A series of metal chlorides with different valences were used during biomass pretreatment and enzymatic saccharification. After pretreatment, the solid substrate (SS), pretreatment liquor (PL), and conversion yield of cellulose (CYC) were characterized. The results showed that the monovalent salts, NaCl and KCl, as well as the divalent salts, MgCl2 and FeCl2, could promote the enzymatic saccharification to a certain extent, while CaCl2 had little influence on the enzymatic saccharification and cellulase activity, and ZnCl2 had an inhibitor effect on them. For a trivalent salt, FeCl3, the removal rate of hemicellulose and the CYC could come up to a high value. The hemicellulose degradation was mainly related to the valences of metal ions. The cellulose models, the enzymatic saccharification, and the enzyme activity assay results showed that most of the metal chlorides had promoter action toward the enzymatic hydrolysis and cellulase activity, with the exception of ZnCl2. Moreover, the metal ions, especially with high valences, had local effects, which could intensify the potentiation or inhibition impacts on the cellulase activities.
Improving the Efficiency of Biomass Pretreatment and Enzymatic Saccharification Process by Metal Chlorides
Dan Huo,a,d,e Danni Xie,a Qiulin Yang,a,b,c,* Qiujuan Liu,a Qingxi Hou,a and Zhengyi Tao e
A series of metal chlorides with different valences were used during biomass pretreatment and enzymatic saccharification. After pretreatment, the solid substrate (SS), pretreatment liquor (PL), and conversion yield of cellulose (CYC) were characterized. The results showed that the monovalent salts, NaCl and KCl, as well as the divalent salts, MgCl2 and FeCl2, could promote the enzymatic saccharification to a certain extent, while CaCl2 had little influence on the enzymatic saccharification and cellulase activity, and ZnCl2 had an inhibitor effect on them. For a trivalent salt, FeCl3, the removal rate of hemicellulose and the CYC could come up to a high value. The hemicellulose degradation was mainly related to the valences of metal ions. The cellulose models, the enzymatic saccharification, and the enzyme activity assay results showed that most of the metal chlorides had promoter action toward the enzymatic hydrolysis and cellulase activity, with the exception of ZnCl2.Moreover, the metal ions, especially with high valences, had local effects, which could intensify the potentiation or inhibition impacts on the cellulase activities.
Keywords: Metal chlorides; Pretreatment; Enzymatic saccharification; Cellulase activity
Contact information: a: Tianjin Key Laboratory of Pulp & Paper, Tianjin University of Science & Technology, Tianjin 300457, China; b: Key Laboratory of Recycling and Eco-treatment of Waste Biomass of Zhejiang Province, Zhejiang University of Science and Technology, Hangzhou 310023, China; c: Key Laboratory of Renewable Energy, Chinese Academy of Sciences, Guangzhou 510640, China; d: Jiangsu Key Lab of Biomass-based Green Fuels and Chemicals, Nanjing Forestry University, Nanjing, 210042, China; e: State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China; *Corresponding author: email@example.com (Q. L. Yang)
Cellulose, the most abundant organic compound on the earth, is one of the three main components in plant cell walls. Cellulose is a linear, unbranched, and natural polymer consisting of 500 to 10000 glucose units, and the long polymer chains are parallel to each other, forming microfibers through hydrogen bonds. Among the hydroxyl groups and oxygen-containing groups within the same, or adjacent molecules, the hydrogen bonds are easily generated and allow many molecules to form a crystal structure. This dense structure makes the cellulose degradation become difficult, and apretreatment process is normally conducted for the lignocellulose prior to enzymatic saccharification.
Metal chlorides are divided into three types, alkaline metal chloride, alkaline-earth metal chloride, and transition metal chloride (Kang et al. 2013). According to the electronic theory of acid and alkali, a Lewis acid is defined as the atom, molecule, ion, or atomic cluster that can accept the external electron pairs, namely the electron-pair acceptor. Thus, the metal chlorides are Lewis acids. For the pretreatment using metal chlorides, the dense structures among cellulose, hemicellulose, and lignin can be destroyed. A proportion, even all of the hemicellulose, can be removed, and the physical-chemical structure can be changed, such as decreasing the crystallinity and degree of polymerization of the cellulose, increasing the porosity and specific surface area of the raw material, etc. (Liu and Wyman 2006; Sathitsuksanoh et al. 2012; Zhang et al. 2016). To date, the actions of the metal chlorides in biomass pretreatment and enzymatic hydrolysis are unclear. This study examined the characteristics of the metal chlorides in biomass pretreatment and enzymatic hydrolysis, which can provide an overall evaluation for the biomass conversion process.
Eucalypt chips were milled and screened to obtain a fraction of 40 to 60 mesh size. 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 were of analytical grade and purchased from Sinopharm Chemical Reagent Co. (Shanghai, China).
Pretreatment with Metal Chlorides
The pretreatment process was conducted in an oil bath, where 5 g of eucalyptus sample was placed into a steel reactor (effective volume 45 mL) with a solid/liquid ratio of 1:6. The metal chloride concentration, pretreatment time, and pretreatment temperature were as follows: 0.3 mol/L of Cl–, 20 min, and 160 °C, respectively. After pretreatment, the reactor was placed into cold water to terminate the reaction. The solid residue from the reactor was filtered with an 80-mesh screen. The filtrate designated as the PL was analyzed to quantify the glucose and xylose contents. The filter residue designated as the SS was washed with deionized water (100 mL) three times. The SS was analyzed to quantify the cellulose, hemicellulose, and lignin contents and then used for enzymatic hydrolysis.
A total of 2 g of the SS was placed into a beaker flask and diluted with a buffer solution (sodium acetate / acetic acid, 0.1 mol/L, pH = 4.8) to a concentration of 2.5% (all based on the oven dried SS, o.d.). Then, 10 FPU/g of cellulase, 15 CBU/g of β-glucosidase, and 3 drops of ethyl acetates were added into the beaker flask. The beaker flask was sealed and put into a shaker for 48 h at 50 °C with a rotational speed of 150 rpm. After hydrolysis, the beaker flask was heated for 5 min at 90 °C in a water bath to deactivate the enzymes.
For the enzymatic hydrolysis, using cellulose model as substrate, the absorbent cotton was also milled and screened to obtain a fraction of 40 to 60 meshes. The metal chlorides were added into the hydrolysis system with a range of 0.05 mol/L to 1.0 mol/L. In particular, sodium acetate and acetic acid were used to readjust the buffer solution (pH 4.8). Other hydrolysis conditions were the same as that of the SS.
Characterization of the Raw Material, the SS, and Hydrolysate
For the raw material and the SS, the NREL LAP method was used (Mosier et al. 2005). The composition of the raw material was as follows: glucan 47.86%, xylan 15.50%, arabinan 1.03%, and lignin 29.15%. The SS yield was calculated as the mass ratio of the SS and eucalyptus chips.
The PL and enzymatic hydrolysate were centrifuged at 10000 rpm for 10 min and filtered through a membrane filter (0.22 μm) prior to detecting. The HPLC (Agilent 1260) equipped with a Bio-Rad Aminex HPX-87H (300 × 7.8 mm) column was used, and detecting conditions were as follows: column temperature 55 ºC, differential detector, mobile phase H2SO4 (0.05 mol/L) with a flow rate of 0.6 mL/min, and a sampling volume of 10 μL. The removal rates of cellulose and hemicellulose were calculated as their loss ratios during the pretreatment. The CYC was calculated as follows:
CYC = 0.9 * (glucose in enzymatic hydrolyzate) / (cellulose in the SS) (1)
Determination of Cellulase Activity
The filter paper activity (Fpase) for cellulase was determined using a buffer solution (pH 4.8) and the DNS method (Ghose 1987; Sluiter et al. 2012). The original cellulase was diluted with the buffer solution prior to detecting. The specific dilution factor, achieved by releasing 2 mg of glucose in the Fpase detection, was used to investigate the influence of metal chlorides on the activity. Consequently, the metal chlorides were dissolved into the buffer solution and formed a serious of solutions (pH 4.8), which replaced the buffer solution, that were used for activity detection.
RESULTS AND DISCUSSION
Pretreatment using Different Metal Chlorides
The raw materials were pretreated under the same conditions, and the addition of metal chlorides was based on the same Cl– concentration. For comparison, the pretreatments using H2O (blank sample) and HCl were conducted. The results are shown in Figs. 1 and 2.
In Fig. 1, the pretreatment using HCl had the highest CYC (96.3%) and the lowest SS yield (39.5%). When the monovalent and divalent salts KCl, NaCl, CaCl2, and MgCl2 were used, the SS yields were all about 85%. However, the SS yields of the pretreatment using ZnCl2 and FeCl2 decreased, indicating that the degradation impact of ZnCl2 and FeCl2 on the raw material was more severe. The pretreatment using divalent salts had a higher CYC than that of the monovalent salts. The pretreatment using MgCl2 not only retained a higher SS yield but also obtained a higher CYC (40.0%), demonstrating that the MgCl2 was more suitable for enzymatic hydrolysis. For pretreatment using the trivalent salt FeCl3, the SS yield was further decreased, while the CYC was as high as 67.9% and was increased 7.74 times compared to the blank sample, meaning that the Fe3+ ions were extremely helpful for enzymatic saccharification, rather than the Fe2+ ions. Though the CYC was very high for the pretreatment using HCl, the removal rate of cellulose came up to 39.5%, indicating that a great amount of the cellulose was degraded into the PL. The pretreatment using metal chlorides mainly caused the hemicellulose degradation compared to the lignin and cellulose degradation, especially for the pretreatments using CaCl2, MgCl2, ZnCl2, FeCl2, and FeCl3, where their hemicellulose losses were all over 30% and cellulose losses were less than 10%. This demonstrated that pretreatment using trivalent and divalent salts were more beneficial for the hemicellulose degradation than that of the monovalent salts. When FeCl3 was used in pretreatment, the hemicellulose and cellulose losses were as high as 75.7% and 12.4%, respectively. Though higher losses could reduce the cellulose mass in the SS, the conversion efficiency (based on the raw material) of the pretreatment using FeCl3 was still higher than that of the other salts. Based on that analysis, the FeCl3 and MgCl2 were more suitable for pretreatment and followed enzymatic saccharification.
For the PL, glucose and xylose concentrations were analyzed (Fig. 2). Specifically, the cellulose and hemicellulose degradation products not only contained monosaccharides, but also contained a series of oligosaccharides. Generally, pretreatment using metal chlorides had a weaker influence on cellulose than hemicellulose, and the glucose content in the PL was correspondingly lower than that of the xylose. When the monovalent, bivalent, and trivalent salts were used in the pretreatment, the xylose contents were about 1.0 mg/mL, 3.9 to 5.7 mg/mL, and as high as 9.1 mg/mL, respectively. Considering the removal rate of hemicellulose, it demonstrated that the hemicellulose degradation was related to the valence of metal ions. That point was also confirmed by the previous research done by Liu et al. (2006), who treated xylose and xylotriose using KCl, NaCl, CaCl2, MgCl2, and FeCl3. The results showed that the degradation of these two types of sugars could be accelerated by these inorganic salts, with the order of FeCl3> MgCl2> CaCl2> KCl> NaCl.
Fig. 1. Effects of the pretreatment using metal chlorides on the SS and the CYC
Fig. 2. Characterization of the PL from the pretreatment using metal chlorides
Enzymatic Hydrolysis using Metal Chlorides
Even though the SS had been washed sufficiently, a portion of the metal ions from the metal chlorides still remained absorbed or chelated onto its surface. To research these metal ions influence, the metal chlorides were added into the enzymatic hydrolysis system, and the cellulose models absorbent cotton was used as substrate. The results are shown in Fig. 3.
Compared to the blank sample, the increased rate of the CYC was more than 20% when the addition of monovalent salts KCl and NaCl came up to 1 mol/L. For the divalent salts, when 1 mol/L of the MgCl2 and FeCl2 were added into the hydrolysis processes, the CYC was increased 56.2% and 115.2%, respectively. However, the CaCl2 had little effect on the enzymatic hydrolysis process, and the ZnCl2 severely restrained the hydrolysis process. When 1 mol/L of ZnCl2 was used, the CYC was decreased by as much as 59.7%. This indicated that the divalent salts had different impacts on enzymatic saccharification, though their metal ions had the same valences. For the trivalent salt FeCl3(1 mol/L), the CYC was increased 233.1%, which could greatly facilitate the hydrolysis process. Thus, the enzymatic hydrolysis process was obviously changed by adding suitable metals ions into the hydrolysis system, especially adding Fe3+ ions. Among these metal chlorides, the changes of enzymatic hydrolysis efficiency were not related to the valence of metal ions, which were mainly caused by the metal ions itself.
Fig. 3. Effects of the metal chlorides on the enzymatic saccharification
Cellulase Activity Assay using Chloride Salts
The metal chloride solutions were used, instead of the buffer solution, for the cellulase activity assay. The results are shown in Fig. 4.
Some metal ions, by serving as an activator, can impact the cellulase activity to a certain extent, and some enzymes also need the metal ion or metal complex to arouse their activities. When the concentration of KCl, CaCl2, NaCl, MgCl2, ZnCl2, FeCl2, and FeCl3 came up to 1 mol/L in the system, the cellulase activities were increased 13.6%, 0.6%, 14.1%, 7.3%, -5.8%, 10.2%, and 17.5%, respectively. Similar to the hydrolysis impacts, the cellulase activity could be promoted by KCl, NaCl, MgCl2, FeCl2, and FeCl3, whereas it could be inhibited by ZnCl2 and not affected by the CaCl2.Unlike other metal ions, Zn2+ is a necessary ion for some enzymes to make up the active center and maintain spatial structure. However, it also could form a coordinate bond with other electronic centers and thereby intercept the combination of enzymes and substrate. Thus, the Zn2+ mainly had a blocking effect on the cellulase activity (McCall et al. 2000).
Moreover, Table 1 shows the growth rates of the CYC and cellulase activity when 1 mol/L of metal chlorides were added into the enzymatic hydrolysis and activity detection processes, respectively. The NaCl and KCl had a certain promoter action on the cellulase activity and enzymatic hydrolysis, where the growth rate of the CYC was only 1.6 to1.7 times higher than that of the cellulase activity. When the divalent salts CaCl2, MgCl2, and FeCl2 were used, the growth rate of the CYC was about 7.0 to 11.0 times higher than that of the cellulase activity. The inhibition effects of the ZnCl2 on enzymatic hydrolysis were also more severe than on the cellulase activity. For the trivalent salt FeCl3, the biggest gap of the growth rate between the CYC and cellulase activity appeared. This showed that the cellulase activity was improved less than the CYC under the same metal ions concentrations, especially the metal ions with high valences. Further analysis found that the absorbent cotton with polar hydroxyl on the surface could be ionized in water, which had negative charges (Horvath and Lindstrom 2007). Unlike the FPase detection, the milled absorbent cotton had a larger specific surface area. The metal ions from the dissolved metal chlorides could be attracted onto the cellulose surface, causing their surface concentration to be raised. Hence, the local effects of the metal ions could strengthen the promotion or inhibitory influences on the cellulase activity. Because of the low valences of the Na+and K+ ions, the promotion action of NaCl and KCl were weak compared to the other metal ions. Thus, it was beneficial for enzymatic saccharification to select suitable metal ions to be added into the hydrolysis system.
Fig. 4. Effects of the metal chlorides on the cellulase activity
Table 1. Changes of the CYC and Cellulase Activity by Adding Metal Chlorides
- The pretreatment using metal chlorides mainly resulted in hemicellulose degradation, where the removal rate of hemicellulose followed a sequence of trivalent salts (FeCl3) > divalent salts (CaCl2/MgCl2/ZnCl2/FeCl2) > monovalent salts (KCl/NaCl), and the MgCl2 and FeCl3 were more suitable for enzymatic hydrolysis.
- Most of the metal chlorides had promoter actions for the enzymatic saccharification and cellulase activity, while the ZnCl2 could inhibit those two processes.
- The metal ions, especially those with high valences, had local effects that could accelerate the enhancement or inhibition actions on the cellulase activities.
The authors are grateful to the financial supports from National Key Research and Development Program of China (2017YFB0307905), Open Foundation of Key Laboratory of Recycling and Eco-treatment of Waste Biomass of Zhejiang Province in Zhejiang University of Science and Technology (2016REWB22), Key Laboratory of Renewable Energy in Chinese Academy of Sciences (Y607kb1001, Y607ka1001), State Key Laboratory of Pulp and Paper Engineering in South China University of Technology (201607, 201451), Jiangsu Key Lab of Biomass-based Green Fuels and Chemicals in Nanjing Forestry University (JSBGFC14011), and Project of Tianjin University of Science and Technology “13th Five-Year” comprehensive investment innovation team.
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Article submitted: December 5, 2017; Peer review completed: January 14, 2018; Revised version received and accepted: January 19, 2018; Published: January 29, 2018.