Enhanced Rate of Enzymatic Saccharification with the Ionic Liquid Treatment of Corn Straw Activated by Metal Ion Solution

Abstract

The aim of this paper was to effectively reduce environmental pollution and further improve the enzymatic hydrolysis rate of corn straw. Thus, a pretreatment method for activating cellulose by using ionic liquid to treat metal ion solution was developed. By investigating the effects of the three factors of substrate mass fraction, reaction temperature, and reaction time, and the interaction between the factors on the pretreatment effect, the response surface design method was used to optimize the conditions of ionic liquid (1-butyl-3-methylimidazolium chloride) treatment of corn straw after activation, and the physicochemical structure and enzymatic hydrolysis efficiency before and after treatment were compared and analyzed. The experimental results showed that the yield of reducing sugar was increased by 157.85% and 150.41%, respectively, compared with the untreated corn straw. The analysis of chemical composition and structure showed that the cellulose content of the material increased significantly by 68.11% and 60.54%, respectively, after ionic liquid treatment. The results of the scanning electron microscope (SEM) observation and X-ray diffraction (XRD) showed that the relative crystallinity of the material decreased after ionic liquid treatment, which was more conducive to the enzymatic hydrolysis of cellulose.

1. Introduction

As the world’s energy demand continues to grow, the overexploitation of fossil fuels has led to increasingly serious environmental pollution [1]. Countries around the world are committed to the development of efficient, pollution-free renewable energy. Biomass is regarded as an ideal source of alternative petrochemical resource materials because of its wide source and short regeneration cycle [2,3]. Lignocellulose is the most abundant renewable biomass resource on earth, which can be converted into energy fuels, chemicals, and materials by biological, physical, or chemical means. It has been reported that ethanol produced from cellulosic ethanol and other biomass resources has the potential to reduce greenhouse gas emissions by 86%, and not only can effectively alleviate the oil crisis, but also avoids the aggravation of environmental pollution [4,5,6,7,8,9]. However, the complex structure of lignocellulose hinders its direct hydrolysis, making it difficult to achieve component separation, hydrolysis and saccharification, making the efficient utilization of lignocellulose biomass challenging [10]. Therefore, the destruction of its crystal structure is the key to improving enzymatic saccharification [11]. Traditional pretreatment methods of lignocellulosic biomass include physical methods [12], chemical methods [13], and biological methods [14]. Various pretreatment techniques can improve the hydrolysis rate by changing the physical and chemical structure of lignocellulosic biomass [15]. However, compared with traditional cellulose solvents, ‘emerging green solvent’ ionic liquids have the advantages of easy separation, recyclability, stable properties, and environmental friendliness, and are widely used in cellulose dissolution, cellulose pretreatment, and cellulose hydrolysis [16,17,18,19,20,21]. Ionic liquids (ILs) are molten salts that consist of bulky organic cations and inorganic/organic anions. They consist of pyridinium, imidazolium cations, and OAc−, HCOO−, or Cl− anions, for example, which have the ability to dissolve cellulose [13,22].

In previous studies, Erdmenger et al. [23] studied the effect of different alkyl chain lengths of 1-alkyl-3-methylimidazolium chloride ([BMIM] Cl) on the solubility of cellulose. The results showed that the solubility obtained using [BMIM] Cl was the highest. On the other hand, FitzPatrick et al. [24] found that the ionic liquid (1-ethyl-3-methylimidazolium acetate) they proposed can dissolve cellulose efficiently, although the viscosity of the organic salt used has a key effect on the whole process. Recent studies have found that wood pulp cellulose was degraded in three phosphate-based ILs, 1-ethyl-3-methylimidazolium dimethyl phosphate ([Emim] DMP), 1-ethyl-3-methylimidazolium diethyl phosphate ([Emim]DEP) and 1-butyl-3-ethylimidazolium diethyl phosphate ([Beim] DEP). The degradation degree increased with the increase in the dissolution temperature and the accumulation of dissolution time [25]. 1-Ethyl-3-methylimidazolium chloride (EMIMCl-) and EMIMCl · acetic acid (AcOH)-based deep eutectic solvents (DESs) exhibited quite different solvent capabilities for cellulose; that is, cellulose solubility was high in EMIMCl [26]. In the acetate-based IL/co-solvent systems, cellulose prefers to form a molecularly dispersed state, while in the chloride-based IL/co-solvent systems, cellulose tends to form an initial solution state of single molecular chains coexisting with aggregates [27]. Existing studies have found that a limitation of 1-alkyl-3-methylimidazolium ([BMIM]) is its high viscosity, which slows down the dissolution of cellulose [28].

Over the past decade, imidazole ionic liquids containing chlorides have attracted great interest from researchers. Using 1-ethyl3-methylimidazolium chloride ([C₂C₁IM] [Cl]) as a model, Chaban et al. [29] were the first to find that highly viscous IL spontaneously and rapidly penetrated into polar carbon nanotubes (CNTs) of any diameter at 363 K and higher temperatures. Ohbad et al. [30] characterized the unique structure of 1-ethyl-3-methylimidazolium chloride in carbon nanotubes. Chlorine anions can migrate across the imidazole ring in a relatively ordered nano-scale framework maintained by 1-ethyl-3-methylimidazolium cations. Despite the high viscosity of [C₂C₁MIM] [Cl], [C₂C₁MIM] [Cl] easily penetrates into CNTs with a width of 1−3 nm at slightly higher temperatures (323–363 K) [31]. However, the method of using ionic liquid to treat metal ion solution to pretreat corn straw has not been reported. It is limited to the use of a single pretreatment method to destroy the structure and cellulose crystallinity of corn straw. Therefore, the combination of various treatment methods can better improve the utilization rate of corn straw and reduce production costs, which has high research value.

In this study, corn straw was used as raw material, and the straw raw material was activated by ion solution pretreatment of metal ion solution. The optimum activation conditions of metal ion solution treated by ionic liquid were determined by response surface experimental design, and the chemical composition, crystallinity, and structure of corn straw before and after treatment under the optimum conditions were analyzed. The research results can provide technical references and theoretical support for the utilization of biomass.

2. Materials and Methods

2.1. Raw Materials

The raw material of corn straw was collected from Zhongliangshan District of Chongqing City, which was dried naturally. After being crushed with a hammer mill, it was screened using a standard sieve. The corn straw material with a particle size of 60 mesh was dried at 105 °C. Cellulase was purchased from Ningxia Heshibi Biotechnology Co., Ltd., (Ningxia, China), ionic liquid [BMIM]Cl was purchased from Moni Chemical Technology (Shanghai, China) Co., Ltd., (Shanghai, China), and ferric chloride solution and aluminum chloride solution were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd (Shanghai, China).

2.2. Metal Ion Solution Pretreatment Experiment

As a uniform experimental design method for pretreatment of corn straw with ferric chloride and aluminum chloride solution, 60.00 g corn straw raw material screened with a standard sieve was pretreated according to the experimental design conditions, the treated material was collected, washed five times with deionized water, and then dried at 105 °C for later use.

2.3. Ionic Liquid Treatment Experiment

Pretreated materials were treated using the ionic liquid method. The substrate mass fraction of 2~6%, reaction temperature of 80~120 °C, and reaction time of 2~6 h were selected as the three factors of the response surface optimization experiment of enzymatic hydrolysis. According to the response, as part of the surface experimental design method, cellulase hydrolysis experiments were carried out on the extracted cellulose materials, as shown in

Table 1. Response surface experimental design factor level.

Factor	Level
	−1	0	1
X1 Substrate mass fraction (%)	2	4	6
X2 Reaction temperature (°C)	80	100	120
X3 Reaction time (h)	2	4	6

.

2.4. Analysis Method

The filter paper activity of cellulase was determined to be 60 FPU/g according to the enzyme activity method [32]. The reducing sugar concentration in the enzymatic hydrolysate of corn straw was determined via 3,5-dinitrosalicylic acid colorimetry [33]; the component content of corn straw material was determined using the Van Soest method [34].

2.5. Structural and Morphological Analysis

XRD (Malvern Panaco intelligent X-ray diffractometer Empyrean), SEM (low vacuum tungsten lamp scanning electron microscope SEM3200), and FTIR (Fourier transform infrared spectroscopy imaging system PerkinElmer) were used to observe and analyze the structure, morphology, and bond changes of intermolecular and intramolecular bonds in corn straw raw materials and metal-ion-solution-treated materials. XRD scanning range: 10~50, acceleration voltage: 40 kV, current: 150 mA, step length: 0.02/2θ, scanning speed: 4/min, SEM acceleration voltage: 10 kV, FTIR scanning range: 400 cm⁻¹~4000 cm⁻¹.

2.6. Statistical Analysis Methods

The data obtained in the experiment were analyzed and processed using Origin 8.0 software processing system and Windows Excel and Word (2003, 2010 editions) Office software. All determinations were carried out in triplicate and the mean values were presented, and the data are reported as the averages of three separate experiments ± SD (n = 3).

3. Results

3.1. Optimization Experiment Results of Response Surface Method

In this study, reducing sugar yield as the response value, according to the response surface design of experimental enzymatic hydrolysis experiments, the results are shown in

Table 2. Solution response surface experimental design and results.

Test Number	Factor			Reducing Sugar Yield (mg·g−1)
	X1	X2	X3
1	−1	1	0	623.76 a, 603.57 b
2	0	0	0	700.67, 679.74
3	−1	−1	0	583.45, 574.12
4	0	1	1	691.23, 669.82
5	1	−1	0	678.76, 656.52
6	1	1	0	685.48, 649.83
7	−1	0	−1	592.13, 566.92
8	0	−1	1	663.21, 627.56
9	0	0	0	701.52, 678.12
10	−1	0	1	623.87, 601.48
11	0	0	0	700.45, 681.25
12	1	0	1	672.43, 658.53
13	0	1	−1	670.52, 641.79
14	0	−1	−1	599.78, 569.44
15	1	0	−1	675.78, 647.52

Note: ^a Ferric chloride solution. ^b Aluminum trichloride solution.

,

Table 3. Significance test of each coefficient of the developed quadratic regression model.

Item	Coefficient	Standard Error	t	P
constant	2334.81 a, 679.703 b	2592.76 a, 9.221 b	16.19 a, 73.713 b	0.0034 a, 0.0013 b
X1	1457.47, 33.289	1457.47, 5.647	65.31, 5.895	0.0005, 0.0021
X2	2656.84, 17.171	2656.84, 5.647	16.59, 3.041	0.0096, 0.0032
X3	1582.88, 16.465	1582.88, 5.647	9.88, 2.916	0.0256, 0.0294
X1 X1	−44.98, −33.617	3.286, 8.312	−13.688, −4.045	0.0001, 0.0102
X2 X2	−17.74, −25.077	3.286, 8.312	−5.398, −3.017	0.0017, 0.0211
X3 X3	−23.71, −27.474	3.286, 8.312	−7.217, −3.305	0.0004, 0.0303
X1 X2	−8.32, −9.035	3.157, 7.986	−2.635, −1.131	0.0388, 0. 0395
X1 X3	−10.42, −5.888	3.157, 7.986	−3.300, −0.737	0.0164, 0.0441
X2 X3	−9.73, −7.522	3.157, 7.986	−3.081, −0.942	0.0216, 0.0492

Note: ^a Ferric chloride solution. ^b Aluminum trichloride solution.

and

Table 4. Variance analysis of regression equation.

Source of Variance	Degree of Freedom	Sum of Squares	Corrects the Sum of Squares	Correct Variance	F	P
Regression	9	23,009.2 a, 22,159.3 b	23,009.2 a, 22,159.3 b	2556.58 a, 2462.14 b	64.13 a, 9.65 b	0.001 a, 0.011 b
Linear	3	12,312.1, 13,392.7	12,312.1, 13,392.7	4104.02, 4464.24	102.95, 17.50	0.001, 0.004
Square	3	9607.8, 8075.0	9607.8, 8075.0	3202.61, 2691.68	80.34, 10.55	0.001, 0.013
Interaction	3	1089.3, 691.5	1089.3, 691.5	363.11, 230.51	9.11, 0.90	0.018, 0.501
Residual Error	5	199.3, 1275.4	199.3, 1275.4	39.86, 255.08
Missing Fit	3	197.5, 1270.5	197.5, 1270.5	65.84, 423.50	72.5, 172.84	0.014, 0.006
Pure Error	2	1.8, 4.9	1.8, 4.9	0.91, 2.45
Total	14	23208.5, 23434.7

Note: ^a Ferric chloride solution. ^b Aluminum trichloride solution.

.

In the Design-Expert. 12.0 software, X1 (substrate mass fraction), X2 (reaction temperature), and X3 (reaction time) were taken as independent variables, and enzymatic hydrolysis reducing sugar yield Y as the response value. The multivariate quadratic regression Equations (1) and (2) were obtained as follows:

$$\begin{gathered} \mathrm{Y}=700.88+36.15\mathrm{X}1+18.22\mathrm{X}2+14.07\mathrm{X}3-36.57\mathrm{X}1\mathrm{X}1-21.44\mathrm{X}2\mathrm{X}2-23.25\mathrm{X}3\mathrm{X}3- \\ 8.40\mathrm{X}1\mathrm{X}2-8.77\mathrm{X}1\mathrm{X}3-10.68\mathrm{X}2\mathrm{X}3 \end{gathered}$$

(1)

$$\begin{gathered} \mathrm{Y}=679.66+33.34\mathrm{X}1+17.16\mathrm{X}2+16.37\mathrm{X}3-33.58\mathrm{X}1\mathrm{X}1-25.07\mathrm{X}2\mathrm{X}2-27.37\mathrm{X}3\mathrm{X}3- \\ 9.03\mathrm{X}1\mathrm{X}2-5.99\mathrm{X}1\mathrm{X}3-7.50\mathrm{X}2\mathrm{X}3 \end{gathered}$$

(2)

The first column number of reducing sugar yield in Table 2 is ferric chloride solution response surface experimental data, the second column number is aluminum chloride solution response surface experimental data. The results of Table 4 show that the F values were 64.13, 9.65, and p (<0.05), indicating that the model was highly significant; among them, X1 and X2 were extremely significant, while X3 was significant; the interaction items X1X2, X1X3, and X2X3 were significant.

Figure 1 and Figure 2 are the response curves of reducing sugar after ferric chloride activation and aluminum chloride activation by ionic liquid treatment, respectively. It can be seen from the a in Figure 1 and Figure 2 that with the increase in reaction temperature, the enzymatic hydrolysis reducing sugar rate tends to be stable, and when the temperature is higher than 80 °C and 100 °C, the enzymatic hydrolysis rate has a downward trend; when the substrate mass fraction is higher than 2%, the enzymatic hydrolysis rate will increase with the increase of the substrate mass fraction. When the substrate mass fraction is greater than 4%, the enzymatic hydrolysis rate will no longer continue to increase. Therefore, the reaction temperature and mass fraction should be controlled at 60–80 °C, 80–100 °C, respectively, and 4–6%, 2–4%, respectively, and the enzymatic hydrolysis rate can reach the maximum value. It can be seen from b in Figure 1 and Figure 2 that the enzymatic hydrolysis rate of corn straw increased with the increase in reaction time and reached the maximum when the reaction time was 4 h and near 4 h. After that, it did not increase any further. When the substrate mass fraction is around 4%, the enzymatic hydrolysis rate reaches the maximum. Therefore, the reaction time and the substrate mass fraction are controlled at 4–6%, 2–4%, respectively, and 2–4 h, 2–4 h, respectively. The enzymatic hydrolysis rate can reach its maximum. It can be seen from c in Figure 1 and Figure 2 that the reaction time and temperature required to reach the maximum value of cellulase hydrolysis should be between 2–4 h, 2–4 h, respectively, and 60–80 °C, 80–100 °C, respectively. In summary, the optimal conditions are as follows: when the mass fraction of substrate is 4.2% and 3.6%, the reaction temperatures are 85 °C and 105 °C, respectively. The reaction times are 185 min and 230 min, respectively. The maximum theoretical value of reducing sugar yield (Y) is 712.66 mg/g and 690.85 mg/g, respectively. To test the accuracy of the model, the verification test was carried out under this condition, and the yield of reducing sugar after enzymatic hydrolysis was 706.52 mg/g, 686.13 mg/g, respectively, which was close to the model prediction results, indicating that the model was effective. At the same time, compared with untreated raw materials, the yield of reduced sugar increased by 157.85% and 150.41%, respectively.

3.2. Analysis of Components

Corn straw raw materials and ionic liquid treatment chemical composition of metal-ion-activated corn straw materials before and after treatment are shown in

Table 5. Composition of raw corn straw samples treated by mental ion and ionic liquids (g).

	Corn Straw	Cellulose	Hemicellulose	Lignin	Soluble Material	Ash
Not Processed	0.600	0.185	0.189	0.046	0.175	0.004
Iron Trichloride Solution + Ionic Liquid Treatment	0.600	0.311	0.155	0.045	0.077	0.010
Aluminum Trichloride Solution + Ionic Liquid Treatment	0.600	0.297	0.157	0.052	0.081	0.011

. The results of Table 5 showed that the content of cellulose increased by 68.11% and 60.54%, respectively, hemicellulose decreased by 17.99% and 16.93%, respectively, and soluble substances decreased by 56.00% and 53.71%, respectively, when the corn straw activated by ferric chloride solution and aluminum chloride solution was treated with ionic liquids.

3.3. Fourier Transformation Infrared Spectroscopy Analysis

Figure 3 is the FTIR diffraction pattern of corn straw before treatment with two metal ion solutions, and Figure 4 is the FTIR diffraction pattern after treatment. It can be seen from Figure 3 that the cellulose spectrum of corn straws treated in different ways is still roughly the same. In this study, we mainly judged whether the cellulose had been derivatized after ionic liquid treatment by infrared spectroscopy and judged the crystal structure of cellulose based on the change of characteristic absorption peaks. From the diagram, it can be seen that the absorption peak intensity of cellulose before and after treatment has changed greatly, but the position of each peak remains unchanged, which indicates that cellulose does not undergo a derivatization reaction in the process of dissolution and regeneration; that is, the dissolution of cellulose by ionic liquid is a direct dissolution process. At the same time, after treatment with ionic liquid, the hydroxyl absorption peak of cellulose is significantly weakened, indicating that cellulose changes from type I to type II structure.

3.4. X-ray Diffraction Analysis

From Figure 5, it can be seen that the diffraction characteristic peaks of the material diffraction pattern change greatly after the corn straw raw material is activated by the ionic liquid, indicating that the [BMIM] Cl treatment destroys the crystal structure of the straw material, thus changing its crystal structure. Referring to the empirical algorithm of Segal et al. [35], the crystallinity of corn straw raw material was calculated to be 88.36°. The crystallinity of ionic-liquid-treated ferric chloride solution and aluminum chloride solution was 38.36° and 49.31°, respectively.

3.5. Scanning Electron Microscope Analysis

The appearance of corn straw changed significantly before and after pretreatment, as shown in Figure 6. Before pretreatment, the surface structure of corn straw raw material was smooth, and the cellulose was uniform, smooth, and orderly. However, after the activation of metal ion solution treated by the ionic liquid, the structure of corn straw raw material changed noticeably. After the activation of two metal ion solutions treated with ionic liquid, the surface structure of corn straw raw material became loose, and obvious holes, pits, and cracks appeared.

4. Discussion

4.1. Effect of Response Surface on Enzymatic Hydrolysis Results

According to the results of cellulase hydrolysis experiments, the yield of reducing sugar was greatly improved compared with the raw materials. The reaction temperature and the mass fraction of the substrate were the decisive factors, followed by the reaction time. When the reaction temperature of ionic liquid was too low, cellulose could not be fully dissolved, and the crystallinity of cellulose decreased little, so the enzymatic hydrolysis rate was low. However, when the reaction temperature of ionic liquid exceeds a certain value, the crystallinity of cellulose will no longer decrease, remaining in a stable state with an optimum condition. It was also found that the yield of reducing sugar via cellulase hydrolysis after activation with ferric chloride solution was higher than that of the other. The reason was that after the corn straw raw material was treated with ferric chloride solution, the adhesive layer structure of the material was destroyed more than that of the other, which was more conducive to the penetration and dissolution of ionic liquids. The crystal structure of cellulose was destroyed significantly, so its enzymatic hydrolysis value was higher. After the corn straw was treated with ferric chloride solution, the structure of the bonding layer was more easily destroyed. The reason is that in the process of metal ion pretreatment, the structure of corn straw is destroyed by hemicellulose under the action of metal ions, and the surface and internal structure became loose, which is conducive to the infiltration of ionic liquids into the interior of straw molecules, disrupting the intermolecular and intramolecular bond energy, so that cellulose is more exposed. Due to the role of metal ions, similar to that of Lewis acid, it can play the role of acid hydrolysis, so that hemicellulose is easy to hydrolyze, resulting in a decrease in its content. It can be seen from the results that the content of cellulose activated by ferric chloride treated with ionic liquid increased the most. Therefore, the surface and internal structure of corn straw treated with ionic liquid were looser, and the content of hemicellulose decreased, resulting in the structure of the bonding layer being more easily destroyed.

4.2. Effect of Ionic Liquids on Components

Compared with the raw material, the content of hemicellulose and soluble substances decreased, and the content of cellulose increased. The main reason was that under the activation of metal-ion solution, the adhesive layer composed of hemicellulose and lignin became loose and the structure was seriously damaged, so that hemicellulose was easily decomposed. At the same time, due to the destruction of corn straw cell structure, many of the intracellular substances were released in greater quantities [36]. The results showed that the raw materials activated by metal ions and then treated with ionic liquids could increase the content of cellulose, so that more hemicellulose could be decomposed. The decomposed hemicellulose could also be used in medicine, chemical industry, and other fields [37], which was conducive to the comprehensive utilization of corn straw. It can also be seen from the results that the content of cellulose activated by metal ions in ionic liquid treatment increased to varying degrees, among which the content of cellulose increased the most after the activation of ferric chloride solution, and the yield of reducing sugar by enzymatic hydrolysis was much higher than that of raw materials. The yield of reducing sugar by enzymatic hydrolysis after activation of aluminum chloride solution was lower than that of ferric chloride solution due to the difference in cellulose content, but still higher than that of untreated raw materials.

4.3. Effect of Ionic Liquids on Crystallinity

The crystalline structure of cellulose in corn straw is one of the key factors affecting the yield of cellulase. After the activation of metal ion solution and the treatment of ionic liquid, the crystal structure of cellulose was destroyed to varying degrees, and the crystallinity decreased. The crystal structure of cellulose was the most destroyed after the activation of ferric chloride solution and the treatment of ionic liquid, so the crystallinity of cellulose was the lowest, which was also consistent with the previous enzymatic hydrolysis results and chemical composition results.

4.4. Effect of Ionic Liquid on Morphology

From the results of scanning electron microscopy, it can be seen that the surface structure of the material became rough and uneven after the activation of the metal ion solution and the treatment of the ionic liquid. After the treatment of metal-ion solution, the surface structure of corn straw became fluffy, and a large number of holes appeared. After the treatment of ionic liquid, the hydrogen bonds between and within the cellulose molecules were destroyed, making it looser, which was conducive to the contact of cellulase. The results of SEM observation further verified the results of XRD analysis, indicating that the dense structure and crystallinity of corn straw raw materials were the key factors restricting the increase in the reduced sugar yield of corn straw.

5. Conclusions

(1)

Response surface methodology was used to optimize the enzymatic hydrolysis conditions of corn straw after metal ion activation by ionic liquid treatment. The optimization results are as follows: ionic liquid treatment of ferric chloride solution activation, substrate mass fraction of 4.2%, reaction temperature of 85 °C, reaction time of 185 min. The aluminum chloride solution was activated by ionic liquid treatment. The substrate mass fraction was 3.6%, the reaction temperature was 105 °C, and the reaction time was 230 min. Through the experiment of enzymatic hydrolysis, it was found that the enzymatic hydrolysis value of the material activated by ionic liquid treatment of ferric chloride solution was the largest, reaching 706.52 mg/g.

(2)

The results of component analysis showed that the cellulose content of corn straw was significantly increased after the activation of metal ions by ionic liquid treatment, and the yield of reducing sugar after cellulase hydrolysis was increased.

(3)

FTIR analysis showed that ionic liquids could dissolve more cellulose, so the hydrogen bonds of cellulose weakened and the spatial structure changed. The XRD results showed that the relative crystallinity of corn straw material was reduced to a certain extent compared with the raw material after the activation of metal ion solution by ionic liquid. The SEM results showed that the treated material became fluffy, the surface was uneven, and more holes appeared, which was more conducive to the enzymatic hydrolysis of cellulase.