Enzymatic hydrolysis of corn stalk in a hollow fiber ultrafiltration membrane reactor
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1 biomass and bioenergy 33 (2009) Available at Technical note Enzymatic hydrolysis of corn stalk in a hollow fiber ultrafiltration membrane reactor Sen Yang a,b, Wenyong Ding a, Hongzhang Chen a, * a State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, P.O. Box 353, Beijing , PR China b The Center of Biomass Engineering, College of Resources and Environment, China Agricultural University, Beijing , PR China article info Article history: Received 8 July 2006 Received in revised form 27 May 2008 Accepted 29 May 2008 Published online 20 August 2008 abstract A hollow fiber ultrafiltration (UF) membrane reactor was set up to investigate the enzymatic hydrolysis of steam-exploded corn stalk. It was found that the hydrolysis rate, as well as the reducing sugar (RS) yield, could be markedly enhanced in the UF membrane reactor due to the continuous removal of inhibitory products. Compared with traditional batch hydrolysis, the hydrolysis rate and RS yield could increase 200% and 206%, respectively. ª 2008 Elsevier Ltd. All rights reserved. Keywords: Membrane reactor Corn stalk Cellulase Enzymatic hydrolysis Ultrafiltration 1. Introduction Corn is a major crop in the northern part of China. Associated with corn production is a corresponding annual production of million tons of corn stalk. The stalk has traditionally been removed from the field by the practice of open-field burning. This practice clears the field for new plantings and cleans the soil of disease-causing agents to a certain extent. However, it also produces visible smoke and has negative effect on air quality. On the other hand, in the search for viable alternative energy, the corn stalk is considered as a source of liquid fuels. Corn stalk can be converted through bioconversion to ethanol, which is a clean-burning transportation-fuel oxygenate. However, utilization of this resource in biotechnology requires that the substrate must first be hydrolyzed to fermentable reducing sugars. The cellulose and hemicellulose in stalk (and lignocellulose in general) are not directly available for bioconversion because of their intimate association with lignin [1,2]. Enzymatic hydrolysis of native lignocellulosics is therefore difficult and the rate is very slow. In order to enhance the enzymatic susceptibility of these carbohydrates, specific pretreatment processes are necessary. Pretreatment methods for lignocellulosic materials have been extensively studied. The pretreatment can be classified as acidic pretreatment [3], alkaline pretreatment [4], physical pretreatment, steaming, steam explosion [5] wet oxidation [6], etc. Steam explosion is one of the best ways of pretreatment. Since the early investigations of * Corresponding author. Tel: þ ; fax: þ addresses: syang@cau.edu.cn (S. Yang), hzchen@home.ipe.ac.cn (H. Chen) /$ see front matter ª 2008 Elsevier Ltd. All rights reserved. doi: /j.biombioe
2 biomass and bioenergy 33 (2009) Mason in 1929, the steam explosion has been a well-known way of disrupting different lignocellulosic plant materials into the main components: cellulose, lignin and hemicellulose [7]. The most important operational conditions in steam explosion pretreatment are time, temperature, and chip size [8]. The factors that affect the enzymatic hydrolysis of cellulose include substrates, cellulase activity, reaction conditions (temperature, ph, as well as other parameters), and a strong product inhibition. The slow reaction rate, exacerbated by the product inhibition, has been recognized as the major obstacle in achieving an economically viable commercial operation of enzymatic hydrolysis of cellulose. The reaction can be significantly accelerated by continuous removal of the produced sugars. To improve the yield and rate of the enzymatic hydrolysis, membrane reactors with different configurations have been investigated [9 12]. The membrane reactor utilizing an UF membrane with an appropriate molecularweight cutoff, keeps the larger components in the reactor while low-molecular-weight molecules, such as sugars, pass through the membrane and leave the reactor as permeate. Thus, recovery of cellulolytic enzymes and removal of inhibiting products can be achieved. The objective of this work was to investigate the enzymatic hydrolysis of steam-exploded corn stalk in a hollow fiber UF membrane reactor. We have chosen the hollow fiber geometry because this reactor type is more useful for industrial applications, owing the high ratio of membrane surface to the working solution volume, a parameter of fundamental relevance in any membrane process. 2. Experimental 2.1. Substrate and enzymes Steam-exploded corn stalk was prepared by treating chopped corn stalk (3 4 cm, containing 15% of water) in a steamexploded vessel (1 m 3 ) at 1.5 MPa for 10 min and then discharge promptly. After the pretreatment, the solid residue was dried without washing and used in the hydrolysis experiments directly. The composition of steam-exploded corn stalk was 6.2% hemicellulose, 50.2% cellulose and 21.5% lignin plus ash. The cellulase extracted from Trichoderma reesei used in this work was obtained from Ningxia xiasheng Co. (China). The cellulase activity and the b-glucosidase activity of this solution were 110 FPU ml 1 and 37 IU ml 1, respectively [13,14] Membrane reactor Membrane reactor consisted of a buffer container, a 250 ml Erlenmeyer flask as main reactor and a hollow fiber UF module outside (Fig. 1). The reactor was kept at 50 C in a water bath, the optimal catalytic temperature for the cellulase. All tubing was wrapped with a glass fiber cloth for thermal isolation. The buffer container was used to continuously feed fresh buffer to the reactor. The pressure applied to the UF module was typically about MPa. Permeate was collected in a measuring cylinder for flow control. An automatic fraction collector was used for permeate collection in long-term experiments. Feeding pump Reservoir Water bath Hydrolysis unit The hollow fiber UF module was made in Research Center for Eco-Environmental Sciences (China). The module consisted of 10 fibers with an internal diameter of 1.0 mm, a wall thickness of 0.1 mm, and a length of 10 cm. The total membrane area was m 2. The fibers were made of polysulfone (PS) with a narrow molecular-weight cutoff (MWCO) of 10,000 Da. As the UF membrane used in the system has no separation selectivity towards reducing sugar permeation, it is assumed that the measured RS concentration in the permeate also represented the sugar concentration in the reactor Enzymatic hydrolysis A weighed amount of pretreated substrate was placed in the reactor and an enzyme solution was added to a final volume. The concentration of corn stalk was calculated as the amount of dry substance per volume of the reaction mixture. Pretreated corn stalk was not additionally washed. The reactions were conducted at a constant ph 4.8, maintained by a sodium acetate buffer. An initial hydrolysis period was conducted in the main reaction container. After 30 min, the recycling pump and the feeding pump were turned on. At this point, the pressure was applied, and a continuous permeate collection started. The hydrolysis stopped after 72 h. The RS concentration in the permeate flow was measured using the dinitrosalicylicacid (DNS) method [15]. 3. Results and discussion 3.1. Effect of enzyme loading Valve Recycling pump Fig. 1 Experimental device. The optimal ration between enzyme and substrate is very important for the efficient use of cellulase enzyme complex [16]. Thus, the effect of enzyme loading on enzymatic hydrolysis was investigated. Substrate concentration was fixed at 100 g L 1, and typical flux at pseudo-steady state was between 7.0 and 9.0 L m 2 h 1. Higher cellulose conversion and higher concentration of RS were occurred at higher enzyme loading (Figs. 2 and 3). The high cost of enzymes, however, makes high dosage impractical for this type of application [17]. For this reason, an enzyme loading of 20 FPU g 1 was selected for the remaining experiments Effect of substrate concentration UF membrane module Permeate The optimal concentration of substrate is very important for the efficient use of cellulase and the RS concentration. Thus, P P
3 334 biomass and bioenergy 33 (2009) Fig. 2 Cellulose conversion of pretreated corn stalk for different enzyme loadings. Fig. 4 Effect of substrate concentration on the RS concentration. the effects of substrate concentration on enzymatic hydrolysis were investigated at fixed enzyme to substrate ratio. The enzyme loading, 20 FPU g 1 (substrate) was kept unchanged, and the reaction volume was maintained by replenishing the reactor with fresh buffer but without fresh substrate. Typical fluxes at pseudo-steady state were between 7.0 and 9.0 L m 2 h 1. The range of substrate concentration was from 20 to 100 g L 1. The membrane reactor cannot be operated with higher substrate concentration due to little solution. Fig. 4 showed the RS concentration in permeate flux with different substrate concentrations. When enzyme loading was fixed, increasing substrate concentration resulted in more cellulose available for hydrolysis and higher enzyme concentration in solution, which means higher RS concentration in hydrolyzates. Similar results have also been observed for other lignocellulosics such as softwood, weeds and bagasse [18]. However, the cellulose conversion was not proportionally increased (Fig. 5). The cellulose conversion amounts the peak (85%) with 85 g L 1 substrate and decreases with higher substrate concentration. The reason is due to endproduct inhibition or insufficient hydrolysis time for the additional cellulose Effect of dilution rate The dilution rate [D (h 1 ) ¼ permeate flow (ml h 1 )/reaction volume (ml)] is thought to be limited by the operating conditions such as the concentration of substrate within the reactor and filtration module. In this system, every 1 g corn stalk could adsorb 5 ml water, so the dilution rate could not increase beyond 0.65 h 1. Hydrolysis of 100 g L 1 dry weight was performed at different dilution rates over 72 h time period. Fig. 6 showed the RS concentration in the permeate as Fig. 3 Effect of enzyme loading on the RS concentration. Fig. 5 Cellulose conversion of pretreated corn stalk at different substrate concentrations.
4 biomass and bioenergy 33 (2009) Fig. 6 Effect of dilution rate on the RS concentration. a function of time. The cellulose conversion was shown in Fig. 7. It has been generally observed that enzyme hydrolysis of cellulose in batch reactor had a rapid initial rate followed by a continuous rate fall which ultimately tends to zero [9]. Compared with the batch operation, the substrate conversion in the continuous operation is greater. When the dilution rate was 0.2, 0.4 and 0.65 h 1, the conversion was calculated to be increased 83%, 91% and 200%, respectively; the average reaction rate in 72 h (Fig. 8) increased 85%, 131% and 206%, respectively. 4. Conclusions Corn stalk was evaluated as a source of fermentable carbohydrate by measuring the yield of sugars obtained after combining the steam explosion pretreatment and hydrolysis with commercial cellulases in a membrane reactor. The rate of hydrolysis, as well as the yield of RS, was markedly enhanced in the UF membrane reactor due to the Fig. 7 Effect of dilution rate on the cellulose conversion. Fig. 8 Effect of dilution rate on the average reaction rate, E 0 [ 20 FPU g L1, S 0 [ 100 g L L1. continuous removal of inhibitory products. Compared with traditional batch hydrolysis, the hydrolysis rate and RS yield could increase 200% and 206%, respectively. As a result, however, the product stream has a very low content of RS, which will require preconcentration before fermentation. Acknowledgements This study was performed thanks to funding by National Basic Research Program of China (2004CB719700), Hi-Tech Research and Development Program of China (2001AA514023) and Knowledge Innovation Programm of Chinese Academy of Sciences (KJCXZ-SW-206-2). references [1] Binder A, Pelloni L, Fiechter A. Delignification of straw with ozone to enhance biodegradability. Eur J Appl Microbiol Biotechnol 1980;11:1 5. [2] Schmidt AS, Thomsen AB. Optimization of wet oxidation pretreatment of wheat straw. Bioresour Technol 1998;64: [3] Saha BC, Iten LB, Cotta MA, Wu YV. Dilute acid pretreatment, enzymatic saccharification and fermentation of wheat straw to ethanol. Process Biochem 2005;40: [4] Zhu SD, Wu YX, Yu ZN, Zhang X, Wang CW, Yu FQ, et al. Production of ethanol from microwave-assisted alkali pretreated wheat straw. Process Biochem 2006;41: [5] Cantarella M, Cantarella L, Gallifuoco A, Spera A, Alfani F. Comparison of different detoxification methods for steamexploded poplar wood as a substrate for the bioproduction of ethanol in SHF and SSF. Process Biochem 2004;39: [6] Bjerre AB, Olesen AB, Fernqvist T, Plöger A, Schmidt AS. Pretreatment of wheat straw using combined wet oxidation and alkaline hydrolysis resulting in convertible cellulose and hemicellulose. Biotechnol Bioeng 1996;49: [7] Saddler JN, Ramos LP, Breuil C. Steam pretreatment of lignocellulosic residues. In: Saddler JN, editor. Bioconversion
5 336 biomass and bioenergy 33 (2009) of forest and agricultural plant residues. Oxon: CBA International; p [8] Duff SJB, Murray WD. Bioconversion of forest products industry waste cellulosics to ethanol: a review. Bioresour Technol 1996;55:1 33. [9] Gan Q, Allen SJ, Taylor G. Design and operation of an integrated membrane reactor for enzymatic cellulose hydrolysis. Biochem Eng J 2002;12: [10] Ohlson I, Tragardh G, Hahnhagerdal B. Enzymatic hydrolysis of sodium-hydroxide-pretreated sallow in an ultrafiltration membrane reactor. Biotechnol Bioeng 1984;26: [11] Ohlson I, Tragardh G, Hahnhagerdal B. Evaluation of UF and RO in a cellulose saccharification process. Desalination 1984; 51: [12] Yang S, Ding WY, Chen HZ. Enzymatic hydrolysis of rice straw in a tubular reactor coupled with UF membrane. Process Biochem 2006;41: [13] Berghem LER, Petterss LG. The mechanism of enzymatic cellulose degradation. Isolation and some properties of a b- glucosidase from Trichoderma viride. Eur J Biochem 1974;46: [14] Mandels M, Andreotti R, Roche C. Measurement of saccharifying cellulase. Biotechnol Bioeng 1976;6: [15] Ghose TK. Measurement of cellulase activities. Pure Appl Chem 1987;59: [16] Lee SG, Kim HS. Optimal operating policy of the ultrafiltration membrane bioreactor for enzymatic hydrolysis of cellulose. Biotechnol Bioeng 1993;42: [17] Vlasenko EY, Ding H, Labavitch JM, Shoemaker SP. Enzymatic hydrolysis of pretreated rice straw. Bioresour Technol 1997;59: [18] Tengborg C, Galbe M, Zacchi G. Reduced inhibition of enzymatic hydrolysis of steam-pretreated softwood. Enzyme Microb Technol 2001;28:
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