Bioresource Technology 193 (2015) 331–336
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Efficient hydrolysis of corncob residue through cellulolytic enzymes from Trichoderma strain G26 and L-lactic acid preparation with the hydrolysate Lulu Xie, Jin Zhao, Jian Wu, Mingfu Gao, Zhewei Zhao, Xiangyun Lei, Yi Zhao, Wei Yang, Xiaoxue Gao, Cuiyun Ma, Huanfei Liu, Fengjuan Wu, Xingxing Wang, Fengwei Zhang, Pengyuan Guo, Guifu Dai ⇑ School of Life Sciences, Zhengzhou University, 100 Kexue Road, Zhengzhou 450001, China
h i g h l i g h t s Trichoderma longibrachiatum G26 can produce high levels of cellulolytic enzymes. The cellulolytic enzymes can overcome the product inhibition. A three-stage enzymatic saccharification strategy for corncob residue is proposed. The hydrolysate of corncob residue is suitable for L-lactic acid fermentation.
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Article history: Received 14 May 2015 Received in revised form 18 June 2015 Accepted 19 June 2015 Available online 25 June 2015 Keywords: Corncob residue Solid-state fermentation Saccharification Trichoderma longibrachiatum Cellulase
a b s t r a c t To prepare fermentable hydrolysate from corncob residue (CCR), Trichoderma strain G26 was cultured on medium containing CCR for production of cellulolytic enzymes through solid-state fermentation (SSF), resulting in 71.3 IU/g (FPA), 136.2 IU/g (CMCase), 85.1 IU/g (b-glucosidase) and 11,344 IU/g (xylanase), respectively. Through a three-stage saccharification strategy, CCR was hydrolyzed by the enzymatic solution (6.5 FPU/ml) into fermentable hydrolysate containing 60.1 g/l glucose (81.2% cellulose was converted at solid loading of 12.5%), 21.4% higher than that by the one-stage method. And then the hydrolysate was used to produce L-lactic acid by a previous screened strain Bacillus coagulans ZX25 in the submerged fermentation. 52.0 g/l L-lactic acid was obtained after fermentation for 44 h, with 86.5% glucose being converted to L-lactic acid. The results indicate that the strains and the hydrolysis strategy are promising for commercial production of L-lactic acid from CCR and other biomass. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction It is well known that lignocelluloses in agricultural residues are the most abundant, low-cost and renewable biomass (Zhang et al., 2007; Ragauskas et al., 2006). High efficient utilization of lignocelluloses has long been a research hot-spot, as sugars produced from lignocelluloses can be biotransformed to liquid biofuel (ethanol, butanol) and other bio-based chemicals, without competition with the increasing food demand. In China, nearly 20 million tons of corncobs are produced annually (Bai et al., 2008) and the hemicellulose fraction of corncobs is generally extracted by dilute-acid treatment for xylose and furfural production (Doiseau et al., 2014; Li et al., 2014), thereby leaves behind the corncob residue (CCR) as a solid waste, which is usually burned as boiler fuels ⇑ Corresponding author. Tel.: +86 371 67767604; fax: +86 371 67784867. E-mail address: [email protected] (G. Dai). http://dx.doi.org/10.1016/j.biortech.2015.06.101 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
(Cheng et al., 2014b), far away from resource utilization. Since the cost of feedstock and pretreatment has been shared by the co-products and CCR contains a significant amount of cellulose (about more than 50%), production of ethanol and other products from CCR with the enzymatic hydrolysis process is a potential strategy in industry scale. However, a process with lower cost and higher productivity is still yet to be developed for the industrial production of bio-based chemicals from CCR. One of the key challenges is the enzymatic hydrolysis of CCR for generating high yield fermentable sugars (Piccolo and Bezzo, 2009) economically. In order to increase the enzymatic hydrolysis efficiency and obtain higher sugars titer, researches about CCR saccharification using various cellulolytic enzymes which are commercially available (Fan et al., 2013), as well as screening strains with high levels of cellulase production, which are suitable for CCR hydrolysis have been taken (Xia and Shen, 2004; Cheng et al., 2009). Moreover, several very useful
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strategies for CCR saccharification, such as utilizing crude extract of cellulase produced by Trichoderma in combination with that from species of Aspergillus or Penicillium for a supplementation of b-glucosidase to overcome the product inhibition (Chen et al., 2010; Baba et al., 2015; Thongpoo et al., 2014), or using delignin CCR or pretreated CCR by dilute acids as substrate of enzymatic saccharification were explored (Cheng et al., 2014a,b; Fan et al., 2013; Chu et al., 2012). Previously, we have made efforts on isolating cellulase producing strains (B-8, FC-1) and screening the cellulases with good performance on CCR saccharification, produced by the strains through submerged fermentation. In this work, the crude extract of the enzymes, which contained high levels of FPA, CMCase and xylanase, produced by a strain G26 identified as Trichoderma longibrachiatum, was prepared through solid-state fermentation (SSF). To get high glucose titer from CCR efficiently, a three-stage enzymatic saccharification strategy was developed. Moreover, the CCR hydrolysate was fermented to L-lactic acid by Bacillus coagulans ZX25, isolated by us previously. The obtained strains and established strategy in this study are useful for commercial L-lactic acid production from lignocellulosic materials in the future.
CCR hydrolysis was carried out, for one-stage method, by adding the indicated amount of substrate (dry weight) into the enzyme solution (50 ml) in each 250 ml Erlenmeyer flask, and then samples were incubated in a shaker at 180 rpm, under the optimal temperature (50 °C) and pH (4.8) for the indicated time. For the multi-stage saccharification experiments, the total volume and concentration of the enzyme solution (as to FPU/ml) used were kept the same with one-stage process, but adjusting the adding procedure as to that 40 ml was added at the beginning, then 10 ml was supplemented at the indicated time, by one time or twice. Samples were taken at indicated time, and separated by centrifugation at 4360g for 5 min. Glucose and reducing sugar in the supernatant were analyzed. The cellulose conversion (as glucose) was calculated by the following equation:
2. Methods
Conversion ð%Þ ¼
2.1. Raw materials
where Cg is the obtained glucose concentration (g/l); V is the volume of enzyme solution (l); M is the total amount of substrate (g).
CCR, composed of 53.3% cellulose, 8.7% hemicellulose, 19.1% lignin, 1.8% ash and 17.1% others, was kindly provided by Jiaozuo Huakang Chemical Co., Ltd., China. Rice straw was obtained from a local farm nearby Zhengzhou city, Henan, China. The above lignocellulosic materials were dried and physically processed using a F120 cutting mill (Beijing Zhongxing Weiye Instrument Co., Ltd., China) and crushed to 100-mesh particles. Wheat bran and soybean powder were purchased from a local market on the outskirts of Zhengzhou. Corn steep liquor was obtained from Henan Lianhua Gourmet Powder Co., Ltd., China. All chemicals used in the experiments were of analytical grade. 2.2. Microorganisms Trichoderma strains were isolated and preserved as reported previously (Fang et al., 2011). B. coagulans ZX25, isolated from soil around the root of a pine tree in Zhengzhou University by us, was maintained on slants with 0.5% (w/v) peptone, 0.3% (w/v) yeast extract, 0.2% (w/v) K2HPO4, 2% (w/v) glucose, and 1.5% (w/v) agar at 4 °C. 2.3. Media, inoculum preparation and solid-state fermentation (SSF) Fungal spores on PDA were harvested and suspended in sterile water, inoculated (106/ml) into each 250 ml Erlenmeyer flask containing 50 ml of 10% (w/v) wheat bran extract which was boiled for 20 min. Cell cultures were harvested, after incubated on a shaker at 28 °C and 180 rpm for 24 h, then inoculated into 250 ml Erlenmeyer flasks containing SSF medium, composed of rice straw 4.0 g, wheat bran 4.0 g, corncob residue 2.0 g and 15.0 ml nutrient solution contained (NH4)2SO4 2.0% (w/v), KH2PO4 1.0% (w/v), soybean cake powder 8.9% (w/v), urea 1.0% (w/v), corn steep liquor 4.0% (v/v), incubated in an incubator at 28 °C for the indicated period. 2.4. Extraction of enzymes After a desired period, 100 ml of 50 mM sodium citrate buffer was added to per flask, then the flasks were shook on a shaker at
180 rpm and 28 °C for 2 h to let enzymes release completely. Then the mixture was centrifuged at 8516g for 10 min at 4 °C and the supernatant (fungal extract) was removed and used as the crude enzyme extract. 2.5. Enzymatic hydrolysis of CCR
C g V 0:9 100 M 53:3%
2.6. L-lactic acid fermentation with CCR hydrolysate To prepare seed culture, the culture of B. coagulans ZX25 grown on slants were transferred to liquid media containing 60.1 g/l glucose, 0.5% (w/v) peptone, 0.3% (w/v) yeast extract, 0.2% (w/v) K2HPO4, at pH 7.0 and incubated at 50 °C for 20 h. This culture was used to provide 6% (v/v) inoculum for fermentation. The L-lactic
acid submerged fermentation was performed in 50 ml Erlenmeyer flasks each filled with 20 ml hydrolysate (60.1 g/l glucose) supplemented with 0.5% (w/v) peptone, 0.3% (w/v) yeast extract, 0.2% (w/v) K2HPO4 and 6% (w/v) CaCO3 for 60 h at 50 °C, pH 7.0 and 140 rpm. Samples were taken with specific time intervals to determine cell mass (OD600nm), residual sugar and L-lactic acid. 2.7. Analytical methods
Activities of filter paper (FPA), carboxymethyl cellulose (CMCase) and b-glucosidase were determined as described in previous paper (Fang et al., 2011). One unit (IU) of enzyme activity was defined as the amount of enzyme that release 1 lmol reducing sugar per minute under the assay conditions. Xylanase activity was determined by measuring total reducing sugar released from 1% (w/v) birch wood xylan (Sigma) in 0.9 ml sodium citrate buffer, 50 mM, pH 4.8, when 0.1 ml suitably diluted enzyme was added. After the mixture was incubated at 50 °C for 15 min, 3 ml dinitrosalicylic acid (DNS) was added to stop the reaction, then the enzyme activity was determined as mentioned by Miller. The reducing sugar yields were determined using the DNS (3,5-dinitrosalicylic acid) method (Miller, 1959). One unit (IU) of xylanase activity was defined as the amount of enzyme that release 1 lmol reducing sugar per minute under the assay conditions. The concentration of glucose produced from corncob residue, as well as the L-lactic acid yield was determined by a SBA-40E Biological Sensor (Shandong Academy of Sciences Institute for Biological Studies, Jinan, China). Sugars in CCR hydrolysate and fermentation broth were analyzed by High-Performance Capillary Electrophoresis (HPCE) method using a Waters Quanta 4000 (Millipore Instruments, Co.
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Ltd., Massachusetts, US). Electrophoretic run was carried out at 10 kV for 25 min, maintaining the capillary temperature at 25 °C, resulting in a current of approximately 108 mA. The running buffer was prepared by 35 mM sodium tetraborate (pH 9.3) for 3 min and 1-phenyl-3-methyl-5-pyrazolone acted as the sugar derivative agent (Liang et al., 2008). Peak identification was carried out by comparing retention times of the unknown peaks to those of the standard compounds. 3. Results and discussion 3.1. Comparison of cellulase producing ability of six Trichoderma fungi in SSF The cellulase producing abilities of the six Trichoderma strains, B-8, B-13, B-19, C1, A6 and G26 in SSF with CCR, wheat bran and rice straw at ratio of 2:4:4 were compared. The results (Fig. 1A–C) showed that the titer of cellulase produced by G26 was the highest among the six strains, resulted in 40.9 IU (FPA), 99.7 IU (CMCase) and 71.8 IU (b-glucosidase) per gram of substrate (dry weight) for 96 h fermentation, respectively, which indicated that G26 had remarkable advantages in production of cellulase, especially in total cellulase (FPA) and endoglucanase (CMCase). Furthermore, the strain G26 was identified as a T. longibrachiatum by morphological observing combined with the sequence analysis for ITS gene of 18SrDNA (results were not listed). Compared with other reported cellulase producing strains of T. longibrachiatum (Gusakov et al., 1985; Gusakov, 2011), G26 showed superiority in b-glucosidase. 3.2. Cellulolytic enzymes preparation by G26 with an optimum medium To improve producing capacity of cellulase, an optimum medium, containing rice straw 4.0 g, wheat bran 4.0 g, corncob residue 2.0 g and 15.0 ml nutrient solution composed of (NH4)2SO4 2.0%
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(w/v), K2HPO4 1.0% (w/v), soybean cake powder 8.9% (w/v), urea 1.0% (w/v) and corn steep liquor 4.0% (v/v) (results were not listed). The enzyme activities for FPA, CMCase and b-glucosidase in SSF at the optimal medium reached 71.3 ± 3.0 IU/g, 136.2 ± 0.6 IU/g and 85.1 ± 0.1 IU/g, which were 174.3%, 136.6% and 118.5% compared with that before optimization, respectively. Considering the important roles of hemicellulases during the hydrolysis of lignincellulosic materials (Berlin et al., 2005; Kumar and Wyman, 2009; Gao et al., 2011), as well as the strain T. longibrachiatum PTCC 5140 with good performance on xylanase production (Azin et al., 2007), the activity of xylanase produced by G26 in the SSF with the above optimal medium was explored. It indicated that besides satisfactory cellulase producing ability, G26 produced high titer of xylanase, reaching 11,344 ± 28 IU /g.
3.3. Enzymatic hydrolysis of CCR As shown in Table 1, CCR was hydrolyzed at different initial FPA (IU/ml) under the solid loading of 8.5% (w/v), 12.5% (w/v) and 16.5% (w/v), respectively. It was found that, though yield of both glucose and reducing sugar improved with the increasing of enzyme dosage and solid loading, the conversion of cellulose to glucose decreased from 62.1% to 54.2% while saccharifying with 6.5 FPU/ml at solid loading from 12.5% (w/v) to 16.5% (w/v), and also the productivity of glucose to one unit of FPU at solid loading of 12.5% (w/v) was remarkably lowered while increasing the cellulase loading to 10.2 FPU/ml. The results for the time course of glucose released (Fig. 2A) from CCR at solid loading of 12.5% (w/v) showed that the hydrolysis efficiency significantly slowed down while increasing the enzyme loading from 6.5 to 12.6 FPU/ml, resulting in the maximal glucose yield from 46 to 56 g/l, indicating conversion of 62.1% and 75.6%, respectively. Moreover, the glucose level could not be further enhanced after 72 h hydrolysis, which did not be significantly improved even while treated with enzyme as high titer as
Fig. 1. Activities of cellulases produced by Trichoderma strains through SSF. FPA (A), CMCase (B) and b-glucosidase (C) were produced by strains cultured with medium containing 1.2 g corncob residue, 2.4 g wheat bran, 2.4 g rice straw, mixed with nutrient solution containing (NH4)2SO4 3.0% (w/v), KH2PO4 1.0% (w/v) and soybean cake power 1.0% (w/v), at solid–liquid ratio of 1:1.5 (w/v), for the indicated hours, respectively. Data shown are means of three replicated experiments ± standard error.
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Table 1 Effect of enzyme dosage and solid loading on the hydrolysis of CCR. FPU/mla
enzymes of G26 was caused by product accumulation. The results showed that the glucose production were 45.3, 44.3, 44.5 and 44.7 g/l with 0, 23, 38 and 55 g/l glucose added at the beginning of hydrolysis, respectively, which indicated that the glucose productivities were little lowered by pre-adding glucose, even while the initial glucose concentration was as high as 55 g/l, indicating that the cellulolytic enzymes produced by G26 is potential to be used for hydrolysate preparation with high yield of fermentable sugars.
Solid loading (w/ v)
Sugar (g/l)b
8.5%
Reducing sugar Glucose
27 ± 0.3
31 ± 1.1
12.5%
Reducing sugar Glucose
–
33 ± 0.7
61 ± 1.0
67 ± 0.8
–
28 ± 0.1
46 ± 0.7
52 ± 0.5
16.5%
Reducing sugar Glucose
–
–
66 ± 1.8
70 ± 1.4
3.4. Improvement of hydrolysis efficiency of CCR by adding surfactants
–
–
53 ± 1.1
57 ± 0.0
Recently, surfactants especially non-ionic surfactants have been proven to enhance the enzymatic hydrolysis conversion from lignocellulosic biomass substrates, and can lower the enzyme requirement for a given yield effectively (Seo et al., 2011; Li et al., 2012). Therefore, we tried to improve the hydrolysis efficiency through adding certain amount of non-ionic surfactants during the hydrolysis of CCR. Significant improvements were observed both with adding (poly) ethylene glycol (PEG) 6000 and with Tween-80 (Fig. 3A and B), and the higher content of the surfactant, the higher yield of sugars. The maximum glucose titer (58 g/l) and reducing sugar (68 g/l) were obtained with 0.05 g PEG 6000/g substrate added, which were 45% and 39% higher than that without surfactant added, respectively. And with the hydrolysis going on, the glucose titer and reducing sugar titer increased slower and the increase ended earlier than that with surfactant added. That was consistent with the results of Kristensen et al. (Kristensen et al., 2007), which maybe because of the increasing of free enzymes in solution due to the reduction of non-productive/irreversible adsorption of cellulase onto lignin and crystalline cellulose (Kristensen et al., 2007; Li et al., 2012). But regretfully, the residual PEG 6000 in the CCR hydrolysate performed an inhibitory effect on the following L-lactic acid fermentation (results were not listed).
1.4
2.8
6.5
24 ± 0.9
31 ± 0.6
36 ± 0.9
21 ± 0.7
10.2 – –
a
The crude enzyme solution used here was extracted by adding 10 times of the extract buffer (v/w; dry weight) into the fermented mash, resulting in 6.5 FPU/ml; the solution with enzyme activity higher than 6.5 FPU/ml was prepared by extracting the fresh fermented mash with the above mentioned enzyme solution containing 6.5 FPU/ml, whereas those lower than 6.5 FPU/ml was obtained by diluting with buffer. b Results are means of three replicates ± standard error.
3.5. Improvement of hydrolysis efficiency of CCR through a multi-stage process Here we developed a three-stage process for the CCR hydrolysis. The results in Fig. 3C showed that the multi-stage process presented an advantage over the traditional one. The yield of sugar increased faster and was significantly higher than that of control and it seemed better if the supplementation carried out for twice, especially supplementation earlier (24 & 48 h) was better, resulting in the maximum production of glucose 60.1 g/l with a conversion of 81.2% at 72 h, which was 21.4% higher than that of the control. 3.6. Analysis of sugar components of CCR hydrolysate
Fig. 2. The time course of CCR hydrolysis with different FPA dosage at the solid loading of 12.5% (w/v) under the optimum conditions. The concentration of glucose (A) and reducing sugar (B) were determined after taking samples at the indicated time, respectively. Data shown are means of three replicated experiments ± standard error.
12.6 FPU/ml. Similar results were observed for the production of reducing sugar (Fig. 2B). As decreased enzymatic efficiency caused by accumulation of glucose or cellobiose is thought to be the main limitation for getting high concentration of fermentable sugars (Gusakov et al., 1985), we compared with the glucose production from CCR, without or with 23, 38 or 55 g/l glucose added at the beginning of hydrolysis, to investigate if the hindered glucose releasing by
Besides sugar titer, the sugar components in CCR hydrolysate is another key factor that influences the production of L-lactic acid, because high level of unavailable sugars for B. coagulans ZX25 will be remained in the fermented broth, which would lower the quality and increase the cost of L-lactic acid. Therefore, we analyzed the sugars in CCR hydrolysate by High-Performance Capillary Electrophoresis (HPEC). Compared with the standard mixture, the hydrolysate was only composed of glucose and very little xylose (results were not listed), whereas maltose, ribose, mannose and galactose were undetectable. 3.7. L-lactic acid fermentation using the CCR hydrolysate Moreover, the CCR hydrolysate contained 60.1 g/l glucose, supplemented with 0.5% (w/v) peptone, 0.3% (w/v) yeast extract and
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Fig. 4. Time course of L-lactic acid production with hydrolysate of CCR. Fermentation was conducted in 50 ml Erlenmeyer flasks containing 20 ml hydrolysate at a 50 °C shaker (140 rpm) for 60 h, pH was controlled with CaCO3. Samples were taken every 4 h, and the yield of L-lactic acid and residual sugar, as well as the optical density (OD600nm) after three times dilution, were measured, respectively. Data shown are means of three replicated experiments ± standard error.
for the 44 h fermentation. In addition, the residual sugar in the broth after 44 h fermentation was little, as shown in Fig. 4. 4. Conclusion A strain T. longibrachiatum G26 with high yield cellulase production in SSF was developed for the preparation of fermentable hydrolysate from CCR, followed by CCR hydrolysis with the crude enzyme extract using a novel three-stage strategy. The results showed that the production of glucose from CCR was 21.4% improved by the three-stage hydrolysis, and the obtained hydrolysate containing 60.1 g/l glucose is suitable for L-lactic acid production. In further studies, lowering the cost of cellulase preparation, decreasing the enzyme loading for CCR hydrolysis, as well as shorting the hydrolysis period are still needed. Acknowledgement This research work was supported by the Bidding Project of Henan Province (China) for Major Public Welfare Scientific Research (NO. 91100910300). Fig. 3. The improvement of CCR hydrolysis efficiency through adding surfactant or multi-stage process. CCR hydrolysis was carried out at the final solid loading of 12.5% (w/v) with 6.5 FPU/ml under the optimum conditions. (A) The glucose and reducing sugar obtained without or with different dosages (0.1–5.0 g/100 g CCR) of PEG 6000 (P) or Tween-80 (T). (B) The comparison of variation for glucose and reducing sugar with the hydrolysis time prolonging between control and the treatment of adding 0.05 g/g CCR of PEG 6000. (C) Improvement through multistage process. For control, 50 ml crude enzyme extract was one-time added at the beginning of the enzymatic reaction; for the treatments of 36 and 48 h, 40 ml enzyme solution were firstly added, and then 10 ml were supplemented at 36 and 48 h, respectively; for the treatments of 24 & 48 h and 36 & 72 h, 10 ml were supplemented by twice, for 5 ml each time, at 24 and 48 h, 36 and 72 h, respectively. Samples were taken at the indicated time, and then glucose concentration was measured before the enzyme supplementation. The glucose yield means the value to the final volume. Data shown are means of three replicated experiments ± standard error.
0.2% (w/v) K2HPO4 was used to prepare L-lactic acid by fermentation with B. coagulans ZX25. The results (Fig. 4) showed that the present CCR hydrolysate was very suitable for ZX25 growth, as well as the accumulation of L-lactic acid, which was time-saving with higher yield and higher conversion, obtaining a maximum yield of 52.0 g/l with the sugar-acid conversion (as to glucose) of 86.5%
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Efficient hydrolysis of corncob residue through cellulolytic enzymes from Trichoderma strain G26 and L-lactic acid preparation with the hydrolysate Lulu Xie, Jin Zhao, Jian Wu, Mingfu Gao, Zhewei Zhao, Xiangyun Lei, Yi Zhao, Wei Yang, Xiaoxue Gao, Cuiyun Ma, Huanfei Liu, Fengjuan Wu, Xingxing Wang, Fengwei Zhang, Pengyuan Guo, Guifu Dai ⇑ School of Life Sciences, Zhengzhou University, 100 Kexue Road, Zhengzhou 450001, China
h i g h l i g h t s Trichoderma longibrachiatum G26 can produce high levels of cellulolytic enzymes. The cellulolytic enzymes can overcome the product inhibition. A three-stage enzymatic saccharification strategy for corncob residue is proposed. The hydrolysate of corncob residue is suitable for L-lactic acid fermentation.
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Article history: Received 14 May 2015 Received in revised form 18 June 2015 Accepted 19 June 2015 Available online 25 June 2015 Keywords: Corncob residue Solid-state fermentation Saccharification Trichoderma longibrachiatum Cellulase
a b s t r a c t To prepare fermentable hydrolysate from corncob residue (CCR), Trichoderma strain G26 was cultured on medium containing CCR for production of cellulolytic enzymes through solid-state fermentation (SSF), resulting in 71.3 IU/g (FPA), 136.2 IU/g (CMCase), 85.1 IU/g (b-glucosidase) and 11,344 IU/g (xylanase), respectively. Through a three-stage saccharification strategy, CCR was hydrolyzed by the enzymatic solution (6.5 FPU/ml) into fermentable hydrolysate containing 60.1 g/l glucose (81.2% cellulose was converted at solid loading of 12.5%), 21.4% higher than that by the one-stage method. And then the hydrolysate was used to produce L-lactic acid by a previous screened strain Bacillus coagulans ZX25 in the submerged fermentation. 52.0 g/l L-lactic acid was obtained after fermentation for 44 h, with 86.5% glucose being converted to L-lactic acid. The results indicate that the strains and the hydrolysis strategy are promising for commercial production of L-lactic acid from CCR and other biomass. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction It is well known that lignocelluloses in agricultural residues are the most abundant, low-cost and renewable biomass (Zhang et al., 2007; Ragauskas et al., 2006). High efficient utilization of lignocelluloses has long been a research hot-spot, as sugars produced from lignocelluloses can be biotransformed to liquid biofuel (ethanol, butanol) and other bio-based chemicals, without competition with the increasing food demand. In China, nearly 20 million tons of corncobs are produced annually (Bai et al., 2008) and the hemicellulose fraction of corncobs is generally extracted by dilute-acid treatment for xylose and furfural production (Doiseau et al., 2014; Li et al., 2014), thereby leaves behind the corncob residue (CCR) as a solid waste, which is usually burned as boiler fuels ⇑ Corresponding author. Tel.: +86 371 67767604; fax: +86 371 67784867. E-mail address: [email protected] (G. Dai). http://dx.doi.org/10.1016/j.biortech.2015.06.101 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
(Cheng et al., 2014b), far away from resource utilization. Since the cost of feedstock and pretreatment has been shared by the co-products and CCR contains a significant amount of cellulose (about more than 50%), production of ethanol and other products from CCR with the enzymatic hydrolysis process is a potential strategy in industry scale. However, a process with lower cost and higher productivity is still yet to be developed for the industrial production of bio-based chemicals from CCR. One of the key challenges is the enzymatic hydrolysis of CCR for generating high yield fermentable sugars (Piccolo and Bezzo, 2009) economically. In order to increase the enzymatic hydrolysis efficiency and obtain higher sugars titer, researches about CCR saccharification using various cellulolytic enzymes which are commercially available (Fan et al., 2013), as well as screening strains with high levels of cellulase production, which are suitable for CCR hydrolysis have been taken (Xia and Shen, 2004; Cheng et al., 2009). Moreover, several very useful
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strategies for CCR saccharification, such as utilizing crude extract of cellulase produced by Trichoderma in combination with that from species of Aspergillus or Penicillium for a supplementation of b-glucosidase to overcome the product inhibition (Chen et al., 2010; Baba et al., 2015; Thongpoo et al., 2014), or using delignin CCR or pretreated CCR by dilute acids as substrate of enzymatic saccharification were explored (Cheng et al., 2014a,b; Fan et al., 2013; Chu et al., 2012). Previously, we have made efforts on isolating cellulase producing strains (B-8, FC-1) and screening the cellulases with good performance on CCR saccharification, produced by the strains through submerged fermentation. In this work, the crude extract of the enzymes, which contained high levels of FPA, CMCase and xylanase, produced by a strain G26 identified as Trichoderma longibrachiatum, was prepared through solid-state fermentation (SSF). To get high glucose titer from CCR efficiently, a three-stage enzymatic saccharification strategy was developed. Moreover, the CCR hydrolysate was fermented to L-lactic acid by Bacillus coagulans ZX25, isolated by us previously. The obtained strains and established strategy in this study are useful for commercial L-lactic acid production from lignocellulosic materials in the future.
CCR hydrolysis was carried out, for one-stage method, by adding the indicated amount of substrate (dry weight) into the enzyme solution (50 ml) in each 250 ml Erlenmeyer flask, and then samples were incubated in a shaker at 180 rpm, under the optimal temperature (50 °C) and pH (4.8) for the indicated time. For the multi-stage saccharification experiments, the total volume and concentration of the enzyme solution (as to FPU/ml) used were kept the same with one-stage process, but adjusting the adding procedure as to that 40 ml was added at the beginning, then 10 ml was supplemented at the indicated time, by one time or twice. Samples were taken at indicated time, and separated by centrifugation at 4360g for 5 min. Glucose and reducing sugar in the supernatant were analyzed. The cellulose conversion (as glucose) was calculated by the following equation:
2. Methods
Conversion ð%Þ ¼
2.1. Raw materials
where Cg is the obtained glucose concentration (g/l); V is the volume of enzyme solution (l); M is the total amount of substrate (g).
CCR, composed of 53.3% cellulose, 8.7% hemicellulose, 19.1% lignin, 1.8% ash and 17.1% others, was kindly provided by Jiaozuo Huakang Chemical Co., Ltd., China. Rice straw was obtained from a local farm nearby Zhengzhou city, Henan, China. The above lignocellulosic materials were dried and physically processed using a F120 cutting mill (Beijing Zhongxing Weiye Instrument Co., Ltd., China) and crushed to 100-mesh particles. Wheat bran and soybean powder were purchased from a local market on the outskirts of Zhengzhou. Corn steep liquor was obtained from Henan Lianhua Gourmet Powder Co., Ltd., China. All chemicals used in the experiments were of analytical grade. 2.2. Microorganisms Trichoderma strains were isolated and preserved as reported previously (Fang et al., 2011). B. coagulans ZX25, isolated from soil around the root of a pine tree in Zhengzhou University by us, was maintained on slants with 0.5% (w/v) peptone, 0.3% (w/v) yeast extract, 0.2% (w/v) K2HPO4, 2% (w/v) glucose, and 1.5% (w/v) agar at 4 °C. 2.3. Media, inoculum preparation and solid-state fermentation (SSF) Fungal spores on PDA were harvested and suspended in sterile water, inoculated (106/ml) into each 250 ml Erlenmeyer flask containing 50 ml of 10% (w/v) wheat bran extract which was boiled for 20 min. Cell cultures were harvested, after incubated on a shaker at 28 °C and 180 rpm for 24 h, then inoculated into 250 ml Erlenmeyer flasks containing SSF medium, composed of rice straw 4.0 g, wheat bran 4.0 g, corncob residue 2.0 g and 15.0 ml nutrient solution contained (NH4)2SO4 2.0% (w/v), KH2PO4 1.0% (w/v), soybean cake powder 8.9% (w/v), urea 1.0% (w/v), corn steep liquor 4.0% (v/v), incubated in an incubator at 28 °C for the indicated period. 2.4. Extraction of enzymes After a desired period, 100 ml of 50 mM sodium citrate buffer was added to per flask, then the flasks were shook on a shaker at
180 rpm and 28 °C for 2 h to let enzymes release completely. Then the mixture was centrifuged at 8516g for 10 min at 4 °C and the supernatant (fungal extract) was removed and used as the crude enzyme extract. 2.5. Enzymatic hydrolysis of CCR
C g V 0:9 100 M 53:3%
2.6. L-lactic acid fermentation with CCR hydrolysate To prepare seed culture, the culture of B. coagulans ZX25 grown on slants were transferred to liquid media containing 60.1 g/l glucose, 0.5% (w/v) peptone, 0.3% (w/v) yeast extract, 0.2% (w/v) K2HPO4, at pH 7.0 and incubated at 50 °C for 20 h. This culture was used to provide 6% (v/v) inoculum for fermentation. The L-lactic
acid submerged fermentation was performed in 50 ml Erlenmeyer flasks each filled with 20 ml hydrolysate (60.1 g/l glucose) supplemented with 0.5% (w/v) peptone, 0.3% (w/v) yeast extract, 0.2% (w/v) K2HPO4 and 6% (w/v) CaCO3 for 60 h at 50 °C, pH 7.0 and 140 rpm. Samples were taken with specific time intervals to determine cell mass (OD600nm), residual sugar and L-lactic acid. 2.7. Analytical methods
Activities of filter paper (FPA), carboxymethyl cellulose (CMCase) and b-glucosidase were determined as described in previous paper (Fang et al., 2011). One unit (IU) of enzyme activity was defined as the amount of enzyme that release 1 lmol reducing sugar per minute under the assay conditions. Xylanase activity was determined by measuring total reducing sugar released from 1% (w/v) birch wood xylan (Sigma) in 0.9 ml sodium citrate buffer, 50 mM, pH 4.8, when 0.1 ml suitably diluted enzyme was added. After the mixture was incubated at 50 °C for 15 min, 3 ml dinitrosalicylic acid (DNS) was added to stop the reaction, then the enzyme activity was determined as mentioned by Miller. The reducing sugar yields were determined using the DNS (3,5-dinitrosalicylic acid) method (Miller, 1959). One unit (IU) of xylanase activity was defined as the amount of enzyme that release 1 lmol reducing sugar per minute under the assay conditions. The concentration of glucose produced from corncob residue, as well as the L-lactic acid yield was determined by a SBA-40E Biological Sensor (Shandong Academy of Sciences Institute for Biological Studies, Jinan, China). Sugars in CCR hydrolysate and fermentation broth were analyzed by High-Performance Capillary Electrophoresis (HPCE) method using a Waters Quanta 4000 (Millipore Instruments, Co.
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Ltd., Massachusetts, US). Electrophoretic run was carried out at 10 kV for 25 min, maintaining the capillary temperature at 25 °C, resulting in a current of approximately 108 mA. The running buffer was prepared by 35 mM sodium tetraborate (pH 9.3) for 3 min and 1-phenyl-3-methyl-5-pyrazolone acted as the sugar derivative agent (Liang et al., 2008). Peak identification was carried out by comparing retention times of the unknown peaks to those of the standard compounds. 3. Results and discussion 3.1. Comparison of cellulase producing ability of six Trichoderma fungi in SSF The cellulase producing abilities of the six Trichoderma strains, B-8, B-13, B-19, C1, A6 and G26 in SSF with CCR, wheat bran and rice straw at ratio of 2:4:4 were compared. The results (Fig. 1A–C) showed that the titer of cellulase produced by G26 was the highest among the six strains, resulted in 40.9 IU (FPA), 99.7 IU (CMCase) and 71.8 IU (b-glucosidase) per gram of substrate (dry weight) for 96 h fermentation, respectively, which indicated that G26 had remarkable advantages in production of cellulase, especially in total cellulase (FPA) and endoglucanase (CMCase). Furthermore, the strain G26 was identified as a T. longibrachiatum by morphological observing combined with the sequence analysis for ITS gene of 18SrDNA (results were not listed). Compared with other reported cellulase producing strains of T. longibrachiatum (Gusakov et al., 1985; Gusakov, 2011), G26 showed superiority in b-glucosidase. 3.2. Cellulolytic enzymes preparation by G26 with an optimum medium To improve producing capacity of cellulase, an optimum medium, containing rice straw 4.0 g, wheat bran 4.0 g, corncob residue 2.0 g and 15.0 ml nutrient solution composed of (NH4)2SO4 2.0%
333
(w/v), K2HPO4 1.0% (w/v), soybean cake powder 8.9% (w/v), urea 1.0% (w/v) and corn steep liquor 4.0% (v/v) (results were not listed). The enzyme activities for FPA, CMCase and b-glucosidase in SSF at the optimal medium reached 71.3 ± 3.0 IU/g, 136.2 ± 0.6 IU/g and 85.1 ± 0.1 IU/g, which were 174.3%, 136.6% and 118.5% compared with that before optimization, respectively. Considering the important roles of hemicellulases during the hydrolysis of lignincellulosic materials (Berlin et al., 2005; Kumar and Wyman, 2009; Gao et al., 2011), as well as the strain T. longibrachiatum PTCC 5140 with good performance on xylanase production (Azin et al., 2007), the activity of xylanase produced by G26 in the SSF with the above optimal medium was explored. It indicated that besides satisfactory cellulase producing ability, G26 produced high titer of xylanase, reaching 11,344 ± 28 IU /g.
3.3. Enzymatic hydrolysis of CCR As shown in Table 1, CCR was hydrolyzed at different initial FPA (IU/ml) under the solid loading of 8.5% (w/v), 12.5% (w/v) and 16.5% (w/v), respectively. It was found that, though yield of both glucose and reducing sugar improved with the increasing of enzyme dosage and solid loading, the conversion of cellulose to glucose decreased from 62.1% to 54.2% while saccharifying with 6.5 FPU/ml at solid loading from 12.5% (w/v) to 16.5% (w/v), and also the productivity of glucose to one unit of FPU at solid loading of 12.5% (w/v) was remarkably lowered while increasing the cellulase loading to 10.2 FPU/ml. The results for the time course of glucose released (Fig. 2A) from CCR at solid loading of 12.5% (w/v) showed that the hydrolysis efficiency significantly slowed down while increasing the enzyme loading from 6.5 to 12.6 FPU/ml, resulting in the maximal glucose yield from 46 to 56 g/l, indicating conversion of 62.1% and 75.6%, respectively. Moreover, the glucose level could not be further enhanced after 72 h hydrolysis, which did not be significantly improved even while treated with enzyme as high titer as
Fig. 1. Activities of cellulases produced by Trichoderma strains through SSF. FPA (A), CMCase (B) and b-glucosidase (C) were produced by strains cultured with medium containing 1.2 g corncob residue, 2.4 g wheat bran, 2.4 g rice straw, mixed with nutrient solution containing (NH4)2SO4 3.0% (w/v), KH2PO4 1.0% (w/v) and soybean cake power 1.0% (w/v), at solid–liquid ratio of 1:1.5 (w/v), for the indicated hours, respectively. Data shown are means of three replicated experiments ± standard error.
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Table 1 Effect of enzyme dosage and solid loading on the hydrolysis of CCR. FPU/mla
enzymes of G26 was caused by product accumulation. The results showed that the glucose production were 45.3, 44.3, 44.5 and 44.7 g/l with 0, 23, 38 and 55 g/l glucose added at the beginning of hydrolysis, respectively, which indicated that the glucose productivities were little lowered by pre-adding glucose, even while the initial glucose concentration was as high as 55 g/l, indicating that the cellulolytic enzymes produced by G26 is potential to be used for hydrolysate preparation with high yield of fermentable sugars.
Solid loading (w/ v)
Sugar (g/l)b
8.5%
Reducing sugar Glucose
27 ± 0.3
31 ± 1.1
12.5%
Reducing sugar Glucose
–
33 ± 0.7
61 ± 1.0
67 ± 0.8
–
28 ± 0.1
46 ± 0.7
52 ± 0.5
16.5%
Reducing sugar Glucose
–
–
66 ± 1.8
70 ± 1.4
3.4. Improvement of hydrolysis efficiency of CCR by adding surfactants
–
–
53 ± 1.1
57 ± 0.0
Recently, surfactants especially non-ionic surfactants have been proven to enhance the enzymatic hydrolysis conversion from lignocellulosic biomass substrates, and can lower the enzyme requirement for a given yield effectively (Seo et al., 2011; Li et al., 2012). Therefore, we tried to improve the hydrolysis efficiency through adding certain amount of non-ionic surfactants during the hydrolysis of CCR. Significant improvements were observed both with adding (poly) ethylene glycol (PEG) 6000 and with Tween-80 (Fig. 3A and B), and the higher content of the surfactant, the higher yield of sugars. The maximum glucose titer (58 g/l) and reducing sugar (68 g/l) were obtained with 0.05 g PEG 6000/g substrate added, which were 45% and 39% higher than that without surfactant added, respectively. And with the hydrolysis going on, the glucose titer and reducing sugar titer increased slower and the increase ended earlier than that with surfactant added. That was consistent with the results of Kristensen et al. (Kristensen et al., 2007), which maybe because of the increasing of free enzymes in solution due to the reduction of non-productive/irreversible adsorption of cellulase onto lignin and crystalline cellulose (Kristensen et al., 2007; Li et al., 2012). But regretfully, the residual PEG 6000 in the CCR hydrolysate performed an inhibitory effect on the following L-lactic acid fermentation (results were not listed).
1.4
2.8
6.5
24 ± 0.9
31 ± 0.6
36 ± 0.9
21 ± 0.7
10.2 – –
a
The crude enzyme solution used here was extracted by adding 10 times of the extract buffer (v/w; dry weight) into the fermented mash, resulting in 6.5 FPU/ml; the solution with enzyme activity higher than 6.5 FPU/ml was prepared by extracting the fresh fermented mash with the above mentioned enzyme solution containing 6.5 FPU/ml, whereas those lower than 6.5 FPU/ml was obtained by diluting with buffer. b Results are means of three replicates ± standard error.
3.5. Improvement of hydrolysis efficiency of CCR through a multi-stage process Here we developed a three-stage process for the CCR hydrolysis. The results in Fig. 3C showed that the multi-stage process presented an advantage over the traditional one. The yield of sugar increased faster and was significantly higher than that of control and it seemed better if the supplementation carried out for twice, especially supplementation earlier (24 & 48 h) was better, resulting in the maximum production of glucose 60.1 g/l with a conversion of 81.2% at 72 h, which was 21.4% higher than that of the control. 3.6. Analysis of sugar components of CCR hydrolysate
Fig. 2. The time course of CCR hydrolysis with different FPA dosage at the solid loading of 12.5% (w/v) under the optimum conditions. The concentration of glucose (A) and reducing sugar (B) were determined after taking samples at the indicated time, respectively. Data shown are means of three replicated experiments ± standard error.
12.6 FPU/ml. Similar results were observed for the production of reducing sugar (Fig. 2B). As decreased enzymatic efficiency caused by accumulation of glucose or cellobiose is thought to be the main limitation for getting high concentration of fermentable sugars (Gusakov et al., 1985), we compared with the glucose production from CCR, without or with 23, 38 or 55 g/l glucose added at the beginning of hydrolysis, to investigate if the hindered glucose releasing by
Besides sugar titer, the sugar components in CCR hydrolysate is another key factor that influences the production of L-lactic acid, because high level of unavailable sugars for B. coagulans ZX25 will be remained in the fermented broth, which would lower the quality and increase the cost of L-lactic acid. Therefore, we analyzed the sugars in CCR hydrolysate by High-Performance Capillary Electrophoresis (HPEC). Compared with the standard mixture, the hydrolysate was only composed of glucose and very little xylose (results were not listed), whereas maltose, ribose, mannose and galactose were undetectable. 3.7. L-lactic acid fermentation using the CCR hydrolysate Moreover, the CCR hydrolysate contained 60.1 g/l glucose, supplemented with 0.5% (w/v) peptone, 0.3% (w/v) yeast extract and
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Fig. 4. Time course of L-lactic acid production with hydrolysate of CCR. Fermentation was conducted in 50 ml Erlenmeyer flasks containing 20 ml hydrolysate at a 50 °C shaker (140 rpm) for 60 h, pH was controlled with CaCO3. Samples were taken every 4 h, and the yield of L-lactic acid and residual sugar, as well as the optical density (OD600nm) after three times dilution, were measured, respectively. Data shown are means of three replicated experiments ± standard error.
for the 44 h fermentation. In addition, the residual sugar in the broth after 44 h fermentation was little, as shown in Fig. 4. 4. Conclusion A strain T. longibrachiatum G26 with high yield cellulase production in SSF was developed for the preparation of fermentable hydrolysate from CCR, followed by CCR hydrolysis with the crude enzyme extract using a novel three-stage strategy. The results showed that the production of glucose from CCR was 21.4% improved by the three-stage hydrolysis, and the obtained hydrolysate containing 60.1 g/l glucose is suitable for L-lactic acid production. In further studies, lowering the cost of cellulase preparation, decreasing the enzyme loading for CCR hydrolysis, as well as shorting the hydrolysis period are still needed. Acknowledgement This research work was supported by the Bidding Project of Henan Province (China) for Major Public Welfare Scientific Research (NO. 91100910300). Fig. 3. The improvement of CCR hydrolysis efficiency through adding surfactant or multi-stage process. CCR hydrolysis was carried out at the final solid loading of 12.5% (w/v) with 6.5 FPU/ml under the optimum conditions. (A) The glucose and reducing sugar obtained without or with different dosages (0.1–5.0 g/100 g CCR) of PEG 6000 (P) or Tween-80 (T). (B) The comparison of variation for glucose and reducing sugar with the hydrolysis time prolonging between control and the treatment of adding 0.05 g/g CCR of PEG 6000. (C) Improvement through multistage process. For control, 50 ml crude enzyme extract was one-time added at the beginning of the enzymatic reaction; for the treatments of 36 and 48 h, 40 ml enzyme solution were firstly added, and then 10 ml were supplemented at 36 and 48 h, respectively; for the treatments of 24 & 48 h and 36 & 72 h, 10 ml were supplemented by twice, for 5 ml each time, at 24 and 48 h, 36 and 72 h, respectively. Samples were taken at the indicated time, and then glucose concentration was measured before the enzyme supplementation. The glucose yield means the value to the final volume. Data shown are means of three replicated experiments ± standard error.
0.2% (w/v) K2HPO4 was used to prepare L-lactic acid by fermentation with B. coagulans ZX25. The results (Fig. 4) showed that the present CCR hydrolysate was very suitable for ZX25 growth, as well as the accumulation of L-lactic acid, which was time-saving with higher yield and higher conversion, obtaining a maximum yield of 52.0 g/l with the sugar-acid conversion (as to glucose) of 86.5%
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