Appl Biochem Biotechnol DOI 10.1007/s12010-014-1242-2
The Effect of Nonenzymatic Protein on Lignocellulose Enzymatic Hydrolysis and Simultaneous Saccharification and Fermentation Hui Wang & Shinichi Kobayashi & Hatsue Hiraide & Zongjun Cui & Kazuhiro Mochidzuki
Received: 22 April 2014 / Accepted: 10 September 2014 # Springer Science+Business Media New York 2014
Abstract Nonenzymatic protein was added to cellulase hydrolysis and simultaneous saccharification and fermentation (SSF) of different biomass materials. Adding bovine serum albumin (BSA) and corn steep before cellulase enhanced enzyme activity in solution and increased cellulose and xylose conversion rates. The cellulose conversion rate of filter paper hydrolysis was increased by 32.5 % with BSA treatment. When BSA was added before cellulase, the remaining activity in the solution was higher than that in a control without BSA pretreatment. During SSF with pretreated rice straw as the substrate, adding 1.0 mg/mL BSA increased the ethanol yield by 13.6 % and final xylose yield by 42.6 %. The results indicated that lignin interaction is not the only mechanism responsible for the positive BSA effect. BSA had a stabilizing effect on cellulase and relieved cumulative sugar inhibition of enzymatic hydrolysis of biomass materials. Thus, nonenzymatic protein addition represents a promising strategy in the biorefining of lignocellulose materials. Keywords Lignocellulose . Saccharification . Nonenzymatic protein . Simultaneous saccharification and fermentation (SSF)
H. Wang : S. Kobayashi : H. Hiraide : K. Mochidzuki (*) Institute of Industrial Science, The University of Tokyo, Tokyo 153-8505, Japan e-mail: [email protected] H. Wang e-mail: [email protected] S. Kobayashi e-mail: [email protected] H. Hiraide e-mail: [email protected] Z. Cui College of Agronomy and Biotechnology/Center of Biomass Engineering, China Agricultural University, Beijing 100193, China e-mail: [email protected]
Appl Biochem Biotechnol
Introduction Ethanol produced from lignocellulosic biomass has a great potential to address vital energy security, trade deficit, environmental, and economic issues that are becoming more urgent in light of the declining petroleum reserves and increasing international demand for transportation fuels [1, 2]. The conversion of cellulose to glucose, which is the initial process of biorefining, has remained a bottleneck [3], restricting the initial conversion rate. High cellulase dosage and prolonged process time increase the final ethanol yield but result in high process costs. Thus, it is important to find methods to reduce the cellulase dosage and shorten the process time without decreasing the conversion rate. Many studies have been aimed to identify and manipulate the inhibiting factors in enzymatic hydrolysis of lignocellulose substrates. It has been shown that the surface area of cellulose is an important substrate characteristic in determining initial rates of hydrolysis [4, 5]. As mentioned in a report by Rollin et al. [6], it has been concluded that increasing cellulose accessibility is a more important pretreatment consideration than delignification for effectively releasing sugars from recalcitrant lignocelluloses at a high yield. Enzyme activity loss because of nonproductive adsorption on lignin surfaces has been identified as an important factor for decreasing enzyme effectiveness, and the effect of surfactants and noncatalytic protein on the enzymatic hydrolysis of cellulose into fermentable sugars has been extensively studied [7–9]. The cellulose conversion rate at 72 h was increased from 9 to 21 % and 1 to 8.5 % for samples with high and low lignin contents by the injection of Tween-20 (0.024– 0.24 mM), respectively [10]. Eriksson et al. [7] tested the effect of the addition of bovine serum albumin (BSA) at a concentration of 17 g/L on the hydrolysis of steampretreated spruce and compared it with the effect of different surfactants. In a previous study, we concluded that surfactants adsorb to lignin and prevent the unproductive adsorption of enzymes [7]. The addition of surfactants and polymers has also been described to exert a stabilizing effect on enzymes. Kaar and Holtzapple [11] suggested that Tween can protect enzymes from thermal denaturation in hydrolysis conducted at higher temperatures. Our recent study showed that BSA improves the stability of cellulase [12]. Most studies have focused on the use of different surfactants, and only few reports are available on the use of BSA and corn steep as additives. Furthermore, the mechanisms underlying the enhancement of enzymatic hydrolysis by additives are unknown. To investigate these mechanisms and identify the substrate characteristics influencing the effects of additives during enzymatic cellulose hydrolysis, the present study compared pure cellulosic substrates (filter paper), natural lignocellulosic materials (rice straw), and noncellulosic biomass (xylan and lignin) by compositional analysis, enzymatic hydrolysis, and simultaneous saccharification and fermentation (SSF) using BSA as a model compound.
Materials and Methods Materials Substrate Pretreated rice straw [2 %, NaOH 1 N, 50 °C 4 h, 25 °C overnight, washed with deionized (DI) water until neutral, dried at 60 °C to constant weight, and milled to pass through a 1-mm mesh]. The compositions of materials used in the present study are shown in Table 1.
Appl Biochem Biotechnol Table 1 Lignocellulosic components of biomass materials Sample
Cellulose %
Hemicellulose %
Lignin %
Rice straw
31.7
27.1
5.2
Pretreated rice straw
47.3
16.1
3.8
Model lignocellulosic biomass component: Cellulose: filter paper (Whatman No. 1, 1×1-cm squares) Hemicellulose: xylan (1 %, from birchwood, Sigma) Lignin: lignin powder (0.5 %, lignin, organosolv, Sigma) Protein: BSA (Cohn, fraction V, pH 7.0, Wako) Cellulase: Acremonium cellulase (AC, derived from A. cellulolyticus; Meiji Seika, Tokyo, Japan; No.: ACCF-4940). Avicelase activity is 1040 u/g (pH 5.7). Dry yeast: Ethanol RedTM (Saccharomyces cerevisiae, Fermentis, France) Corn steep: Solvlys 095 E (Roquette Freres)
Methods BSA Pretreatment Biomass substrates were put into a 50 mM citric buffer (pH 4.8) in a shaking flask (filter paper 2 g dry weight/100 mL, xylan 1 g dry weight/100 mL, lignin 0.5 g dry weight/100 mL, pretreated rice straw 2 g dry weight/100 mL) and autoclaved at 121 °C for 20 min. After cooling to room temperature, BSA was placed in the flasks to make the initial BSA concentration of 0.5, 1.0, or 1.5 mg/mL, and the flasks were then placed in a shaking incubator with shaking at 150 rpm at 50 °C. Aliquots of 0.5 mL were taken at 0, 0.5, 1, 3, 6, 12, 24, 48, and 72 h and immediately centrifuged at 10,000 rpm for 5 min. The supernatants were passed through a 0.22-μm filter paper and assayed for free BSA concentration. Enzymatic Hydrolysis All experiments with enzymes were performed in 50 mM citric buffer (pH 4.8). Enzymatic hydrolysis of the substrate was performed at 15 filer paper units (FPU) cellulase/g substrate, with and without 24-h BSA pretreatment. Incubation conditions were the same as those for BSA pretreatment (150 rpm, 30 or 50 °C). Samples of 1 mL were collected at 0, 0.5, 1, 3, 6, 12, 24, 48, and 72 h; chilled on ice; and centrifuged at 10,000 rpm for 5 min. The supernatants were passed through a 0.22-μm filter paper. Glucose concentration, free protein concentration, and free cellulase activity were measured in the supernatants as described below. All the experimental results are the average of two replicates, unless specified otherwise. SSF All SSF runs were performed in 300-mL flasks (working volume 150 mL) in a shaking incubator with shaking at 130 rpm at 35 °C. The SSF reaction mixture contained 6 g of substrates, and the enzyme dosage was 15 FPU/g substrate. The basal medium contained 10 g/L yeast extract and 20 g/L peptone. Corn steep SSF used a basal medium containing only 50 g/L corn steep. The basal medium was autoclaved at 121 °C for 20 min. SSF experiment protocols employed standard biomass analytical methods of the National Renewable Energy Laboratory (NREL) (NREL/TP-510-42630).
Appl Biochem Biotechnol
Analytical Methods Protein Concentration Determination The Protein Assay Lowry Kit (Nacalai Tesque No. 29470) was used for protein quantitative analysis using the Lowry method [13]. Cellulase Activity An enzyme activity assay using Whatman No. 1 filter paper as a substrate was performed. NREL FPU (NREL/TP-510-42628) was used for enzyme activity analysis. Supernatants from samples collected after concentration and filter treatment were used to measure free cellulose activity. Sugar and ethanol concentrations in the solution were measured by high-performance liquid chromatography (SHIMADZU LC-10AD, with refractive index detector RID-10A). The column was SUGAR SH1011 (SHODEX). The components of materials were analyzed using a fiber analyzer (Model ANKOM220, USA) as described previously [14].
Results Effect of Nonenzymatic Protein on Enzymatic Hydrolysis The effect of nonenzymatic protein on enzyme activity was evaluated by measuring enzyme activity (in FPU) remaining free in the solution during enzymatic hydrolysis. Based on a relative activity of 100 % for the enzyme in the solution at time 0, when nonenzymatic protein was added before cellulase, the remaining solution activity was higher than that in the control without nonenzymatic protein presoaking (Fig. 1). After hydrolysis of pretreated rice straw for 48 h, the relative enzyme activity was 43.67 % in the control. With BSA and corn steep treatment, at least 52.73 and 58.02 %, respectively, of the initial activity was retained. The effects of BSA on enzymatic hydrolysis of different materials are shown in Fig. 2. Enzymatic hydrolysis of each biomass material was performed with AC. With the addition of BSA, the production concentration increased in all the experiments, although not all increases were statistically significant. The cellulose conversion of filter paper hydrolysis was increased by 32.5 % (from 74.90 to 98.93 %). Initially, the effect of BSA on the yield of enzymatic hydrolysis was insignificant. After a 24-h enzymatic hydrolysis of pretreated rice straw, glucose conversion increased by 17.9 % (from 69.01 to 81.35 %) with 15 FPU/g substrate, and after a 72-h hydrolysis, it increased by 15.3 % (from 71.37 to 82.29 %). Corn steep also increased the glucose yield from enzymatic hydrolysis of pretreated rice straw, particularly at a low enzyme dosage (Fig. 3). Absorption Behaviors of BSA onto Biomass Materials To determine the adsorption isotherms of different substrates, BSA preparations with various concentrations were tested and protein content in supernatants was determined. The data shown in Fig. 4 indicate that the adsorption isotherms on filter paper, xylose, and lignin were well fitted to a linear correlation (R2 >0.9). The data again revealed pronounced differences between pure biomass materials (filter paper, xylan, and lignin) and natural lignocellulosic materials (rice straw). We also investigated the adsorption behavior of BSA on different substrates at 50 °C. BSA adsorption varied according to the biomass material: lignin ≥ xylan > pretreated rice straw > filter paper (Fig. 5). The adsorbed amounts of BSA and cellulase were measured during the time course of hydrolysis. After BSA presoaking, the adsorption behavior of enzyme varied across different
Appl Biochem Biotechnol
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Fig. 2 Effect of BSA on the enzymatic hydrolysis of biomass materials at 50 °C. a Substrate: filter paper and xylan. b Substrate: rice straw (substrate concentration: filter paper 2 % w/v, xylan 1 % w/v, rice straw 2 % w/v; enzyme dosage: 15 FPU/g substrate)
Appl Biochem Biotechnol
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unpretreated rice straw - 7.5 FPU unpretreated rice straw - corn steep - 15 FPU
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Time ( Hour) Fig. 3 Effect of corn steep on glucose yield of pretreated and unpretreated rice straw at 35 °C with different enzyme dosages (substrate concentration: 2 % w/v, enzyme dosage: 7.5 and 15 FPU/g substrate)
biomass substrates. In xylan hydrolysis, enzyme adsorption was much lower than that without BSA presoaking at the beginning of hydrolysis. However, after 72 h, in contrast to 1.0- and 1.5-mg/mL BSA presoaking treatments, enzyme adsorption in the 0.5-mg/mL BSA presoaking treatment was slightly greater than that in the control (Fig. 6a). During lignin hydrolysis, enzyme adsorption with BSA presoaking was lower than that without BSA presoaking (Fig. 6b). Figure 6c shows that during the hydrolysis of rice straw, the dynamics of enzyme and BSA adsorption was similar to that of xylan hydrolysis. Interestingly, enzyme adsorption on xylan was the highest among all biomass materials. These results show that BSA increased the amount of free enzyme during the enzymatic hydrolysis of biomass materials; however, this effect diminished in the course of hydrolysis. Effect of Protein Additives on SSF Figure 7a shows the effect of 1 mg/mL BSA on SSF of filter paper and pretreated rice straw. The highest ethanol concentration was obtained after approximately 48 h for filter paper and pretreated rice straw. The addition of 1.0 mg/mL BSA increased the ethanol yield by 13.6 % (from 7.41 to 8.31 g/L) with pretreated rice straw as the substrate. In SSF of filter paper, the final ethanol concentrations were 16.30 g/L with BSA pretreatment and 16.03 g/L without BSA pretreatment. However, as shown in Fig. 7b, the use of corn steep as a protein additive and presoaking for 24 h before SSF did not markedly affect the ethanol yield in SSF of filter
Appl Biochem Biotechnol 30
a
60
b y = 67.173x + 1.6352 R² = 0.9433
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Fig. 4 Adsorption isotherm of BSA at 50 °C (a adsorption isotherm of filter paper, b adsorption isotherm of xylan, c adsorption isotherm of lignin, d adsorption isotherm of rice straw)
paper. With pretreated rice straw as the substrate, the final ethanol concentration was increased by 5.0 % (from 8.05 to 8.48 g/L) by corn steep presoaking for 24 h. However, during SSF of different materials, the final xylose yield increased by 42.6 % (from 2.19 to 3.13 g/L) with BSA addition and 38.6 % (from 2.19 to 3.04 g/L) with corn steep presoaking (Fig. 8). When glucose and xylose were added to the enzymatic hydrolysis process, decreased cellulose and xylan conversion was observed. Following the addition of 10 and 5 mg/mL glucose to the enzymatic hydrolysis of filter paper, glucose concentrations ranged from 21.60 (with BSA) to 18.81 g/L and from 20.39 (with BSA) to 18.05 g/L, respectively (Fig. 9a). Following the addition of 0.4 and 0.2 mg/mL xylose to the enzymatic hydrolysis of xylan, xylose concentrations ranged from 3.43 (with BSA) to 1.88 g/L and from 3.23 (with BSA) to 3.0 g/L, respectively (Fig. 9b).
Discussion As shown in Figs. 1 and 2, BSA and corn steep presoaking improved the enzymatic hydrolysis of biomass materials at various stages. BSA and other proteins adsorb competitively and
Appl Biochem Biotechnol
Fig. 5 Quantity of BSA absorbed on different biomass substrates at 50 °C
irreversibly to lignin and thereby improve the effectiveness of cellulase, consequently increasing the enzymatic hydrolysis rate of biomass materials [6, 8]. In the present study, BSA markedly improved the cellulose conversion rate in the hydrolysis of pure cellulose material (filter paper). Lignin interaction is not the only mechanism responsible for the positive effect of BSA on the enzymatic hydrolysis of cellulose and xylan. Our previous results showed that the effect of BSA on the remaining enzyme activity was much higher at 50 °C than at 35 °C [12] and that yield improvements were observed only after prolonged reaction times for biomass materials. As described in previous studies [12], the mechanism of “lignin blocking” by nonenzymatic protein was not responsible for the increased enzyme performance. We also measured the enzyme activity during the incubation of enzyme and BSA without the addition of a biomass substrate in a previous study [12] and showed that BSA has a stabilizing effect on an enzyme/substrate complex. The results of xylan conversion showed that the effect of BSA on hydrolysis was more pronounced for natural biomass material (rice straw) than for pure biomass material (xylan). Lignocellulose is a highly complex structure with a range of characteristics that influence and limit the hydrolysis of carbohydrate polymers into fermentable sugars. Noncellulose substrates play an important role in the positive effect of BSA on enzymatic hydrolysis. The relationship between BSA and lignocellulose characteristics needs to be studied further. Therefore, the adsorption behavior of BSA on biomass materials was the focus of the present study. Besides BSA, corn steep was also used as a nonenzymatic protein in SSF of pretreated rice straw. Corn steep was used as an additive because of its high protein content and its nutrient composition, which makes it useful for microbial culture. In addition, corn steep is an
Appl Biochem Biotechnol
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Appl Biochem Biotechnol
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inexpensive alternative to materials such as BSA. However, its variable composition is a disadvantage in investigating the mechanism. 4
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Appl Biochem Biotechnol
Fig. 9 Effect of BSA to enzymatic hydrolysis of filter paper and xylan under sugar addition (a substrate: filter paper, b substrate: xylan)
The components of substrates made a crucial difference in the absorption behavior of BSA. In contrast to previous studies focusing on the lignin content [6], the effect of BSA on xylan hydrolysis was also studied. Xylose, the major unit of hemicellulose, is the second most abundant sugar in rice straw after glucose, and its efficient conversion is important for lignocellulose utilization. During hydrolysis and SSF process, the decomposition of biomass materials leads to a decreased adsorption surface, and at the same time, adsorbed BSA is desorbed. In contrast, lignin cannot be hydrolyzed by cellulase and its component does not change during enzymatic hydrolysis. The adsorption of BSA on lignin was stable and showed almost no change during hydrolysis. It is thought that lignin is a primary cause of the loss of enzyme protein [7]. BSAwas found to moderately increase xylan conversion. In addition, hemicellulose forms a protective physical barrier to enzymatic attack [15]. These results showed that the positive performance of BSA results from multiple factors and that the mechanism differs among biomass materials. During simple saccharification of filter paper and pretreated rice straw, cellulose conversion was increased by 32.5 and 15.3 %, respectively. For the production of ethanol on the basis of enzymatic hydrolysis, this result suggests that if protein additives exert the same effect on simple enzymatic hydrolysis and SSF, the final ethanol yield should markedly increase. However, the finding that the addition of commercial protein (BSA) or natural protein (corn steep) before SSF did not significantly affect final ethanol yield but sharply increased the final xylose yield indicates that the mechanisms underlying nonenzymatic protein effects in simple enzymatic hydrolysis are different from those in SSF and that other mechanisms are perhaps equally important. During SSF, glucose is removed from the system as soon as it is formed and its inhibitory effect on the cellulase system is diminished. However, xylose as a pentose sugar is not fermentable by ordinary yeast and accumulates during the SSF process. We accordingly speculated that the protein additives relieved cumulative feedback inhibition of enzymatic hydrolysis during SSF, possibly explaining why additives led to marked increases in the final xylose yield during SSF of rice straw. Various factors influence the yields of ethanol from lignocelluloses. The hydrolysis rate in separate hydrolysis and fermentation (SHF) is strongly affected by end-product inhibition [16].
Appl Biochem Biotechnol
In SSF, this inhibition is decreased because glucose is consumed by a fermenting organism as soon as it is formed. Slow xylose consumption during fermentation may lead to the presence of compounds that inhibit the growth and fermentation activity of the microorganism [17, 18]. These findings suggest that the advantages of the addition of protein such as BSA and corn steep can be attributed to their action in relieving production inhibition in enzymatic hydrolysis of biomass materials. Research has been conducted to elucidate the mechanism underlying end-product inhibition of cellulase enzyme systems during cellulose hydrolysis [19]. When glucose and mannose were added to fermented prehydrolysate in the enzymatic hydrolysis step, a small reduction in cellulose conversion from 76.3 to 72.8 % was observed [20]. Although inhibition by glucose and cellobiose is substantially reduced in SSF, the effect of ethanol on cellulase activity remains. The severity of inhibition was in the order cellulobiose > glucose > ethanol (on a same-weight basis) [21]. However, there have been few attempts to determine the effect of protein additives on sugar inhibition during the enzymatic hydrolysis of lignocellulosic materials. Because of the intricate nature of lignocellulosic material, most mechanisms proposed for pure cellulose substrates, such as Avicel and filter paper, are typically not representative of those for natural lignocellulosic substrates with various degrees of noncellulosic contaminants. In addition, there is a lack of information on mechanisms of production inhibition in enzymatic hydrolysis and SSF of biomass materials. The mechanism underlying the effect of BSA on SSF and the factors influencing BSA and product inhibition need to be studied in the future. In conclusion, BSA and corn steep increased the enzymatic conversion of cellulose filter paper, xylan, and pretreated rice straw. BSA effect strength varied with biomass type. BSA enhanced the enzymatic hydrolysis of filter paper, thereby improving enzyme stability and prolonging enzyme life, and moderately increased xylan conversion. BSA adsorption affinity was similar for lignin and hemicelluloses. Results of rice straw hydrolysis indicate that BSA prevents enzyme adsorption to lignin. BSA effect on efficiency was lower for SSF than for enzymatic hydrolysis. BSA and corn steep increased the final xylose yield, and BSA relieved the cumulative sugar inhibition of hydrolysis. Acknowledgments This work was supported by JST/JICA-SATREPS, “Sustainable Integration of Local Agriculture and Biomass Industries,” and a grant from the National High Technology Research and Development Program of China (863 Program) (No. 2012AA101803). The English language was reviewed by Enago (www.Enago.jp).
References 1. Zaldivar, J., Nielsen, J., & Olsson, L. (2001). Applied Microbiology and Biotechnology, 56, 17–34. 2. Yang, B., & Wyman, C. E. (2008). Biofuels, Bioproducts and Biorefining, 2, 26–40. 3. Lynd, L. R., Laser, M. S., Bransby, D., Dale, B. E., Davison, B., Hamilton, R., Keller, M., McMillan, J. D., Sheehan, J., & Wyman, C. E. (2008). Nature Biotechnology, 26, 169–172. 4. Mooney, C. A., Mansfield, S. D., Touhy, M. G., & Saddler, J. N. (1998). Bioresource Technology, 64, 113–119. 5. Ramos, L. P., Nazhad, M. M., & Saddler, J. N. (1993). Enzyme and Microbial Technology, 15, 821–831. 6. Rollin, J. A., Zhu, Z., Sathitsuksanoh, N., & Zhang, Y. P. (2011). Biotechnology and Bioengineering, 108, 22–30. 7. Eriksson, T., Börjesson, J., & Tjerneld, F. (2002). Enzyme and Microbial Technology, 31, 353–364. 8. Kim, S., & Holtzapple, M. T. (2006). Bioresource Technology, 97, 583–591. 9. Yang, B., & Wyman, C. E. (2006). Biotechnology and Bioengineering, 94, 611–617. 10. Seo, D., Fujita, H., & Sakoda, A. (2011). Bioresource Technology, 102, 9605–9612. 11. Kaar, W. E., & Holtzapple, M. T. (1998). Biotechnology and Bioengineering, 59, 419–427. 12. Wang, H., Mochidzuki, K., Kobayashi, S., Hiraide, H., Wang, X., & Cui, Z. (2013). Applied Biochemistry and Biotechnology, 170, 541–551.
Appl Biochem Biotechnol 13. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Journal of Biological Chemistry, 193, 265–275. 14. Guo, P., Wang, X., Zhu, W., Yang, H., Cheng, X., & Cui, Z. (2008). Journal of Environmental Sciences, 20, 109–114. 15. Öhgren, K., Bura, R., Saddler, J., & Zacchi, G. (2007). Bioresource Technology, 98, 2503–2510. 16. Philippidis, G. P., Smith, T. K., & Wyman, C. E. (1993). Biotechnology and Bioengineering, 41, 846–853. 17. Katahira, S., Mizuike, A., Fukuda, H., & Kondo, A. (2006). Applied Microbiology and Biotechnology, 72, 1136–1143. 18. Buaban, B., Inoue, H., Yano, S., Tanapongpipat, S., Ruanglek, V., Champreda, V., Pichyangkura, R., Rengpipat, S., & Eurwilaichitr, L. (2010). Journal of Bioscience and Bioengineering, 110, 18–25. 19. Ghosh, P., Pamment, N. B., & Martin, W. R. B. (1982). Enzyme and Microbial Technology, 4, 425–430. 20. Tengborg, C., Galbe, M., & Zacchi, G. (2001). Enzyme and Microbial Technology, 28, 835–844. 21. Gusakov, A. V., & Sinitsyn, A. P. (1992). Biotechnology and Bioengineering, 40, 663–671.
The Effect of Nonenzymatic Protein on Lignocellulose Enzymatic Hydrolysis and Simultaneous Saccharification and Fermentation Hui Wang & Shinichi Kobayashi & Hatsue Hiraide & Zongjun Cui & Kazuhiro Mochidzuki
Received: 22 April 2014 / Accepted: 10 September 2014 # Springer Science+Business Media New York 2014
Abstract Nonenzymatic protein was added to cellulase hydrolysis and simultaneous saccharification and fermentation (SSF) of different biomass materials. Adding bovine serum albumin (BSA) and corn steep before cellulase enhanced enzyme activity in solution and increased cellulose and xylose conversion rates. The cellulose conversion rate of filter paper hydrolysis was increased by 32.5 % with BSA treatment. When BSA was added before cellulase, the remaining activity in the solution was higher than that in a control without BSA pretreatment. During SSF with pretreated rice straw as the substrate, adding 1.0 mg/mL BSA increased the ethanol yield by 13.6 % and final xylose yield by 42.6 %. The results indicated that lignin interaction is not the only mechanism responsible for the positive BSA effect. BSA had a stabilizing effect on cellulase and relieved cumulative sugar inhibition of enzymatic hydrolysis of biomass materials. Thus, nonenzymatic protein addition represents a promising strategy in the biorefining of lignocellulose materials. Keywords Lignocellulose . Saccharification . Nonenzymatic protein . Simultaneous saccharification and fermentation (SSF)
H. Wang : S. Kobayashi : H. Hiraide : K. Mochidzuki (*) Institute of Industrial Science, The University of Tokyo, Tokyo 153-8505, Japan e-mail: [email protected] H. Wang e-mail: [email protected] S. Kobayashi e-mail: [email protected] H. Hiraide e-mail: [email protected] Z. Cui College of Agronomy and Biotechnology/Center of Biomass Engineering, China Agricultural University, Beijing 100193, China e-mail: [email protected]
Appl Biochem Biotechnol
Introduction Ethanol produced from lignocellulosic biomass has a great potential to address vital energy security, trade deficit, environmental, and economic issues that are becoming more urgent in light of the declining petroleum reserves and increasing international demand for transportation fuels [1, 2]. The conversion of cellulose to glucose, which is the initial process of biorefining, has remained a bottleneck [3], restricting the initial conversion rate. High cellulase dosage and prolonged process time increase the final ethanol yield but result in high process costs. Thus, it is important to find methods to reduce the cellulase dosage and shorten the process time without decreasing the conversion rate. Many studies have been aimed to identify and manipulate the inhibiting factors in enzymatic hydrolysis of lignocellulose substrates. It has been shown that the surface area of cellulose is an important substrate characteristic in determining initial rates of hydrolysis [4, 5]. As mentioned in a report by Rollin et al. [6], it has been concluded that increasing cellulose accessibility is a more important pretreatment consideration than delignification for effectively releasing sugars from recalcitrant lignocelluloses at a high yield. Enzyme activity loss because of nonproductive adsorption on lignin surfaces has been identified as an important factor for decreasing enzyme effectiveness, and the effect of surfactants and noncatalytic protein on the enzymatic hydrolysis of cellulose into fermentable sugars has been extensively studied [7–9]. The cellulose conversion rate at 72 h was increased from 9 to 21 % and 1 to 8.5 % for samples with high and low lignin contents by the injection of Tween-20 (0.024– 0.24 mM), respectively [10]. Eriksson et al. [7] tested the effect of the addition of bovine serum albumin (BSA) at a concentration of 17 g/L on the hydrolysis of steampretreated spruce and compared it with the effect of different surfactants. In a previous study, we concluded that surfactants adsorb to lignin and prevent the unproductive adsorption of enzymes [7]. The addition of surfactants and polymers has also been described to exert a stabilizing effect on enzymes. Kaar and Holtzapple [11] suggested that Tween can protect enzymes from thermal denaturation in hydrolysis conducted at higher temperatures. Our recent study showed that BSA improves the stability of cellulase [12]. Most studies have focused on the use of different surfactants, and only few reports are available on the use of BSA and corn steep as additives. Furthermore, the mechanisms underlying the enhancement of enzymatic hydrolysis by additives are unknown. To investigate these mechanisms and identify the substrate characteristics influencing the effects of additives during enzymatic cellulose hydrolysis, the present study compared pure cellulosic substrates (filter paper), natural lignocellulosic materials (rice straw), and noncellulosic biomass (xylan and lignin) by compositional analysis, enzymatic hydrolysis, and simultaneous saccharification and fermentation (SSF) using BSA as a model compound.
Materials and Methods Materials Substrate Pretreated rice straw [2 %, NaOH 1 N, 50 °C 4 h, 25 °C overnight, washed with deionized (DI) water until neutral, dried at 60 °C to constant weight, and milled to pass through a 1-mm mesh]. The compositions of materials used in the present study are shown in Table 1.
Appl Biochem Biotechnol Table 1 Lignocellulosic components of biomass materials Sample
Cellulose %
Hemicellulose %
Lignin %
Rice straw
31.7
27.1
5.2
Pretreated rice straw
47.3
16.1
3.8
Model lignocellulosic biomass component: Cellulose: filter paper (Whatman No. 1, 1×1-cm squares) Hemicellulose: xylan (1 %, from birchwood, Sigma) Lignin: lignin powder (0.5 %, lignin, organosolv, Sigma) Protein: BSA (Cohn, fraction V, pH 7.0, Wako) Cellulase: Acremonium cellulase (AC, derived from A. cellulolyticus; Meiji Seika, Tokyo, Japan; No.: ACCF-4940). Avicelase activity is 1040 u/g (pH 5.7). Dry yeast: Ethanol RedTM (Saccharomyces cerevisiae, Fermentis, France) Corn steep: Solvlys 095 E (Roquette Freres)
Methods BSA Pretreatment Biomass substrates were put into a 50 mM citric buffer (pH 4.8) in a shaking flask (filter paper 2 g dry weight/100 mL, xylan 1 g dry weight/100 mL, lignin 0.5 g dry weight/100 mL, pretreated rice straw 2 g dry weight/100 mL) and autoclaved at 121 °C for 20 min. After cooling to room temperature, BSA was placed in the flasks to make the initial BSA concentration of 0.5, 1.0, or 1.5 mg/mL, and the flasks were then placed in a shaking incubator with shaking at 150 rpm at 50 °C. Aliquots of 0.5 mL were taken at 0, 0.5, 1, 3, 6, 12, 24, 48, and 72 h and immediately centrifuged at 10,000 rpm for 5 min. The supernatants were passed through a 0.22-μm filter paper and assayed for free BSA concentration. Enzymatic Hydrolysis All experiments with enzymes were performed in 50 mM citric buffer (pH 4.8). Enzymatic hydrolysis of the substrate was performed at 15 filer paper units (FPU) cellulase/g substrate, with and without 24-h BSA pretreatment. Incubation conditions were the same as those for BSA pretreatment (150 rpm, 30 or 50 °C). Samples of 1 mL were collected at 0, 0.5, 1, 3, 6, 12, 24, 48, and 72 h; chilled on ice; and centrifuged at 10,000 rpm for 5 min. The supernatants were passed through a 0.22-μm filter paper. Glucose concentration, free protein concentration, and free cellulase activity were measured in the supernatants as described below. All the experimental results are the average of two replicates, unless specified otherwise. SSF All SSF runs were performed in 300-mL flasks (working volume 150 mL) in a shaking incubator with shaking at 130 rpm at 35 °C. The SSF reaction mixture contained 6 g of substrates, and the enzyme dosage was 15 FPU/g substrate. The basal medium contained 10 g/L yeast extract and 20 g/L peptone. Corn steep SSF used a basal medium containing only 50 g/L corn steep. The basal medium was autoclaved at 121 °C for 20 min. SSF experiment protocols employed standard biomass analytical methods of the National Renewable Energy Laboratory (NREL) (NREL/TP-510-42630).
Appl Biochem Biotechnol
Analytical Methods Protein Concentration Determination The Protein Assay Lowry Kit (Nacalai Tesque No. 29470) was used for protein quantitative analysis using the Lowry method [13]. Cellulase Activity An enzyme activity assay using Whatman No. 1 filter paper as a substrate was performed. NREL FPU (NREL/TP-510-42628) was used for enzyme activity analysis. Supernatants from samples collected after concentration and filter treatment were used to measure free cellulose activity. Sugar and ethanol concentrations in the solution were measured by high-performance liquid chromatography (SHIMADZU LC-10AD, with refractive index detector RID-10A). The column was SUGAR SH1011 (SHODEX). The components of materials were analyzed using a fiber analyzer (Model ANKOM220, USA) as described previously [14].
Results Effect of Nonenzymatic Protein on Enzymatic Hydrolysis The effect of nonenzymatic protein on enzyme activity was evaluated by measuring enzyme activity (in FPU) remaining free in the solution during enzymatic hydrolysis. Based on a relative activity of 100 % for the enzyme in the solution at time 0, when nonenzymatic protein was added before cellulase, the remaining solution activity was higher than that in the control without nonenzymatic protein presoaking (Fig. 1). After hydrolysis of pretreated rice straw for 48 h, the relative enzyme activity was 43.67 % in the control. With BSA and corn steep treatment, at least 52.73 and 58.02 %, respectively, of the initial activity was retained. The effects of BSA on enzymatic hydrolysis of different materials are shown in Fig. 2. Enzymatic hydrolysis of each biomass material was performed with AC. With the addition of BSA, the production concentration increased in all the experiments, although not all increases were statistically significant. The cellulose conversion of filter paper hydrolysis was increased by 32.5 % (from 74.90 to 98.93 %). Initially, the effect of BSA on the yield of enzymatic hydrolysis was insignificant. After a 24-h enzymatic hydrolysis of pretreated rice straw, glucose conversion increased by 17.9 % (from 69.01 to 81.35 %) with 15 FPU/g substrate, and after a 72-h hydrolysis, it increased by 15.3 % (from 71.37 to 82.29 %). Corn steep also increased the glucose yield from enzymatic hydrolysis of pretreated rice straw, particularly at a low enzyme dosage (Fig. 3). Absorption Behaviors of BSA onto Biomass Materials To determine the adsorption isotherms of different substrates, BSA preparations with various concentrations were tested and protein content in supernatants was determined. The data shown in Fig. 4 indicate that the adsorption isotherms on filter paper, xylose, and lignin were well fitted to a linear correlation (R2 >0.9). The data again revealed pronounced differences between pure biomass materials (filter paper, xylan, and lignin) and natural lignocellulosic materials (rice straw). We also investigated the adsorption behavior of BSA on different substrates at 50 °C. BSA adsorption varied according to the biomass material: lignin ≥ xylan > pretreated rice straw > filter paper (Fig. 5). The adsorbed amounts of BSA and cellulase were measured during the time course of hydrolysis. After BSA presoaking, the adsorption behavior of enzyme varied across different
Appl Biochem Biotechnol
100 BSA Corn steep Control
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Fig. 2 Effect of BSA on the enzymatic hydrolysis of biomass materials at 50 °C. a Substrate: filter paper and xylan. b Substrate: rice straw (substrate concentration: filter paper 2 % w/v, xylan 1 % w/v, rice straw 2 % w/v; enzyme dosage: 15 FPU/g substrate)
Appl Biochem Biotechnol
4
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unpretreated rice straw - 7.5 FPU unpretreated rice straw - corn steep - 15 FPU
1
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Time ( Hour) Fig. 3 Effect of corn steep on glucose yield of pretreated and unpretreated rice straw at 35 °C with different enzyme dosages (substrate concentration: 2 % w/v, enzyme dosage: 7.5 and 15 FPU/g substrate)
biomass substrates. In xylan hydrolysis, enzyme adsorption was much lower than that without BSA presoaking at the beginning of hydrolysis. However, after 72 h, in contrast to 1.0- and 1.5-mg/mL BSA presoaking treatments, enzyme adsorption in the 0.5-mg/mL BSA presoaking treatment was slightly greater than that in the control (Fig. 6a). During lignin hydrolysis, enzyme adsorption with BSA presoaking was lower than that without BSA presoaking (Fig. 6b). Figure 6c shows that during the hydrolysis of rice straw, the dynamics of enzyme and BSA adsorption was similar to that of xylan hydrolysis. Interestingly, enzyme adsorption on xylan was the highest among all biomass materials. These results show that BSA increased the amount of free enzyme during the enzymatic hydrolysis of biomass materials; however, this effect diminished in the course of hydrolysis. Effect of Protein Additives on SSF Figure 7a shows the effect of 1 mg/mL BSA on SSF of filter paper and pretreated rice straw. The highest ethanol concentration was obtained after approximately 48 h for filter paper and pretreated rice straw. The addition of 1.0 mg/mL BSA increased the ethanol yield by 13.6 % (from 7.41 to 8.31 g/L) with pretreated rice straw as the substrate. In SSF of filter paper, the final ethanol concentrations were 16.30 g/L with BSA pretreatment and 16.03 g/L without BSA pretreatment. However, as shown in Fig. 7b, the use of corn steep as a protein additive and presoaking for 24 h before SSF did not markedly affect the ethanol yield in SSF of filter
Appl Biochem Biotechnol 30
a
60
b y = 67.173x + 1.6352 R² = 0.9433
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Fig. 4 Adsorption isotherm of BSA at 50 °C (a adsorption isotherm of filter paper, b adsorption isotherm of xylan, c adsorption isotherm of lignin, d adsorption isotherm of rice straw)
paper. With pretreated rice straw as the substrate, the final ethanol concentration was increased by 5.0 % (from 8.05 to 8.48 g/L) by corn steep presoaking for 24 h. However, during SSF of different materials, the final xylose yield increased by 42.6 % (from 2.19 to 3.13 g/L) with BSA addition and 38.6 % (from 2.19 to 3.04 g/L) with corn steep presoaking (Fig. 8). When glucose and xylose were added to the enzymatic hydrolysis process, decreased cellulose and xylan conversion was observed. Following the addition of 10 and 5 mg/mL glucose to the enzymatic hydrolysis of filter paper, glucose concentrations ranged from 21.60 (with BSA) to 18.81 g/L and from 20.39 (with BSA) to 18.05 g/L, respectively (Fig. 9a). Following the addition of 0.4 and 0.2 mg/mL xylose to the enzymatic hydrolysis of xylan, xylose concentrations ranged from 3.43 (with BSA) to 1.88 g/L and from 3.23 (with BSA) to 3.0 g/L, respectively (Fig. 9b).
Discussion As shown in Figs. 1 and 2, BSA and corn steep presoaking improved the enzymatic hydrolysis of biomass materials at various stages. BSA and other proteins adsorb competitively and
Appl Biochem Biotechnol
Fig. 5 Quantity of BSA absorbed on different biomass substrates at 50 °C
irreversibly to lignin and thereby improve the effectiveness of cellulase, consequently increasing the enzymatic hydrolysis rate of biomass materials [6, 8]. In the present study, BSA markedly improved the cellulose conversion rate in the hydrolysis of pure cellulose material (filter paper). Lignin interaction is not the only mechanism responsible for the positive effect of BSA on the enzymatic hydrolysis of cellulose and xylan. Our previous results showed that the effect of BSA on the remaining enzyme activity was much higher at 50 °C than at 35 °C [12] and that yield improvements were observed only after prolonged reaction times for biomass materials. As described in previous studies [12], the mechanism of “lignin blocking” by nonenzymatic protein was not responsible for the increased enzyme performance. We also measured the enzyme activity during the incubation of enzyme and BSA without the addition of a biomass substrate in a previous study [12] and showed that BSA has a stabilizing effect on an enzyme/substrate complex. The results of xylan conversion showed that the effect of BSA on hydrolysis was more pronounced for natural biomass material (rice straw) than for pure biomass material (xylan). Lignocellulose is a highly complex structure with a range of characteristics that influence and limit the hydrolysis of carbohydrate polymers into fermentable sugars. Noncellulose substrates play an important role in the positive effect of BSA on enzymatic hydrolysis. The relationship between BSA and lignocellulose characteristics needs to be studied further. Therefore, the adsorption behavior of BSA on biomass materials was the focus of the present study. Besides BSA, corn steep was also used as a nonenzymatic protein in SSF of pretreated rice straw. Corn steep was used as an additive because of its high protein content and its nutrient composition, which makes it useful for microbial culture. In addition, corn steep is an
Appl Biochem Biotechnol
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Fig. 6 Changes in the amount of adsorbed BSA and enzyme on biomass substrates during enzymatic hydrolysis process at 50 °C (a substrate: xylan, b substrate: lignin, c substrate: rice straw)
Appl Biochem Biotechnol
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inexpensive alternative to materials such as BSA. However, its variable composition is a disadvantage in investigating the mechanism. 4
Xylose (g / L)
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Time ( Hour) Fig. 8 Effect of nonenzymatic protein on production of xylose during SSF
Appl Biochem Biotechnol
Fig. 9 Effect of BSA to enzymatic hydrolysis of filter paper and xylan under sugar addition (a substrate: filter paper, b substrate: xylan)
The components of substrates made a crucial difference in the absorption behavior of BSA. In contrast to previous studies focusing on the lignin content [6], the effect of BSA on xylan hydrolysis was also studied. Xylose, the major unit of hemicellulose, is the second most abundant sugar in rice straw after glucose, and its efficient conversion is important for lignocellulose utilization. During hydrolysis and SSF process, the decomposition of biomass materials leads to a decreased adsorption surface, and at the same time, adsorbed BSA is desorbed. In contrast, lignin cannot be hydrolyzed by cellulase and its component does not change during enzymatic hydrolysis. The adsorption of BSA on lignin was stable and showed almost no change during hydrolysis. It is thought that lignin is a primary cause of the loss of enzyme protein [7]. BSAwas found to moderately increase xylan conversion. In addition, hemicellulose forms a protective physical barrier to enzymatic attack [15]. These results showed that the positive performance of BSA results from multiple factors and that the mechanism differs among biomass materials. During simple saccharification of filter paper and pretreated rice straw, cellulose conversion was increased by 32.5 and 15.3 %, respectively. For the production of ethanol on the basis of enzymatic hydrolysis, this result suggests that if protein additives exert the same effect on simple enzymatic hydrolysis and SSF, the final ethanol yield should markedly increase. However, the finding that the addition of commercial protein (BSA) or natural protein (corn steep) before SSF did not significantly affect final ethanol yield but sharply increased the final xylose yield indicates that the mechanisms underlying nonenzymatic protein effects in simple enzymatic hydrolysis are different from those in SSF and that other mechanisms are perhaps equally important. During SSF, glucose is removed from the system as soon as it is formed and its inhibitory effect on the cellulase system is diminished. However, xylose as a pentose sugar is not fermentable by ordinary yeast and accumulates during the SSF process. We accordingly speculated that the protein additives relieved cumulative feedback inhibition of enzymatic hydrolysis during SSF, possibly explaining why additives led to marked increases in the final xylose yield during SSF of rice straw. Various factors influence the yields of ethanol from lignocelluloses. The hydrolysis rate in separate hydrolysis and fermentation (SHF) is strongly affected by end-product inhibition [16].
Appl Biochem Biotechnol
In SSF, this inhibition is decreased because glucose is consumed by a fermenting organism as soon as it is formed. Slow xylose consumption during fermentation may lead to the presence of compounds that inhibit the growth and fermentation activity of the microorganism [17, 18]. These findings suggest that the advantages of the addition of protein such as BSA and corn steep can be attributed to their action in relieving production inhibition in enzymatic hydrolysis of biomass materials. Research has been conducted to elucidate the mechanism underlying end-product inhibition of cellulase enzyme systems during cellulose hydrolysis [19]. When glucose and mannose were added to fermented prehydrolysate in the enzymatic hydrolysis step, a small reduction in cellulose conversion from 76.3 to 72.8 % was observed [20]. Although inhibition by glucose and cellobiose is substantially reduced in SSF, the effect of ethanol on cellulase activity remains. The severity of inhibition was in the order cellulobiose > glucose > ethanol (on a same-weight basis) [21]. However, there have been few attempts to determine the effect of protein additives on sugar inhibition during the enzymatic hydrolysis of lignocellulosic materials. Because of the intricate nature of lignocellulosic material, most mechanisms proposed for pure cellulose substrates, such as Avicel and filter paper, are typically not representative of those for natural lignocellulosic substrates with various degrees of noncellulosic contaminants. In addition, there is a lack of information on mechanisms of production inhibition in enzymatic hydrolysis and SSF of biomass materials. The mechanism underlying the effect of BSA on SSF and the factors influencing BSA and product inhibition need to be studied in the future. In conclusion, BSA and corn steep increased the enzymatic conversion of cellulose filter paper, xylan, and pretreated rice straw. BSA effect strength varied with biomass type. BSA enhanced the enzymatic hydrolysis of filter paper, thereby improving enzyme stability and prolonging enzyme life, and moderately increased xylan conversion. BSA adsorption affinity was similar for lignin and hemicelluloses. Results of rice straw hydrolysis indicate that BSA prevents enzyme adsorption to lignin. BSA effect on efficiency was lower for SSF than for enzymatic hydrolysis. BSA and corn steep increased the final xylose yield, and BSA relieved the cumulative sugar inhibition of hydrolysis. Acknowledgments This work was supported by JST/JICA-SATREPS, “Sustainable Integration of Local Agriculture and Biomass Industries,” and a grant from the National High Technology Research and Development Program of China (863 Program) (No. 2012AA101803). The English language was reviewed by Enago (www.Enago.jp).
References 1. Zaldivar, J., Nielsen, J., & Olsson, L. (2001). Applied Microbiology and Biotechnology, 56, 17–34. 2. Yang, B., & Wyman, C. E. (2008). Biofuels, Bioproducts and Biorefining, 2, 26–40. 3. Lynd, L. R., Laser, M. S., Bransby, D., Dale, B. E., Davison, B., Hamilton, R., Keller, M., McMillan, J. D., Sheehan, J., & Wyman, C. E. (2008). Nature Biotechnology, 26, 169–172. 4. Mooney, C. A., Mansfield, S. D., Touhy, M. G., & Saddler, J. N. (1998). Bioresource Technology, 64, 113–119. 5. Ramos, L. P., Nazhad, M. M., & Saddler, J. N. (1993). Enzyme and Microbial Technology, 15, 821–831. 6. Rollin, J. A., Zhu, Z., Sathitsuksanoh, N., & Zhang, Y. P. (2011). Biotechnology and Bioengineering, 108, 22–30. 7. Eriksson, T., Börjesson, J., & Tjerneld, F. (2002). Enzyme and Microbial Technology, 31, 353–364. 8. Kim, S., & Holtzapple, M. T. (2006). Bioresource Technology, 97, 583–591. 9. Yang, B., & Wyman, C. E. (2006). Biotechnology and Bioengineering, 94, 611–617. 10. Seo, D., Fujita, H., & Sakoda, A. (2011). Bioresource Technology, 102, 9605–9612. 11. Kaar, W. E., & Holtzapple, M. T. (1998). Biotechnology and Bioengineering, 59, 419–427. 12. Wang, H., Mochidzuki, K., Kobayashi, S., Hiraide, H., Wang, X., & Cui, Z. (2013). Applied Biochemistry and Biotechnology, 170, 541–551.
Appl Biochem Biotechnol 13. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Journal of Biological Chemistry, 193, 265–275. 14. Guo, P., Wang, X., Zhu, W., Yang, H., Cheng, X., & Cui, Z. (2008). Journal of Environmental Sciences, 20, 109–114. 15. Öhgren, K., Bura, R., Saddler, J., & Zacchi, G. (2007). Bioresource Technology, 98, 2503–2510. 16. Philippidis, G. P., Smith, T. K., & Wyman, C. E. (1993). Biotechnology and Bioengineering, 41, 846–853. 17. Katahira, S., Mizuike, A., Fukuda, H., & Kondo, A. (2006). Applied Microbiology and Biotechnology, 72, 1136–1143. 18. Buaban, B., Inoue, H., Yano, S., Tanapongpipat, S., Ruanglek, V., Champreda, V., Pichyangkura, R., Rengpipat, S., & Eurwilaichitr, L. (2010). Journal of Bioscience and Bioengineering, 110, 18–25. 19. Ghosh, P., Pamment, N. B., & Martin, W. R. B. (1982). Enzyme and Microbial Technology, 4, 425–430. 20. Tengborg, C., Galbe, M., & Zacchi, G. (2001). Enzyme and Microbial Technology, 28, 835–844. 21. Gusakov, A. V., & Sinitsyn, A. P. (1992). Biotechnology and Bioengineering, 40, 663–671.
