant temperature of 37 oC for 25 h. Clostridium beijerinckii zzu-01 conserved in the freezer was taken out and inoculated into seed culture medium in proportion of 6% (same as above), and cultured anaerobically at a constant temperature of 37 oC for 28 h.
Screening bacterial strain
Clostridium beijerinckii zzu-01 and Clostridium acetobutylicum zzu-02 were inoculated, respectively, into the screening medium. At the end of fermentation, the butanol yields produced by two strains were checked and compared. The strain that generated higher yield of butanol was selected as the bacterial strain for this study.
The fermentation conditions were: Sugar concentrations in enzymatically hydrolyzed solution of steam exploded corn straws, 57.5 g/L; initial pH, 6.3; the amount of added CaCO3, 5g/L; the bacterial inoculation concentration to enzymatically hydrolyzed solution, 6%; fermentation temperature, 37 oC, the amounts of the added nutritional elements, i.e. yeast extract, CH3COONH4, KH2PO4, and C6H6N2O, 0.8, 6.0, 0.5, and 0.25 g/L, respectively.
Optimization of butanol fermentation conditions
Because the major components in the enzymatically hydrolyzed supernatant of steam-exploded corn straw are mainly sugars, supplementation of certain amounts of nutrients for bacterial growth can enable the bacterial cells to grow and ferment normally. In this study, in addition to testing bacterial strains, effects of initial pH value, the amounts of added CaCO3 and the sugar concentrations in the enzymatically hydrolyzed supernatant on the butanol yield were also examined. Moreover, the effects of nutritional elements including CH3COONH4 (A), yeast extract (B), KH2PO4 (C) and C6H6N2O (D) were examined using an orthogonal experiment, which was designed by using L9 (34) orthogonal experimental table (Table 1). Each orthogonal experiment was repeated three times.
Table 1. Orthogonal Factor Level Table
Fermentation of enzymatically hydrolyzed supernatant of steam-exploded corn straw
Three-hundred milliliters of fermentation culture medium was transferred to a 500 mL-conical flask. The prepared bacterial seed solution was inoculated into the fermentation culture medium in a proportion of 6% and incubated at a constant temperature of 37 oC for 70 h.
Measurement of the reducing sugar contents
The reducing sugar contents were measured by using 3,5-dinitrosalicylic acid (DNS) method as described previously (Ghose 1987).
Measurement of butanol content
Butanol content was measured by using gas chromatography with a GC7980 gas chromatograph (Shanghai SiMo Analytical Instruments LLC, Shanghai, China). Capillary column was used, and the temperature at sample injection port was 230 oC, column temperature was 70 oC, and the running time was 6 min. The program was set without increasing temperature and without splitting injection. The injection volume was 1 μL, the flow rate of H2 gas was 30 mL/min, the air flow rate was 300 mL/min, and the internal standard used was isobutanol.
Calculation of conversion rates
The conversion rates of butanol and total solvent were calculated using the following equations:
For both equations, the concentration units for butanol, total solvent and sugar residues were all expressed as g/L.
RESULTS AND DISCUSSION
Temperature is the important influencing factor for fermentation. The suitable fermented temperature of Clostridium acetobutylicum is 37 ℃ according to several studies (Xue et al. 2016; Dong et al. 2018; Luo et al. 2018). Suitable growth temperature for Clostridium acetobutylicum zzu-02 was also 37 ℃ in each experiment. Thus, corn stover enzymolysis liquid was fermented with Clostridium acetobutylicum zzu-02 at 37 ℃.
Screening Bacterial Strains
The bacterial strains were screened mainly based on the butanol yield produced by the tested strains. Analyses of the butanol yield produced by two strains after fermentation revealed that the butanol yield produced from the enzymatically hydrolyzed supernatant of steam-exploded corn straws containing 52 g/L reducing sugars by fermentation with Clostridium acetobutylicum zzu-02 and Clostridium beijerinckii zzu-01 was 6.925 and 5.238 g/L, respectively. The butanol yield produced by fermentation withClostridium acetobutylicum zzu-02 was significantly higher than that produced by fermentation with Clostridium beijerinckii–zzu-01. The difference in butanol yield between two strains may be because higher concentrations of carbon sources may cause significant inhibition of the growth of Clostridium beijerinckii (Lee et al. 2008). Thus, Clostridium acetobutylicum zzu-02 was selected as the experimental strain for this study.
Effects of Sugar Concentrations in Enzymatic Hydrolysis Solution on Butanol Yield
In order to enhance the butanol production intensity, the sugar concentration is required to be increased as high as possible in such circulation that no product inhibition occurs and the butanol yield is not reduced. An amount 15% (w/w) of steam exploded corn straws in a ratio of corn straw/deionized water of 3/20 was enzymatically hydrolyzed. The hydrolyzed solution was centrifuged at 1500 rpm for 10 min. The supernatant was saved and its sugar concentration was measured to be 57.5 g/L, which was relatively low. After fermentation, the concentration of sugar residues was low, indicating that the sugars in the fermented solution had almost been used up.
Fig. 1. Solvent concentrations in various concentrated supernatants after fermentation
In order to increase the sugar concentration, an attempt was made to concentrate the enzymatically hydrolyzed supernatant. The supernatant solution was concentrated to 75 (mass fraction, the same as below), 67, and 50% of the original supernatant, respectively. After concentration, the sugar concentrations reached 76.7, 86.2, and 115 g/L, respectively (Table 2). The concentrated supernatants with different sugar contents were fermented, and the changes in the butanol yield and total solvent yield were monitored and compared.
There were relatively large differences in butanol concentrations and total solvent concentrations in various concentrated supernatants after enzymatic hydrolysis (Fig. 1). The butanol concentration and total solvent concentration in the enzymatically hydrolyzed supernatants of the original supernatant were the highest. The higher the concentration degree was, the lower were the butanol concentration and total solvent concentration.
Table 2. Concentrations of Reducing Sugars in Various Concentrated Supernatants before and after Fermentation
There were also differences in reducing sugar contents in various concentrated supernatants before and after fermentation (Table 2). The higher the content of reducing sugar content was in the enzymatic hydrolysis solution of the original supernatant, the lower were the amounts of sugars that were consumed during fermentation.
Fig. 2. Effects of initial pH values on butanol yield
Fig. 3. Effects of the concentrations of the added CaCO3 on butanol yield
Because of the presence of substances that inhibit the bacterial growth in the enzymatically hydrolyzed solution of stream-exploded corn straw, with the increase in the sugar contents of concentrated solutions, the sugar content was increased and the concentrations of the inhibitory substances were also increased. Thus, when the enzymatically hydrolyzed supernatant was concentrated to 50% of the original supernatant, the butanol concentration was 1.13 g/L, which was very low. Thus, in order to increase the production intensity, appropriate pretreatment of corn straw is needed first before enzymatic hydrolysis. The inhibitory substances generated during enzymatic hydrolysis of steam-exploded corn straws were found to be mainly formic acid (4.05g/L) and furfural (3.86 g/L) etc. Pretreatment of the corn straws by washing with water before enzymatic hydrolysis can reduce the contents of formic acid and furfural and thus, can increase the butanol yield.
Effects of Initial pH Value, Amount of Added CaCO3 and Bacterial Inoculum Concentration on Butanol Yield
Another key factor that affects the butanol yield is the initial pH value. If the initial pH value is too low, it is not favorable for bacterial growth, whereas if the initial pH value is too high, it is not favorable for solvent production. After being detoxified, the pH value of the enzymatically hydrolyzed supernatant of steam-exploded corn straw was around 5.95±0.05. The pH values were adjusted with anhydrous ammonia. The effects of the changes in pH values on the butane yield were examined to determine the optimal initial pH value at which the butanol yield produced by fermentation of steam -exploded corn straw was the highest one. The effects of the initial pH values within the range of 5.9 to 7.0 on fermentation of steam-exploded corn straws were examined. It was found that within this initial pH range, there were not big differences in butanol yield and that when the initial pH value was 6.3, the butanol yield was slightly higher (Fig. 2).
When butanol is produced by pilot fermentation, during the fermentation process, the butanol yield reaches the maximal value only when the pH value was maintained in the range of 4.5 to 5.0. CaCO3 is an important buffering substance for butanol fermentation with steam-exploded corn straws. When corn straw was utilized to produce butanol, if CaCO3 was not added, the pH value would be reduced to 3.78 during fermentation, and this resulted in the fermentation solution that was too acidic, which was not favorable for the growth of Clostridium acetobutylicum and thus, affected the butanol yield. The effects of changes in CaCO3 concentrations at the range of 1 to 6 g/L in the fermentation solution on the butanol yield were investigated. The results showed that when the concentration of the added CaCO3 reached 5g/L, the butanol yield was the highest (Fig. 3). When its concentration was too high or too low, CaCO3reduced the butanol yield.
Fig. 4. Effects of inoculation concentrations on butanol yield
The bacterial inoculation concentrations also caused effects on butanol yield to a certain extent. The butanol yield was increased with the increase in inoculation concentrations. When the proportion of the inoculation concentration reached 6%, butanol yield was not further increased but was kept stable (Fig. 4). Thus, the proportion of 6% was selected as the inoculation concentration for fermentation in this study.
Effects of Supplement of Nutritional Elements on the Butanol Yield
The results of orthogonal experimentation showed that the influence degrees of different nutritional elements, i.e. CH3COONH4 (A), yeast extract (B), KH2PO4 (C), and C6H6N2O (D), which were supplemented to the enzymatically hydrolyzed supernatant on butanol yield were in the order (from high to low) of A>C>B>D. Among the nutritional elements, the effect of CH3COONH4 on butane yield was the highest one (Table 3). Analysis on the results of orthogonal experiment revealed that the combination of A2B3C1D2 was the optimal one. Under these conditions, the butanol concentration in enzymatically hydrolyzed supernatant was 9.726 g/L (Fig. 5).
Table 3. Results of L9（34）Orthogonal Experiment
When Clostridium beijerinckii was used as the bacterial strain and yeast extract was used as the bacterial nutrient, the yield of solvent production from the hydrolysis solution of wheat straw reached 0.6 g/(L·h) (Qureshi et al. 2007). Yeast extract is also suitable to be used as the nutrient for the fermentation of steam-exploded corn straw with of Clostridium acetobutylicum. Supplementation of CH3COONH4provided the nitrogen source for the growth of Clostridium acetobutylicum in the enzymatically hydrolyzed supernatant of steam-exploded corn straws. CH3COOH was also an important chemical for ABE fermentation, which was closely related to the effective expression of enzymes during the processes of acid production and solvent production (Gu et al. 2009). Thus, supplementing of the proper amount of CH3COONH4 was favorable for fermentation and could increase the butanol yield and the utilization rate of sugars. Among the trace nutritional elements, K+ is essential for cellular growth. When K+ concentration was zero, fermentation did not produce solvents but produced only acids. With the increase in K+ concentrations, the yield of solvent production was increased. However, when K+ concentration reached certain level, the yield of solvent production was not further increased but was kept constant (Yi et al. 2018). Thus, supplement of the appropriate amount of KH2PO4 not only provided a phosphate source but also provided K+, which was beneficial for increasing the yield of solvent production.
C6H6N2O is an important component for the formation of co-enzymes NAD and NADP. Both NAD and NADP are the co-enzymes of a number of dehydrogenases. The nicotinamide moiety of two co-enzymes has the functions of reversible hydrogenization and dehydrogenization, which plays an important role in transferring electrons and protons in the redox reactions during many biological oxidation processes (Lee et al. 2018). Within the butanol metabolism pathway, from the steps of acetyl-CoA to butanol, four dehydrogenases, i.e. 3-hydroxybutyrate dehydrogenase, isobutyryl coenzyme-A dehydrogenase, butyraldehyde dehydrogenase, and butanol dehydrogenase, are involved in these oxidation-reduction reactions. Thus, supplement of a small amount of C6H6N2O can accelerate butanol pathway and thus, is favorable for butanol production.
Fig. 5. Butanol from steam-exploded corn stover
Verification of the Experimental Results under the Optimized Conditions
In order to verify the reliability of the optimized fermentation conditions, the yield of solvent production with steam-exploded corn straws was compared to those with several other sugar sources including corn starch, enzymatically hydrolyzed solution of bran, and glucose solution.
It was found that the butanol yields produced by Clostridium acetobutylicum fermentation with steam-exploded corn straws, enzymatically hydrolyzed solution of bran and glucose solution were all around 10g/L. The yield produced with steam-exploded corn straws was slightly lower than that with corn starch but slightly higher than that with enzymatically hydrolyzed solution of bran (Table 4). These results indicate that the optimized conditions are suitable for butanol production by Clostridium acetobutylicumfermentation with steam-exploded corn straws as the raw materials.
Table 4. The Yields of Solvent Production by Clostridium Acetobutylicum Fermentation with Different Sources of Raw Materials
- Clostridium acetobutylicum zzu-02 was selected, which was capable of fermenting steam-exploded corn straws and producing higher butanol yield.
- The optimal conditions for fermentation by this strain with steam exploded corn straws were determined, and the butanol yield reached 9.88 g/L under these conditions.
- According to the butanol metabolism pathway, supplementation of a small amount of C6H6N2O can effectively increase the butanol yield
This work was financially supported by the National Key Research and Development Program of China (2017YFD0301102-5), the Doctoral Research Fund of North China University of Water Resources Electric Power, China (4001/40534), and the science and technology research project of Henan province (182102110029).
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Article submitted: February 15, 2018; Peer review completed: May 19, 2018; Article received and accepted: May 30, 2018; Published: June 11, 2018.