Enhanced Enzymatic Hydrolysis of Corncob by Synthesized Enzyme-Mimetic Magnetic Solid Acid Pretreatment in an Aqueous Phase.

A novel magnetic carbon-based solid acid catalyst (C350-Cl) was synthesized through a simple impregnation-carbonization process and used for the pretreatment of corncob in an aqueous medium. Under the optimized pretreatment reaction conditions, the yield of pentose reached 91.6% with a hemicellulose removal rate of 91.7%, and the subsequent enzymatic digestibility of the pretreated corncob residue reached 90.0% at 48 h. C350-Cl is a magnetic enzyme-mimetic solid acid catalyst, and its catalytic behavior is similar to those of enzymes. In addition, the catalyst is also an excellent carrier for Fe and Cl in that the Fe3+ and Cl-can be released slowly in the pretreatment to assist the hydrolysis of lignocellulose. Compared with the traditional method with other catalysts, this hydrolysis process is suitable for the effective and sustainable saccharification of lignocellulose for producing fermentable sugar.

Introduction

The overuse of conventional fossil fuel has resulted in problems such as climate change, air pollution, and energy shortages; therefore, exploration of alternative renewable resources and the development of technologies for using such resources have drawn increasing attention worldwide.[1] Lignocellulose biomass is a renewable and clean resource that can be used as an ideal substitute for fossil fuel resources to synthesize chemicals and fuels.[2−4]

The depolymerization of lignocellulose into saccharide and further fermentation are important pathways for lignocellulose utilization. The coupling of pretreatment with enzymatic hydrolysis is an effective way to degrade the hemicellulose and cellulose in lignocellulose. However, lignocellulose is complicated and consists of hemicellulose, cellulose, and lignin,[5] and how to reduce chemically stable lignocellulose through a sustainable and green process is a research hotspot.[6]

To date, many methods have been developed for the pretreatment of lignocellulose. Among them, pretreatment with homogeneous acid (including HCl, H2SO4, and H3PO4) as catalyst is the most effective saccharification method and has been widely used in industrial processes.[7−9] Lloyd et al. reported that a xylose yield of up to 32.4% was obtained when sulfuric acid (0.98%, w/w) was used as the catalyst to pretreat corn stover at 140 °C for 40 min.[10] The subsequent enzymatic digestibility of the pretreated residue reached 51.8% at an enzyme loading of 60 FPU/g (here, FPU stands for filter paper units; one FPU is defined as the amount of cellulase required for producing 1 μmol reducing sugars per minute).[10] However, the usage of liquid acids as catalysts results in a series of problems, such as equipment corrosion and environmental pollution. To avoid these problems, mild pretreatment methods involving a metal salt (FeCl3) that can acidify the reaction system were tested and achieved high hemicellulose removal rates with high enzymatic digestibility.[11,12] Almost all the hemicellulose in corn stover was removed after pretreatment with 0.1 mol/L FeCl3 solution at 160 °C for 20 min, and a high enzymatic hydrolysis yield of 91.6% was obtained with 60 FPU/g of cellulose loaded.[13] These reports indicated that the Cl– released from FeCl3 combines with H+ to generate HCl under aqueous hydrothermal conditions. Although this method can efficiently pretreat lignocellulose, removing the Cl– from the waste is difficult, which limits the utilization of this process. LHW (liquid hot water) pretreatment,[14−16] without the addition of any chemicals, has been considered a green method for promoting the enzymatic digestibility of lignocellulose. With an enzyme loading of 20 FPU/g substrate, up to 90% of the glucose from the sugar bagasse could be recovered by enzymatic hydrolysis after LHW pretreatment at 180 °C for 30 min.[15] However, the pretreatment product is composed of polysaccharides (e.g., xylo-, arabino-, and galacto-oligosaccharides) with a random degree of polymerization, which makes subsequent utilization difficult.

Recently, the pretreatment of lignocellulose with a solid acid (including molecular sieves,[17] metal oxides,[18] and carbon-based solid acids[19−21]) has attracted attention because solid acids have the advantage of being separable and recyclable after the reaction.[22] Among the catalysts listed above, carbon-based solid acids are economical, their structures can be controlled, and they are stable under hydrothermal conditions, making them well applicable for biomass hydrolysis.

Enzymatic hydrolysis is an effective method for converting cellulosic substrates into monomeric fermentable sugars. Cellulase is a protein consisting of exo-β-glucanase, endo-β-glucanase, and β-glucosidase (formed by basic protein groups −NH2, −COOH, −OH, and −SH), which can effectively hydrolyze the glycosidic linkages in cellulosic substrates.[23,24] Thus, enzyme-mimetic carbon-based solid acids are effective catalysts for hemicellulose degradation, and they consist of a binding domain and a catalytic domain, simulating the synergistic catalytic behaviors of enzymes for lignocellulose hydrolysis.[25] Shuai et al.[26] synthesized a sulfonated chloromethyl polystyrene resin (CP–SO3H) that contains both a cellulase binding domain (−CH2Cl) and a catalytic domain (−SO3H). The −CH2Cl moieties form strong hydrogen bonds with −OH moieties on the cellulose chain, and the acidic groups (−SO3H) cleave the β-1,4-glycosidic linkages (−O−) between the cellulose units. The activation energy for hydrolyzing crystalline cellulose catalyzed by a cellulase-mimetic solid acid (83 kJ/mol)[26] is lower than that when using sulfuric (170 kJ/mol) or other solid acid catalysts (110 kJ/mol),[19] indicating that the cellulase-mimetic sold acid is more effective for cellulose depolymerization under mild reaction conditions. For carbon-based solid acid catalysts, carboxyl groups (−COOH) are typically formed during carbonization of lignocellulose as a precursor.[27] The −COOH groups in the catalyst are acidic catalytic functional groups.[28] Therefore, if a solid acid catalyst containing Cl– and −COOH moieties can be synthesized, it could suitably enhance the mass transfer process between the catalyst and lignocellulose, which can promote the reaction rate.

In this study, the preparation method of a novel magnetic enzyme-mimetic carbon-based solid acid catalyst from microcrystalline cellulose and its application in the pretreatment (hydrolysis) of corncob under hydrothermal conditions were reported. This catalyst not only showed high catalytic activity in the hydrolytic pretreatment of corncob with a significantly enhanced enzymatic digestibility but could also be easily separated from the mixture by a magnet. Furthermore, the catalyst could retard the release of chloride ions during the pretreatment reaction to enhance the hydrolysis of lignocellulose and be reused multiple times with a slight decrease in catalytic activity for pentose yield. This two-step hydrolysis, pretreatment with the solid catalyst combined with enzymatic hydrolysis, provides a sustainable and effective method for the saccharification of lignocellulose into reducing sugars.

Results and Discussion

Characterization of C350-Cl and C350

The surface morphologies of C350-Cl and its precursor (oven-dried microcrystalline cellulose immersed in FeCl3) are shown in Figure . As shown in Figure a, some particles were distributed on the smooth surface of the precursor, and the particles were mainly ferric hydroxide (FeO(OH)), which formed during hydrothermal coprecipitation and drying.[32] By comparing Figure a and Figure b, there are obvious changes due to the carbonization process; these mainly include the surface of the microcrystalline cellulose becoming rough and porous due to the dehydration reaction, and the particles became micromorphologically clustered due to FeO(OH) being converted into Fe3O4 during the carbonization process.[33]

Figure 1

SEM of C350-Cl and its precursor: (a) image of the C350-Cl precursor and (b) image of C350-Cl.

The elemental compositions of C350-Cl and its precursor, shown in Table , are CH0.7O0.32Cl0.09Fe0.03 and CH1.72O0.59Cl0.11Fe0.02, respectively. Comparison of the elemental data shows that Cl and Fe have been successfully bound to the catalyst. In addition, the decreases in the H and O contents verified the occurrence of the dehydration reaction.

Table 1

Elemental Compositions of C350-Cl and Its Precursor

element	C (%)	H (%)	O (%)	Cl (%)	Fe (%)
precursor	40.95	5.86	32.15	12.27	4.23
C350-Cl	59.36	3.47	25.01	15.24	6.43

To illustrate the details, XRD, FT-IR, and XPS were used to analyze prepared C350-Cl and C350. The XRD patterns of C350 and C350-Cl exhibited broad but weak diffraction peaks at 2θ from 15 to 30°, and this peak can be attributed to amorphous carbon composed of aromatic carbon sheets in a considerably random orientation,[32] as shown in Figure a. At the same time, the characteristic peak of Fe3O4 is shown in Figure a, and the diffraction peaks at 2θ = 30.1, 35.4, 43.1, 56.9, and 62.5° were attributed to the (2 2 0), (3 1 1), (4 0 0), (5 1 1), and (4 4 0) lattice planes of Fe3O4, respectively,[33] which also confirms the formation of Fe3O4 as a product indicated by SEM analysis.

Figure 2

(a) Structure and functional group analysis of C350-Cl and C350XRD patterns; (b) FT-IR spectra.

The FT-IR spectrum of the catalysts is shown in Figure b. The absorption peak at 3386 cm–1 is attributed to the O–H stretching vibration of −COOH and phenolic −OH groups.[27] The adsorption peak at 1702 cm–1 is attributed to C=O in −COOH,[19] and the peak at 1610 cm–1 is assigned to the C=C stretch in the carbon skeleton. The adsorption peak at 1431 cm–1 was assigned to C–H bonds in the saturated hydrocarbon, and the peak at 1260 cm–1 was attributed to C–O stretching vibrations of −COOH and phenol moieties.[34] The FT-IR observations reveal that both C350-Cl and C350 contain −COOH and phenolic −OH groups.

The XPS spectra of C350-Cl and C350 are shown in Figure . The double peaks at a binding energy of 706–710 eV are from Fe 2p 3/2 and Fe 2p 1/2, and the peaks at 530–534, 282–290, and 196–202 eV are assigned to O, C, and Cl, respectively.[35] From the survey results of C350-Cl and C350 shown in Figure a, the C350-Cl catalyst possesses four main elements, Fe, O, C, and Cl, and C350 mainly contains O and C. This result is consistent with the results shown by XRF and FT-IR. The deconvolutions of the C 1s and Cl 2p spectra are shown in Figure b,c. The C 1s spectrum of C350-Cl can be deconvoluted into three single peaks (Figure b). The binding energy of 284.5 eV for C 1s confirms the presence of C–C bonds (carbon skeleton), the binding energy at 286 eV is characteristic of C–O (in the form of −COOH or C–OH), and that at 288 eV is indicative of C=O bonds (mainly in the form of −COOH).[36] The Cl 2p signals can be deconvoluted into two double peaks, which come from the overlap of chloride signals at 200 and 197 eV and the C–Cl bonds of C350-Cl at 201.5 and 199.5 eV.[37]

Figure 3

(a) X-ray photoelectron spectra of C350-Cl and C350; (b) C 1s spectrum of C350-Cl; (c) Cl 2p spectrum of C350-Cl.

Based on these characterization results, the properties and differences of C350-Cl and C350 catalysts could be distinguished, and the carbon structures were proposed as shown in Figure . C350-Cl and C350 are amorphous carbon structures composed of uniform nanographene sheets bearing −COOH and −OH groups in a considerably random pattern. In addition, C350-Cl also consists of C–Cl groups and Fe3O4 magnetic particles. These groups make it distinct from conventional solid acids with single functional groups, and the roles of these groups in the catalytic saccharification of hemicellulose will be discussed later.

Figure 4

Schematic of C350 and C350-Cl. The schematic does not represent the real amounts and distribution of functional groups.

Pretreatment of Corncob Catalyzed by C350-Cl

To obtain a higher xylose yield, the hydrolysis temperature, hydrolysis time, ratio of corncob to catalyst, and water content were optimized. As shown in Figure a, the highest xylose and xylan yields of 77.4 and 14.2% were obtained from the reaction at 150 °C for 2 h. The reaction temperature was increased from 110 to 190 °C, and the highest yields of pentose and xylose were obtained at 150 °C. This result can be attributed to the increase in the reaction temperature promoting the saccharification of hemicellulose and xylan while also promoting further degradation of the formed monosaccharide.[38] When the temperature was further increased, no xylan was detected, and the yield of xylose decreased. Therefore, a reaction temperature of approximately 150 °C was best for these consecutive reactions under these reaction conditions. At the same time, the increase in the furfural concentration obtained above 150 °C also confirmed the occurrence of side reactions, and the subsequent decrease was due to another aldol reaction.[39,40]

Figure 5

Effects of the (a) hydrolysis temperature, (b) hydrolysis time, (c) ratio of catalyst to corncob, and (d) water content on the sugar yield in corncob pretreatment catalyzed by C350-Cl. In (a)–(d), only one hydrolysis parameter was varied according to 150 °C, 2 h, 2 g of corncob, 2 g of C350-Cl, and 50 mL of deionized water as the other parameters.

Figure b shows the effect of the pretreatment reaction time on the reducing sugar yield. The reactions were performed from 0.5 to 2.5 h at 150 °C with 2 g of corncob, 2 g of catalyst, and 50 mL of deionized water. With the reaction time increasing from 0.5 to 2.5 h, the yield of xylose obviously increased from 61.14 to 77.39%, the yield of xylan decreased from 27.76 to 14.47%, and the yield of total reducing sugars decreased at 2 h. This result implies that along with corncob hydrolysis into xylose, xylose underwent substantial degradation at longer reaction times due to its concentration. Thus, 2 h was selected as the optimum reaction time for further research.

Figure c shows the effect of catalyst loading on the yield of reducing sugar. Catalyst loadings from 1 to 2.5 g were investigated at 150 °C for 2 h with 2 g of corncob and 50 mL of deionized water. When the catalyst loading increased from 1 to 1.5 g, the yields of total reducing sugars were similar, but the yield of xylose was significantly increased. This result was attributed to the catalyst having more catalytic activity in the degradation of the saccharide of hemicellulose or the oligomer rather than the degradation of xylose. When the catalyst loading was increased from 1.5 to 2 g, the total yield of reducing sugars obviously increased from 77.2 to 91.6%, which was due to the higher catalyst loading increasing the number of acid sites in the reaction system. However, a further increase of the catalyst loading resulted in a decrease in both the yields of total reducing sugars and xylose for two main reasons; first, more acid sites are present in the reaction system, and they can also catalyze the degradation of xylose, and second, the increase in the amount of solid in the system leads to a decrease in mass transfer efficiency, which can decrease the generating rate of hemicellulose and insoluble oligomer saccharification but also enhance the degrading rate of xylan and xylose.[41] Thus, a catalyst loading of 2 g was selected as the optimum amount for further studies.

Figure d shows the influence of water amount on the yield of total reducing sugars. Amounts of water from 30 to 90 mL were investigated at 150 °C for 2 h with 2 g of corncob and 2 g of catalyst. As the amount of water was increased from 30 to 90 mL, the yield of total reducing sugars increased from 75.7 to 91.6% (at 50 mL) and then decreased significantly to 43.9% (at 90 mL). This result was due to the lower liquid-to-solid ratio hindering mass transfer in the system, and the higher ones result in a lower concentration of acid sites, which essentially decreases the amount of catalyst. Therefore, 50 mL of water was selected as the optimal amount.

Based on the above investigation, the optimized reaction conditions for hemicellulose saccharification and total reducing sugar yield were identified as 2 g of corncob, 2 g of catalyst, and 50 mL of deionized water at 150 °C for 2 h. In addition, glucose and glucan, the products of cellulose saccharification, could be obtained in the pretreatment process of corncob. Since glucose and glucan are not the main products of the pretreatment, their reaction behaviors are not discussed here.

The catalytic activity of C350-Cl for the pretreatment of corncob under the optimum reaction conditions was compared with those of other solid acid catalysts (Table ). These catalysts can be divided into three types: the first is Amberlyst-15, a synthetic resin bearing only −SO3H groups; the second is the catalyst bearing −COOH, −OH, and −SO3H groups; and the third is the catalyst synthesized in this work bearing −COOH, −OH, and C–Cl groups. Compared with Fe3O4/C-SO3H,[42] the yield of xylose from C350-Cl is much higher than that with Fe3O4/C-SO3H under mild reaction conditions. Compared with C-SO3H[41] and Gp-SO3H-H2O2,[43] a similar xylose yield was achieved in a shorter reaction time. In addition, the catalytic activity in this work is better than that of Amberlyst-15,[43] and a higher xylose yield can be obtained in a shorter reaction time. These results imply that the enzyme-mimetic synergistic effect of the bound functional groups plays an important role in the hydrolysis of the hemicellulose of corncob. The details of the enzyme-mimetic synergistic effect among the −COOH, −OH, and C–Cl groups bound to this catalyst will be discussed later.

Table 2

Catalytic Activity Comparison of C350-Cl and Other Catalysts

catalyst	temperature (°C)	time (h)	xylose yield (%)	glucose yield (%)
C350-Cla (this work)	150	2	77.4	3.6
Fe3O4/C-SO3Hb[42]	160	16	44.3
C-SO3Hc[41]	140	6	78.1	7.4
Amberlyst-15d[43]	140	12	60.2	5.8
Gp-SO3H-H2O2c[43]	140	12	78.4	6.1

Optimal conditions: 2 g of catalyst, 2 g of corncob, and 50 mL of water.

Optimal conditions: 1.0 g of catalyst, 0.5 g of corncob, and 50 mL of water.

Optimal conditions: 0.25 g of catalyst, 0.5 g of corncob, and 25 mL of water.

Optimal conditions: 0.5 g of catalyst, 0.25 g of corncob, and 25 mL of water.

Enzymatic Hydrolysis of the Pretreated Corncob Residue

The component changes of the corncob after the pretreatment process are shown in Table . Through the pretreatment process, the proportions of glucan in the corncob residue increased from 36.9 to 61.3%, while the proportion of xylan decreased from 32.2 to 6.3%. Almost all the hemicellulose and a portion of the lignin in the corncob were removed with removal rates of 91.7 and 26.3%, respectively. In addition, the retention rate of cellulose in pretreated corncob was 70.6%, which meets the aim of the pretreatment process.

Table 3

Changes in the Components of Corncob before and after Optimum Pretreatment

	component (%)			removal rate (%)
corncob	glucan	xylan	lignin	hemicellulose	cellulose	lignin
natural	36.9	32.2	14.3	91.7	29.4	26.3
pretreated	61.3	6.3	24.8

Figure shows the enzymatic hydrolysis of natural and pretreated corncob. The comparison indicates that the enzymatic digestibility of the pretreated corncob residue is much higher than that of natural corncob. With an enzyme loading of 40 FPU/g, the enzymatic digestibility of natural corncob was 59.3% at 48 h and 64.7% at 72 h, whereas that of the pretreated corncob residue was 90.0% at 48 h and 94.8% at 72 h. The total sugar yield from corncob of 86.0% was obtained after pretreatment and enzymatic hydrolysis of 72 h. The enzymatic digestibility of pretreated corncob at 48 and 72 h increased by 30.7 and 30.1%, respectively, which indicates that pretreatment with C350-Cl could effectively enhance the enzymatic digestibility of corncob.

Figure 6

Enzymatic digestibility of natural and pretreated corncob. Pretreatment conditions: 150 °C, 2 h, 2 g of corncob, 2 g of C350-Cl, and 50 mL of deionized water. Enzymatic hydrolysis conditions: 5% substrate concentration (g/mL) at 50 °C.

The enzymatic digestibility of the residues pretreated with C350-Cl is comparable to those following other pretreatment methods (i.e., other solid acids, liquid hot water, and dilute acid and alkali pretreatment), as shown in Table . The enzymatic digestibility values of corncob[44] and sugarcane bagasse[45] were 82.4 and 77.9%, respectively, after LHW pretreatment at 180 °C for 20 min. The enzymatic digestibility of sugarcane bagasse was only 76.6% with the addition of 1.25% HCl during the LHW pretreatment process.[46] Higher enzymatic digestibility (98.5%) was obtained after the addition of pressurized 25% NH3.[45] Sugarcane bagasse pretreated with 0.5 M NaOH solution before LHW pretreatment also showed a higher enzymatic hydrolysis, and an enzymatic digestibility of 97.5% was obtained after 72 h.[47] However, these methods are not only complicated but also not friendly to the environment. In contrast, pretreatment with solid acid C350-Cl provided an enzymatic digestibility of 94.8% after 72 h, which is better than that achieved with LHW pretreatment, and the developed method also has no concomitant device corrosion or environmental pollution. Furthermore, the solid acid can be easily recycled and reused due to its magnetic property.

Table 4

Comparison of Enzymatic Digestibility after Different Pretreatment Methods

raw material	pretreatment	time (h)	enzymatic digestibility (%)
corncob (this work)	C350-Cla	72	94.8
corncob[44]	LHWb	48	82.4
sugarcane bagasse[45]	LHWc	72	77.9
sugarcane bagasse[45]	LHW + NH3d	72	98.5
sugarcanebagasse[46]	LHW + HCle	72	76.6
sugarcane bagasse[47]	LHW + NaOHf	72	97.5

Enzymatic hydrolysis (40 FPU/g) after optimal pretreatment conditions: 2 g of C350-Cl, 2 g of corncob, 50 mL of water, 150 °C, 2 h.

Enzymatic hydrolysis (40 FPU/g) after optimal pretreatment conditions: 12.5% (w/v) solid loading, 180 °C, 20 min.

Enzymatic hydrolysis (40 FPU/g) after optimal pretreatment conditions: LHW: 30 g of sugarcane bagasse, 600 mL of water, 4 MPa, 180 °C, 20 min.

Enzymatic hydrolysis (40 FPU/g) after optimal pretreatment conditions: 30 g of sugarcane bagasse, 600 mL of water, 25% NH3 at 160 °C, 6 MPa, 60 min.

Enzymatic hydrolysis (40 FPU/g) after optimal pretreatment conditions: 35 g of sugarcane bagasse, 12.5% solid loading, 1.25% HCl, 130 °C, 10 min.

Enzymatic hydrolysis (40 FPU/g) after optimal pretreatment conditions: 1 g of sugarcane bagasse, 20 mL of 0.5 M NaOH solvent, 80 °C for 3 h, then 30 g of pretreated sugarcane bagasse (5% w/v in water), 180 °C, 30 min

To explain the enhanced enzymatic digestibility of corncob after pretreatment, XRD and SEM were used to analyze the changes in the natural and pretreated corncob, as shown in Figures S1 and S2. In the pattern of the pretreated corncob residue, the intensity of the signal at 2θ = 18°, which represents the amorphous area, is almost the same as that of corncob. However, the intensity of the diffraction peak at 2θ = 22.5°, which represents the crystalline part, indicates an obvious change due to pretreatment. The crystallinity index (CrI) increased from 41.5 to 55.1%, indicating that the amorphous cellulose was maintained during pretreatment. In addition, this result not only agrees with the component analysis but also illustrates the reason for the enhanced enzymatic digestibility.

The surface morphology of the natural corncob was smooth and dense (Figure S2a), whereas the structure was deconstructed after pretreatment with C350-Cl, and the surface became loose and shattered. The porous structure of the pretreated corncob is also shown in Figure S2b, and this morphology indicates that the pretreatment process destroyed the structure and improved the accessibility of the cellulose to the cellulase.

Recyclability Performance

Easy separation from the reaction system for reuse is an important property of a solid acid catalyst. C350-Cl is a magnetic enzyme-mimetic carbon-based solid acid catalyst that can be separated from the reaction system by a magnet after the pretreatment reaction, as shown in Figure .

Figure 7

Separation of the catalyst from the reaction mixture with a magnet (the figure was taken by a co-author of this manuscript (G.L.)).

After the recovery of C350-Cl, it was reused in the pretreatment of corncob as shown in Figure a. After five cycles of reuse, the total yield of xylose and xylan decreased slightly from 91.6 to 80.2%, and the total yield of pentose was steady after the third cycle of reuse. In contrast, the yield of xylose decreased significantly and then became steady. Analysis of the pretreated hydrolysate indicated that the concentrations of Fe3+ and Cl– were high in the first usage (440.5 and 1077.6 mg/L, respectively, corresponding to 17% of the ferric iron and chloride initially bound to C350-Cl) of the catalytic reaction and became stable after the second cycle (approximately 20 mg/L ferric iron and 200 mg/L chloride (Table S1), corresponding to 0.8% of the ferric iron and 3.3% of the chloride initially bound to C350-Cl). There are two main reasons for the detected Fe3+ and Cl–. First, a portion of the FeCl3 was not converted into Fe3O4 during the carbonization process but became bound to the catalyst as FeCl3 or in other forms. Therefore, the attached FeCl3 was dissolved into the hydrolysate during the first pretreatment process. The second reason was the dissolution of the formed Fe3O4 in the acidic system.

Figure 8

Reusability of C350-Cl. (a) Sugar yield in the catalyst reused cycle for corncob pretreatment. (b) Enzyme digestion rate of natural and pretreated corncob. Pretreatment conditions: 150 °C, 2 h, 2 g of C350-Cl, 2 g of corncob, and 50 mL of deionized water. N.C. represents natural corncob, and P.C. represents pretreated corncob.

The aim of pretreatment of corncob is to deconstruct its structure and enhance the enzymatic digestibility of pretreated residue. The enzymatic hydrolysis of the corncob residue pretreated with the recycle catalyst was carried out, and its enzymatic digestibility is shown in Figure b. Through the comparison of Figure a and Figure b, the enzymatic digestibility of the pretreated residue indicated a similar trend to the yield of sugar. The decrease of catalytic activity of C350-Cl results in the decrease in the yield of xylose during the pretreatment process and also in a decrease of the enzymatic digestibility of the residue. Fortunately, although the enzymatic digestibility of the corncob residue pretreated with the recycled catalyst declined, the enzymatic digestibility was stable and much higher than that of natural corncob.

To illustrate the key catalytic factors in the reaction, some comparative experiments with different additives related to components of the catalyst or the reaction system (according to the tested values of each component on the catalyst or in the hydrolysate) were carried out using the reaction conditions shown in Figure . FeCl3 is capable of disrupting the ether and ester linkages of hemicellulose.[13] To confirm the influence of FeCl3, 0.022 g of FeCl3 in 50 mL of deionized water (the same Fe3+concentration as that in the hydrolysate catalyzed by fresh C350-Cl) was used as a control experiment for FeCl3 (Figure ). FeCl3 provided 16.37% xylose and 81.36% pentose yields, which are both lower than those obtained with C350-Cl (77.39% xylose and 91.56% pentose, Figure ). This means that although Fe3+ and Cl– are released after the first use of C350-Cl, this concentration is not high enough to catalyze the degradation of corncob xylan into xylose.[11] The lowest yield of pentose (55.97%) was obtained from the hydrothermal pretreatment of corncob (as the No cat result). The yields of pentose increased slightly when C350 and Fe3O4 were added as the catalysts, and the catalytic activity of C350 (62.38% yield of pentose) was higher than that of Fe3O4 (60.23% yield of pentose). The catalytic activity of Fe3O4 might be due to the ionized H+ in the hydrothermal system causing partial dissolution of Fe3O4,[48] and Fe3+ can catalyze the hydrolysis of corncob hemicellulose.[49] Two functional groups (−OH and −COOH) are bound to the catalyst based on the characterization of C350. Carboxyl (−COOH) moieties are able to hydrolyze the β-1,4-glycosidic bonds of cellulose,[19] which may be the catalytic pathway of C350 in the hydrolysis of corncob, allowing it to achieve a higher yield of pentose than that using liquid hot water (62.38% yield of pentose with C350 versus 55.97% with no catalyst).

Figure 9

Experiments with different catalysts at 150 °C for 120 min. The reaction conditions are as follows: FeCl3, 0.022 g of FeCl3, 2 g of corncob, 50 mL of water; C350, 2 g of C350, 2 g of corncob, 50 mL of water; No cat, 2 g of corncob with no catalyst (liquid hot water), 50 mL of water; Fe3O4, 0.5 g of Fe3O4, 2 g of corncob, 50 mL of water.

Since the acidity of the solid acid is an important factor that affects corncob hydrolysis,[33] the total populations of acid sites in the first, second, and fifth cycles of C350-Cl were detected and are shown in Table S2. The result shows that the trend in the amount of total acid has no correlation with the trend in the xylose yield. Thus, the acidity of C350-Cl is not the main reason for the higher xylose yield. In conclusion, all the evidence indicates that the unique C–Cl moieties of C350-Cl are the key to generating the xylose product.

Based on the results of these reuse and comparison experiments, a possible mechanism of corncob hydrolysis catalyzed by C350-Cl was proposed, as shown in Figure . The hydrolysis pretreatment of corncob catalyzed by C350-Cl in an aqueous medium is a reaction on the surface of reactants, and the mass transfer between the corncob and catalyst is the main factor controlling the reaction. Cl, O, and H are the main components of the corncob and the catalyst, and positively charged H ions can interact with the negatively charged O and Cl ions. Therefore, hydrogen bonds formed between the −OH and −O– groups in the corncob and C–Cl groups in the catalyst,[26,50] similar to the binding domain of enzymes, enhancing the mass transfer between the corncob and the catalyst. Then, the acidic functional groups (−OH and −COOH) cleave the glycosidic linkages[24] of hemicellulose or xylan to gradually produce xylose, similar to the catalytic domain of enzymes. When Cl is reduced due to the breakage of the C–Cl bonds during the recycling of the catalyst, the hydrogen bonds between the hemicellulose chains and the catalyst become weaker, resulting in a decrease in the proportion of xylose in the obtained pentose. The catalytic behavior of this catalyst is similar to the cellulase-mimetic solid acid CP-SO3H synthesized by Shuai et al.,[26] which also contained both a binding domain and a catalytic domain.

Figure 10

Schematic of the catalytic mechanism between C350-Cl and lignocellulose (it does not represent the real amount and distribution of functional groups).

In conclusion, the existence of C–Cl on the catalyst can enhance the mass transfer during the pretreatment process because of the formed hydrogen bond between lignocellulose and the catalyst, and they have a synergistic effect on the saccharification of lignocellulose. With the release of Fe3+ and Cl–, the selectivity for xylose decreased dramatically, but the yield of pentose became stable after the second time usage. In the future research work, we must focus on how to make the C–Cl bond and Fe3O4 particles stable on loading onto the catalyst, which can maintain the high catalytic activity and selectivity of the catalyst.

Conclusions

A novel magnetic enzyme-mimetic solid acid catalyst (C350-Cl) was synthesized without the traditional sulfonation process. The catalyst showed high catalytic activity for the pretreatment of corncob, with a xylose yield of 77.4% and a xylan yield of 14.2% under the optimum reaction conditions (150 °C, 2 h, 2 g of corncob, 2 g of catalyst, and 50 mL of deionized water), and the catalyst could easily be separated from the residue by an external magnet. Because of the high removal rate of hemicellulose in corncob (up to 91.7%), the accessibility of the cellulose to the enzyme was obviously enhanced, and the enzymatic digestibility of the pretreated corncob residue was much higher than that of natural corncob. The synergistic effect between the C–Cl and −COOH moieties of the catalyst was found. During the pretreatment of corncob with C350-Cl, the C–Cl groups form hydrogen bonds with the hemicellulose in the corncob, and these interactions promote the accessibility between the catalyst and substrate, similar to what occurs in the binding domain of enzymes; the acidic group (−COOH) breaks the glycosidic linkages in hemicellulose and xylan to gradually degrade them into xylose. This work not only suggests the potential of a lignocellulose biorefinery process but also indicates a new platform for the design of carbon-based solid acid catalysts for the pretreatment of lignocellulose.

Experimental Section

Materials

Corncob (40–60 mesh) was obtained from a farm in Shandong province, China, and dried at 80 °C to a constant weight, and the material contained 36.9% cellulose, 32.2% lignocellulose, and 14.3% lignin according to the NREL analysis method.[29] Iron (III) chloride (CP) was purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Microcrystalline cellulose (GR) and anhydrous ferric chloride (AR) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Cellulase (190 FPU/g) was purchased from Imperial Jade Bio-Technology Co., Ltd. (Ningxia, China).

Catalyst Preparation

Microcrystalline cellulose (10 g) was added into FeCl3 solution (1000 mL) with a concentration of 10 mmol/L and stirred (500 rpm) at room temperature for 5 h. Then, the mixture was heated to evaporate the water and dried at 105 °C to a constant weight. The obtained black solid was carbonized in a tube furnace with nitrogen (50 mL/min) protection at 350 °C (heating rate: 3 °C/min) and kept at the temperature for 1 h. The carbonized black solid was then ground into a powder (<100 mesh) and washed with ethanol and hot water (> 80 °C) until no Fe3+ and Cl– were detected upon treatment with KSCN and AgNO3 solutions. Finally, the catalyst was dried to a constant weight in an oven at 105 °C and is denoted C350-Cl. For comparison, a carbon catalyst (denoted C350) was synthesized following the same procedure without the addition of FeCl3.

Characterization of the Catalyst, Corncob, and Pretreated Residues

The functional groups on the catalysts were detected by Fourier transform infrared (FT-IR) spectroscopy (TENSOR27 from Bruker, Karlsruhe, Germany) from 400 to 4000 cm–1 using the standard KBr pellet method. The micromorphologies of the catalysts were characterized by cold field scanning electron microscopy (Cryo-SEM, S-4800 from Hitachi, Tokyo, Japan). The crystal structures of C350-Cl and C350 were examined by X-ray diffraction (XRD) using an X’Pert Pro MPD system (PANalyticAlmele, the Netherlands). The operating conditions were 40 kV and 40 mA using a Cu Kα radiation source, and the scanning angle (2θ) was from 5 to 80° with a scanning rate of 4°/min. The elemental composition of the catalyst was analyzed by X-ray photoelectron spectroscopy (XPS) with an ESCALAB 250Xi system (Thermo, Waltham, USA). The contents of C, O, and H in the catalysts were detected using a Vario EL Cube elemental analysis system (Elementar, Frankfurt, Germany), and the contents of Fe and Cl in the catalysts were detected by X-ray fluorescence spectrometry (XRF) with an AXIOSmAX-PETRO system (PANalytical B.V. manufacturer, the Netherlands).

The acid amount of catalyst was detected by acid–base titration.[30] The catalyst (0.2 g) and 20 mL of 0.05 mol/L NaOH were mixed in a 50 mL centrifuge tube and treated with an ultrasonic apparatus for 1 h. Then, 15 mL of the supernatant of the centrifuged mixture was titrated with 0.05 mol/L HCl using methyl orange solution as an indicator.

XRD and SEM analyses of natural and pretreated corncob were also carried out using the same instruments under the same conditions mentioned above.

Two-Step Corncob Hydrolysis

The corncob pretreatment (hydrolysis) was conducted in a 100 mL autoclave with a stirring apparatus (CJF-0.1, Dalian Tongda Reactor Factory, China). The catalyst (1–2.5 g), corncob (2 g), and deionized water (30–90 mL) were added into the reactor and mixed at a constant stirring rate of 500 rpm. The reactor temperature was detected using a thermocouple and maintained from 110 to 190 °C. The reaction time was from 30 to 150 min. After the reaction, the catalyst was separated from the reaction medium with an external magnet for reuse, and the hydrolysate was collected for further analysis. The pretreated residue was separated from the reactant, washed, and dried at 50 °C to a constant weight for enzymatic hydrolysis.

Enzymatic hydrolysis was as follows: 1 g of pretreated corncob residue (under the optimum reaction conditions) was hydrolyzed at 50 °C for 12–72 h in a 10 mL centrifuge tube with a shaking speed of 150 rpm. The concentration of substrate was 5% (w/v) relative to cellulose, and the cellulase loading was 40 FPU/g of dried residue. The hydrolysate of the enzymatic hydrolysis was collected for analysis.

Analytical Procedures and Calculation

The reducing sugars and byproducts (furfural, 5-hydroxymethyl furfural, acetic acid, formic acid, and d-glucuronic acid) in the hydrolysate were detected by high-performance liquid chromatography (HPLC, Waters 2695, Milford, USA) with a Shodex sugar SH-1011 chromatographic column at a column temperature of 50 °C. The mobile phase was 5 mmol/L H2SO4, and the flow rate was 0.5 mL/min. The sugar oligomers were treated and measured according to the method in a previous report.[15]

The concentrations of Fe3+ and Cl– in the hydrolysate were determined by inductively coupled plasma emission spectrometry (OPTIMA 8000, Perkin Elmer, USA) and three-channel ion chromatography (Model 883, Metrohm, Switzerland), respectively.

The yields of monomeric and oligomeric sugars from the pretreatment process were calculated using eqs and 2:where Nmono is the mole of the corresponding sugar in the pretreated hydrolysate, Noligo and Ntotal are the moles of the corresponding oligomer and total reducing sugars in the pretreated hydrolysate, respectively, and M is the mole of the corresponding sugar in the natural corncob.

The enzymatic digestibility of the natural and pretreated corncob is defined aswhere n is the mole number of glucose in the enzymatic hydrolysate and m is the mole number of glucose in the natural or pretreated corncob.

The total yield of sugar from the pretreatment of the corncob and subsequent enzymatic hydrolysis of the pretreated corncob residue is defined by eq .where a and b are the mass qualities of xylose and glucose obtained in the pretreatment of corncob, respectively, c and d are the mass qualities of xylose and glucose obtained in the enzymatic hydrolysis of pretreated corncob residue, respectively, and e and f are the mass qualities of hemicellulose and cellulose in corncob.

The crystallinity indices of natural and pretreated corncob (CrI)[31] are defined by eq .where I2θ = 22° is the intensity of the (002) diffraction plane at 2θ = 22° in the XRD pattern and I2θ = 18° is the intensity of the baseline at 2θ = 18° in the XRD pattern.

The removal rates of hemicellulose and lignin in corncob are defined by eqs and 7, respectively.where j is the mass quality of lignin of corncob and g and h are the mass qualities of cellulose and lignin in pretreatment corncob, respectively.