Enhanced Enzymatic Hydrolysis of Rice Straw Pretreated by Oxidants Assisted with Photocatalysis Technology

This work evaluated the effectiveness of rice straw pretreatment using a TiO2/UV system in the presence of oxidants. The effects of TiO2 concentrations, pH and photocatalysis time were investigated. Inorganic oxidants including H2O2, K2S2O8, and KIO4 were added to further enhance the effect on enzymatic hydrolysis of rice straw. The TiO2/UV/ H2O2 pretreatment showed a higher amount of released reducing sugar (8.88 ± 0.10 mg/mL, compared to 5.47 ± 0.03 mg/mL in untreated sample). Composition analyses of rice straw after the TiO2/UV/H2O2 pretreatment showed partial lignin and hemicellulose removal. Moreover, structural features of untreated and pretreated rice straw were analyzed through FE-SEM, FT-IR, and XRD. This work suggests that H2O2 is an efficient addition for photocatalysis pretreatment of rice straw.


Introduction
Lignocellulosic biomass is an abundant and sustainable energy resource for producing of biofuels and valuable chemicals, such as bioethanol, biobutanol, xylitol and lactic acid [1]. The use of lignocellulosic biomass as an alternative fuel is considered one of the best solutions to the current energy crisis. Furthermore, utilizing lignocellulosic biomass reduces greenhouse gas emissions, promotes rural area economic development, and enhances energy security [2,3]. Despite these advantages, the biomass recalcitrance due to the cuticle and epicuticular waxes, structural heterogeneity, degree of lignification and crystalline cellulose of lignocellulosic biomass is a challenge in the search to obtain high bioethanol production [4,5]. Herein, finding a pretreatment that can break down biomass recalcitrance is the imperative for all forms and types of lignocellulosic biomass [6,7].
A suitable pretreatment technology should be both financially viable and effective [8]. It could also facilitate lignin or hemicellulose removal, disrupt hydrogen bonds in crystalline cellulose and increase enzymatic access to cellulose [7,9]. Among several pretreatments, photocatalytic oxidation has attracted significant attention for the depolymerization of lignin under mild conditions [10]. For a potential photocatalyst, there has been increasing interest in environmental applications of titanium dioxide (TiO 2 ) due to its commercial availability and chemical stability [11]. It is capable of indirect photolysis or heterogeneous photocatalysis when TiO 2 combines with ultraviolet (UV) light [12]. It is generally accepted that OH radical oxidation and electron-hole reaction occur due to the short wavelength of UV light to complete the photolysis process [13], and show very fast reaction rates [14,15]. Several groups have used the pretreatment technology of TiO 2 /UV to degrade organic contaminants [16,17], and the TiO 2 /UV process can successfully pretreat paper mill effluent, black liquor, and olive mill waste water [10,18]. Moreover, some attempts have been made to depolymerize lignin with a simpler structure [11]. However, TiO 2 as a photocatalyst produces hydroxyl radicals that cannot be infinitely increased, and adding an excess amount of titanium dioxide alone will disperse the ultraviolet light in the solution.
To reduce the probability of recombing photogenerated electrons and holes, more holes and increasing hydroxyl radical concentration can be provided for oxidative degradation of organic contaminants. Some researchers have found that the presence of oxidants can promote the performance of TiO 2 /UV process [19]. Gözmen [20] observed that increasing periodate ion concentration (1-5 mM) enhances the degradation of basic dyes in the TiO 2 /UV system. Inorganic oxidants such as IO 4 − , BrO 3 − , S 2 O 8 2− , and H 2 O 2 can quench conduction band electrons and form reactive radical intermediates, so they are used as additives applied in TiO 2 /UV [19]. Photocatalysis, are considered to have mild pretreatment conditions and provide an environmentally friendly alternative. The TiO 2 /UV process has been successfully applied to pretreat organic species [10]. Related research has focused mainly on using the TiO 2 /UV system to degrade commercial lignin powder and lignin solutions [11]. Compared with native biomass, commercial lignin has a less complicated structure. To the best of our knowledge, there have been very few studies based on native biomass, such as rice straw. This study utilized oxidants such as H 2 O 2 , K 2 S 2 O 8 and KIO 4 as additives in a TiO 2 /UV system. They have high polarizability so that they can capture the electrons ejected from TiO 2 [21] and are more receptive to price. This study: (i) investigated the effects of TiO 2 concentrations, pH, and photocatalysis time; (ii) determined the effectiveness of oxidants such as KIO 4 , K 2 S 2 O 8 and H 2 O 2 as additives for rice straw pretreatment with a TiO 2 /UV system; (iii) investigated oxidants can improve the pretreatment efficacy and how they function during the process; and (iv) investigated the optimal oxidant to achieve the desirable enzymatic hydrolysis efficiency.

Materials and Chemical Reagents
Rice straw was supplied by the Chinese Academy of Sciences (Xiamen, China). It was cut into small pieces, dried at 60 • C, and then ground and screened to 40 mesh size particles for further use. TiO 2 (99.9%) was purchased from Sigma Aldrich Co., Ltd. (St. Louis, MO, USA) The photocatalytic device was purchased from BiLon Co., Ltd. (Xian, China) The UV lamp (36W, BiLon Co., Ltd., Xian, China) with irradiation wavelength 254 nm. KIO 4 , K 2 S 2 O 8 , and H 2 O 2 [30% (w/w)] were obtained from Guangzhou Chemical Reagent Factory (Guangzhou, China). All other reagents were analytical grade.

Photocatalysis Pretreatment
The rice straw powder was first dewaxed in acetone at 20 • C for 16 h [22]. The dewaxed rice straw was cleaned with deionized water and oven dried at 60 • C for 24 h. Sample preparation consisted of soaking 1.0 g rice straw in 40 mL TiO 2 solution. The initial pH (3, 4, 6, 7, 8, and 10) was adjusted by addition of appropriate amounts of HCl (1N) or NaOH (1N). TiO 2 concentrations were 0%, 0.05%, 0.25%, 0.50%, 1.0%, and 2.0% (w/v). The sample preparation was transferred into a 500-mL beaker and exposed to UV irradiation in a device at ambient temperature (30 ± 1 • C), with a magnetic stirrer. The UV lamp was positioned in a quartz glass vessel to maintain a uniform distribution of UV light during pretreatment. The UV lamp was in a fixed position in the center of the bottom of the beaker. UV lamp irradiation times ranged from 0 to 8 h (i.e., 0, 1, 2, 3, 4, 6 and 8 h).

Enzymatic Hydrolysis of Rice Straw
Untreated and pretreated samples were tested at a solid loading of 2.5% (w/v) in 25-mL conical flasks. Each sample enzymatic hydrolysis reaction contained 0.05 M sodium citrate buffer (pH = 4.8), 0.02% sodium azide, cellulase complex (Novozyme NS220086, 250 FPU mL −1 , Copenhagen, Denmark) at a loading of 50 FPU/g and β-glucosidase (Novozyme NS221118, 320 CBU mL −1 ) at a loading of 40 CBU/g. The reactions were carried out in a horizontal shaker incubator (150 rpm, Jinghong Experimental Equipment Co., Ltd., Shanghai, China) for 48 h at 50 • C. Samples taken after 0 and 48 h of hydrolysis and centrifuged to remove the residue at 7000 rpm were analyzed for released reducing sugar by the dinitrosalicylic acid (DNS) method [23].

Addition of Oxidants in a TiO 2 /UV System
For experiments with TiO 2 /UV/oxidants, TiO 2 concentrations were 0.50% (w/v). Three types of oxidants were used only for rice straw pretreatment in the TiO 2 /UV system: KIO 4 , K 2 S 2 O 8 , and H 2 O 2 (1-16 mM). The UV irradiation time was 3 h.

Characterization Analyses
The morphologies of untreated and pretreated rice straw were performed using JSM-7001F Field-Emission Scanning Electron Microscope (JEOL, Tokyo, Japan). The samples were fixed with conductive tape on brass sample stubs and then coated with Au before observation. Images were acquired at a 15-KV acceleration voltage and magnification × 500.
The untreated and pretreated samples for Fourier Transform Infrared (FTIR) spectroscopic analysis were determined using a Nicolet 6700 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) over a wavenumber range from 500-4000 cm −1 with 20 scans and a resolution of 4 cm −1 . The samples were dispersed of spectroscopic grade potassium bromide (KBr) and pressed into disks to determined.
X-ray diffraction analysis was tested using an Ultima X-ray diffractometer of Rigaku Corporation (40 KV, 20 mA, Tokyo, Japan) with Cu Kα radiation. The 2θ range was from 5 • to 40 • in steps of 0.02 • at a rate of 8 • min −1 [24]. The crystallinity index (CrI) of untreated and pretreated samples was calculated according to Equation (1) [25].
where I 002 is the intensity of the crystalline regions (2θ = 22.5 • ) and I am is the intensity of amorphous portion (2θ = 18.7 • ).

Optimization of Conditions for Photocatalysis Pretreatment
The variables used in the pretreatment were TiO 2 concentrations, pH solution, and photocatalysis irradiation time. To optimize pretreatment conditions, the selected TiO 2 concentrations were 0%, 0.05%, 0.25%, 0.50%, 1.0%, and 2.0% (w/v). The effect of TiO 2 concentration on enzymatic hydrolysis of rice straw is illustrated in Figure 1a, in which the released amount of reducing sugar increased with the TiO 2 concentration from 0.05% to 1.0% (w/v). The highest released reducing sugar was 8.04 ± 0.15 mg/mL, observed at TiO 2 concentration of 1.0% (w/v). Higher TiO 2 concentration absorbed more energy during UV irradiation process. However, adding an excess amount of TiO 2 alone will disperse the ultraviolet light in the solution, markedly decreasing the enzymatic hydrolysis efficiency. Consequently, optimal TiO 2 concentration was 1.0% (w/v). Photocatalysis times and their effects on enzymatic hydrolysis of rice straw were investigated. Figure 1c shows that the released reducing sugar increased when irradiation time ranged from 0 to 8 h. The TiO2/UV pretreatment time was prolonged from 1 to 3 h. As the irradiation time increased to 8 h, enzymatic hydrolysis of rice straw was reduced. Therefore, optimal conditions for the TiO2/UV pretreatment were 3 h, pH 8, and TiO2 concentration 1.0% (w/v).

Effect of Added Oxidants in TiO2/UV Pretreatment on Enzymatic Hydrolysis of Rice Straw
In subsequent experiments, the reducing dosage of TiO2 was chosen as a concentration of 0.50%  The solution pH has a significant effect on photocatalysis reactions taking place on the TiO 2 surface. Thus, the pH of solution was studied for enzymatic hydrolysis of rice straw in the pH range of 3-10 at a fixed concentration of TiO 2 (1.0% (w/v)). Mechanisms of the TiO 2 /UV process on the effect of pH are complex and the solution pH dictates the surface charge properties of the photocatalyst [26]. TiO 2 is an amphoteric metal oxide, and, at more acidic values, the surface will be positively charged. Conversely, the surface is negatively charged. A pH that is too high or too low is not suitable for TiO 2 /UV on enzymatic hydrolysis of rice straw, as shown in Figure 1b. The pH value of 8 was selected as the most suitable photocatalysis pretreatment.
Photocatalysis times and their effects on enzymatic hydrolysis of rice straw were investigated. Figure 1c shows that the released reducing sugar increased when irradiation time ranged from 0 to 8 h. The TiO 2 /UV pretreatment time was prolonged from 1 to 3 h. As the irradiation time increased to 8 h, enzymatic hydrolysis of rice straw was reduced. Therefore, optimal conditions for the TiO 2 /UV pretreatment were 3 h, pH 8, and TiO 2 concentration 1.0% (w/v).

Effect of Added Oxidants in TiO 2 /UV Pretreatment on Enzymatic Hydrolysis of Rice Straw
In subsequent experiments, the reducing dosage of TiO 2 was chosen as a concentration of 0.50% (w/v). The added oxidants in a TiO 2 /UV system function as electronic scavengers, as well as the oxidant itself. The released reducing sugar was 5.47 ± 0.03 mg/mL of TiO 2 /UV pretreatment, for which TiO 2 concentration was 0.50% (w/v) without oxidant added. As indicated in Figure 2, adding inorganic oxidants increased the released reducing sugar of rice straw in the TiO 2 /UV system. The optimal released reducing sugar for TiO 2 /UV/K 2 S 2 O 8 , TiO 2 /UV/KIO 4 and TiO 2 /UV/H 2 O 2 pretreatments were 6.80 ± 0.10 mg/mL, 6.90 ± 0.20 mg/mL and 8.88 ± 0.10 mg/mL, respectively. These results confirm previous reports that TiO 2 /UV/oxidant provided better photocatalysis than TiO 2 /UV alone. For increasing hydrolysis, H 2 O 2 was more effective than K 2 S 2 O 8 or KIO 4 , and excess K 2 S 2 O 8 and KIO 4 reduced the hydrolysis efficiency of rice straw in a TO 2 /UV system. The excess of K 2 S 2 O 8 inhibited photocatalysis process, due to the increase of sulfate radical anion in solution, was adsorbed on the TiO 2 surface. The adsorbed sulfate radical anion also reacted with hydroxyl radicals, thus reducing the photocatalytic activity [27]. Further increase of KIO 4 decreased the photocatalytic efficiency, as the excess periodate ion in solution may scavenge hydroxyl radicals [19]. Released reducing sugar increased with increasing H 2 O 2 concentration up to 13 mM, but exceeding 13 mM of H 2 O 2 concentration decreased the hydrolysis efficiency. In particular, the addition of 13 mM H 2 O 2 had a greater impact than the TiO 2 concentration of 0.50% in the TiO 2 /UV system. The released reducing sugar from TiO 2 /UV/H 2 O 2 pretreatment was greater than TiO 2 /UV/K 2 S 2 O 8 and TiO 2 /UV/KIO 4 pretreatment. The addition of oxidants in a TiO 2 /UV system are generally applied for treating reactive dyes and organic contaminants in industrial wastewater [19,28]. Prior studies have proposed that the role of oxidants in TiO 2 /UV system increased the formation of hydroxyl radicals and impeded electron-hole recombination. Hence, a proper concentration of H 2 O 2 was important for enzymatic hydrolysis of rice straw. Table 1 summarizes the compositions of untreated and pretreated rice straw. Optimum conditions were chosen for the three oxidants to analyze chemical compositions and selected the corresponding control group of oxidants for comparison. The contents of untreated rice straw were determined as lignin (18.68 ± 0.30%), hemicellulose (32.98 ± 1.10%), and cellulose (37.47 ± 0.57%), similar to previous studies [29,30]. The H 2 O 2 alone, H 2 O 2 /UV pretreatment and TiO 2 /UV/H 2 O 2 pretreatment of lignin contents were 17.04 ± 0.74%, 17.45 ± 0.99% and 16.10 ± 0.67%, respectively. Lignin levels of TiO 2 /UV/K 2 S 2 O 8 and TiO 2 /UV/KIO 4 pretreatments were 18.41 ± 0.33% and 17.69 ± 0.54%, respectively. This demonstrated that the TiO 2 /UV/H 2 O 2 pretreatment improved enzymatic hydrolysis of rice straw. Some research has proposed lignin removal as an essential factor for enzymatic saccharification of lignocellulosic biomasses [31]. Hemicellulose contents by TiO 2 /UV/K 2 S 2 O 8 and TiO 2 /UV/KIO 4 pretreatment were 31.46 ± 0.28% and 32.63 ± 0.31% compared with 32.98 ± 1.10% for the untreated. Öhgren [32] reported that hemicellulose hinders enzyme access to cellulose, reducing the cellulase accessibility of lignocellulose biomass. As noted above, the components of rice straw after TiO 2 /UV/K 2 S 2 O 8 and TiO 2 /UV/KIO 4 pretreatment showed no significant difference in the content of lignin, cellulose and hemicellulose. Composition analysis demonstrated that TiO 2 /UV/H 2 O 2 pretreatment improved enzymatic efficiency more than pretreatment with other oxidants to support TiO 2 /UV.  Table 1 summarizes the compositions of untreated and pretreated rice straw. Optimum conditions were chosen for the three oxidants to analyze chemical compositions and selected the corresponding control group of oxidants for comparison. The contents of untreated rice straw were determined as lignin (18.68 ± 0.30%), hemicellulose (32.98 ± 1.10%), and cellulose (37.47 ± 0.57%), similar to previous studies [29,30]. The H2O2 alone, H2O2/UV pretreatment and TiO2/UV/H2O2 pretreatment of lignin contents were 17.04 ± 0.74%, 17.45 ± 0.99% and 16.10 ± 0.67%, respectively. Lignin levels of TiO2/UV/K2S2O8 and TiO2/UV/KIO4 pretreatments were 18.41 ± 0.33% and 17.69 ± 0.54%, respectively. This demonstrated that the TiO2/UV/H2O2 pretreatment improved enzymatic hydrolysis of rice straw. Some research has proposed lignin removal as an essential factor for enzymatic saccharification of lignocellulosic biomasses [31]. Hemicellulose contents by TiO2/UV/K2S2O8 and TiO2/UV/KIO4 pretreatment were 31.46 ± 0.28% and 32.63 ± 0.31% compared with 32.98 ± 1.10% for the untreated. Öhgren [32] reported that hemicellulose hinders enzyme access to cellulose, reducing the cellulase accessibility of lignocellulose biomass. As noted above, the components of rice straw after TiO2/UV/K2S2O8 and TiO2/UV/KIO4 pretreatment showed no significant difference in the content of lignin, cellulose and hemicellulose. Composition analysis demonstrated that TiO2/UV/H2O2 pretreatment improved enzymatic efficiency more than pretreatment with other oxidants to support TiO2/UV.

FE-SEM, XRD and FTIR Profile of Untreated and Pretreated Rice Straw
FE-SEM was performed to observe the morphological structures of untreated and pretreated rice straw and determine structural changes of the sample surfaces after photocatalysis pretreatment. Untreated rice straw (Figure 3a) presented flat and well-ordered surface structure. FE-SEM images show that the TiO 2 /UV/H 2 O 2 pretreated sample (Figure 3b) has a different morphology, disrupting the extensive, well-ordered surface of rice straw. Moreover, the surface has loose and irregular structure, with some cracks on the surface (marked with red circle). Changes of pretreated rice straw were beneficial to the enzymatic hydrolysis process due to the enhancement of cellulose accessibility to the enzyme. FTIR spectroscopy identified the changes of organic groups of untreated and pretreated rice straw, as shown in Figure 4 (marked with red circle). An untreated sample of rice straw was observed and compared with a pretreated sample obtained at the conditions of TiO 2 /UV/H 2 O 2 . The band at 3415 cm −1 was subject to O-H stretching of the hydroxyl group. Among the peaks, 2918 cm −1 (C-H stretching of methyl and methyene groups), 1059 cm −1 (C-O stretching vibration) and 898 cm −1 (C-H deformation vibration) were assigned as the characteristic peaks of cellulose and hemicellulose. A decrease was observed in the absorbance of the 1641 cm −1 of conjugated carbonyl stretch in lignin [33]. Moreover, lignin-related peaks had some changes in the vibration at the absorbance bands of 1514 cm −1 (aromatic skeletal vibrations) and 1319 cm −1 (C-O vibration in the syringyl ring). With TiO 2 /UV/H 2 O 2 , the rice straw was weaker than the untreated sample, indicating greater lignin removal.
FE-SEM was performed to observe the morphological structures of untreated and pretreated rice straw and determine structural changes of the sample surfaces after photocatalysis pretreatment. Untreated rice straw (Figure 3a) presented flat and well-ordered surface structure. FE-SEM images show that the TiO2/UV/H2O2 pretreated sample (Figure 3b) has a different morphology, disrupting the extensive, well-ordered surface of rice straw. Moreover, the surface has loose and irregular structure, with some cracks on the surface (marked with red circle). Changes of pretreated rice straw were beneficial to the enzymatic hydrolysis process due to the enhancement of cellulose accessibility to the enzyme. FTIR spectroscopy identified the changes of organic groups of untreated and pretreated rice straw, as shown in Figure 4 (marked with red circle). An untreated sample of rice straw was observed and compared with a pretreated sample obtained at the conditions of TiO2/UV/H2O2. The band at 3415 cm −1 was subject to O-H stretching of the hydroxyl group. Among the peaks, 2918 cm −1 (C-H stretching of methyl and methyene groups), 1059 cm −1 (C-O stretching vibration) and 898 cm −1 (C-H deformation vibration) were assigned as the characteristic peaks of cellulose and hemicellulose. A decrease was observed in the absorbance of the 1641 cm −1 of conjugated carbonyl stretch in lignin [33]. Moreover, lignin-related peaks had some changes in the vibration at the absorbance bands of 1514 cm −1 (aromatic skeletal vibrations) and 1319 cm −1 (C-O vibration in the syringyl ring). With TiO2/UV/H2O2, the rice straw was weaker than the untreated sample, indicating greater lignin removal.  The CrI of untreated sample was 43.90%, and the CrI of TiO 2 /UV/H 2 O 2 pretreatment was 46.63%. TiO 2 /UV/H 2 O 2 pretreatment increased the intensity of the removal of amorphous components such as hemicellulose and lignin. Additionally, this pretreatment influenced enzymatic hydrolysis that released reducing sugar. There were similar observations of increased crystallinity index for biomass pretreatments such as previous surfactant-assisted ultrasound-ionic liquid-based pretreatment and concentrated phosphoric acid plus hydrogen peroxide pretreatment, where the crystallinity index increased by deconstruction of the crystalline cellulose [34,35].    Figure 5 shows XRD diffraction of untreated and TiO2/UV/H2O2 pretreated rice straw. The CrI of untreated and TiO2/UV/H2O2 pretreatment rice straw was calculated according to Equation (1). The CrI of untreated sample was 43.90%, and the CrI of TiO2/UV/H2O2 pretreatment was 46.63%. TiO2/UV/H2O2 pretreatment increased the intensity of the removal of amorphous components such as hemicellulose and lignin. Additionally, this pretreatment influenced enzymatic hydrolysis that released reducing sugar. There were similar observations of increased crystallinity index for biomass pretreatments such as previous surfactant-assisted ultrasound-ionic liquid-based pretreatment and concentrated phosphoric acid plus hydrogen peroxide pretreatment, where the crystallinity index increased by deconstruction of the crystalline cellulose [34,35].

Conclusions
This work suggests adding oxidants can boost lignin removal and improve the release of reducing sugar of rice straw. Among the oxidants, H2O2 addition in the TiO2/UV system for pretreated rice straw was most effective to reduce the dosage of TiO2 under the same pretreatment conditions. Optimal TiO2/UV/H2O2 pretreatment conditions for rice straw were adding 13 mM H2O2 at pH = 4, irradiation time of 3 h and 0.50% (w/v) concentration of TiO2. Results of the composition and characterization analyses indicated that TiO2/UV/H2O2 pretreatment efficiently provided lignin removal and significant changes in morphology.

Conclusions
This work suggests adding oxidants can boost lignin removal and improve the release of reducing sugar of rice straw. Among the oxidants, H 2 O 2 addition in the TiO 2 /UV system for pretreated rice straw was most effective to reduce the dosage of TiO 2 under the same pretreatment conditions. Optimal TiO 2 /UV/H 2 O 2 pretreatment conditions for rice straw were adding 13 mM H 2 O 2 at pH = 4, irradiation time of 3 h and 0.50% (w/v) concentration of TiO 2 . Results of the composition and