Huo, D., Wang, D., Yang, Q., Si, C., Liu, Q., Li, B., and Zhang, F. (2018). "Enzymatic saccharification of eucalyptus chips with a pretreatment process using NH4Cl," BioRes. 13(3), 6332-6340.
NH4Cl was used to optimize the pretreatment conditions for biomass pretreatment to improve enzymatic saccharification and hemicellulose degradation of eucalyptus chips. After pretreatment, the solid substrate (SS) and pretreatment liquor (PL) were characterized, and the SS was enzymatically hydrolyzed to detect the conversion yield of cellulose (CYC). For the pretreatment using NH4Cl, the removal rate of hemicellulose reached up to 100% in some cases, but a great proportion of cellulose remained in the SS. The optimized conditions for pretreatment using NH4Cl were 0.3 M NH4Cl at 200 °C for 25 min. A comprehensive evaluation found that the most suitable severity parameter for pretreatment and enzymatic saccharification was 4.5, although a higher severity parameter could increase the CYC. XRD and FTIR analysis showed that the pretreatment had little influence on the cellulose crystalline region, and the lignin was well-retained in the pretreatment process.
The chloride salt NH4Cl dissolves easily in water to form a weakly acidic solution, and the acidity can be increased by heating. Kang et al. (2013a) pretreated Miscanthus straw using NH4Cl to investigate xylan hydrolysis. The results show that the xylan in the raw material can be completely hydrolyzed when 2% NH4Cl is used at 185 °C for 15 min (Kang et al. 2013a,b), which demonstrates that NH4Cl has strong degradation effects on hemicellulose and can be used as a pretreatment for biomass conversion. Moreover, the PL, being rich in carbohydrates and NH4Cl, is an excellent liquid fertilizer and has little influence on the environment.
In this study, milled eucalyptus chips were pretreated using NH4Cl to explore the degradation of cellulose and hemicellulose, and the obtained SS was hydrolyzed using enzymes to calculate the CYC.
Eucalyptus chips provided by Jingui Paper Co. Ltd. (Guangxi, China) were milled and screened to obtain a fraction of 40 mesh to 60 mesh. Cellulase (Celluclast 1.5 L, 50.97 FPU/mL) and β-glucosidase (Novozyme 188, 1290.69 CBU/mL) were obtained from Novozymes (Beijing, China). All other reagents used were of analytical grade and purchased from Sinopharm Chemical Reagent Co. (Shanghai, China).
The pretreatment process was performed in custom-built tubular 316-L stainless steel reactors 45 mL in capacity. The eucalyptus powder was placed in the reaction kettle with a solid-to-liquid ratio (NH4Cl solution) of 1:6. NH4Cl concentration, pretreatment holding time, and pretreatment temperature were controlled in the ranges of 0.05 M to 0.5 M, 5 min to 60 min, and 140 °C to 220 °C, respectively. After pretreatment, the reactor was instantly placed in fluid cool water to terminate the reaction. The solid residue from the reactor was filtered by vacuum filtration to obtain the PL and the SS.
The components of the raw material and the SS were analyzed according to the NREL LAP method (Sluiter et al. 2008). The components of the raw material were as follows: 47.86% glucan, 15.50% xylan, 1.03% arabinan, and 29.15% lignin. The SS yield was designated as the mass ratio of the SS to that of the raw material.
The SS was diluted with a buffer solution (0.1 M sodium acetate and 0.1 M acetic acid, pH 4.8) to keep the concentration at 2.5% (all based on the oven dried SS, o.d.). Then, 30 FPU/g of cellulase, 37.5 CBU/g of β-glucosidase, and three drops of ethyl acetate were added to the diluted substrate. The hydrolysis process was conducted in a cultivation shaker for 48 h at 50 °C, with a rotational speed of 150 rpm. After hydrolysis, the enzymatic slurry was heated for 5 min at 90 °C in a water bath to deactivate the enzymes.
where I002 is the maximum intensity of the (002) lattice diffraction and Iam is the intensity diffraction at 2θ = 18 ° (Segal et al. 1959).
As shown in Table 1, when the pretreatment time was extended, the SS yield clearly declined, and its downtrend changed slowly when the pretreatment time was greater than 30 min, which indicates that most of the degradation reaction was completed in a short time. For the PL, the maximum contents of the xylose and glucose (12.38 mg/mL and 5.65 mg/mL, respectively) emerged when pretreated for 30 min. However, when the pretreatment time was prolonged further, their contents decreased remarkably. Saeman (1945) found that the degradation of hemicellulose is accomplished according to a specific pathway: hemicellulose → oligosaccharides → monosaccharides → degradation byproducts. The sustained increase in the furfural and 5-HMF contents confirmed that some xylose and glucose was dehydrated in the pretreatment process. Especially for glucose, the 5-HMF content was as high as 15.29 mg/mL when pretreated for 60 min. This demonstrated that prolonging the pretreatment time was not conducive for retaining the cellulose in the SS. Correspondingly, the converted glucose mass also decreased. The production of formic acid and acetic acid were mostly related to the hemicellulose. Their contents always increased during the pretreatment, which also explained the increasing hemicellulose degradation in the pretreatment. Based on the above analysis, the optimized pretreatment time was approximately 25 min, where the CYC reached 92.6%. In the meantime, the cellulose loss was at a relatively low level.
As shown in Figs. 1 and 2, when the pretreatment temperature was increased from 140 to 220 °C, the SS yield decreased by 22.4%. Most of the degradation reactions were accomplished at a relatively lower pretreatment temperature, as the SS yield changed little when the pretreatment temperature was above 210 °C. Figure 1 shows that the effects of the pretreatment temperature on hemicellulose degradation were more severe than those on cellulose. As the pretreatment temperature increased towards 220 °C, nearly all of the hemicellulose was destroyed. At that temperature, the cellulose loss was as high as 20.7%. An obvious inflection point emerged where the cellulose loss accelerated when the pretreatment temperature was greater than 190 °C, which indicates that maintaining a lower temperature is beneficial for reserving the cellulose.
Figure 2 shows that the maximum contents of xylose and glucose in the PL occurred at 220 °C. Correspondingly, the CYC was as high as 99.5%. Higher pretreatment temperatures not only resulted in a great amount of cellulose degradation, but also destroyed the crystal structure of cellulose; thus, the CYC was at a very high level. Unlike the glucose and xylose in the PL, when the pretreatment temperature was greater than 200 °C, the increasing trend of the CYC slowed, as the CYC nearly reached the maximum value and was difficult to increase. This demonstrated that higher pretreatment temperature was not beneficial to the CYC, as it not only caused massive hemicellulose degradation, but also resulted in rapid cellulose degradation. Hence, a pretreatment temperature of approximately 200 °C was suitable, and the CYC at this temperature was approximately 90%.
The influence of NH4Cl concentration is shown in Figs. 3 and 4. The NH4Cl concentration had smaller impacts on SS yield and cellulose loss than it did on hemicellulose loss. When the NH4Cl concentration of the pretreatment system increased from 0.1 M to 0.5 M, the SS yield, cellulose loss, and hemicellulose loss increased by 2.5%, 12.3%, and 59.0%, respectively. More than 86% of the hemicellulose in the raw material was degraded when the NH4Cl concentration approached 0.5 M, which indicated that NH4Cl concentration remarkably affected hemicellulose degradation.
As shown in Fig. 4, similar to the trends for pretreatment time and pretreatment temperature, the xylose content in the PL was greater than that of the glucose. When NH4Cl concentration was greater than 0.4 M, the xylose contents changed slightly, while the cellulose loss continuously increased with increasing NH4Cl concentration, which indicates that higher dosages of NH4Cl only caused more cellulose degradation. For the CYC, a noticeable inflection point emerged when 0.3 M NH4Cl was used, which indicates that further increasing the NH4Cl concentration had little influence on the CYC. Thus, the optimal NH4Cl concentration for the pretreatment and enzymatic hydrolysis was approximately 0.3 M, at which point the CYC reached 85.5%.
The pH values of the pretreatment system were adjusted with HCl and NaOH to determine the effect of pH on the effect of pretreatment of ammonium chloride pretreatment system. The results are shown in Table 2. The generating rates of monosaccharides are accelerated by a low pH, so that amounts of furfural and 5-HMF are generated (Marzialetti et al. 2008). From pH 3 to pH 5, the SS yield, hemicellulose loss, and glucose and xylose contents in the PL changed slightly, which demonstrates that the pretreatment process was mostly affected by the NH4Cl itself, not the pH value. During this process, large amount of ammonium ion in the solution could react with the lignocellulosic materials by the ammonolysis of ester crosslinks of the xylan units and by cleaving the bonds linking between hemicellulose and lignin in LCC at high temperature (Wang et al. 1967). When the pH was less than 3, the major reaction was the hemicellulose hydrolyzed by hydronium ion when more hydrogen ions were present in the solution (similar to the dilute acid pretreatment) in addition to the chloride salts themselves, and more cellulose was degraded and dissolved into the PL. However, a notable influence on the CYC was caused by the pH values, which indicates that both the hydrogen ions and NH4+ had an important role in enzymatic scarification. Overall, it was suitable for the pretreatment when the pH value was adjusted to 4, and the CYC was increased to 92.0%. More importantly, these pH values could be reached using a small number of acids.
The raw material and the SS (pretreated using 0.3 M NH4Cl at 170 °C for 20 min) were detected by FTIR and XRD to explore their microscopic variation. The results are shown in Figs. 6 and 7. The chemical composition for the raw material and the pretreatment SS are shown in Table 3. After pretreated by NH4Cl, most of the hemicellulose was hydrolyzed into the hydrolysate. With the degradation of the hemicellulose, the cellulose obviously increased from 40.39% to 63.29%. The relative content of lignin was increased slightly because of the degradation of hemicellulose and the solution of water extractions.
In Fig. 5, the crystallinities of the raw material and the SS were 51.5% and 63.3%, respectively. In connection with the removal of the amorphous substance of the hemicellulose degradation from the raw material, the SS mostly contained cellulose and lignin, and its crystallinity was obviously higher than that of the raw material. The crystal structure of cellulose was difficult to destroy in the NH4Cl pretreatment process, and only a proportion of amorphous cellulose was degraded. However, the CYC was obviously improved by the pretreatment, which indicated that the crystallinity was not the main factor that impacted the enzymatic hydrolysis process. The spectral assignments of FTIR were given in accordance with previous research (Yang et al. 2007; Zhang et al. 2016b).
In Fig. 6, the band at 3300 cm-1 to 2800 cm-1 was assigned to the stretching of asymmetric and symmetric methyl as well as methylene groups of cellulose, where the absorbance intensity in the SS was clearly stronger than that in the raw material. The band at 1710 cm-1to 1730 cm-1 was the characteristic peak of hemicellulose, and it was assigned to carbonyl stretching in unconjugated ketones and carbonyl groups. The absorbance intensity in the SS was obviously weaker than that in the raw material, which demonstrates that a great amount of the hemicellulose was degraded in pretreatment. The band at 1329 cm-1 was assigned to condensed syringyl and guaiacyl rings, and the band at 1267 cm-1 corresponded to the signal of the guaiacyl ring. The raw material and the SS had strong absorption peaks in these two bands, which indicate that the pretreatment had little influence on lignin.
The NH4Cl concentration, pH value, pretreatment time, and pretreatment temperature strongly affected the enzymatic hydrolysis of eucalyptus chips. In addition, longer pretreatment times and higher pretreatment temperatures were favorable for cellulose degradation, which caused a great amount of 5-HMF and furfural to be generated. NH4Cl concentration had marginal impact on the SS yield and the cellulose degradation. Depending on the pH value, the NH4Cl and hydrogen ions could combine to impact the pretreatment process. Thus, the optimal pretreatment conditions were 0.3 M NH4Cl at 200 °C for 25 min.
The authors are grateful for the financial support of the National Key Research and Development Plan of China (2017YFB0307900), the Open Foundation of Key Laboratory of Renewable Energy, Chinese Academy of Sciences (Y607kb1001, Y607ka1001), the Jiangsu Province Biomass Energy and Materials Laboratory in Institute of Chemical Industry of Forest Products, CAF (JSBEM201811), the State Key Laboratory of Pulp and Paper Engineering in South China University of Technology (201607), and the Project of Tianjin University of Science and Technology “13th Five-Year” Comprehensive Investment Innovation Team, Innovation Foundation for Young Teachers in Tianjin University of Science & Technology (2014CXLG14).
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Article submitted: April 12, 2018; Peer review completed: May 29, 2018; Revised version received: June 29, 2018; Accepted July 1, 2018; Published: July 5, 2018.