Effect of particle size on the rate of enzymatic hydrolysis of cellulose
Introduction
Being the most abundant polysaccharide on the earth, cellulose is generally utilized in food, fuel, biomaterial and energy (Gan, Allen, & Taylor, 2003). In food, cellulose is generally recognized as fiber, which is the most sought information on nutrition labels (Todd & Variyam, 2008) due to growing interest in its health benefits. In US, the entire fiber market is worth $192.8 millions in 2004. Insoluble fiber dominates the market with a market share of $176.2 millions (Heller, 2008). In addition to health benefits, cellulose can be converted to biofuel by a multistep process that includes pretreatment, enzymatic hydrolysis, and fermentation. Pretreatment is an important and necessary step that opens up the tightly structured cell wall, thereby, allowing carbohydrolytic enzymes access to cellulose (Zeng, Mosier, Huang, Sherman, & Ladisch, 2007). Owing to the refractory structure of cellulose, hydrolysis is the key process for the biological conversion of cellulosic materials. Thus, increasing the yield of glucose from cellulose is helpful for developing bioethanol without competing with agricultural crops.
Enzymatic hydrolysis of cellulosic biomass depends on many factors: physical properties of the substrate (composition, crystallinity, degree of polymerization, etc.), enzyme synergy (origin, composition, etc.), mass transfer (substrate adsorption, bulk and pore diffusion, etc.), and intrinsic kinetics (Zhang & Lynd, 2004a). The enzymatic kinetics of cellulose degradation has been studied intensively in recent 50 years. Nevertheless, kinetics of cellulose degradation is still poorly understood because of competing effects such as physical properties of the substrate, enzyme synergy, and mass transfer to the intrinsic kinetics (Peri, Karra, Lee, & Karim, 2007). The structural heterogeneity and complexity of cell-wall constituents such as microfibrils and matrix polymers are part of reasons causing the recalcitrance of cellulosic materials (Himmel et al., 2007). The cellulose-hydrolysing enzymes (i.e., cellulases) are divided into three major groups: endo-glucanases, cellobiohydrolases (exo-glucanases), and β-glucosidases. The endo-glucanases catalyze random cleavage of internal bonds of the cellulose chain, while cellobiohydrolases attack the chain ends, releasing cellobiose. The enzymes of β-glucosidases are only active on cello-oligosaccharides and cellobiose, and release glucose monomers units from cellobiose (Kumar, Singh, & Singh, 2008). Therefore, glucose and cellobiose are two major products from enzymatic hydrolysis of cellulose by cellulase. Two major steps (including adsorption of enzymes onto surfaces of cellulose and breakage of β-1,4-glucosidic bond between glucose) are involved in enzymatic hydrolysis of cellulose (Peri et al., 2007). Langmuir isotherm was generally used to describe the adsorption of cellulase onto the surface of cellulose due to its simplicity and good fitting to experimental data. However, the necessity of detail characteristics of the adsorption phenomena constrained its applications. Michaelis–Menten equation (Bezerra and Dias, 2007, Michaelis and Menten, 1913) was the most widely used to describe enzymatic kinetics. The initial rate of hydrolysis (v0) can be expressed as:where Vmax denotes the maximum rate of hydrolysis and [S]0 is the initial concentration of substrate, Km is the Michaelis–Menten constant and physically represents the concentration of substrate as the hydrolysis rate reaches Vmax/2, and it is also considered as an index of affinity between substrate and enzyme. Either increasing Vmax or decreasing Km enhances the reaction rate.
In some enzymatic reactions, the products inhibit the reaction via three different ways: competitive, noncompetitive or uncompetitive. Some investigators (Bezerra and Dias, 2004, Gruno et al., 2004) have revealed the presence of product competitive inhibition in enzymatic hydrolysis of cellulose using cellulase prepared from Trichoderma reesei. With the introduction of competition inhibition, Eq. (1) can be modified as (Gusakov, Sinitsyn, & Klyosov, 1985):where [P] is the product concentration, and Ki denotes the inhibition constant. Apparently, v0 obtained from Eq. (2) is less than that obtained from Eq. (1). To understand the effect of inhibition on hydrolysis rate by using initial rate method, the product concentration can be considered as a constant for a short time period when a finite quantity of product is added. During this short time period, the formation of product from substrate is assumed negligible. The concentration of product ([P]) can be considered as a constant ([P]0) in Eq. (2) which is rearranged as:whereIn a plot of 1/v0 versus 1/[S]0, the intercept on ordinate is the reciprocal of Vmax and the intercept on abscissa is negative reciprocal of Kmapp. The plot is known as the Lineweaver–Burk (L–B) plot (Lineweaver & Burk, 1934). In this study, L–B plot was employed to examine the product inhibition behavior by altering the concentration of [P]0 during the hydrolysis of cotton fiber by cellulase prepared from T. reesei.
Reduction in particle size of cellulose could enhance the affinity between cellulose and enzyme and thus increase the hydrolysis rate. The hydrolysis rate has been doubled in 10-h reaction when the average size was reduced from 82 to 38 μm (Gan et al., 2003). The size reduction also enhances the production of glucose or reducing sugars. Reducing size from 590 to 33 μm resulted in 55% increase in glucose production in 72-h hydrolysis of cellulose (Dasari & Berson, 2007). It appears that size reduction is an attractive method to increase the yield of hydrolysates from cellulose. However, literatures concerned with the effect of reduction of size to submicron scale on the hydrolysis of cellulose are limited. This study was attempted to explore the effect of size reduction of cellulose on hydrolysis rate, kinetic parameters and yield of glucose. The change in crystallinity associated with size reduction was also discussed.
Section snippets
Materials
Microcrystalline cotton cellulose (designated as unmilled cellulose, UC) (Sigma Cellulose, Type 20), cellulase (EC 3.2.1.4 prepared from T. reesei ATCC 26921, lyophilized powder, 6.0 unit/mg solid as labeled. One unit will liberate 1.0 μmol of glucose from cellulose in one hour at pH of 5.0 at 37 °C.), glucose and cellobiose were purchased from Sigma–Aldrich Inc. (St. Louis, MO, USA). Distilled deionized water (DDW) was used in the preparation of suspension.
Media milling
A semi-batch type media mill (MiniPur,
Particle size distribution (PSD)
The PSD of UC exhibited a unimodal distribution ranged from 1.83 to 90.13 μm with a volume-averaged diameters of 25.52 μm. During the milling, large particles were broken into small ones; volume percentage of small particles was increased with milling time, in the meanwhile, the percentage of large particles was decreased. Thus, the unimodal distribution of UC would turn to milti-modal ones. For example, the milled cellulose (MC-3-120) exhibited bimodal PSD, which 74 vol.% percentage of particles
Conclusions
The particle size of cellulose has been reduced to submicron scale by media milling. In the mean while, the crystallinity was also significantly reduced. In this study, the smallest average particle size (0.78 μm by volume) and the largest specific surface area (25.50 m2/g) were obtained at low concentration (3%) after being milled for 120 min. At high concentration (7%), the particles were greater than those at low concentration at the same milling time. Media milling generally reduced the
Acknowledgment
This study is part of a research project sponsored by the National Science Council of the Republic of China (Project No. NSC-93-2214-E-002-028). The financial support is greatly appreciated.
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