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An environment friendly and efficient process for xylitol bioconversion from enzymatic corncob hydrolysate by adapted Candida tropicalis
Chemical Engineering Journal 263 (2015) 249–256
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Chemical Engineering Journal journal homepage: www.elsevier.com/locate/cej
An environment friendly and efficient process for xylitol bioconversion from enzymatic corncob hydrolysate by adapted Candida tropicalis Zhe Li 1, Xiaoxiao Guo 1, Xudong Feng, Chun Li ⇑ School of Life Science, Beijing Institute of Technology, Beijing 100081, PR China
h i g h l i g h t s A green process was developed for hydrolysate preparation and xylitol production. Different conditions were investigated to improve xylanolytic enzymes production. Different parameters were optimized to prepare corncob hemicellulose hydrolysate. High xylitol yield was obtained from hydrolysate by adapted Candida tropicalis.
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Article history: Received 17 August 2014 Received in revised form 22 October 2014 Accepted 1 November 2014 Available online 8 November 2014 Keywords: Xylanolytic enzymes production Corncob hemicellulose hydrolysate Enzymatic hydrolysis Xylitol fermentation Candida tropicalis
a b s t r a c t This study reported an environment friendly process for xylitol bioconversion from corncob hemicellulose hydrolysate prepared through xylanolytic enzymes by adapted Candida tropicalis. In order to improve the production of xylanolytic enzymes from Aspergillus terreus, the fermentation medium and growth conditions were optimized. Maximum xylanase (722 U/g) and b-xylosidase (196 U/g) production were exhibited on wheat bran and corncob (mass ratio of 7:3) medium supplemented with 2% (v/v) NaNO3 and 0.05% (v/v) Tween 80 at pH 6.8, 1:1 (v/w) moisture level, 20% inoculum level and 30 °C for 120 h of cultivation. For preparation of corncob hemicellulose hydrolysate, the optimum enzymatic hydrolysis conditions contained enzyme dosage (30.6 U/g), water addition (19.2 mL/g), temperature (48.01 °C), pH (5.75). Under these conditions, the sugars of 18.03 g/L xylose and 4.87 g/L glucose were obtained in 8 h. The corncob hemicellulose hydrolysate was used for xylitol fermentation and the maximum xylitol yield of 75.14% was obtained by adapted C. tropicalis after the optimization of fermentation. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction Xylitol is a five-carbon sugar polyalcohol which can be applied to the food, medicine and other fields [1–3]. Because of its properties, such as similarity in sweetness to sucrose, no insulin requirement, inhibition of dental caries and low calories, xylitol is widely used as a sugar substitute [4,5]. Additionally, xylitol is identified as one of the twelve high added-value chemicals that can be produced from biomass. Xylitol can serve as a valuable synthetic building block for various chemical compounds like xylaric acid or glycols [6]. The traditional production of xylitol is through chemical hydrogenation of xylose over a Raney-Nickel catalyst [7], which includes
⇑ Corresponding author. Tel./fax: +86 10 68913171. 1
E-mail address: [email protected] (C. Li). These authors contributed equally to this study.
http://dx.doi.org/10.1016/j.cej.2014.11.013 1385-8947/Ó 2014 Elsevier B.V. All rights reserved.
high pressure and temperature, as well as expensive separation and purification steps. Compared with this process, bioconversion of xylitol from hemicelluloses hydrolysate by microorganisms represents a renewable process with moderate reaction conditions and low energy requirements, which ensures high product selectivity, low cost and safety [8]. It has been shown that Candida tropicalis is a desirable microorganism for xylitol production with high yield and volumetric productivity from xylose-containing hydrolysate [9]. Corncob is one of the most abundant agricultural wastes containing about 30% xylan-type hemicelluloses [10], which is recognized as satisfactory sources for xylitol synthesis [11]. However, corncob cannot be directly utilized by microorganisms, it should be pretreated by hydrolysis to produce sugars that are available for xylitol fermentation [12]. Autohydrolysis [13,14], high-pressure CO2–H2O treatment [15,16], steam explosion [17] and dilute sulfuric acid treatment [18] have been widely used to destroy the lignocellulosic structure and release the hemicelluloses sugars.
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However, hydrothermal treatments are time consuming and the remaining xylo-oligosaccharides cannot be completely hydrolyzed to xylose [19]. Moreover, the harsh conditions of these treatments could degrade the hemicelluloses sugars [20]. On the other hand, sulfuric acid used for hemicellulose hydrolysate preparation could cause environmental problems and damage the equipment [21]. In addition, acidic hydrolysate of hemicellulose comprises a complex mixture of components which have been recognized as fermentation inhibitors, such as organic acid (acetic, formic and levulinic acid), furan derivatives (furfural and 5-hydroxymethylfurfural) and phenolic compounds [22], necessitating complicated detoxification and purification treatments before xylitol fermentation which could raise the production cost [23]. As a consequence, chemical processes are gradually replaced by enzymatic ones which are specific and environment friendly. It has been demonstrated that endo-1,4-b-xylanase (EC 3.2.1.8) and b-xylosidase (EC 3.2.1.37) play a very important role in degrading xylan: xylan is firstly hydrolyzed into xylo-oligosaccharide by endo-1,4-bxylanase and then xylo-oligosaccharide is further hydrolyzed into xylose by b-xylosidase [24]. Many bacterial and fungal species are able to utilize xylan as a carbon source, among which Aspergillus terreus is an efficient producer of xylanolytic enzymes [25]. In this paper, in order to overcome the shortcomings observed in previous studies, an environment friendly xylitol producing bioprocess from enzymatic hemicellulose hydrolysate was developed which had moderate reaction conditions and low pollution. Production of xylanolytic enzymes from A. terreus was investigated and xylanolytic enzymes were then used to prepare hydrolysate. Xylitol fermentation from the hydrolysate by adapted C. tropicalis was evaluated. This bioprocess of xylitol production left out the use of toxic catalyst, the expensive detoxification and purification steps of xylose, and had the benefits of energy saving and environmental protection. 2. Materials and methods 2.1. Strains and media Xylanolytic enzymes producing strain A. terreus Li-20 and xylitol producing strain C. tropicalis BIT-Xol-1 were both originally isolated in Shihezi, Xinjiang, China and stored in laboratory. A. terreus Li-20 was maintained on PDA slants (sliced potatoes 200 g/L, dextrose 20 g/L, agar powder 20 g/L) at 4 °C and subcultured once every 6 weeks. C. tropicalis was maintained on YPD slants (10 g/L yeast extract, 20 g/L peptone and 20 g/L glucose) at 4 °C and sub-cultured once every 6 weeks. 2.2. Solid-state fermentation The lignocellulosic materials (corncob and wheat bran) were locally collected after harvesting (Southern suburb of Beijing, China). They were air-dried in the sun, milled into 10 mesh particles and stored until use. Solid-state fermentation (SSF) was performed for xylanolytic enzymes production by A. terreus Li-20. The experiments were conducted in 250 mL Erlenmeyer flasks which were sterilized at 121 °C for 20 min. In each flask, 10 g of corncob and wheat bran mixture (in solid state) with the mass ratio of 1:1 was used as basal solid carbon source, unless stated otherwise. 2% (NH4)2SO4 solution was used as nitrogen source. In order to moisten the solid substrate, the initial ratio of water to material was 1:1 (v/w). Different nitrogen sources (peptone, yeast extract, NaNO3, NH4NO3, NH4Cl and urea), different concentrations of Tween 80 (0.05%, 0.1% and 0.15%) and different ratios of water to material (0.5:1, 1:1, 1.5:1, 2:1 and 2.5:1) were also tested to find
the optimum fermentation conditions for xylanolytic enzymes production. To prepare the spore suspension, A. terreus Li-20 was cultured for 4–5 d at 30 °C on PDA slopes. Then, a solution of 0.1% Tween 80 (v/v) was added to each slope, and the spore suspension was made by lightly brushing the mycelium with a sterile wire loop. The spore suspension was then centrifuged, washed twice in distilled water and finally suspended in distilled water to give a final spore count of 107 spores/mL. The density of the suspension was measured using a haemocytometer. To each flask, different ratios (5, 10, 20, 30, v/w) of A. terreus Li20 spore suspension were inoculated in sterilized medium, stirred uniformly and incubated for 6 d at different temperatures (20 °C, 25 °C, 30 °C, 35 °C) and different initial pH which was adjusted by citrate acetate buffer (3.8, 4.8) and phosphate buffer solution (5.8, 6.8, 7.8). The flasks were gently tapped intermittently to prevent solidification of the medium. 1 mL of sterile water supplement was added every 24 h to keep the humidity of the culture medium. 2.3. Enzyme extraction After suitable periods of fermentation, xylanolytic enzymes were extracted from the solid materials with 10-fold (v/w) tap water by shaking (170 rpm) at room temperature for 60 min. The suspended materials and fungal biomass were separated by filtering through a nylon cloth and centrifugation (12,000g for 15 min at 4 °C). The clarified supernatant was used as crude enzymes. 2.4. Preparation of corncob hemicellulose hydrolysate The basal hydrolysis conditions for preparing corncob hemicellulose hydrolysate were as follows: enzyme dosage of 25 U/g corncob, water addition of 25 mL/g corncob, temperature of 50 °C, pH 5.0, and reaction time of 6 h. Then different enzyme dosages (5–50 U/g corncob), water additions (10–30 mL/g corncob), temperatures (40–60 °C), pH (4–8) and hydrolysis time (1–12 h) were tested to find the optimum conditions for preparation of corncob hemicellulose hydrolysate. 2.5. Experimental design To further optimize the hydrolysis conditions for preparing corncob hemicellulose hydrolysate, response surface methodology (RSM) based on Box–Behnken design was employed to get an integrated hydrolysis condition. The effects of enzyme dosage, water addition, temperature and pH on hydrolysate preparation were evaluated and selected in the four variable, three level RSM design (Table 1). Twenty-nine trials including twenty-four factorial points and five replicates of the central point were arranged by Design Expert 8.0 (Stat-Ease, Inc., MN, US). To certify the credibility of the RSM model, the analysis of variance (ANOVA) was applied. The experiment was carried out in a standard order.
Table 1 Independent variables and levels of RSM. Variables
Symbol
Levels
Enzyme dosage (U/g) Water addition (mL/g) Temperature pH
X1 X2 X3 X4
25 15 45 5.0
1
0
1
30 20 50 6.0
35 25 55 7.0
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2.6. Cell adaptation cultivation The cell adaptation cultivation was performed in 100 mL Erlenmeyer flask with 40 mL of corncob hemicellulose hydrolysate. A single colony of C. tropicalis was inoculated and the culture was grown for 24 h at 35 °C and 170 rpm. An aliquot of the resulting culture was used to inoculate in fresh corncob hemicellulose hydrolysate and the culture was grown for 24 h at 35 °C and 170 rpm. After 25 batches of adaptation cultivation, the culture was spread on corncob hemicellulose hydrolysate plate and the adapted cell which grown fast was checked for their ability to produce xylitol. 2.7. Xylitol bioconversion conditions A single colony of C. tropicalis and adapted C. tropicalis was inoculated into 100 mL Erlenmeyer flask containing 40 mL of YPD medium (10 g/L yeast extract, 20 g/L peptone and 20 g/L glucose), respectively, and the culture was grown for 12 h at 35 °C and 170 rpm. An aliquot of the resulting culture was used to inoculate in 1000 mL Erlenmeyer flask containing 400 mL of corncob hemicellulose hydrolysate (with 10 g/L extra glucose supply) with initial optical density (OD) at 660 nm of 0.5. Fermentations were performed at 35 °C and 170 rpm for 2 days. The hydrolysate was concentrated in a rotary vacuum evaporator at 60 °C and then used for fermentation. Two-stage fermentation was performed at 35 °C and 170 rpm for initial 6 h, then the shaking speed was change to 120 rpm. 2.8. Xylanase activity assay All enzyme activities were assayed using birchwood xylan as the substrate according to the method of Bailey et al. [26]. Briefly, the 1 mL reaction mixture consisting of 0.1 mL of appropriately diluted crude enzyme and 0.9 mL of 0.2 M sodium phosphate buffer (pH 7.0) containing 1% (w/v) birchwood xylan was incubated at 50 °C for 10 min. The reaction was stopped by adding 1.5 mL of 3,5-dinitrosalicylic acid reagent. The mixture was then boiled for 5 min followed by immediate chilling on ice. The amount of reducing sugars released was determined by the standard dinitrosalicylic acid method [27]. One unit of xylanase activity was defined as the amount of enzyme producing 1 lmol of reducing sugar (xylose) from the substrate solution per minute under the assay conditions. 2.9. b-Xylosidase activity assay The enzyme activity of b-xylosidase was determined by measuring pNP released from pNPX at 50 °C [28]. The reaction mixture consisting of 5 mM pNPX in 50 mM phosphate buffer (pH 6.0) was incubated with the enzyme for 10 min in a total volume of 0.25 ml. The reaction was stopped by the addition of 0.75 ml of 2 M Na2CO3, and the amount of pNP released was determined by measuring the absorbance at 410 nm. One unit of b-xylosidase activity was defined as the amount of enzyme required to release 1 lmol of pNP per minute under the conditions described above. 2.10. HPLC analysis The fermentation culture for the xylitol bioconversion (Section 2.6) was centrifuged at 10,000g for 5 min and the supernatant was used for determination of glucose, xylose, xylitol, acetic acid and ethanol. The concentrations of glucose, xylose, xylitol, acetic acid and ethanol were determined by HPLC system, model Shimadzu SCL-10A (Kyoto, Tokyo, Japan) with a RID-10A detector and a Bio-Rad Aminex HPX-87H (300 mm 7.8 mm) column. A
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5 mM H2SO4 solution was used as mobile phase at a flow rate of 0.6 mL/min and the analysis was carried out at 65 °C. 3. Results and discussion 3.1. Optimization of fermentation medium for xylanolytic enzymes production from A. terreus A. terreus which could produce xylanolytic enzymes was isolated from Xinjiang Province in China. In order to improve the production of xylanolytic enzymes, the medium of solid-state fermentation was optimized. Wheat bran and corncob are considered as efficient carbon source which could induce production of xylanolytic enzymes [29,30]. As shown in Fig. 1A, using wheat bran or corncob as solo carbon source could not induce xylanolytic enzymes efficiently. However, the mixture of corncob and wheat bran was the better inducer for xylanolytic enzymes production. When the ratio of wheat bran to corncob was 7:3, the highest xylanase activity of 631 U/g and the second highest xylosidase activity of 63 U/g were obtained, respectively. Following that, different nitrogen sources were selected to improve the xylanolytic enzymes production. As can be seen from Fig. 1B, NaNO3 was the best nitrogen source for xylanase production and the second best for xylosidase production. Although the strain could produce the highest xylosidase using yeast extract, the xylanase production was much lower than that using NaNO3. Furthermore, yeast extract was uneconomical for large scale production of xylanolytic enzymes due to its high cost; therefore, NaNO3 was the most suitable nitrogen source for strain to produce xylanolytic enzymes. Tween 80 is a good surface active agent which could enhance the xylanolytic enzymes production [31], as shown in Fig. 1C, Tween 80 with the concentration of 0.05% (v/v) could increase the xylanase and xylosidase production by 19.3% and 173.6%, respectively. As the moisture content of the medium has a critical effect on SSF [32], different initial water content in the medium was tested. As shown in Fig. 1D, when the ratio of water to material was 1:1 (v/w), the strain could produce the highest xylanolytic enzymes. In summary, the optimum nutrient medium composition was as follows: the ratio of wheat bran to corncob 7:3, 2% (v/v) NaNO3 with 0.05% (v/v) Tween 80 and the ratio of water to material 1:1 (v/w). 3.2. Optimization of growth conditions of A. terreus for xylanolytic enzymes production The growth conditions of A. terreus are also very important for the production of xylanolytic enzymes. Firstly, the effect of temperature on xylanolytic enzymes production was examined, as shown in Fig. 2A, xylanolytic enzymes production increased with the increase of temperature from 20 to 30 °C. As the temperature further increased to 37 °C, a decline in xylanolytic enzymes activity (28% and 22% for xylanase and xylosidase, respectively) was observed. This result was similar to some references, where the highest xylanase titers in fungal systems are obtained at temperature that is optimum for growth of cultures in SSF [33]. Then, the effect of initial pH of the medium on xylanolytic enzymes production was also investigated. As can be seen from Fig. 2B, the optimum pH for strain to produce xylanolytic enzymes was 6.8, it was similar to other strain reported [30]. It has been revealed that environmental parameters can influence the levels of xylanolytic enzymes production; moreover, the effect of inoculum level on the production of xylanolytic enzymes was also studied ranging from 5% to 30% (v/w). The maximal xylanolytic enzymes yields were obtained with 20% (v/w) of inoculum, although a marked difference was not found at lower or higher inoculum concentration (10% and 30%) (Fig. 2C). As xylanolytic enzymes were induced
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Fig. 1. Effects of medium contents on xylanase and xylosidase production. (A) Xylanase and xylosidase production under SSF with different carbon sources. (B) Xylanase and xylosidase production under SSF with different nitrogen sources. (C) Xylanase and xylosidase production under SSF with different concentration of Tween 80. (D) Xylanase and xylosidase production under SSF with different ratio of water to material in the medium.
during the strain growth, cultivation time was a crucial fermentation parameter affecting xylanolytic enzymes production. The result indicated the optimum cultivation time was 120 h, and further increase in incubation time did not show any increment in enzyme production (Fig. 2D). Under the optimum growth conditions with the optimum nutrient medium aforementioned, the xylanase and xylosidase activities were 722 U/g and 196 U/g, respectively, which were 31% and 202% higher in comparison to the activities obtained with the basic medium and growth condition. 3.3. Preparation of corncob hemicellulose hydrolysate The xylanolytic enzymes produced by A. terreus were used to hydrolyze corncob hemicellulose for xylitol synthesis, and different conditions of hydrolysis were investigated. Firstly, the xylanase dosage was tested, as shown in Fig. 3A, the concentration of monosaccharide increased with the increase of dosage from 5 to 30 U/g corncobs, further increase in the enzyme dosage could not improve the hydrolysis of the corncob, and therefore the proper dosage was 30 U/g for the hydrolysis. Water addition was another factor which could affect the hydrolysis, as can be seen from Fig. 3B, the maximum monosaccharide was obtained with water addition of 20 mL/g corncob. Fig. 3C showed the concentration of monosaccharide obtained at different temperatures. Temperature had a significant effect on the production of monosaccharide: the maximum monosaccharide was obtained at 50 °C and the concentration of monosaccharide decreased dramatically as the change of temperature. Then, the pH of hydrolysis was also tested. It was found that the optimum pH for the hydrolysis of corncob was 6 (Fig. 3D). In addition, glucose and xylose showed a very different production
time course: the glucose concentration reached stable after 3 h of hydrolysis, and xylose concentration kept increasing till 8 h. In order to further optimize the preparation of hydrolysate, response surface methodology (RSM) was used for investigation of the interactive effects among the parameters. The designed twentynine trials were displayed along with the results (Supplementary Table S1). According to the RSM mathematical model, the optimal conditions were found as enzyme dosage of 30.6 U/g, water addition of 19.2 mL/g, temperature of 48.01 °C and pH of 5.75, under such circumstances, 18.03 g/L xylose and 4.87 g/L glucose could be obtained after 8 h hydrolysis (Details in Supplementary material). The common methods used for hydrolysis of lignocellulosic materials include autohydrolysis [13,14], high-pressure CO2–H2O treatment [15,16], steam explore [17], alkali [34] and dilute sulfuric acid treatment [35]. The processes of autohydrolysis, high-pressure CO2–H2O treatment and steam explore need no chemicals (such as acid or alkali), but they are typically conducted at high temperature and pressure. Furthermore, xylo-oligosaccharides were the major liquor components using these methods for hydrolysis of lignocellulosic materials; therefore, the hydrolysates are not suitable for fermentation by microorganisms [13–17]. On the other hand, the xylose production of the alkali treatment is relative low [34]. Compared with other hydrolysis methods, dilute sulfuric acid treatment is especially useful for the conversion of xylan to xylose which can be further fermented to xylitol or ethanol by many microbial strains [18]. However, tradition chemical process for hemicellulose hydrolysate preparation includes acid, high pressure and temperature causing severe environmental pollution and equipment corrosion [36]. Moreover, a number of fermentation inhibitors such as acetic acid, formic acid, furfural,
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Fig. 2. Effects of growth conditions on xylanase and xylosidase production. (A) Influence of temperature on xylanase and xylosidase production under SSF. (B) Influence of pH on xylanase and xylosidase production under SSF. (C) Influence of inoculation amount on xylanase and xylosidase production under SSF. (D) Influence of cultivation time on xylanase and xylosidase production under SSF.
hydroxymethylfurfural (HMF) and phenolic compounds are generated during the acid hydrolysis process [37], which causes critical problems for the following bioconversion through fermentation. By comparison, enzymatic hydrolysis of lignocellulosic material used in this study had a lower environmental impact, less hazardous process chemicals, moderate process conditions and generated less inhibitor to fermentation later. Therefore, enzymatic hydrolysis could be an alternative environment friendly process for hemicellulose hydrolysate preparation. 3.4. Xylitol conversion using hemicellulose hydrolysate The corncob hemicellulose hydrolysate prepared by xylanolytic enzymes was used for xylitol conversion by C. tropicalis. As shown in Fig. 4A, the fermentation process generally had three stages and the highest xylitol concentration of 2.67 g/L was obtained at 12 h. In the first stage, the glucose was consumed within 9 h and ethanol was quickly produced by the C. tropicalis. In the middle stage, part of xylose and ethanol were used as carbon source for the cell growth and the xylitol accumulated quickly. In the last stage, the xylose and ethanol were completely consumed and xylitol started to decrease since it was used as carbon source for the metabolism of the cell. In general, the yield of xylitol was very low and it was only 14.81%. In addition, it should be noted that there was relatively high concentration of acetic acid in hydrolysate which could affect the xylitol conversion and cause the low yield of xylitol [38]. Apart from this, no other inhibitors could be detected. In order to increase the tolerance capacity of C. tropicalis to acetic acid, cell adaptation cultivation was adopted. According to the results, the xylitol concentration was increased to 8.18 g/L after 25 batches of cell adaptation cultivation and the corresponding yield was increased to 45.37%, which were about twice higher compared
with that of the wild C. tropicalis (Fig. 4B). Because glucose was consumed quickly and its concentration was low in hydrolysate; the xylose was used as carbon source for the cell growth instead of xylitol conversion which affected the yield of xylitol. Therefore, additional glucose was added into the hydrolysate to improve the xylitol conversion. As shown in Fig. 4B, the xylitol concentration was 11.74 g/L and the yield was 65.11%, respectively, increased by 43.5% compared to that without glucose addition. Although the addition of glucose improved the xylitol production, the cost could also increase due to the price of glucose. In order to obtain the higher concentration of glucose which could help the cell growth, the hydrolysate was concentrated, moreover the higher xylose concentration could benefit the xylitol conversion [39]. As can be seen from Fig. 4B, the adopted C. tropicalis produced 30.52 g/L xylitol with the concentrated hydrolysate, and the yield was 61.04% which was slightly lower than that of fermentation with glucose addition, however, this process was more economical because of no nutrients supplementation in hydrolysate. For the xylitol producing strain, oxygen is the most critical factor to the conversion from xylose to xylitol. It is evident that semi-aerobic condition is the best for the xylitol formation, under which NADPH produced by the pentose-phosphate pathway (PPP) is almost entirely addressed to xylitol formation [40]. Therefore, two-stage fermentation [41] was employed for the improvement of xylitol production. As shown in Fig. 4B, the xylitol concentration was 37.57 g/L and the yield was 75.14%, respectively. From the results, it was indicated that the two-stage fermentation was the optimum process for xylitol bioconversion directly from the hydrolysate. The industrial production of xylitol is currently based on the chemical hydrogenation process using nickel as the catalyst [7]. However, the drawbacks of the chemical conversion process are relatively low xylitol yields because chemical reduction produces
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Fig. 3. Effects of enzyme dosage, ratio of water to corncob mass, temperature, pH and hydrolysis time on preparation of corncob hemicellulose hydrolysate. (A) Production of glucose and xylose in the corncob hemicellulose hydrolysate with different enzyme dosage. (B) Production of glucose and xylose in the corncob hemicellulose hydrolysate with different ratio of water to corncob mass. (C) Production of glucose and xylose in the corncob hemicellulose hydrolysate with different temperature. (D) Production of glucose and xylose in the corncob hemicellulose hydrolysate with different pH. (E) Production of glucose and xylose in the corncob hemicellulose hydrolysate with different hydrolysis time.
byproducts [42]. Therefore, direct xylitol production from hemicellulose hydrolysate appears to be a promising alternative by the natural xylose-fermenting yeast. In this study, C. tropicalis was used for xylitol production from enzymatic hemicellulose hydrolysate, after the cell adaptation cultivation and optimization of fermentation, the yield of xylitol could reach 75.14% which was the same level as previous reports [43–45]. Furthermore, by
comparison with these methods, the process used in this study was simpler because the detoxification of hydrolysate was not necessary, and no nutrients supplementation was needed in the fermentation. Therefore, fermentation with enzymatic corncob hydrolysate was an environment friendly, economical and efficient method to produce xylitol, demonstrating a wide potential application in xylitol bioconversion.
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Fig. 4. (A) The xylitol production by C. tropicalis using corncob hemicellulose hydrolysate prepared by xylanolytic enzymes. d, xylose; j, xylitol; , glucose; N, ethanol; ., acetic acid. (B) Results of xylitol production by C. tropicalis (j), adapted C. tropicalis (d), adapted C. tropicalis with extra supply of glucose (N), adapted C. tropicalis with concentrated hydrolysate (.) and adapted C. tropicalis with concentrated hydrolysate under two-stage fermentation ().
4. Conclusions In this study, an environment friendly process was developed for enzymatic corncob hemicellulose hydrolysate preparation and xylitol bioconversion. Under the optimum conditions, maximum xylanase (722 U/g) and b-xylosidase (196 U/g) were produced by A. terreus. In order to prepare corncob hemicellulose hydrolysate, different parameters were optimized, and 18.03 g/L xylose and 4.87 g/L glucose were obtained. After adaption cultivation of C. tropicalis and optimization of fermentation, the xylitol yield of 75.14% was obtained. This process was demonstrated to be a green way to obtain corncob hemicellulose hydrolysate and a highly efficient method for xylitol production. Acknowledgements This work was financially supported by the National High Technology Research and Development Program of China (863 Program) (No. 2012AA02A704), the Major State Basic Research Development Program of China (973 Program) (No. 2013CB733900), National Natural Science Foundation of China
(Nos. 21176028, 21376028) and The National Research Foundation for the Doctoral Program of Higher Education of China (No. 20121101110050).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cej.2014.11.013.
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Chemical Engineering Journal journal homepage: www.elsevier.com/locate/cej
An environment friendly and efficient process for xylitol bioconversion from enzymatic corncob hydrolysate by adapted Candida tropicalis Zhe Li 1, Xiaoxiao Guo 1, Xudong Feng, Chun Li ⇑ School of Life Science, Beijing Institute of Technology, Beijing 100081, PR China
h i g h l i g h t s A green process was developed for hydrolysate preparation and xylitol production. Different conditions were investigated to improve xylanolytic enzymes production. Different parameters were optimized to prepare corncob hemicellulose hydrolysate. High xylitol yield was obtained from hydrolysate by adapted Candida tropicalis.
a r t i c l e
i n f o
Article history: Received 17 August 2014 Received in revised form 22 October 2014 Accepted 1 November 2014 Available online 8 November 2014 Keywords: Xylanolytic enzymes production Corncob hemicellulose hydrolysate Enzymatic hydrolysis Xylitol fermentation Candida tropicalis
a b s t r a c t This study reported an environment friendly process for xylitol bioconversion from corncob hemicellulose hydrolysate prepared through xylanolytic enzymes by adapted Candida tropicalis. In order to improve the production of xylanolytic enzymes from Aspergillus terreus, the fermentation medium and growth conditions were optimized. Maximum xylanase (722 U/g) and b-xylosidase (196 U/g) production were exhibited on wheat bran and corncob (mass ratio of 7:3) medium supplemented with 2% (v/v) NaNO3 and 0.05% (v/v) Tween 80 at pH 6.8, 1:1 (v/w) moisture level, 20% inoculum level and 30 °C for 120 h of cultivation. For preparation of corncob hemicellulose hydrolysate, the optimum enzymatic hydrolysis conditions contained enzyme dosage (30.6 U/g), water addition (19.2 mL/g), temperature (48.01 °C), pH (5.75). Under these conditions, the sugars of 18.03 g/L xylose and 4.87 g/L glucose were obtained in 8 h. The corncob hemicellulose hydrolysate was used for xylitol fermentation and the maximum xylitol yield of 75.14% was obtained by adapted C. tropicalis after the optimization of fermentation. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction Xylitol is a five-carbon sugar polyalcohol which can be applied to the food, medicine and other fields [1–3]. Because of its properties, such as similarity in sweetness to sucrose, no insulin requirement, inhibition of dental caries and low calories, xylitol is widely used as a sugar substitute [4,5]. Additionally, xylitol is identified as one of the twelve high added-value chemicals that can be produced from biomass. Xylitol can serve as a valuable synthetic building block for various chemical compounds like xylaric acid or glycols [6]. The traditional production of xylitol is through chemical hydrogenation of xylose over a Raney-Nickel catalyst [7], which includes
⇑ Corresponding author. Tel./fax: +86 10 68913171. 1
E-mail address: [email protected] (C. Li). These authors contributed equally to this study.
http://dx.doi.org/10.1016/j.cej.2014.11.013 1385-8947/Ó 2014 Elsevier B.V. All rights reserved.
high pressure and temperature, as well as expensive separation and purification steps. Compared with this process, bioconversion of xylitol from hemicelluloses hydrolysate by microorganisms represents a renewable process with moderate reaction conditions and low energy requirements, which ensures high product selectivity, low cost and safety [8]. It has been shown that Candida tropicalis is a desirable microorganism for xylitol production with high yield and volumetric productivity from xylose-containing hydrolysate [9]. Corncob is one of the most abundant agricultural wastes containing about 30% xylan-type hemicelluloses [10], which is recognized as satisfactory sources for xylitol synthesis [11]. However, corncob cannot be directly utilized by microorganisms, it should be pretreated by hydrolysis to produce sugars that are available for xylitol fermentation [12]. Autohydrolysis [13,14], high-pressure CO2–H2O treatment [15,16], steam explosion [17] and dilute sulfuric acid treatment [18] have been widely used to destroy the lignocellulosic structure and release the hemicelluloses sugars.
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However, hydrothermal treatments are time consuming and the remaining xylo-oligosaccharides cannot be completely hydrolyzed to xylose [19]. Moreover, the harsh conditions of these treatments could degrade the hemicelluloses sugars [20]. On the other hand, sulfuric acid used for hemicellulose hydrolysate preparation could cause environmental problems and damage the equipment [21]. In addition, acidic hydrolysate of hemicellulose comprises a complex mixture of components which have been recognized as fermentation inhibitors, such as organic acid (acetic, formic and levulinic acid), furan derivatives (furfural and 5-hydroxymethylfurfural) and phenolic compounds [22], necessitating complicated detoxification and purification treatments before xylitol fermentation which could raise the production cost [23]. As a consequence, chemical processes are gradually replaced by enzymatic ones which are specific and environment friendly. It has been demonstrated that endo-1,4-b-xylanase (EC 3.2.1.8) and b-xylosidase (EC 3.2.1.37) play a very important role in degrading xylan: xylan is firstly hydrolyzed into xylo-oligosaccharide by endo-1,4-bxylanase and then xylo-oligosaccharide is further hydrolyzed into xylose by b-xylosidase [24]. Many bacterial and fungal species are able to utilize xylan as a carbon source, among which Aspergillus terreus is an efficient producer of xylanolytic enzymes [25]. In this paper, in order to overcome the shortcomings observed in previous studies, an environment friendly xylitol producing bioprocess from enzymatic hemicellulose hydrolysate was developed which had moderate reaction conditions and low pollution. Production of xylanolytic enzymes from A. terreus was investigated and xylanolytic enzymes were then used to prepare hydrolysate. Xylitol fermentation from the hydrolysate by adapted C. tropicalis was evaluated. This bioprocess of xylitol production left out the use of toxic catalyst, the expensive detoxification and purification steps of xylose, and had the benefits of energy saving and environmental protection. 2. Materials and methods 2.1. Strains and media Xylanolytic enzymes producing strain A. terreus Li-20 and xylitol producing strain C. tropicalis BIT-Xol-1 were both originally isolated in Shihezi, Xinjiang, China and stored in laboratory. A. terreus Li-20 was maintained on PDA slants (sliced potatoes 200 g/L, dextrose 20 g/L, agar powder 20 g/L) at 4 °C and subcultured once every 6 weeks. C. tropicalis was maintained on YPD slants (10 g/L yeast extract, 20 g/L peptone and 20 g/L glucose) at 4 °C and sub-cultured once every 6 weeks. 2.2. Solid-state fermentation The lignocellulosic materials (corncob and wheat bran) were locally collected after harvesting (Southern suburb of Beijing, China). They were air-dried in the sun, milled into 10 mesh particles and stored until use. Solid-state fermentation (SSF) was performed for xylanolytic enzymes production by A. terreus Li-20. The experiments were conducted in 250 mL Erlenmeyer flasks which were sterilized at 121 °C for 20 min. In each flask, 10 g of corncob and wheat bran mixture (in solid state) with the mass ratio of 1:1 was used as basal solid carbon source, unless stated otherwise. 2% (NH4)2SO4 solution was used as nitrogen source. In order to moisten the solid substrate, the initial ratio of water to material was 1:1 (v/w). Different nitrogen sources (peptone, yeast extract, NaNO3, NH4NO3, NH4Cl and urea), different concentrations of Tween 80 (0.05%, 0.1% and 0.15%) and different ratios of water to material (0.5:1, 1:1, 1.5:1, 2:1 and 2.5:1) were also tested to find
the optimum fermentation conditions for xylanolytic enzymes production. To prepare the spore suspension, A. terreus Li-20 was cultured for 4–5 d at 30 °C on PDA slopes. Then, a solution of 0.1% Tween 80 (v/v) was added to each slope, and the spore suspension was made by lightly brushing the mycelium with a sterile wire loop. The spore suspension was then centrifuged, washed twice in distilled water and finally suspended in distilled water to give a final spore count of 107 spores/mL. The density of the suspension was measured using a haemocytometer. To each flask, different ratios (5, 10, 20, 30, v/w) of A. terreus Li20 spore suspension were inoculated in sterilized medium, stirred uniformly and incubated for 6 d at different temperatures (20 °C, 25 °C, 30 °C, 35 °C) and different initial pH which was adjusted by citrate acetate buffer (3.8, 4.8) and phosphate buffer solution (5.8, 6.8, 7.8). The flasks were gently tapped intermittently to prevent solidification of the medium. 1 mL of sterile water supplement was added every 24 h to keep the humidity of the culture medium. 2.3. Enzyme extraction After suitable periods of fermentation, xylanolytic enzymes were extracted from the solid materials with 10-fold (v/w) tap water by shaking (170 rpm) at room temperature for 60 min. The suspended materials and fungal biomass were separated by filtering through a nylon cloth and centrifugation (12,000g for 15 min at 4 °C). The clarified supernatant was used as crude enzymes. 2.4. Preparation of corncob hemicellulose hydrolysate The basal hydrolysis conditions for preparing corncob hemicellulose hydrolysate were as follows: enzyme dosage of 25 U/g corncob, water addition of 25 mL/g corncob, temperature of 50 °C, pH 5.0, and reaction time of 6 h. Then different enzyme dosages (5–50 U/g corncob), water additions (10–30 mL/g corncob), temperatures (40–60 °C), pH (4–8) and hydrolysis time (1–12 h) were tested to find the optimum conditions for preparation of corncob hemicellulose hydrolysate. 2.5. Experimental design To further optimize the hydrolysis conditions for preparing corncob hemicellulose hydrolysate, response surface methodology (RSM) based on Box–Behnken design was employed to get an integrated hydrolysis condition. The effects of enzyme dosage, water addition, temperature and pH on hydrolysate preparation were evaluated and selected in the four variable, three level RSM design (Table 1). Twenty-nine trials including twenty-four factorial points and five replicates of the central point were arranged by Design Expert 8.0 (Stat-Ease, Inc., MN, US). To certify the credibility of the RSM model, the analysis of variance (ANOVA) was applied. The experiment was carried out in a standard order.
Table 1 Independent variables and levels of RSM. Variables
Symbol
Levels
Enzyme dosage (U/g) Water addition (mL/g) Temperature pH
X1 X2 X3 X4
25 15 45 5.0
1
0
1
30 20 50 6.0
35 25 55 7.0
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2.6. Cell adaptation cultivation The cell adaptation cultivation was performed in 100 mL Erlenmeyer flask with 40 mL of corncob hemicellulose hydrolysate. A single colony of C. tropicalis was inoculated and the culture was grown for 24 h at 35 °C and 170 rpm. An aliquot of the resulting culture was used to inoculate in fresh corncob hemicellulose hydrolysate and the culture was grown for 24 h at 35 °C and 170 rpm. After 25 batches of adaptation cultivation, the culture was spread on corncob hemicellulose hydrolysate plate and the adapted cell which grown fast was checked for their ability to produce xylitol. 2.7. Xylitol bioconversion conditions A single colony of C. tropicalis and adapted C. tropicalis was inoculated into 100 mL Erlenmeyer flask containing 40 mL of YPD medium (10 g/L yeast extract, 20 g/L peptone and 20 g/L glucose), respectively, and the culture was grown for 12 h at 35 °C and 170 rpm. An aliquot of the resulting culture was used to inoculate in 1000 mL Erlenmeyer flask containing 400 mL of corncob hemicellulose hydrolysate (with 10 g/L extra glucose supply) with initial optical density (OD) at 660 nm of 0.5. Fermentations were performed at 35 °C and 170 rpm for 2 days. The hydrolysate was concentrated in a rotary vacuum evaporator at 60 °C and then used for fermentation. Two-stage fermentation was performed at 35 °C and 170 rpm for initial 6 h, then the shaking speed was change to 120 rpm. 2.8. Xylanase activity assay All enzyme activities were assayed using birchwood xylan as the substrate according to the method of Bailey et al. [26]. Briefly, the 1 mL reaction mixture consisting of 0.1 mL of appropriately diluted crude enzyme and 0.9 mL of 0.2 M sodium phosphate buffer (pH 7.0) containing 1% (w/v) birchwood xylan was incubated at 50 °C for 10 min. The reaction was stopped by adding 1.5 mL of 3,5-dinitrosalicylic acid reagent. The mixture was then boiled for 5 min followed by immediate chilling on ice. The amount of reducing sugars released was determined by the standard dinitrosalicylic acid method [27]. One unit of xylanase activity was defined as the amount of enzyme producing 1 lmol of reducing sugar (xylose) from the substrate solution per minute under the assay conditions. 2.9. b-Xylosidase activity assay The enzyme activity of b-xylosidase was determined by measuring pNP released from pNPX at 50 °C [28]. The reaction mixture consisting of 5 mM pNPX in 50 mM phosphate buffer (pH 6.0) was incubated with the enzyme for 10 min in a total volume of 0.25 ml. The reaction was stopped by the addition of 0.75 ml of 2 M Na2CO3, and the amount of pNP released was determined by measuring the absorbance at 410 nm. One unit of b-xylosidase activity was defined as the amount of enzyme required to release 1 lmol of pNP per minute under the conditions described above. 2.10. HPLC analysis The fermentation culture for the xylitol bioconversion (Section 2.6) was centrifuged at 10,000g for 5 min and the supernatant was used for determination of glucose, xylose, xylitol, acetic acid and ethanol. The concentrations of glucose, xylose, xylitol, acetic acid and ethanol were determined by HPLC system, model Shimadzu SCL-10A (Kyoto, Tokyo, Japan) with a RID-10A detector and a Bio-Rad Aminex HPX-87H (300 mm 7.8 mm) column. A
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5 mM H2SO4 solution was used as mobile phase at a flow rate of 0.6 mL/min and the analysis was carried out at 65 °C. 3. Results and discussion 3.1. Optimization of fermentation medium for xylanolytic enzymes production from A. terreus A. terreus which could produce xylanolytic enzymes was isolated from Xinjiang Province in China. In order to improve the production of xylanolytic enzymes, the medium of solid-state fermentation was optimized. Wheat bran and corncob are considered as efficient carbon source which could induce production of xylanolytic enzymes [29,30]. As shown in Fig. 1A, using wheat bran or corncob as solo carbon source could not induce xylanolytic enzymes efficiently. However, the mixture of corncob and wheat bran was the better inducer for xylanolytic enzymes production. When the ratio of wheat bran to corncob was 7:3, the highest xylanase activity of 631 U/g and the second highest xylosidase activity of 63 U/g were obtained, respectively. Following that, different nitrogen sources were selected to improve the xylanolytic enzymes production. As can be seen from Fig. 1B, NaNO3 was the best nitrogen source for xylanase production and the second best for xylosidase production. Although the strain could produce the highest xylosidase using yeast extract, the xylanase production was much lower than that using NaNO3. Furthermore, yeast extract was uneconomical for large scale production of xylanolytic enzymes due to its high cost; therefore, NaNO3 was the most suitable nitrogen source for strain to produce xylanolytic enzymes. Tween 80 is a good surface active agent which could enhance the xylanolytic enzymes production [31], as shown in Fig. 1C, Tween 80 with the concentration of 0.05% (v/v) could increase the xylanase and xylosidase production by 19.3% and 173.6%, respectively. As the moisture content of the medium has a critical effect on SSF [32], different initial water content in the medium was tested. As shown in Fig. 1D, when the ratio of water to material was 1:1 (v/w), the strain could produce the highest xylanolytic enzymes. In summary, the optimum nutrient medium composition was as follows: the ratio of wheat bran to corncob 7:3, 2% (v/v) NaNO3 with 0.05% (v/v) Tween 80 and the ratio of water to material 1:1 (v/w). 3.2. Optimization of growth conditions of A. terreus for xylanolytic enzymes production The growth conditions of A. terreus are also very important for the production of xylanolytic enzymes. Firstly, the effect of temperature on xylanolytic enzymes production was examined, as shown in Fig. 2A, xylanolytic enzymes production increased with the increase of temperature from 20 to 30 °C. As the temperature further increased to 37 °C, a decline in xylanolytic enzymes activity (28% and 22% for xylanase and xylosidase, respectively) was observed. This result was similar to some references, where the highest xylanase titers in fungal systems are obtained at temperature that is optimum for growth of cultures in SSF [33]. Then, the effect of initial pH of the medium on xylanolytic enzymes production was also investigated. As can be seen from Fig. 2B, the optimum pH for strain to produce xylanolytic enzymes was 6.8, it was similar to other strain reported [30]. It has been revealed that environmental parameters can influence the levels of xylanolytic enzymes production; moreover, the effect of inoculum level on the production of xylanolytic enzymes was also studied ranging from 5% to 30% (v/w). The maximal xylanolytic enzymes yields were obtained with 20% (v/w) of inoculum, although a marked difference was not found at lower or higher inoculum concentration (10% and 30%) (Fig. 2C). As xylanolytic enzymes were induced
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Fig. 1. Effects of medium contents on xylanase and xylosidase production. (A) Xylanase and xylosidase production under SSF with different carbon sources. (B) Xylanase and xylosidase production under SSF with different nitrogen sources. (C) Xylanase and xylosidase production under SSF with different concentration of Tween 80. (D) Xylanase and xylosidase production under SSF with different ratio of water to material in the medium.
during the strain growth, cultivation time was a crucial fermentation parameter affecting xylanolytic enzymes production. The result indicated the optimum cultivation time was 120 h, and further increase in incubation time did not show any increment in enzyme production (Fig. 2D). Under the optimum growth conditions with the optimum nutrient medium aforementioned, the xylanase and xylosidase activities were 722 U/g and 196 U/g, respectively, which were 31% and 202% higher in comparison to the activities obtained with the basic medium and growth condition. 3.3. Preparation of corncob hemicellulose hydrolysate The xylanolytic enzymes produced by A. terreus were used to hydrolyze corncob hemicellulose for xylitol synthesis, and different conditions of hydrolysis were investigated. Firstly, the xylanase dosage was tested, as shown in Fig. 3A, the concentration of monosaccharide increased with the increase of dosage from 5 to 30 U/g corncobs, further increase in the enzyme dosage could not improve the hydrolysis of the corncob, and therefore the proper dosage was 30 U/g for the hydrolysis. Water addition was another factor which could affect the hydrolysis, as can be seen from Fig. 3B, the maximum monosaccharide was obtained with water addition of 20 mL/g corncob. Fig. 3C showed the concentration of monosaccharide obtained at different temperatures. Temperature had a significant effect on the production of monosaccharide: the maximum monosaccharide was obtained at 50 °C and the concentration of monosaccharide decreased dramatically as the change of temperature. Then, the pH of hydrolysis was also tested. It was found that the optimum pH for the hydrolysis of corncob was 6 (Fig. 3D). In addition, glucose and xylose showed a very different production
time course: the glucose concentration reached stable after 3 h of hydrolysis, and xylose concentration kept increasing till 8 h. In order to further optimize the preparation of hydrolysate, response surface methodology (RSM) was used for investigation of the interactive effects among the parameters. The designed twentynine trials were displayed along with the results (Supplementary Table S1). According to the RSM mathematical model, the optimal conditions were found as enzyme dosage of 30.6 U/g, water addition of 19.2 mL/g, temperature of 48.01 °C and pH of 5.75, under such circumstances, 18.03 g/L xylose and 4.87 g/L glucose could be obtained after 8 h hydrolysis (Details in Supplementary material). The common methods used for hydrolysis of lignocellulosic materials include autohydrolysis [13,14], high-pressure CO2–H2O treatment [15,16], steam explore [17], alkali [34] and dilute sulfuric acid treatment [35]. The processes of autohydrolysis, high-pressure CO2–H2O treatment and steam explore need no chemicals (such as acid or alkali), but they are typically conducted at high temperature and pressure. Furthermore, xylo-oligosaccharides were the major liquor components using these methods for hydrolysis of lignocellulosic materials; therefore, the hydrolysates are not suitable for fermentation by microorganisms [13–17]. On the other hand, the xylose production of the alkali treatment is relative low [34]. Compared with other hydrolysis methods, dilute sulfuric acid treatment is especially useful for the conversion of xylan to xylose which can be further fermented to xylitol or ethanol by many microbial strains [18]. However, tradition chemical process for hemicellulose hydrolysate preparation includes acid, high pressure and temperature causing severe environmental pollution and equipment corrosion [36]. Moreover, a number of fermentation inhibitors such as acetic acid, formic acid, furfural,
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Fig. 2. Effects of growth conditions on xylanase and xylosidase production. (A) Influence of temperature on xylanase and xylosidase production under SSF. (B) Influence of pH on xylanase and xylosidase production under SSF. (C) Influence of inoculation amount on xylanase and xylosidase production under SSF. (D) Influence of cultivation time on xylanase and xylosidase production under SSF.
hydroxymethylfurfural (HMF) and phenolic compounds are generated during the acid hydrolysis process [37], which causes critical problems for the following bioconversion through fermentation. By comparison, enzymatic hydrolysis of lignocellulosic material used in this study had a lower environmental impact, less hazardous process chemicals, moderate process conditions and generated less inhibitor to fermentation later. Therefore, enzymatic hydrolysis could be an alternative environment friendly process for hemicellulose hydrolysate preparation. 3.4. Xylitol conversion using hemicellulose hydrolysate The corncob hemicellulose hydrolysate prepared by xylanolytic enzymes was used for xylitol conversion by C. tropicalis. As shown in Fig. 4A, the fermentation process generally had three stages and the highest xylitol concentration of 2.67 g/L was obtained at 12 h. In the first stage, the glucose was consumed within 9 h and ethanol was quickly produced by the C. tropicalis. In the middle stage, part of xylose and ethanol were used as carbon source for the cell growth and the xylitol accumulated quickly. In the last stage, the xylose and ethanol were completely consumed and xylitol started to decrease since it was used as carbon source for the metabolism of the cell. In general, the yield of xylitol was very low and it was only 14.81%. In addition, it should be noted that there was relatively high concentration of acetic acid in hydrolysate which could affect the xylitol conversion and cause the low yield of xylitol [38]. Apart from this, no other inhibitors could be detected. In order to increase the tolerance capacity of C. tropicalis to acetic acid, cell adaptation cultivation was adopted. According to the results, the xylitol concentration was increased to 8.18 g/L after 25 batches of cell adaptation cultivation and the corresponding yield was increased to 45.37%, which were about twice higher compared
with that of the wild C. tropicalis (Fig. 4B). Because glucose was consumed quickly and its concentration was low in hydrolysate; the xylose was used as carbon source for the cell growth instead of xylitol conversion which affected the yield of xylitol. Therefore, additional glucose was added into the hydrolysate to improve the xylitol conversion. As shown in Fig. 4B, the xylitol concentration was 11.74 g/L and the yield was 65.11%, respectively, increased by 43.5% compared to that without glucose addition. Although the addition of glucose improved the xylitol production, the cost could also increase due to the price of glucose. In order to obtain the higher concentration of glucose which could help the cell growth, the hydrolysate was concentrated, moreover the higher xylose concentration could benefit the xylitol conversion [39]. As can be seen from Fig. 4B, the adopted C. tropicalis produced 30.52 g/L xylitol with the concentrated hydrolysate, and the yield was 61.04% which was slightly lower than that of fermentation with glucose addition, however, this process was more economical because of no nutrients supplementation in hydrolysate. For the xylitol producing strain, oxygen is the most critical factor to the conversion from xylose to xylitol. It is evident that semi-aerobic condition is the best for the xylitol formation, under which NADPH produced by the pentose-phosphate pathway (PPP) is almost entirely addressed to xylitol formation [40]. Therefore, two-stage fermentation [41] was employed for the improvement of xylitol production. As shown in Fig. 4B, the xylitol concentration was 37.57 g/L and the yield was 75.14%, respectively. From the results, it was indicated that the two-stage fermentation was the optimum process for xylitol bioconversion directly from the hydrolysate. The industrial production of xylitol is currently based on the chemical hydrogenation process using nickel as the catalyst [7]. However, the drawbacks of the chemical conversion process are relatively low xylitol yields because chemical reduction produces
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Fig. 3. Effects of enzyme dosage, ratio of water to corncob mass, temperature, pH and hydrolysis time on preparation of corncob hemicellulose hydrolysate. (A) Production of glucose and xylose in the corncob hemicellulose hydrolysate with different enzyme dosage. (B) Production of glucose and xylose in the corncob hemicellulose hydrolysate with different ratio of water to corncob mass. (C) Production of glucose and xylose in the corncob hemicellulose hydrolysate with different temperature. (D) Production of glucose and xylose in the corncob hemicellulose hydrolysate with different pH. (E) Production of glucose and xylose in the corncob hemicellulose hydrolysate with different hydrolysis time.
byproducts [42]. Therefore, direct xylitol production from hemicellulose hydrolysate appears to be a promising alternative by the natural xylose-fermenting yeast. In this study, C. tropicalis was used for xylitol production from enzymatic hemicellulose hydrolysate, after the cell adaptation cultivation and optimization of fermentation, the yield of xylitol could reach 75.14% which was the same level as previous reports [43–45]. Furthermore, by
comparison with these methods, the process used in this study was simpler because the detoxification of hydrolysate was not necessary, and no nutrients supplementation was needed in the fermentation. Therefore, fermentation with enzymatic corncob hydrolysate was an environment friendly, economical and efficient method to produce xylitol, demonstrating a wide potential application in xylitol bioconversion.
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Fig. 4. (A) The xylitol production by C. tropicalis using corncob hemicellulose hydrolysate prepared by xylanolytic enzymes. d, xylose; j, xylitol; , glucose; N, ethanol; ., acetic acid. (B) Results of xylitol production by C. tropicalis (j), adapted C. tropicalis (d), adapted C. tropicalis with extra supply of glucose (N), adapted C. tropicalis with concentrated hydrolysate (.) and adapted C. tropicalis with concentrated hydrolysate under two-stage fermentation ().
4. Conclusions In this study, an environment friendly process was developed for enzymatic corncob hemicellulose hydrolysate preparation and xylitol bioconversion. Under the optimum conditions, maximum xylanase (722 U/g) and b-xylosidase (196 U/g) were produced by A. terreus. In order to prepare corncob hemicellulose hydrolysate, different parameters were optimized, and 18.03 g/L xylose and 4.87 g/L glucose were obtained. After adaption cultivation of C. tropicalis and optimization of fermentation, the xylitol yield of 75.14% was obtained. This process was demonstrated to be a green way to obtain corncob hemicellulose hydrolysate and a highly efficient method for xylitol production. Acknowledgements This work was financially supported by the National High Technology Research and Development Program of China (863 Program) (No. 2012AA02A704), the Major State Basic Research Development Program of China (973 Program) (No. 2013CB733900), National Natural Science Foundation of China
(Nos. 21176028, 21376028) and The National Research Foundation for the Doctoral Program of Higher Education of China (No. 20121101110050).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cej.2014.11.013.
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