An Effective Process for Pretreating Rice Husk To Enhance Enzyme

Article pubs.acs.org/IECR

An Effective Process for Pretreating Rice Husk To Enhance Enzyme Hydrolysis Ana Belén Díaz,* Ana Blandino, Csaba Belleli, and Ildefonso Caro Department of Chemical Engineering and Food Technology, Faculty of Sciences, International Agri-Food Campus of Excellence (CeiA3), University of Cádiz, Polígono Rı ́o San Pedro s/n, Puerto Real 11510, Spain S Supporting Information *

ABSTRACT: Numerous pretreatment strategies that have been developed to degrade or remove lignin from lignocellulosic residues to promote the production of sugars by enzymatic saccharification have been described in the literature. Among them, alkaline hydrogen peroxide (AHP) at atmospheric pressure has been described as an efficient method. In this work, a new pretreatment that combines in one stage a process at high pressure (10−30 bar) and alkaline hydrogen peroxide (HPAHP) has been developed. For experiments performed at atmospheric pressure, the maximum hydrolysis yield (72.91 ± 5.93%) was obtained at 7.5% w/v H2O2, with a reaction time of 2 h. With the proposed high-pressure method, a maximum hydrolysis yield of 98.50 ± 6.28% was reached with a reduced peroxide concentration of 3% w/v, a reaction time of 30 min, and a pressure of 30 bar. These HPAHP pretreatment conditions were optimized using a statistical design of experiments. Results showed that the operation variables with the most significant effects on global yield were temperature and peroxide concentration. The response surface model predicts that the optimized conditions for global yield are 3% w/v H2O2, 30 min, 28 bar, and 90 °C. In conclusion, HPAHP pretreatment could be an interesting option for the industrial delignification of rice husk or other lignocellulosic biomass.

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INTRODUCTION Lignocellulosic agricultural residues are promising raw materials for sugar platform biorefinery on a large scale. As they are residues and wastes, they do not compete with primary food production. However, few biorefinery processes based on sugar platforms are cost-competitive in current markets because of the low efficiency and high cost of enzymatic conversion processes.1 Rice husk, which represents 20% of the dry weight in harvested rice, can serve as a low-cost and abundant feedstock for the production of fuel.2 It is considered a waste material because of its low value as animal feed due to its low digestibility, low bulk density, high ash/silica contents, and abrasive characteristics. It can be easily collected from riceprocessing sites and contains about 36% cellulose and 12% hemicellulose. However, rice husk also contains high quantities of lignin (16%) and ash (20%), which complicate its use as a lignocellulosic feedstock for conversion into ethanol.3 Lignin is thought to prevent cellulose degradation mainly by acting as a physical barrier between the cellulolytic enzymes and their substrate.4 Consequently, the rate and extent of enzymatic cellulose degradation in agricultural residues is inversely related to the lignin content, with maximum degradation occurring only when 50% or more of the lignin has been removed. However, even when lignin levels are low, the hydrolysis of cellulose can be limited by the physical properties of the polysaccharide itself; amorphous regions of cellulose are hydrolyzed more easily than microcrystalline regions.4 Numerous pretreatments have been developed to degrade or remove the rigid lignin to promote the production of sugars by enzymatic saccharification. These processes can be physical, chemical, biological, or a combination of them. Although many © 2014 American Chemical Society

different types of pretreatments were tested in different conditions over the past years, advances are still needed for overall costs to become competitive.5 Most pretreatment methods disrupt cell walls to expose the sugar polymers but do not remove much lignin.6−8 Moreover, these pretreatments have limited effectiveness and can generate side products that inhibit later fermentations.9 Hydrogen peroxide (H2O2) is commonly used in pulping for oxidative cellulose bleaching.10 This method can be considered promising for lignin removal as decomposition of H2O2 forms molecular oxygen and other radical species (e.g., HO• and HOO•), which in turn may react with lignin in a variety of ways, making the total oxidation reaction mechanism extremely complicated.10 In this process the pH is one of the most important parameters for efficient application of peroxide.11−14 Depending on the conditions employed for delignification with alkaline peroxide, different changes in the chemical structure of lignin can be observed. Basically, hydrogen peroxide is only able to attack the aliphatic part of lignin, and no degradation of phenolic rings is produced.15 However, when this oxidizing agent is in alkaline conditions and heated at relatively high temperatures, such as 90 °C, it reacts with the phenolic rings of lignin, which are opened, and carboxylic groups are added to the macromolecular structure.11 These modifications increase the water solubility by the insertion of polar groups into the molecule. Therefore, alkaline peroxide pretreatment not only produces selective removal of lignin and xylan without having a Received: Revised: Accepted: Published: 10870

April 2, 2014 June 18, 2014 June 19, 2014 June 19, 2014 dx.doi.org/10.1021/ie501354r | Ind. Eng. Chem. Res. 2014, 53, 10870−10875

Industrial & Engineering Chemistry Research

Article

The solid residue was collected by filtration, washed thoroughly with distilled water until neutral pH of the filtrate, and dried at 60 °C overnight. Subsequently, it was weighed to determine mass loss, which corresponds to the lignin content and other solubilized compounds. After each pretreatment, a liquid sample was taken for RS determination. Pretreatment yield (YP) was calculated as the quotient between the grams of reducing sugars measured in the liquid after the pretreatment and the grams of reducing sugars that should have been measured if all cellulose and hemicellulose contained in the original solid had been completely hydrolyzed (eq 1). The conversion factor from polymer mass to sugar monomer mass has been taken equal to 1.10.

large effect on cellulose but also decreases cellulose crystallinity and swelling of biomass.16 As a consequence, it decreases the recalcitrance of lignocelluloses and enhances the enzymatic digestibility of cellulose. It also has been reported that no furfural or hydroxymethylfurfural (inhibitory sugar degradation products) was detected after this pretreatment.17 Hydrogen peroxide pretreatment can be combined with other methods to increase the hydrolysis yield. For instance, there are references that show its combination with a fungal pretreatment using white-rot fungi and leading to better results compared to those measured after the sole peroxide.18 Thereby, in a previous work,5 we studied the pretreatment of rice husks with alkaline peroxide at atmospheric pressure and now we study the application of high pressure to this process. Thus, the main objective of this work is to improve the effectiveness of the alkaline peroxide method and simultaneously decrease the process time and chemicals concentrations. These achievements will have a positive impact on the sustainability and environmental analysis of the industrial use of rice husk to produce bioethanol.

YP (%) =

mass of RS in the liquid after pretreatment × 100 1.10 × mass of sugars in the raw material

(1)

After selection of the peroxide concentration that provided the highest global yield, 0.25, 1, 2, and 4 h times of reaction were also evaluated. High-Pressure Alkaline Hydrogen Peroxide (HPAHP) Pretreatment. Pretreatments at high pressure were carried out in a reactor supplied by Thar Technology (Pittsburgh, PA, USA; model SF100), which includes a 100 mL vessel. Temperature control was achieved by the thermostatic jacket of the reactor. The operating methodology involved loading the vessel with 3 g of rice husk and 50 mL of hydrogen peroxide solution (at different concentrations depending on the experiment), the pH of which had been adjusted previously to 11.5 with NaOH tablets. The reactor was closed and heated to the desired temperature, at which time the nitrogen was introduced into the vessel to set the pressure. Once the reaction time was finished, the heater was switched off and pressure was released gradually by an automatic pressure control valve. Then, the reactor was opened to collect the solid, which was treated as in atmospheric pressure experiments. After the pretreatment, a liquid sample was collected from the output valve and kept at −70 °C for future RS analysis. Several experiments at high pressure with hydrogen peroxide were set up, using the same H2O2 concentrations as tested at atmospheric pressure (3, 5, and 7.5% w/v) to determine if higher hydrolysis yields were attained. Furthermore, the concentrations of 0.5 and 1% w/v were assayed to examine if it was possible to decrease the H2O2 concentration. For the purpose of comparing only the effect of pressure, these experiments were also accomplished at 90 °C at atmospheric conditions but now a pressure of 30 bar and a reduced time of 30 min. Each experiment was made in duplicate. The following analysis steps were the same as carried out at atmospheric pressure. Enzyme Hydrolysis. The washed and dried water-insoluble residues of rice husk obtained after each pretreatment were hydrolyzed using six different cocktails of Novozyme’s cellulosic ethanol enzyme kit, to ensure that no lack of enzymes was taking place.5 Each solid was introduced in a 100 mL sterile wide-mouth flask and 29.1 mL of citrate buffer (0.05 M, pH5), 0.3 mL of sodium azide (2%), 0.3 mL of the NS22086 enzyme mixture, and 0.06 mL of each of the other five Novozyme enzyme mixtures were added (NS22083, NS22118, NS22119, NS22002, NS22035). Flasks were incubated at 50 °C, 150 rpm, and 72 h. Liquid samples were withdrawn before and after the enzyme hydrolysis and stored at −70 °C for future RS analysis.

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MATERIAL AND METHODS Raw Material. Rice husks were provided by the Spanish company Herba Rice Mills (Sevilla, Spain). The solid was milled and sieved, discarding particles smaller than 500 μm and bigger than 1 mm. The remaining particles were stored in plastic bags at room temperature until used for the experiments. The composition of untreated rice husks was measured according to the method proposed by Sluiter et al., obtaining the following results: 29.8% cellulose, 16.6% hemicellulose, and 22.7% lignin.19 Analysis Method. As an indirect pretreatment efficiency indicator, reducing sugars (RS) were estimated after the pretreatment and before and after the enzyme hydrolysis. With this purpose, the dinitrosalicylic acid (DNS) method in microtiter plate was carried out by using the methodology developed by Gonçalves et al.20 Before the analysis, samples were centrifuged for 10 min, at 10000 rpm and 20 °C. The DNS reagent was prepared by dissolving 5 g of DNS in 250 mL of distilled water at 80 °C. When this solution reached room temperature, 100 mL of 2 N NaOH and 150 g of potassium sodium tartarate-4-hydrate were added, and the volume was completed with distilled water to 500 mL. The reaction was carried out in wells of 340 μL, adding 25 μL of DNS reagent to 25 μL of sample or distilled water (blank). Subsequently, to perform the reaction, the microtiter plate (96 flat test plate of Orange Scientific, made of crystal polyester), with cap, was placed in a thermoblock for 10 min at 105 °C. Then, it was placed on the freezer for 4 min, and 250 μL of distilled water was immediately added to each well. The absorbance of each well was read in a microplate reader (ELx800, Biotek) with a 540 nm filter, using glucose as standard for the calibration curve. Alkaline Hydrogen Peroxide (AHP) Pretreatment. For the pretreatment 3 g of rice husks were slurried in 50 mL of hydrogen peroxide solutions at different concentrations (3, 5, and 7.5% w/v), depending on the experiment, in 250 mL flasks, and the pH was adjusted to 11.5 with NaOH tablets. Each pretreatment was carried out in triplicate. Flasks were covered in aluminum foil and closed with silicone stopples. They were introduced in a water bath at 90 °C for 2 h. To determine the sole effect of peroxide, a blank with 3 g of rice husk and 50 mL of water was also stored at the same temperature. 10871

dx.doi.org/10.1021/ie501354r | Ind. Eng. Chem. Res. 2014, 53, 10870−10875


Article

Hydrolysis yield (YH) was calculated as the quotient between the grams of reducing sugars measured in the liquid after the hydrolysis and the sugars remaining in the solid after the pretreatment (eq 2). The conversion factor from polymer mass to sugar monomer mass has been taken equal to 1.10.

Table 1. Rice Husk Oxidation at Atmospheric Pressure: Effect of [H2O2] [H2O2] (% w/v) 0 3.0 5.0 7.5

mass of RS in the liquid after hydrolysis YH (%) = × 100 1.10 × mass of sugars in the pretreated material (2)

Global yield (YG) was calculated as the quotient between the total reducing sugars produced and the grams that should have been measured if all cellulose and hemicellulose contained in the original solid had been completely hydrolyzed (eq 3).The total reducing sugars produced is the sum of reducing sugars measured after the pretreatment and after the enzyme hydrolysis steps. YG (%) =

Experimental Design and Statistical Analysis. To optimize the HPAHP operation conditions, a D-optimal quadratic statistical experimental design was followed, thus evaluating the effects of the H2O2 concentration, reaction time, pressure, and temperature on the hydrolysis global yield obtained. Initial levels of variables to be analyzed were established according to a preliminary studied and ranged from 1.5 to 3% w/v H2O2, from 10 to 30 min, from 10 to 30 bar, and from 50 to 90 °C, respectively. The D-optimal experimental design allows adequately quantifying a response surface with a reasonable number of tests. In this case, statistical theory leads to a set of 27 experiments with a central point. They were performed in random order for minimizing the effect of any unexplained variability due to external factors. The central point was replicated three times to calculate the experimental error. The significance of effects and two-factors interaction was estimated by ANOVA method. The model was based on response surface methodology (RSM) using the statistical software package Modde 9.0 (Umetrics). In the optimization process, the response level is related to a selected group of factors by the following quadratic equation: YG (%) = β0 +

4

4

i=1

i=1

i=1 j=i+1

βijxixj

± ± ± ±

0.00 0.30 0.44 0.07

global yield (%)

± ± ± ±

3.52 7.77 ± 1.72 12.32 ± 3.69 72.91 ± 5.93

2.55 5.84 10.07 71.15

0.00 1.47 3.40 5.57

Table 2. Rice Husk Oxidation at Atmospheric Pressure: Effect of Pretreatment Time time (h) 0.25 1 2 4

3

∑ βi xi + ∑ βiixi 2 + ∑ ∑

0.99 2.05 2.51 6.10

hydrolysis yield (%)

highest H2O2 concentration. These were 12 times higher than the compounds solubilized in the control. Therefore, as long as the peroxide concentration rises from 0 to 7.5% w/v, there was more solubilization of lignin, improving cellulose and hemicellulose enzymatic attack, presumably as a consequence of the reduction of lignin and cellulose crystallinity. Another factor to be considered is the high silica content of rice husk compared to other residues such as wood, wheat straw, or corn stover. This silica layer may be, as lignin, a physical barrier to limit the enzymatic hydrolysis of glucan and xylan. However, it must have been dissolved in the alkaline peroxide solution, as it is easily dissolved when the pH is >10.21,22 Given these results, it would have been interesting to know if a higher peroxide concentration provides a higher yield. However, it was not possible to test these conditions with the available laboratory equipment in a safe way, due to the high pressure reached in the flasks under such conditions. In addition to this, the use of high H2O2 loadings would be economically challenging to industrial implementation due to the cost of H2O2. Moreover, high concentrations of chemicals should be avoided to minimize environmental impact. In another experiment the effect of the pretreatment time was also evaluated, fixing the H2O2 concentration at 7.5%, which provided the highest yield in the previous study.5 The results obtained are shown in Table 2. As can be seen, with a reaction time of 1 h a hydrolysis yield of 81.60 ± 2.63% is reached, and this value is not improved when the time is increased.

total mass of RS in the liquids after the two steps × 100 1.10 × mass of sugars in the raw material (3)

4

pretreatment yield (%)

(4)

YG is the response factor (global hydrolysis yield) and β0, βi, βii, and βij are the scaled and centered model coefficients (zeroorder, first-order, second-order, and mixed interaction, respectively). The fit of the model was evaluated by R2 and Q2 values.

pretreatment yield (%) 6.36 4.88 6.10 4.99

± ± ± ±

0.11 0.52 0.07 0.25

hydrolysis yield (%) 69.01 81.60 71.15 76.80

± ± ± ±

5.78 2.63 5.57 7.46

global yield (%) 70.99 82.49 72.91 80.63

± ± ± ±

5.37 2.58 5.93 9.70

In all previous results, yields after the pretreatment are negligible compared to those reached in the hydrolysis step. Thus, the results revealed that polysaccharides are not degraded to sugars during the pretreatment, being practically obtained in the hydrolysis stage. However, pretreatment greatly increases lignocelluloses’ susceptibility to enzyme attack, releasing reducing sugars in the following hydrolysis. High-Pressure Alkaline Hydrogen Peroxide Pretreatment. With the aim of improving the pretreatment yield of the alkaline hydrogen peroxide method and simultaneously decreasing the chemical concentration and reaction time, the influence of pressure was studied. For this purpose, experiments at different hydrogen peroxide concentrations were repeated at 30 bar, 90 °C, and a reduced reaction time of 30 min. Therefore, the same H2O2 concentrations assayed at atmos-

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RESULTS AND DISCUSSION Alkaline Hydrogen Peroxide Pretreatment. Table 1 shows the yields obtained when rice husks were pretreated at atmospheric pressure for 2 h with alkaline solution (pH 11.5) at different peroxide concentrations.5 To study this effect, a flask control without H2O2 was treated in the same conditions. As a result, it can be observed that hydrolysis yield increased as long as more concentrated solutions were used, obtaining the best yield (71.15 ± 5.57%) with the highest concentration of peroxide tested (7.5%). These results agree with the solubilized compounds released, which were highest (1.58 ± 0.07 g) at the 10872



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Table 3. Rice Husk Oxidation at High Pressure (30 bar, 90°C, 30 min): Effect of [H2O2] [H2O2] (% w/v)

solubilized compounds (g)

0 0.5 1.0 3.0 5.0 7.5

0.1921 0.4231 0.6343 1.5546 1.5396 1.6204

± ± ± ± ± ±


0.0527 0.0572 0.0273 0.1827 0.0542 0.2302

1.27 1.06 1.41 1.61 1.71 1.15

± ± ± ± ± ±


0.22 0.09 0.29 0.04 0.03 0.03

9.75 19.89 34.82 98.47 97.77 79.43

± ± ± ± ± ±

8.70 3.12 2.83 6.35 4.45 6.14

global yield (%) 10.89 20.74 35.74 98.50 97.22 79.67

± ± ± ± ± ±

8.59 3.15 2.60 6.28 3.48 6.14

Table 4. Rice Husk Oxidation: Effect of Pressure, Pressure−pH, and Peroxide−pH on Rice Husk Pretreatment (90 °C, 30 min) [H2O2] (% w/v)

pH

P (bar)

solubilized compounds (g)



global yield (%)

0 0 2.5

7 11.5 11.5

30 30 0

0.2679 ± 0.0973 0.1779 ± 0.1102 0.2905 ± 0.0604

1.01 ± 0.15 1.07 ± 0.11 1.25 ± 0.11

7.94 ± 0.36 9.86 ± 1.71 18.05 ± 0.79

8.86 ± 0.49 10.82 ± 1.66 19.09 ± 0.86

pheric pressure (3, 5, and 7.5% w/v) were performed in this case. Furthermore, concentrations of 0.5 and 1% w/v were also included to examine if it was possible to decrease the H2O2 concentration without declining yields. Experiments were made in duplicate, with the results shown in Table 3. As can be observed, hydrolysis yield increases as does hydrogen peroxide concentration up to 3%, losing 50% of the total weight of the solid. However, above 5% it decreases slightly. Probably, in these conditions, sugars could have been degraded to other derivatives. Thus, a yield near 100% is attained when 3 g of rice husks are pretreated with 3% alkaline H2O2 at 90 °C and 30 bar for 30 min. However, it was necessary to define if this yield was really the result of the combination of alkaline H2O2 and high pressure, at that temperature, or was solely the result of high pressure. For this purpose, experiments shown in Table 4 were carried out. In the first experiment, the sole effect of pressure is evaluated (row 1 of the table); in the second one, the combination of pressure and alkalinity (row 2); and in the last one, the sole effect of alkaline peroxide (row 3). In none of these three experiments did the hydrolysis yield exceed 20%, and at most only 10% of the original material is solubilized with the pretreatment. Moreover, by comparing these experiments with the previous ones carried out at atmospheric pressure (AHP), it can be inferred that pressure significantly improves hydrolysis yields. Among all of the results obtained, the most prominent result is achieved at 3% H2O2 and 30 bar, as the hydrolysis yield increases around 17 times over the less effective condition and the reaction time is reduced to one-fourth. Thus, it can be concluded that it is really the combined effect of alkaline hydrogen peroxide and adequate pressure and temperature that cause the effectiveness of the proposed pretreatment to reach those maximum hydrolysis yields. Subsequently, HPAHP pretreatment of rice husk was optimized using statistical design of experiments (DOE). In the statistical study, the factors identified for eq 4 were x1, hydrogen peroxide concentration (1.5−3% w/v); x2, time (10− 30 min); x3, pressure (10−30 bar); and x4, temperature (50−90 °C). All of these factors must be scaled and centered. For DOE, global yield was selected as the response variable to analyze the effect of the previous independent variables in the model, and the following quadratic equation was obtained:

Y = 58.3979 + 12.1371x1 + 3.32281x 2 + 7.74327x3 + 12.7966x4 − 0.359536x12 − 0.451732x 22 − 8.33825x32 − 7.05193x42 + 3.77621x1x 2 + 4.275x1x3 + 3.66425x1x4 − 0.121677x 2x3 + 1.55313x 2x4 + 1.12448x3x4 (5) 2

The values of the regression parameters obtained (R = 0.993 and Q2 = 0.944) indicate that it was a very accurate model with excellent predictive power. The plot of the experimental responses versus the polynomial predicted values is given in Figure1.

Figure 1. Predicted versus observed global yield.

Finally, the ANOVA procedure provides the goodness of the model fit. Particularly, in this statistical study the values of the ANOVA parameters obtained demonstrate that the model is significant at the 5% level. Lastly, the β coefficients of eq 4 were normalized to compare the relative importance of the model ́ variables. Basically, all of the linear terms turned out to have positive coefficients, meaning that the response (global yield) increases when they rise (temperature, H2O2 concentration, pressure, or reaction time). This effect can be easily understood as a result of the positive effects of these factors on the quantity of lignin 10873



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(SE), temperatures of 150 or 224 °C are, respectively, reported.27,28 On the contrary, with the exception of the alkaline peroxide assisted wet air oxidation (APAWAO) pretreatment, which requires a first stage of AHP attack of the biomass overnight,16 the reaction time is significantly shorter in the pressurized pretreatments than in the AHP performed at atmospheric pressure. Therefore, for example, when AHP is combined with AFEX, the reaction time is reduced to 10 min.27 Finally, with regard to pressure, reported values in the cited works16,23 are in the range of 5−30 bar, and pressure data of the AHP−AFEX combined pretreatments are typically in the range of 7−30 bar. As a consequence, by comparison of all these processes with the one proposed in this work, it must be empphasized that the latter employs a reaction time and a pressure of the same order of magnitude as those reported, but using much lower reactant concentration and temperature instead. This is a very important factor for the economic viability of the industrial production of bioethanol from rice husk.

removed from the original material, decreasing its recalcitrance and thus easing enzyme hydrolysis. On the other hand, pressure and temperature quadratic terms are negative, indicating the presence of a maximal value for each variable and a negative effect beyond those. This can also be understood as a result of the presence of subsequent degradation routes of sugars to derivatives, which are also positively influenced by these factors, thus decreasing the apparent global yield. Finally, considering the confidence intervals obtained, only the interaction between the H2O2 concentration and the other three factors has significant relevance (positive synergistic influence). This implies that the alkaline peroxide is the key variable, but the other ones enhance the effect to its actual relevance. Once the surface response model was validated, the operating conditions of the process were optimized on the basis of the resultant equation. Thus, the optimization of the global yield was carried out on the basis of the four mentioned variables, which were in the range of the experimental runs. The software predicts that the optimal conditions for the highest global yield (92.87%) are the following: a temperature of 90 °C, 3% w/v alkaline peroxide concentration, a pressure of 28 bar and a reaction time of 30 min. These data correspond to 294 mg of total reducing sugars per gram of dried residue treated. Results obtained by other authors were compared with those of the present study. In general, the majority of works performed at atmospheric pressure employ high H2O2 loadings, as this type of oxidation requires high reactant concentration to attain significant lignin removal from biomass. In relation to this, an important aspect that should be considered in all of these processes is the alkaline liquor generated in the pretreatments, which has to be treated before being recycled or released to the environment. As is well-known, chemicals after alkaline processes can be separated from biomass by washing and regenerated through the lime kiln technology. It can also be observed that pretreatment times are quite high (>6 h), and they can be reduced only when the temperature is raised above 50 °C. Therefore, it is confirmed that peroxide concentration, temperature, and reaction time are the key factors which determine the effectiveness of the AHP pretreatment. On the other hand, these variables not only strongly influence the extent of sugars release and profitability of the treatment but also greatly affect the overall cost of the process. For example, lower H2O2 concentrations or low temperatures result in decreases of the reactant or energy costs, whereas high reaction rates result in lower capital or labor costs, associated with less reactor volume due to the decreased residence time.24 Another important factor to be considered is the range of the solid/liquid ratio (SLR) charged into the pretreatment and hydrolysis reactors. A wide range of SLR, in the range from 0.04 to 0.3 g/mL, is reported. Performing pretreatment and hydrolysis at high solids ratios with no subsequent washing imparts a number of process benefits, including a decrease of process water usage, a decrease in required reactor volumes, and an increase in sugar titer from hydrolysis.24 It is generally described in the literature that when AHP is combined with pressure, the H2O2 concentration can be significantly reduced.25,26 However, the temperature has to be increased drastically. Thus, when AHP is combined with wet air oxidation (WAO), the temperature of this process is reported to be as high as 150 °C16 or 185 °C.23 For AHP assisted with ammonia fiber explosion (AFEX) or with steam explosion

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CONCLUSIONS In this work a new rapid pretreatment for rice husk enzymatic hydrolysis is proposed. This method combines in one stage high pressure and alkaline hydrogen peroxide (HPAHP). Compared with the classic alkaline hydrogen peroxide pretreatment at atmospheric pressure (AHP), in which an average global yield around 75% is obtained, the optimized HPAHP pretreatment allows the concentration of peroxide to be reduced from 7.5 to 3% and the reaction time from 2 h to 30 min, obtaining the maximum yield of 92.87% at 28 bar. In addition to this, the HPAHP method is performed at 90 °C, which is a temperature much lower than the ones established for similar processes. In conclusion, HPAHP pretreatment could be an interesting option for industrial delignification of rice husk or other recalcitrant lignocellulosic biomass. However, further studies would be needed to become cost-competitive by decreasing even more the hydrogen peroxide concentration but without decreasing global yields.

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ASSOCIATED CONTENT

S Supporting Information *

ANOVA plot for global yield, standardized values of effects for global yields, and tables comparing the pretreatment conditions and global yields of the present work with others from literature. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(A.B.D.) Phone: +34956016376. E-mail: anabelen.diaz@uca. es. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Spanish Ministerio de Ciencia e Innovación for financial support of this study (CTQ2010-15452). We also thank Herba Rice Mills for providing the rice husk and Novozymes for granting the cellosic ethanol enzyme kit. 10874



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An Effective Process for Pretreating Rice Husk To Enhance Enzyme

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