Article pubs.acs.org/EF
Enzyme Adsorption and Cellulose Conversion during Hydrolysis of Dilute-Acid-Pretreated Corn Stover Chao Tai and Deepak R. Keshwani* Department of Biological Systems Engineering, University of NebraskaLincoln, Lincoln, Nebraska 68583, United States ABSTRACT: The aim of this study was to investigate the changes in enzyme adsorption to solids and cellulose conversion during the hydrolysis of pretreated corn stover with different substrate and enzyme loadings. Enzyme adsorption achieves equilibrium in the early stage and is stable throughout the hydrolysis process. Enzyme adsorption decreases with the increasing loading of enzyme, except for a system with 15% (w/v) substrate. The inhibition on cellulose digestibility brought by higher substrate loading cannot be easily relieved through elevation of enzyme loading. Glucose does not have an inhibition effect on cellulase adsorption to the solids fraction, while the enzyme is unable to transition from partially degraded cellulose to liquid but can be adsorbed again by lignin. The glucose concentration and ratio of cellulose and lignin are two factors that influence enzyme distribution.
1. INTRODUCTION Bioconversion of lignocellulosic biomass to ethanol is currently being assessed to supplement the use of current fossil-fuelderived petrol because of a lack of resource sustainability of fossil fuels as well as negative environmental influences.1 On the basis of availability, it has been suggested that a possible biomass material for the bioconversion process would be an already available agricultural residue, such as corn stover.2 The biochemical route for conversion of lignocellulose materials to biofuels includes pretreatment to reduce the recalcitrance of substrate, hydrolysis by enzymes, fermentation of the resulting sugars to ethanol, and ethanol recovery.3 A typical batch cellulose hydrolysis initially starts at a relatively rapid rate, followed by a decreasing rate of hydrolysis, and often ending with the incomplete hydrolysis of the substrate, unless an excessive amount of enzymes is used.4 Many hypotheses have been proposed to explain the decreasing rate in the enzymatic hydrolysis process, including thermal instability of cellulase,5 cellulase inactivation,6 end-product inhibition by liberated glucose,7 a potential increase in the substrate recalcitrance over time, substrate reactivity, or competition between the various cellulase components for reactive sites on the substrate.8 Although the reason is still uncertain for the widely observed decreasing conversion rate in hydrolysis of cellulose, most proposed reasons are related to enzyme adsorption and accessibility to the substrate. Barriers to cellulose accessibility include non-cellulosic polymers, such as hemicellulose and lignin,9,10 and covalent associations between cell-wall polymers through ether and ester linkages between lignin and polysaccharides.11 It was found that, on a representative lignocellulosic substrate, cellulase adsorbs to both cellulose and lignin in the substrate.12 The link between changes in the cell-wall structure and digestibility is ultimately dependent upon improved access to the cellulose.13 It was also shown that the pretreatment method of the lignocellulosic substrate had a significant effect on the rate and extent of cellulase adsorption. Of the many pretreatment technologies, dilute acid pretreatment stands out as having been © 2014 American Chemical Society
examined in many studies as well as appearing more economically feasible at larger scale than other current pretreatment technologies.14,15 In this study, the adsorption of cellulase protein on dilute-acid-pretreated corn stover was investigated through the 72 h of hydrolysis with various substrate and cellulase loadings. Factorial and regression models were built to analyze the data on cellulase adsorption and cellulose conversion.
2. EXPERIMENTAL SECTION 2.1. Materials. Corn stover was collected from Rogers Memorial Farm (Lincoln, NE) in 2012; it was air-dried, milled, screened through a 2.36 mm sieve, and homogenized in a single lot. The enzyme preparation used in this work was Cellic CTec2, which was kindly provided by Novozymes North America, Inc. 2.2. Pretreatment. Corn stover samples were pretreated with 1.75% (w/v) sulfuric acid in sealed flasks in an autoclave at 135 °C for 160 min. The solid/liquid ratio was 1:10. The pretreated biomass recovered by filtration through a porcelain Buchner funnel was washed with distilled water until pH was 7. The wet solids were completely transferred to a preweighed plastic bag, weighed, and stored sealed at 4 °C for the enzymatic hydrolysis later. A small portion of the wet pretreated biomass was weighed and dried for composition analysis. 2.3. Enzymatic Hydrolysis. Batch enzymatic hydrolysis experiments were conducted in 50 mL total volume in 125 mL screw-top Erlenmeyer flasks with substrate loadings varied from 5 to 15% (w/v). For each substrate loading, five enzyme loading dosages were applied, ranging from 5 to 60 filter paper units (FPU)/g of cellulose. The activity of CTec2 was 1.04 FPU/mg of protein, determined by the standard procedure developed by the National Renewable Energy Laboratory (NREL).16 A total of 0.05 mol/L sodium citrate buffer was used to maintain pH 5.0, and tetracycline (0.004%, w/v) and cycloheximide (0.003%, w/v) were added to the hydrolysis mixture to prevent microbial growth. The hydrolysis was carried out at 50 °C and 150 rpm for 72 h in a controlled environmental incubator shaker (model I26, New Brunswick Scientific). Aliquots of 0.3 mL were taken at specified time intervals during hydrolysis and centrifuged at 10 000 Received: October 31, 2013 Revised: February 27, 2014 Published: February 27, 2014 1956
dx.doi.org/10.1021/ef402163p | Energy Fuels 2014, 28, 1956−1961
Energy & Fuels
Article
Table 1. Chemical Composition of Raw and Dilute-Acid-Pretreated Corn Stover glucan (%) raw corn stover pretreated corn stover a
38.41 (±1.50) 56.20 (±0.35)
c
xylan (%)
ASLa (%)
AILb (%)
15.98 (±0.56) 0.82 (±0.06)
1.95 (±0.09) 0.90 (±0.07)
19.53 (±0.33) 32.41 (±1.54)
ASL = acid-soluble lignin. bAIL = acid-insoluble lignin. cData in parentheses are the standard deviation based on experimental triplicates.
Figure 1. Enzyme adsorption on the solids fraction during enzymatic hydrolysis of pretreated corn stover. and autoclaved at 121 °C for 20 min for complete protein hydrolysis. After cooling to room temperature, the solution was neutralized by 500 μL of 100% acetic acid while mixing well, followed by adding 500 μL of 2% ninhydrin reagent (Sigma-Aldrich, St. Louis, MO). After boiling for 10 min at 100 °C and cooling to room temperature, the solution was diluted by adding 4 mL of 50% ethanol. After shaking vigorously, 200 μL of the solution was transferred to a 96-well plate; the absorbance of the solution was read by a microplate reader (model Multiskan FC, Fisher Scientific, Hampton, NH) at the wavelength of 570 nm. The protein mass concentration in CTec2 was 177.43 mg/ mL, and the enzyme adsorption on the solids fraction in hydrolysis was calculated by subtracting the free protein concentration from the initial added protein concentration. 2.6. Statistical Analysis. Statistical analysis was conducted in SAS (version Enterprise 4.3, SAS Institute, Cary, NC) to analyze the key variables for enzyme adsorption and cellulose conversion during hydrolysis with different loading features. All hydrolysis experiments were performed in triplicates; when data have been collected, the response variable (yi) was fitted to a quadratic regression model describing the treatment effects as in eq 1
rpm for 10 min; the supernatant were used for sugar and protein analyses. 2.4. Composition and Sugar Analysis. The chemical composition of raw and pretreated corn stover was analyzed using standard analytical procedures developed by the NREL.17,18 Sugars in the hydrolysate were measured in a high-performance liquid chromatography (HPLC) system (model UltiMate 3000, Dionex) with a Bio-Rad Aminex HPX-87P column (300 × 7.8 mm), a Bio-Rad de-ashing guard column, and a refractive index detector. The mobile phase was HPLC-grade water at a flow rate of 0.6 mL/min, and the column temperature was 85 °C. Cellulose conversion was calculated on the basis of the NREL procedure,19 which involves determining the total grams of cellulose digested (on the basis of the glucose concentration in the hydrolysis supernatant) and dividing that by the total grams of cellulose present in the biomass. 2.5. Protein Analysis. The protein mass concentration was measured by the modified ninhydrin method,20,21 and bovine serum albumin (BSA) standard solution (Sigma-Aldrich, St. Louis, MO) was used as the reference standard. In this method, 100 μL of the supernatant sample was first mixed with 300 μL of 10 mol/L NaOH 1957
dx.doi.org/10.1021/ef402163p | Energy Fuels 2014, 28, 1956−1961
Energy & Fuels yi = β0 +
∑ βi xi + ∑ βiixi 2 + ∑ βijxixj
j = 1, 2, ..., k
Article
Table 2. Statistical Analysis of the Effect of the Comparison of Enzyme and Substrate Loadings on Enzyme Adsorption during 72 h of Hydrolysis
i = 1, 2, ..., k ; (1)
where yi represented the enzyme adsorption and cellulose conversion in hydrolysis, β0 was the overall mean, xi represented the enzyme and substrate loadings, βixi was the linear effect, βiixi2 was the quadratic effect, and βijxixj was the interaction effect.
effect level enzyme 5 5 5 10 10 10 20 20 20 40 40 40 60 60 60
3. RESULTS AND DISCUSSION 3.1. Enzyme Adsorption in Batch Hydrolysis. The most significant effect of dilute acid pretreatment of biomass is the hydrolysis of hemicellulose, with possible degradation of the hydrolyzed hemicellulose sugars.22 The removal of the hemicellulose structure appears to have a linear correlation with enzymatic digestibility up to approximately 80% conversion, at which point other factors play a strong role in cellulose accessibility.23 The effect of dilute acid pretreatment at a moderate temperature is shown in Table 1. A total of 11.59% glucan and 81.47% xylan based on raw biomass were removed during pretreatment, making the pretreated biomass consist of 56.20% glucan and 33.31% lignin. If there are no adsorption competitors or inhibitors, enzyme distribution is highly dependent upon substrate composition. Hence, glucan and lignin become key factors that influence enzyme adsorption and cellulose conversion. Figure 1 shows the enzyme adsorption profile during the whole 72 h of hydrolysis, with substrate loadings from 5 to 15% and enzyme loadings varying from 5 to 60 FPU/g of cellulose. For each of the enzyme loadings, adsorption profiles achieve certain equilibriums. However, there were some variations within the first 12 h of hydrolysis with 15% substrate, which was probably due to insufficient mixing energy at high solid loading initially in the system. On the other hand, the adsorption percentage of enzyme to solids fraction in hydrolysis declines with the increase of the enzyme loading, for both 5 and 10% substrate loadings. However, for 15% substrate in the system, no significant difference was found in the enzyme adsorption equilibrium for initial enzyme loadings ranging from 5 to 40 FPU/g of cellulose but there was a sharp increase when the enzyme was loaded at 60 FPU/g of cellulose. The variation of adsorption during the 72 h of hydrolysis was larger for 15% substrate loading than the other two substrate loadings. Statistical analysis also showed that the interaction effect of enzyme and substrate loadings was significant (p value < 0.0001). These interaction effects are summarized in Table 2. The statistical analysis indicates that, when enzyme loading was at or below 20 FPU/g of cellulose, enzyme adsorption was not significantly different for 5 and 10% substrate loading. These differences become significant at higher enzyme loadings. At the same time, with the increase of enzyme loading, the difference in adsorption between 10 and 15% substrate decreased, from 11.81% (statistically significant) to 3.34% (statistically insignificant), while the adsorption with 15% substrate was significantly higher at 40 FPU/g of cellulose. The equilibrium and decreasing trend of enzyme adsorption with increasing enzyme loading is consistent with previously reported work,12,20,24 but the higher adsorption with 15% substrate was unexpected. However, Xiao et al.25 observed a reduced impact of end-product inhibition on cellulose conversion at higher solids content with a constant inhibitor concentration. In their study, the additional quantity of enzyme adsorbed at higher solids content may explain the reduced impact of end-product inhibition. Kumar and Wyman26
a
substrateb
substrate
estimatec
p value
5 5 10 5 5 10 5 5 10 5 5 10 5 5 10
10 15 15 10 15 15 10 15 15 10 15 15 10 15 15
−2.7233 9.0867 11.8100 −3.7000 6.9733 10.6733 4.8367 8.1767 3.3400 8.6233 2.5533 −6.0700 9.5800 −23.1567 −32.7367
0.2648 0.0007
