Optimization of Alkaline Sulfite Pretreatment and Comparative Study

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Optimization of Alkaline Sulfite Pretreatment and Comparative Study with Sodium Hydroxide Pretreatment for Improving Enzymatic Digestibility of Corn Stover Huan Liu,†,§ Bo Pang,§ Haisong Wang,*,†,§ Haiming Li,† Jie Lu,† and Meihong Niu*,† †

Liaoning Key Laboratory of Pulp and Papermaking Engineering, Dalian Polytechnic University, Dalian, 116034 Liaoning, China CAS Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, 266101 Shandong, China

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ABSTRACT: In this study, alkaline sulfite pretreatment of corn stover was optimized. The influences of pretreatments on solid yield, delignification, and carbohydrate recovery under different pretreatment conditions and subsequent enzymatic hydrolysis were investigated. The effect of pretreatment was evaluated by enzymatic hydrolysis efficiency and the total sugar yield. The optimum pretreatment conditions were obtained, as follows: the total titratable alkali (TTA) of 12%, liquid/solid ratio of 6:1, temperature of 140 °C, and holding time of 20 min. Under those conditions, the solid yield was 55.24%, and the removal of lignin was 82.68%. Enzymatic hydrolysis rates of glucan and xylan for pretreated corn stover were 85.38% and 70.36%, and the total sugar yield was 74.73% at cellulase loading of 20 FPU/g and β-glucosidase loading of 10 IU/g for 48 h. Compared with sodium hydroxide pretreatment with the same amount of total titratable alkali, the total sugar yield was raised by about 10.43%. Additionally, the corn stover pretreated under the optimum pretreatment conditions was beaten by PFI at 1500 revolutions. After beating, enzymatic hydrolysis rates of glucan and xylan were 89.74% and 74.06%, and the total sugar yield was 78.58% at the same enzymatic hydrolysis conditions. Compared with 1500 rpm of PFI beating after sodium pretreatment with the same amount of total titratable alkali, the total sugar yield was raised by about 14.05%. KEYWORDS: corn stover, alkaline sulfite pretreatment, enzymatic hydrolysis, total sugar yield

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INTRODUCTION With the development of the economy and the rapid increase of population, the problems of energy shortage and environment pollution have driven humans to pay more attention to renewable and clean energy.1 Biomass can be converted into energy, materials, or chemicals to replace nonrenewable fossil resources. It is one of the hot research topics that catch the attention of governments and scientists around the world.2,3 It also helps reduce carbon dioxide emissions and the greenhouse effect.4 Corn stover is one of the most abundant agricultural residues in areas with high levels of corn production, and it represents an ideally cheap, renewable, widely available feed stock for bioconversion to fuels and chemicals.5,6 Bioconversion of lignocellulosic biomass involves two main processes: enzymatic hydrolysis of the lignocellulosic biomass to produce reducing sugars and fermentation of the sugars to produce ethanol or other chemicals.7 Biomass pretreatment is one of the most important processes in bioethanol production. The chemical barriers such as hemicellulose and lignin greatly inhibit the accessibility of enzyme to the cellulose substrate.8−10 In recent years, a variety of physical (mechanical comminution,11 milling, and grinding), chemical (acid, alkali,12 and organic solvents13), physicochemical (steam explosion14 and ammonia fiber explosion15), and biological16 pretreatment methods have been investigated. Each pretreatment method has different effects on the lignocellulosic structure and the formation of inhibitory compounds for microbial processes.17 Alkali-based pretreatment can efficiently remove lignin and various uronic acid substitutions on © XXXX American Chemical Society

hemicellulose, with relatively low polysaccharide loss compared with acid or hydrothermal pretreatment.18 Also, over 90% of pulp mills are alkali-based.19 Thus, one viable approach is to integrate the alkali-based pretreatment with an existing pulp mill by using the equipment and chemical recovery system already developed in the pulp industrial setting. Therefore, alkali-based pretreatment is considered one of the most promising pretreatment methods.20 But the carbohydrates inevitably dissolve in the black liquor during alkali-based pretreatment process and are difficult to recycle thus affecting the total sugar yield of enzymatic hydrolysis. Our previous study indicated that a high total sugar yield (glucose and xylose) of 0.48 g/g of original biomass could be achieved after alkaline sulfite pretreatment, and it was higher than that from sodium hydroxide pretreatment under the same conditions.21 However, the optimization of total alkali change, liquid/solid ratio, temperature, and holding time has not been researched in the previous study. In this study, the optimal pretreatment conditions were explored on the foundation of the previous study, and the mechanism for improving the total sugar yield was also analyzed. Received: November 11, 2014 Revised: March 13, 2015 Accepted: March 15, 2015

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DOI: 10.1021/jf505433q J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. Effect of TTA charge on delignification rate, polysaccharide recovery, enzymatic hydrolysis rate of polysaccharides, and total sugar yield.

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analysis was carried out using external standard calibration. Duplicate experiments were conducted, and the mean values were reported. FTIR Analysis. The FTIR spectra of the untreated and pretreated corn stover were determined on a FTIR spectrometer (Nicolet 6700, Thermo Fisher Scientific Inc.). The samples were prepared by KBr pellet (the ratio of the samples to the KBr was 1:100). The resolution of the spectra was 4 cm−1 in the range of 400−4000 cm−1, and 32 scans were taken for per sample. SEM Analysis. The SEM analyses of the samples were conducted by scanning electron microscopy (S-4800, Hitachi, Japan) at different magnifications. The oven-dried dried samples were coated with platinum and treated with accelerating voltage of 3.0 kV.

MATERIALS AND METHODS

Materials. The corn stover used in the present study was obtained from Qingdao, Shandong Province, China. Air-dried corn stover was milled using a plant grinder, and the particles between the sizes of 5 and 40 mesh were collected. The screened corn stover was stored in sealed plastic bags at room temperature. Sodium hydroxide and sodium sulfite were purchased from Sinopharm Chemical Reagent Co. Ltd. The enzymes used for the enzymatic hydrolysis were Celluclast 1.5L (cellulase, enzyme activity 192 FPU/mL) and Novozyme 188 (β-glucosidase, enzyme activity 741 IU/mL) provided by Sigma-Aldrich China Inc. The enzymes activities were determined according to the method reported by Ghose.22 All chemicals and enzymes were used as received. Pretreatment of Corn Stover. The pretreatment of corn stover (50 g oven-dried) was carried out in a pot with a total volume of 1.5 L. Corn stover was subjected to pretreatment using sodium hydroxide (NaOH)−sodium sulfite (Na2SO3) with total titratable alkali charge of 6%, 8%, 10%, 12%, and 14%. The molar ratio of NaOH−Na2SO3 was 1:1. The ratio of pretreatment liquor to corn stover (oven-dried) charge was 4:1, 6:1, 8:1, 10:1, and 12:1, respectively. Both solution and corn stover were heated to the desired temperature in 20 min from 30 °C. The pretreatments were done at five levels in the range of 120− 160 °C at holding time ranging from 10 to 50 min. After pretreatment, the bombs were cooled in water to room temperature. The solids were collected and washed with deionized water to remove residual chemicals and dissolved compounds and then used for determination of total solid, lignin, and polysaccharides prior to enzymatic hydrolysis. PFI Refining. The pulp pretreated with alkali was beaten in a PFI mill. Beating conditions were as follows: pulp consistency 10%, beating gap 0.3 mm, revolutions in the range 1500−6000 rpm and increments of 1500 revolutions. The refined pulp was collected and stored in the refrigerator. Enzymatic Hydrolysis. Enzymatic hydrolysis of the substrates was conducted on a 0.4 g (oven-dried) basis in a serum bottle with substrate consistency of 2% (W/V) at 50 °C using an incubator shaker at 95 rpm for 48 h. Sodium citrate buffer (pH = 4.8) was added to maintain the pH at 4.8, while 0.02% sodium azide was used in the mixture to inhibit the microbial infections. An enzyme cocktail of cellulase (20 PFU/g of dry biomass) and β-glucosidase (10 IU/g of dry biomass) was used for enzymatic hydrolysis. Analysis Methods. Chemical Component Analysis. Cellulose, hemicellulose, and lignin contents of corn stover were determined according to the procedures described by National Renewable Energy Laboratory (NREL).23 Acid and enzymatic hydrolysates were examined by high performance liquid chromatography (HPLC; model 1200, Agilent, USA). The HPLC system was equipped with a Bio-Rad Aminex HPX-87H column (300 mm × 7.8 mm) and refractive index detector. The column was run at 55 °C with sulfuric acid (0.005 M) as a mobile phase (0.5 mL/min), and the quantitative

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RESULTS AND DISCUSSION Optimization of the Alkali Dosage. The rates of delignification and polysaccharide retention after different pretreatments are presented in Figure 1a. Lignin is one of the major barriers for the enzymatic hydrolysis of lignocellulosic biomass. The efficiency of enzymatic hydrolysis was usually improved by the lignin removal.24−26 The removal of lignin not only exposes more accessible cellulose but also reduces the strong surface interaction between lignin and enzyme. Figure 1a shows that the removal of lignin was improved by increasing the total titratable alkali (TTA) charge. When TTA charge was 14%, the removal of lignin was as high as 87.66%. Sodium hydrate has a certain effect on delignification. Sulfite also plays an important role in alkaline sulfite pretreatment process. The nucleophilic sulfite (SO32−) in the pretreatment liquor lead to the sulfonation of lignin, as well as the break of α-benzyl ether linkages, α-alkyl ether linkages, and β-benzyl ether linkages on phenolic lignin units.18 One can also observe that glucan and xylan decrease with the increase of TTA charge, and the degradation of glucan is less than that of xylan. One can infer that cellulose is more stable than hemicellulose under the alkaline sulfite pretreatment condition because of its high degree of polymerization and crystalline nature.27 This is the main disadvantage of alkali-based pretreatment at present. So how to promote the deprivation of lignin and maximum preservation of hemicellulose will be an important research topic. The enzymatic hydrolysis rates of polysaccharides and total sugar yield at different chemical charge are illustrated in Figure 1b. Undoubtedly, higher chemical charge will enhance the enzymatic hydrolysis rates of polysaccharides. When the TTA charge increased from 6% to 14%, the enzymatic hydrolysis rates of glucan and xylan increased more than one times. This B

DOI: 10.1021/jf505433q J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 2. Effect of liquid/solid ratio on delignification rate, polysaccharide recovery, enzymatic hydrolysis rate of polysaccharides, and total sugar yield.

Figure 3. Effect of temperature on delignification rate, polysaccharide recovery, enzymatic hydrolysis rate of polysaccharides, and total sugar yield.

hydrolysis rates of polysaccharides increased first and then decreased, and there was little difference when liquid/solid ratio ranged from 6 to 10. Total sugar yield showed the same trends. For example, the total sugar yields were 70.07% and 73.53%, respectively, when liquid/solid ratio was 4:1 and 6:1. In addition, the total sugar yield was only 74.28−75.56% when liquid/solid ratio ranged from 6 to 10. Therefore, the liquid/ solid ratio of 6:1 was optimal. Optimization of Temperature. Figure 3a shows the effect of temperature on the delignification rates and polysaccharide retention under the conditions of TTA charge at 12% and liquid/solid ratio of 6:1. The data show that lignin removal increased with the pretreatment temperature. For example, the removal of lignin increased from 80.19% to 91.66% when the temperature rose from 120 to 160 °C. This indicates that pretreatment temperature plays an important role in delignification. However, the retention of polysaccharides reduced with increasing pretreatment temperature. The degradation of xylan is higher than that of glucan in the same pretreatment conditions. This demonstrates that hemicellulose in corn stover is easier to degrade than cellulose in alkaline sulfite pretreatment. The enzymatic hydrolysis rates of polysaccharides, increased first and then decreased, can be seen from Figure 3b. It is obvious that the enzymatic hydrolysis rate at 140 °C is much higher than that at 120 °C. This is mainly caused by the limited removal of hemicellulose and lignin at relatively low temper-

results from the destruction of cell walls and the dissolution of hemicellulose and lignin. It is obvious that the total sugar yield showed a rapid increase trend with the increase of alkali dosage and reached the maximum value when the TTA charge was 12%. However, the total sugar yield did not increase when we continued to increase the TTA charge to 14%. This was because the retention rate of glucan and xylan decreased in different degrees though the enzymatic hydrolysis rates of glucan and xylan increased 3.52% and 2.45%, respectively, when we continued to increase the TTA charge. Based on the data from Figure 1, one can conclude that 12% is the optimal dosage of chemicals for alkaline sulfite pretreatment. Optimization of the Liquid/Solid Ratio. Figure 2a shows the impacts of liquid/solid ratio on the delignification rates and polysaccharide retention when TTA charge is 12%. About 85% of the lignin in the raw material was removed when the liquid/ solid ratio was 6:1. However, when liquid/solid ratio was 4:1, the removal of lignin was only 53.48%. A high concentration of lignin in the pretreatment liquid can inhibit the dissolution of lignin in the raw material. But if the liquid/solid ratio is too high, the high liquid/solid ratio will reduce the concentration of chemicals and increase the difficulty of subsequent processing. In addition, the data showed that the role of liquid/solid ratio on polysaccharide retention is not significant. According to the curves of enzymatic hydrolysis rates of polysaccharides and total sugar yield in Figure 2b, some details can be seen. With the increase of liquid/solid ratio, enzymatic C

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Figure 4. Effect of holding time on delignification rate, polysaccharide recovery, enzymatic hydrolysis rate of polysaccharides, and total sugar yield.

ature, which does not provide a loose enough structure of corn stover.28 According to the curve of total sugar yield of pretreated corn stover in Figure 3, the highest sugar yield is obtained at 140 °C, which is 74.28%. Furthermore, temperature higher than 140 °C was not necessary nor recommended because higher temperature consumes more energy. Hence, 140 °C was recommended as the optimum pretreatment temperature. Optimization of the Holding Time. In order to investigate the effect of holding time on delignification rates, polysaccharide retention, enzymatic hydrolysis rates, and total sugar yield, the pretreatments were done at holding time ranging from 10 to 50 min. Figure 4a shows the effect of holding time on and polysaccharide retention. One can observe that the delignification rates were increased with the increase of the holding time while the polysaccharides retention decreased. As seen in Figure 4b, increasing the holding time from 10 to 50 min only led to 2% increase in the enzymatic hydrolysis rates of glucan. When holding time was more than 20 min, the total sugar yield decreased because of the reduction in polysaccharide retention. The holding time of 20 min can obtain a better effect. Comparison of Pretreatment between Alkaline Sulfite and Sodium Hydroxide. Optimum pretreatment conditions were obtained through the above optimization experiments: total titratable alkali of 12%, liquid/solid ratio of 6:1, temperature of 140 °C, and holding time of 20 min. Figure 5 shows the comparison between alkaline sulfite pretreatment and sodium hydroxide pretreatment under the optimum conditions. Delignification rates and polysaccharide retention rates of corn stover pretreated with alkaline sulfite are higher than those when we used sodium hydroxide alone. This is because the nucleophilic sulfite (SO32−) and bisulfite (HSO3−) lead to the sulfonation of lignin. Sulfonation makes the lignin more hydrophilic. In addition, a lower sodium hydroxide charge reduces the degradation of cellulose and hemicellulose, which enhances the retention of carbohydrate. For example, compared with sodium hydroxide pretreatment, the retention of xylan is improved by more than 10%. Figure 6 shows the enzymatic hydrolysis rates and total sugar yield of the corn stover pretreated by two different methods and then beaten with a PFI mill. One can observe that both the enzymatic hydrolysis rate of polysaccharides and total sugar yield are improved with the increase of revolutions. The shearing action in mechanical refining results in more internal delamination and surface fibrillation, which will disrupt the

Figure 5. Comparison of alkaline sulfite pretreatment and sodium hydroxide pretreatment.

Figure 6. Effect of refining on enzymatic hydrolysis of polysaccharides and total sugar yield.

crystalline structure of the cellulose microfibrils, increase the accessible specific areas, and lead to a more efficient biomass digestibility.29 Xu et al. found that beating degree of PFI refined substrates was strongly linear with final total sugar yields for the given pretreated corn stover.30 Furthermore, it can achieve a relatively high enzymatic hydrolysis rate of polysaccharides with PFI refining at 1500 revolutions, and the total sugar yield was 78.58%. Compared with the sodium hydroxide pretreatment, the total sugar yield was increased by about 9.68%. D

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Journal of Agricultural and Food Chemistry Mechanism Analysis for the Improvement of Corn Stover Enzymatic Hydrolysis Rate. FTIR Analysis. FTIR spectra of untreated and pretreated corn stover are shown in Figure 7. In particular, all the spectra are dominant by signals at

Figure 7. FTIR spectra of untreated and pretreated corn stover.

3400 and 2920 cm−1 due to the stretching vibrations of O−H and C−H corresponding to the aliphatic moieties in lignin and polysaccharides.31 A shoulder at 1740 cm−1 is attributed to the acetyl and uronic ester groups on hemicellulose or linkages in lignin and ester hemicellulose feurilic and p-coumaric acid carboxylic groups.32 The band at 1740 cm−1 being almost absent indicated that the pretreatment nearly cleaved this ester band from the hemicellulose and lignin. The lignin-associated peaks at 1510 and 795 cm−1 that were assigned to aromatic ring stretch vibrations in lignin were decreased, indicating the removal of the lignin after pretreatment. Furthermore, the band at 1510 cm−1 after alkaline sulfite pretreatment almost vanishes, which also indicates that the alkaline sulfite pretreatment is more efficient than sodium hydroxide on delignification. The carbohydrate-related peaks at 1380 and 1161 cm−1 were assigned to the C−H deformation in carbohydrate and C−O− C vibration in cellulose and hemicellulose.33 After pretreatment, the intensity of these bands was decreased due to the degradation of cellulose and hemicellulose, and after sodium hydroxide pretreatment, the intensity decreased more severely than after alkaline sulfite pretreatment, which corresponded to the results obtained by sugar analysis. In addition, the peak at 897 cm−1 was associated with the β-glucosidic bonds of cellulose, and its increase after pretreatment was also because of the removal of lignin and hemicelluloses.34 As can be seen from Figure 7, the band at 897 cm−1 after alkaline sulfite pretreatment is stronger than that after sodium hydroxide pretreatment, which indicated that the degradation of lignin and hemicellulose were relatively stronger in alkaline sulfite pretreatment. The decrease of the peak at 465 cm−1 (Si−O) was due to the removal of ash. SEM Analysis. Pretreatment affects not only the chemical composition of the corn stover but also the physical appearance of the corn stover at the microscopic level. The scanning electron micrograph (SEM) images of untreated and pretreated corn stover are shown in Figure 8 at different magnifications. The surface of the corn stover that is untreated shown in Figure 8a,b is smooth and contiguous without pores. The tissue surface and cell wall were altered after pretreatment. Figure 8c,d

Figure 8. SEM of untreated and pretreated corn stover: (a, b) raw corn stover without pretreatment; (c, d) samples pretreated with NaOH pretreatment; (e, f) samples pretreated with alkali sulfite pretreatment. Pretreatment was performed at 140 °C with the liquid/ solid ratio of 6 and 12% alkali.

shows images of corn stover after NaOH pretreatment. The smooth, contiguous surface of untreated stover has been perforated by the pretreatment processes. Alkaline pretreatments disrupt the lignocellulosic structure by dissolving lignin and hemicellulose. Treatment with Na2SO3 significantly alters the fibrillar structure. Coarse surfaces and porous areas are very evident. The microfibrils are separated from the initial connected structure and fully exposed. This would increase the external surface area and the porosity of the biomass.35 These pores may increase the enzyme accessible surface area, which increases the enzyme digestibility of the corn stover.36 Conclusion. The optimum conditions of alkaline sulfite pretreatment for corn stover were as follows: total titratable alkali of 12%, liquid/solid ratio of 6:1, temperature of 140 °C, and holding time of 20 min. The delignification rate and the total sugar yield at the optimal condition were 82.45% and 74.73%, respectively, which was higher than those under only NaOH pretreatment, 79.20% and 67.67%. The corn stover pretreated under the optimum pretreatment conditions was beaten by PFI for 1500 rotations. After beating, the total sugar yield was 78.58%. Compared with 1500 rotations of PFI beating after sodium hydroxide pretreatment at the same amount of total titratable alkali, the total sugar yield improvement rate was 14.05%. Furthermore, lignin dissolved in the alkaline sulfite pretreatment exists in the form of lignosulfonate, which is a potential high-value coproduct. Thus, alkaline sulfite pretreatment is a suitable process for corn stover conversion to fermentable sugars. E

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AUTHOR INFORMATION

Corresponding Authors

*H. Wang. Phone: +86 532 80662725. Fax: +86 532 80662725. E-mail: [email protected]. *M. Niu. E-mail: [email protected]. Funding

This research was funded by the Natural Science Foundation of China (Grant Nos. 21206184, 31370582, and 31370584) and the National High Technology Research and Development Program (“863” program) of China (Grant No. 2012AA022301). Notes

The authors declare no competing financial interest.

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DOI: 10.1021/jf505433q J. Agric. Food Chem. XXXX, XXX, XXX−XXX