Introduction

Owing to a limited supply of petroleum oil, increased environmental concerns, and an awareness of global warming, bioproduction of automotive fuels from renewable feedstocks has become a global priority [1]. Lignocellulosic biomass is the most abundant raw material on the planet for biofuel production. The low-carbon alcohols (such as ethanol and butanol) production by biological processes from lignocellulosic biomass has been proven to be technologically feasible.

Lignocellulose is composed of cellulose, hemicellulose, and lignin, which is not available to most of alcohol production microorganisms [2, 3]. Therefore, prior to fermentation, the carbohydrates contained in biomass should be hydrolyzed. Dilute acid hydrolysis is a very effective method, and the hydrolysate contains various sorts of monosaccharides, disaccharides, oligosaccharides, and small amounts of inhibitors [4]. The major monosaccharide components of lignocellulosic hydrolysate are glucose and xylose. Saccharomyces cerevisiae is considered to be the most effective ethanol producer with high ethanol yield, tolerance, and productivity. However, substantial amounts of pentose generated by hydrolysis of hemicellulose cannot be metabolized by wild type of S. cerevisiae due to the lack of xylose reductase and xylitol dehydrogenase activities, and the economy of ethanol production was affected [5].

Butanol production by acetone-butanol-ethanol (ABE) fermentation is now receiving more and more attention because of the chemical advantages of butanol as a new type of biofuel. First, the calorific value of butanol is higher than ethanol. Second, the noncorrosive property of butanol allows it to be transported with existing equipment for gasoline transportation. Third, butanol can be blended with gasoline at any ratio without modification of engines [6, 7]. Compared with bioethanol and biodiesel, biobutanol can be produced from wider material sources [8]. Clostridium acetobutylicum, an important solvents-producing organism, is capable of metabolizing a variety of carbohydrates [9]. Glucose and xylose in the lignocellulosic hydrolysate can be metabolized into acetone, butanol, and ethanol. However, butanol production by ABE fermentation from lignocellulosic hydrolysate is often limited by the poor solvents yield, the low solvent productivity, and the low final solvent titers [10].

In this study, the feasibility of mixed fermentation of S. cerevisiae and C. acetobutylicum CH02 from a mixture of glucose and xylose for solvents production was investigated. The aim of this work was to overcome the defect of xylose utilization in ethanol fermentation and to improve the solvents yield and productivity in ABE fermentation.

Materials and Methods

Strains, Media, and Seed Culture

Laboratory stocks of C. acetobutylicum CH02 spore suspensions were maintained in 5 % corn meal medium at 4 °C. C. acetobutylicum CH02 spores were heat-shocked at 100 °C for 90 s followed by cooling in cold water for 5 min. 0.5 mL of heat-shocked spores were inoculated in 10 mL of 5 % corn meal medium and then incubated at 37 °C for 24 h, after which 0.5 mL of actively growing culture was transferred to 10 mL of 5 % corn meal medium for seed culture at 37 °C for 12 h. It should be pointed out that the strain C. acetobutylicum CH02 was screened by adaptation which can grow well in the medium without sparging oxygen-free nitrogen gas before inoculation.

S. cerevisiae (purchased from Angel Yeast Co., Ltd.) was maintained on a YPD agar medium (yeast extract 10 g/L, peptone 20 g/L, glucose 20 g/L, and agar 20 g/L) slant in a 20-mL test tube at 4 °C. A loop of slant culture was inoculated into 50 mL of YPD medium (yeast extract 10 g/L, peptone 20 g/L, and glucose 20 g/L); subsequently, the medium was incubated for seed culture at 30 °C and 160 rpm for 24 h.

The media for ABE fermentation, ethanol fermentation, and mixed fermentation were the same, which composed of 25 g/L glucose and 25 g/L xylose. Wheat bran, which contains an adequate amount of nutrients, was found to be an ideal source of nitrogen and phosphorus in ABE, ethanol, and mixed fermentation. Wheat bran was used as nitrogen and phosphate sources in all batch fermentations without mineral supplement, and the supplementation level of wheat bran was based on ABE fermentation.

Batch Fermentation

Batch fermentations were carried out in 250-mL conical flasks filled with 200-mL medium incubated in a static incubator. The inoculation levels of C. acetobutylicum CH02 and S. cerevisiae were controlled at 5 % for batch ethanol, ABE, and mixed fermentations, and the temperature was controlled at 37 °C.

Analytical Methods

Solvents (acetone, butanol, and ethanol) were analyzed using gas chromatograph (Agilent 7890A GC), and sugars (xylose and glucose) were analyzed using high-performance liquid chromatography (Waters 2685 HPLC) according to our previous study [11].

Results and Discussion

Results of ABE Fermentation Under Different Wheat Bran Supplementation Conditions

Wheat bran is an available side product of the wheat milling industry. Its major constituents are nonstarch polysaccharides (46 %), starch (10–20 %), protein (15–22 %), lignin (4–8 %), and minor constituents [12]. Wheat bran has been used mainly as animal feed stock until now. There are reports about butanol production from wheat bran by ABE fermentation [13]. In this work, wheat bran was found to be an ideal crude nitrogen and phosphorus source for ABE fermentation. In the media optimization, a series of batch fermentations with the media supplemented with wheat bran ranging from 5.50 to 19.25 g/L was conducted, and the results are shown in Table 1.

Table 1 Results of ABE fermentation under different wheat bran supplementation conditions
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As can been observed in Table 1, ABE titers reached the highest level of 18.45 g/L when the addition of wheat bran was 11.00 g/L. When the addition of wheat bran was lower than 11.00 g/L, the residual sugars were high, which means the nitrogen and phosphorus source is not sufficient. ABE titers declined when the addition of wheat bran was higher than 11.00 g/L, indicating an effect of inhibitory on ABE fermentation. Under the best wheat bran supplementation condition, the ABE yield of 0.38 g/g was achieved.

Solvents Production and Sugars Utilization During ABE and Ethanol Fermentation

Profiles of sugars utilization and solvents production by ABE fermentation were illustrated in Fig. 1a; glucose was consumed quickly within 60 h, during which no xylose uptake was detected. Xylose uptake began at 60 h, which suggested the effect of CCR, and the effect has been reported by other investigators [14]. The solvents were not detected at 12 h, which suggested the transition from acidogenesis to solventogenesis occurred between 12 and 24 h. The ABE titers reached 18.45 g/L at the end of the fermentation at 120 h.

Fig. 1
figure 1

Sugars utilization and solvents production during ABE and ethanol fermentation (a, ABE fermentation; b, ethanol fermentation)

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Profiles of sugars utilization and ethanol production during ethanol fermentation were illustrated in Fig. 1b; glucose was metabolized and ethanol was produced quickly within 24 h, which suggested the metabolic rate of glucose in S. cerevisiae was much higher than that in C. acetobutylicum CH02. Xylose uptake was not observed during the whole fermentation process, which suggested that S. cerevisiae was unable to utilize xylose. A final ethanol titer of 11.46 g/L was obtained at 24 h.

Solvents Production and Sugars Utilization During Mixed Fermentation

Batch mixed fermentations with different inoculation methods were conducted, and the results were listed in Table 2. Ethanol yield from glucose by ethanol fermentation was 0.46 g/g, 90.20 % of the maximum theoretical yield (0.51 g/g). Total solvents yield from mixed sugars by ABE fermentation was 0.38 g/g, which was lower than the yield of ethanol fermentation. For mixed fermentation with different inoculation methods, the total solvents yields ranged from 0.37 to 0.41 g/g. The total solvent yields increased when the S. cerevisiae was inoculated earlier. The highest total solvents yield was achieved when S. cerevisiae was inoculated 24 h earlier than C. acetobutylicum CH02, with the inoculation levels of 5 %. At this condition, the total solvents yield was 0.41 g/g, compared to 0.38 g/g in ABE fermentation, increased of 7.9 %. Without doubt, more glucose was consumed by S. cerevisiae when S. cerevisiae was inoculated earlier, and consequently, higher solvents yield was achieved. Compared with ethanol fermentation, the residual xylose concentrations in mixed fermentations were much lower, which suggested that the xylose utilization defect was overcome in mixed fermentation. The final solvents titers in mixed fermentations ranged from 17.50 to 19.62 g/L, which were much higher than ethanol titer of 11.45 g/L in ethanol fermentation.

Table 2 Results of mixed fermentation with different inoculation methods
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The profiles of sugars utilization and solvents production during the mixed fermentation processes are shown in Fig. 2. In these inoculation methods, S. cerevisiae was inoculated 24, 12, and 0 h before C. acetobutylicum, respectively, and the seed culture conditions and inoculation levels of S. cerevisiae and C. acetobutylicum in these inoculation methods were the same as described in “Materials and Methods.” The fermentation time for mixed fermentation with the inoculation methods of SC-CA-24 h, SC-CA-12 h, and SC-CA-0 h was 96, 72, and 60 h, respectively. The fermentation time was prolonged when the S. cerevisiae was inoculated later than C. acetobutylicum CH02 (data not shown). The total solvents productivities obtained from the mixed fermentation with the inoculation methods of SC-CA-24 h, SC-CA-12 h, and SC-CA-0 h were 0.20, 0.27, and 0.32 g/L/h, compared with the productivity of 0.15 g/L/h obtained from ABE fermentation, increased of 33 %, 80 %, and 113 %, respectively. The CCR in mixed fermentation was alleviated when the glucose was metabolized quickly by S. cerevisiae, and consequently, the productivities were improved dramatically.

Fig. 2
figure 2

Sugars utilization and solvents production during mixed fermentation with different inoculation methods (a, SC-CA-24 h; b, SC-CA-12 h; c, SC-CA-0 h)

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Conclusions

In this work, the feasibility of mixed fermentation of S. cerevisiae and C. acetobutylicum CH02 from a mixture of glucose and xylose for solvents production was investigated, and the results showed that the method was successful. The xylose utilization defect in ethanol fermentation was overcome by mixed fermentation. The fast utilization of glucose by S. cerevisiae alleviated the CCR in mixed fermentation, and therefore, the fermentation time was reduced and the solvents productivity was improved. The solvents yield in mixed fermentation was improved as a result of the high ethanol yield from glucose by S. cerevisiae.