Abstract
The stability of cellulase in the presence of ionic liquids (ILs) is pivotal for the in situ saccharification of cellulose. This study investigated the effects of various carbon sources on the cellulase production of Paenibacillus sp. LLZ1. The results showed that the activities of both the total cellulase and endoglucanase induced by microcrystalline cellulose were higher than those induced by other examined carbon sources. Simultaneously, a zymography analysis revealed the presence of seven protein bands (carboxymethylcelluloses 3, 4 and 6 through 10) with endoglucanase activity when MCC was used as the carbon source for culturing Paenibacillus sp. LLZ1. The endoglucanase induced by MCC showed stability in a 25% solids 1-ethyl-3-methylimidazolium diethylphosphate ([Emim]DEP) solution. Furthermore, the addition of metal ions and surfactants increased the endoglucanase activity in the [Emim]DEP solution, where 0.5 mM of Fe2+ and 0.1% of polysorbate (PSM 60) led to 11% and 29% increases in filter paper activity, respectively. Finally, three types of biomass (MCC, bagasse cellulose, and bagasse) were used as raw materials for in situ enzymatic hydrolysis when involved 5% [Emim]DEP and 0.1% polyoxyethylene (60) sorbitan monostearate (PSM 60). Fourier-transform infrared spectroscopy and specific surface area measurements were used to explain the effects of PSM 60 on the hydrolysis efficiency of these three biomasses.
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Diversity of Paenibacillus sp. LLZ1 Cellulase and its Improved Enzyme Activity and Stability in the Ionic Liquid 1-Ethyl-3-methylimidazolium Diethyl Phosphate
Wei Yang, Min Zhou, Lishi Yan, Xin Ju, and Liangzhi Li *
The stability of cellulase in the presence of ionic liquids (ILs) is pivotal for the in situ saccharification of cellulose. This study investigated the effects of various carbon sources on the cellulase production of Paenibacillus sp. LLZ1. The results showed that the activities of both the total cellulase and endoglucanase induced by microcrystalline cellulose were higher than those induced by other examined carbon sources. Simultaneously, a zymography analysis revealed the presence of seven protein bands (carboxymethylcelluloses 3, 4 and 6 through 10) with endoglucanase activity when MCC was used as the carbon source for culturing Paenibacillus sp. LLZ1. The endoglucanase induced by MCC showed stability in a 25% solids 1-ethyl-3-methylimidazolium diethylphosphate ([Emim]DEP) solution. Furthermore, the addition of metal ions and surfactants increased the endoglucanase activity in the [Emim]DEP solution, where 0.5 mM of Fe2+ and 0.1% of polysorbate (PSM 60) led to 11% and 29% increases in filter paper activity, respectively. Finally, three types of biomass (MCC, bagasse cellulose, and bagasse) were used as raw materials for in situ enzymatic hydrolysis when involved 5% [Emim]DEP and 0.1% polyoxyethylene (60) sorbitan monostearate (PSM 60). Fourier-transform infrared spectroscopy and specific surface area measurements were used to explain the effects of PSM 60 on the hydrolysis efficiency of these three biomasses.
Keywords: Biomass; Cellulase; Endoglucanase activity; Ionic liquid; Paenibacillus sp. LLZ1; Surfactant
Contact information: School of Chemistry, Biology, and Material Engineering, Suzhou University of Science and Technology, Suzhou 215009, P.R. China; *Corresponding author: liliangzhi01@163.com
INTRODUCTION
The conversion of cellulosic materials to soluble sugars is one of the major approaches for sustainable energy generation (Zheng et al. 2017). However, cellulose is recalcitrant to enzymatic hydrolysis due to the extensive networks of inter- and intramolecular hydrogen bonds as well as van der Waals interactions (He et al. 2015; Hu et al. 2016a). Presently, ionic liquids (ILs) have been applied in lignocellulosic pretreatments as designable solvents to increase the accessibility to the enzyme, thereby promoting the hydrolysis efficiency of cellulose (Perez-Pimienta et al. 2013). However, the activity of a majority of the cellulolytic enzymes is impeded due to the presence of ILs (Hu et al. 2016a), and the enzymes may be partially or completely inactivated in the presence of low concentrations of ILs.
Therefore, an industrially feasible pretreatment and hydrolysis process requires robust cellulases that are stable and active in the presence of small amounts of ILs co-precipitated with recovered cellulose or for saccharification in the presence of ILs. To develop cellulases for enhanced hydrolysis of cellulose into monosaccharides in non-natural IL environments, several IL-tolerant strains were selected from chemically polluted microhabitats or from culture media containing IL environments (Xu et al. 2016). Genomic studies have revealed that most of the microorganisms, such as Trichoderma reesei and Aspergillus niger, can produce a wide spectrum of cellulase (Brunecky et al. 2017). Moreover, cellulase-producing bacteria often produce diverse varieties of cellulase that can adapt to changing environmental conditions (Leis et al. 2017). Although several reports have been published on the diversity of cellulase, only a few studies addressed the IL tolerance of cellulases induced by different environmental conditions. To resolve the contradiction between the different effects of ILs on cellulose and cellulase, the immobilization of cellulase has been widely investigated. For example, the immobilization of cellulase onto PEGylated graphene oxide (GO) nanosheets was successfully achieved, and the PEGylated GO-Cellulase retained 61% of the initial activity in 25% (w/v) 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) (Xu et al. 2016). In addition, different types of surfactants combined with IL pretreatment were also investigated. Recently, the nonionic surfactants were found to enhance the enzymatic hydrolysis of microcrystalline cellulose (Avicel PH101) by reducing the cellulase deactivation (Lou et al. 2018). To the best of the authors’ knowledge, the hydrolysis of lignocellulose is catalyzed by microbial cellulases, including endoglucanase, β-glucosidase, and cellobiohydrolase. Another study suggested that a cellulase-producing strain, Talaromyces amestolkiae, could secrete high levels of β-glucosidase, β-1,4-endoglucanase, and exoglucanase when cultured in the presence of different carbon sources (Eugenio et al. 2017). In addition, various carbon sources induced the formation of various cellulases. Surprisingly, previous studies have seldom focused on the IL tolerance of cellulases induced by different carbon sources.
The inactivation mechanism of Paenibacillus sp. LLZ1 cellulases (PCs) in the presence of 1-ethyl-3-methylimidazolium diethylphosphate ([Emim]DEP) has been preliminarily investigated (Hu et al. 2016a, 2016b). Herein, further attempts were made to investigate the influence of various carbon sources including microcrystalline cellulose (MCC), sucrose, carboxymethylcellulose (CMC), cellobiose, starch, and glycerol on the production of cellulase by Paenibacillus sp. LLZ1. Sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) was used to reveal the diversity of Paenibacillus sp. LLZ1 endoglucanase induced via different carbon sources. Subsequently, the [Emim]DEP tolerance of cellulases induced by different carbon sources was explored. Finally, the addition of metal ions and surfactants improved the cellulase activity in the [Emim]DEP solution. Under optimal conditions, the MCC, bagasse, and bagasse cellulose (BC) were used as raw materials for in situ enzymatic hydrolysis in the reaction system with the [Emim]DEP solution and surfactant. Fourier-transform infrared spectroscopy (FTIR) and specific surface area were used to explore the underlying mechanism.
EXPERIMENTAL
Materials
The [Emim]DEP with > 99% purity was purchased from Chenjie Chemical Co., Ltd. (Shanghai, China). Both the microcrystalline cellulose (MCC) and Whatman Grade No. 1 filter paper were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Bagasse was obtained from the Guangdong province, China. It was washed with distilled water, dried to constant weight at 85 °C, crushed, and passed through 40-mesh sieve before usage; the purity was approximately 46%. Bagasse cellulose was extracted with 90% purity following procedures described by Hu et al. (2016a) with some modifications. All other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
Bacteria strain and cellulase
The Paenibacillus sp. LLZ1 used in this study was previously isolated by the authors from soil samples (Hu et al. 2016a), and the strain was cultured in a liquid medium that contained (g/L): 3.0 dibasic sodium phosphate (Na2HPO4), 0.8 ammonium nitrate (NH4NO3), 0.5 magnesium sulfate heptahydrate (MgSO4·7H2O), 0.5 calcium chloride (CaCl2), 2.0 yeast extract, and 5.0 of a carbon source. The six carbon sources selected were MCC, sucrose, CMC, cellobiose, starch, and glycerol. The strain was cultured for 7 days at 30 ℃ and 200 rpm, centrifuged at 6000 g for 10 min, and filtered through 0.22-μm nylon filters. The cell-free supernatant was used as a crude cellulase.
Methods
Enzyme assay
The filter paper activity (FPA; representing total cellulase activity) was determined by incubating the crude enzyme with Whatman No. 1 filter paper for 1 h at 40 ℃. A typical reaction mixture contained 0.5 mL of crude enzyme and 0.5 mL of 0.1 M acetic acid buffer (pH 6.0) soaked with 50 mg of Whatman No. 1 filter paper as a substrate. The crude cellulase was incubated with 2% CMC as the substrate at 40 ℃ for 30 min to analyze the CMC activity (CMCA; representing endoglucanase activity). The reaction mixture contained 0.5 mL of crude cellulase and 0.5 mL of 2% CMC substrate solubilized in 0.1 M of acetic acid buffer (pH 6.0). After incubation, the amount of liberated reducing sugar was quantified using the 3,5-dinitrosalicylic acid (DNS) method: Diluted in moderation, 1 mL of the diluted mixture was added to a 3 mL DNS boiling water bath for 5 min, and the absorbance was measured at 540 nm after cooling down to room temperature. The reducing sugar content obtained via the enzymatic reaction was calculated from the standard curve.
Electrophoresis and zymogram analysis of cellulase
A total of 50 μL of crude cellulase solution was incubated at 40 ℃ for 1 h. The samples for SDS-PAGE were prepared by mixing 8 μL of incubated cellulase solution and 2 μL of gel loading buffer without a reducing agent. The protein samples were resolved on 8% separating gel containing 0.1% CMC-Na as a substrate using the method described by Raddadi et al.(2013) with some modifications. After electrophoresis, the gel was incubated in renaturation buffer (0.1 M acetic acid buffer (pH 6.0) containing 2% polyethylene glycol octylphenol ether (Triton X-100) at 4 °C for 1 h and then soaked in the same buffer without Triton X-100 for 12 h at 4 °C. Finally, the gel was incubated at 40 °C for 1 h, stained in 0.1% Congo red for 30 min, and de-stained using 1 M of NaCl. The cellulolytic bands appeared as clear zones on the red background.
Stability of PCs in [Emim]DEP solution
First, the PCs were produced in different culture media with various carbon sources. To investigate the stability of cellulase in ionic liquids, a certain amount of [Emim]DEP was solubilized in 0.1 M of acetate buffer. Then, 0.5 mL of the crude enzyme solution was added to 0.5 mL of the acetate buffer containing [Emim]DEP to achieve the IL concentrations of 0%, 5%, 10%, 15%, 20%, and 25% (v/v). Subsequently, 50 mg of filter paper was incubated in the reaction system for 1 h at 40 ℃. The altered FPA with different concentrations of IL in the hydrolytic process was determined by the above enzyme analysis method. Similarly, the CMCA was measured, and the effect of ILs on the endoglucanase inactivation was evaluated.
Effect of metallic ions and surfactants on PCs
To investigate the effects of metal ions (Fe2+ and Zn2+) on PCs, filter paper and CMC were selected as substrates in the reaction system within ILs. In this study, the FPA and CMCA were determined. The metal ions were added at concentrations of 0.5 or 1 mM. Moreover, the influence of a hydrophilic nonionic surfactant (polysorbate 60 (PSM 60; PSM 60) and alcohol ethoxylate-9 (AEO-9)) on PCs was evaluated with the addition of 0.05% and 0.1% (v/v), respectively.
In situ hydrolysis of biomass under optimal conditions
The conditions of the in situ hydrolysis of biomass were optimized in the authors’ previous study (Hu et al. 2016a). The reaction temperature was 40 ℃ in the presence of 5% (v/v) [Emim]DEP concentration and 0.1% (v/v) of PSM 60 was used for the in situ hydrolysis of biomass. Herein, MCC, BC, and bagasse were selected as in situ enzymatic substrates. Before the in situ hydrolysis of biomass, a substantial amount of [Emim]DEP was used for the pretreatment of the above three biomasses with the addition of 0.1 M acetate buffer (pH 6.0), and the final [Emim]DEP concentration was 5% (v/v) in the reaction mixture. Specifically, 850 μL of the mixture was added to 150 µL of crude enzyme solution, and the final substrate content was 1 g/L. The process was monitored by withdrawing the samples from the reaction mixture at regular intervals. The conversion rate of cellulose was calculated according to Eqs. 1 through 3:
Conversion rate (%) = 0.9 × [glucose weight (g)] × 100 / [cellulose weight (g)] (1)
BC: [cellulose weight (g)] = [BC weight (g)] × 90% (2)
Bagasse: [cellulose weight (g)] = [Bagasse weight (g)] × 46% (3)
Analysis methods
The glucose released through in situ hydrolysis was quantified using a SBA-40C biological sensing analyzer (Biology Institute of Shandong Academy of Sciences, Jinan, China).
The variances of functional groups towards cellulose substrates MCC, bagasse, and BC were obtained using a Nicolet iS10 FTIR spectrometer (Nicolet Co., Waltham, MA, USA). The spectra were recorded in the range of 500 to 4000 cm-1 at a resolution of 0.5 cm-1.
The specific surface areas of MCC, bagasse, and BC were calculated using the Quantachrome Autosorb® IQ surface and pore area analyzer (Quantachrome, Palm Beach County, FL, USA) by degassing the sample under vacuum at 90 °C for 12 h before analysis. The specific surface area was calculated using the Brunauer-Emmet-Teller (BET) method.
RESULTS AND DISCUSSION
Effects of Carbon Source on Cellulase Production by Paenibacillus sp. LLZ1
Cellulase is a complex enzyme system composed of various hydrolases, including endoglucanase, exoglucanase, and β-glucanosidase, which are diverse and inducible, and the biosynthesis of cellulase can be induced via polysaccharides, oligosaccharides, and monosaccharides (Shida et al. 2015). The present study aimed to examine the impact of various carbon sources (MCC, sucrose, CMC, cellobiose, starch, and glycerol) on the production of PCs. Figure 1 shows that CMCA (U/mL) was found in the following order: 1.19 MCC > 0.77 sucrose > 0.65 CMC > 0.53 cellobiose > 0.4 soluble starch > 0.33 glycerol. Furthermore, the order of FPA was similar to that of CMCA, and the FPA (U/mL) was measured at 0.53 MCC, 0.38 sucrose, 0.35 CMC, 0.31 cellobiose, 0.25 soluble starch, and 0.18 glycerol. The data in Fig. 1 indicated that the activities of both total cellulase (FPA) and endoglucanase (CMCA) induced by MCC were higher than those induced by the other examined carbon sources. However, using starch or glycerol as carbon sources induced low cellulase activity. This result was in agreement with a previous report; however, the difference in the CMC-induced cellulase activity was higher than that of MCC (Hong et al. 2013). In contrast, compared to the MCC as a carbon source, the activities of endoglucanases induced by sucrose, CMC, and cellobiose were approximately 65%, 55%, and 45%, respectively (Fig. 1). For all of the tested carbon sources, the CMCA was approximately twice as high as the FPA. This phenomenon was similar to the results previously described, wherein the endoglucanase activity induced by different carbon sources (such as CMC, ball-milling cellulose, or filter paper) was approximately 1.8-fold of FPA (Sohail et al. 2016).
Fig. 1. Effects of different carbon sources on the cellulase activity; the error bars indicate the standard deviation from three independent experiments
In addition, cellulases have a natural diversity. For example, Pason et al. (2006) reported 17 proteins produced by Bacillus curdlanolyticus B-6, and the zymogram analysis revealed 9 CMCases. Similarly, in this study, zymogram analysis revealed the existence of multiple protein bands with endoglucanase activity. The results in Fig. 2 indicated that at least eight endoglucanases (CMC1 through CMC10) were detected from the gel. Strikingly, the different carbon sources induced the expression of cellulose-producing genes in Trichoderma rimanniiand produced different types of cellulases (Margolles-Clark et al. 1997). Herein, at least seven endoglucanases were produced when the carbon source was MCC (CMC3, CMC4, and CMC6 through CMC10). Additionally, the protein bands with endoglucanase activity in the media-containing sucrose, CMC, cellobiose, starch, and glycerol were 5 (CMC3 and CMC6 through CMC9), 4 (CMC5 and CMC7 through CMC9), 1 (CMC10), 4 (CMC1 through CMC3 and CMC8), and 3 (CMC4 through CMC6), respectively. Previous studies suggested that different protein bands in SDS-PAGE represent various endoglucanases, and the corresponding endoglucanase activities might be evaluated by the intensity of the protein bands (Raddadi et al. 2013; Hu et al. 2016b). Figure 2 shows only one bright band for when cellobiose was used as the carbon source. Moreover, the bands of endoglucanases induced by starch and glycerol were also few and fuzzy. These results were similar to those of the enzymatic activity obtained via endoglucanase analysis (Fig. 1) using the same three carbon sources (cellobiose, starch, and glycerol). Overall, the current results confirmed that various endoglucanases could be produced by Paenibacillus sp. LLZ1 when using different carbon sources in the fermentation medium. Similar results were also found by Pardo and Magnelli (1996), wherein the maximal yield of cellulase was achieved using MCC as the carbon source; however, CMC-Na induced the endoglucanase production. Therefore, the exploration and regulation of carbon sources seem to be plausible for improving the expression and activity of cellulase.
Fig. 2. Effects of different carbon sources on endoglucanase diversity of Paenibacillus sp. LLZ1
Stability of PCs in [Emim]DEP Solution
Although several studies have explored the enzymatic hydrolysis of biomass in systems with ILs, only a few studies found that cellulase could withstand a high concentration of ILs (Jaeger et al. 2015). In a previous study, a novel environmentally friendly aqueous-dimethyl sulfoxide/[Emim]DEP-cellulase system was established for the efficient in situ saccharification of cellulose (Hu et al. 2016a). Herein, the stability of PCs in the reaction system with various concentrations of [Emim]DEP was investigated. Figure 3 shows the altered activity of PCs in the reaction system with different concentrations of [Emim]DEP; six carbon sources were selected for the production of PCs. Similar to a majority of the cellulases, the activity of PCs induced via different carbon sources decreased with an increase in [Emim]DEP concentration (Fig. 3). The relative FPA induced by MCC was reduced 32% (0.36 U/mL) in 5% (v/v) [Emim]DEP; however, the amount of relative FPA reduction induced by the other examined carbon sources was approximately 40%. When the [Emim]DEP concentration increased to 25% (v/v), the relative FPA induced via glycerol as the carbon source was reduced 92% (0.016 U/mL); however, the relative FPA induced via MCC was 19% (0.1 U/mL). Thus, compared with the FPA induced by the other carbon sources, the FPA induced via MCC was stable in the reaction system with a specific concentration of [Emim]DEP.
Fig. 3. Stability of Paenibacillus sp. LLZ1 cellulase induced by different carbon sources in [Emim]DEP solution; the error bars indicate the standard deviation from three independent experiments
Various effects were exerted on the individual cellulase components by the IL. A previous study indicated that the relative activities of endoglucanase, cellobihydrolase, and β-glucosidase in the presence of 10% 1,3-dimethylimidazolium dimethylphosphate ([Dmim]DMP) were 63%, 60%, and 34%, respectively (Engel et al. 2012). Therefore, the present study focused on the investigation of endoglucanase stability in the enzymatic hydrolysis system with [Emim]DEP.
Fig. 4. Stability of Paenibacillus sp. LLZ1 endoglucanase induced by three carbon sources in [Emim]DEP; the error bars indicate the standard deviation from three independent experiments
Figure 4 shows the changes in CMCA in the reaction system with different concentrations of [Emim]DEP, and the endoglucanase was induced by MCC, sucrose, and CMC. Furthermore, when the [Emim]DEP concentration was 5% (v/v), the relative CMCAs were 94% (1.12 U/mL, MCC), 93% (0.72 U/mL, sucrose), and 91% (0.59 U/mL, CMC). However, the corresponding FPA was < 70%, which showed that Paenibacillus sp. LLZ1 endoglucanase was stable in 5% [Emim]DEP solution. For example, by increasing the [Emim]DEP concentration from 5% to 25%, the residual relative CMCA persisted at > 58%. The residual CMCA induced via MCC was 67% (0.80 U/mL) in 25% [Emim]DEP solution, which demonstrated the tolerance of ILs among the three tested carbon sources.
To the best of the authors’ knowledge, there has been a lack of studies demonstrating that endoglucanase obtained from commercial or microbial organisms is stable in ILs. Table 1 shows the stability data of several endoglucanases in different ILs. Interestingly, the Paenibacillus sp. LLZ1 endoglucanase showed an improved IL tolerance. For example, compared to the results of Johnson et al. (2016), the decrease in the specific activity of enzyme E1 was 39% in 25% [Emim]DEP solution, while that induced via MCC was only 33% (Table 1). In addition, compared with several other endoglucanases in the table, the endoglucanase produced in this experiment showed relatively good stability despite the different types of ionic liquids used. Thus, Paenibacillus sp. LLZ1 endoglucanase exists stably in 25% [Emim]DEP solution and has a better prospect for industrial application.
Table 1. Recently Published Stability of Endoglucanase in Ionic Liquids
Stability of PCs in 5% [Emim]DEP Solution with Addition of Metal Ions or Surfactants
The cellulase induced via MCC showed enhanced stability in the [Emim]DEP solution. Moreover, some metal ions or surfactants effectively improved the cellulase activity under harsh conditions as previously described (Bharmoria et al. 2014; Akimkulova et al. 2016). The effects of metal ions (Fe2+ and Zn2+) and nonionic surfactants (PSM 60 and AEO-9) on cellulases in the presence of 5% [Emim]DEP solution as the ionic liquid were investigated. As shown in Fig. 5, Fe2+, PSM 60, and AEO-9 effectively promoted the FPA; however, Zn2+ slightly inhibited FPA. Compared to the control, when Fe2+ was added at 0.5 mM in the reaction system with 5% [Emim]DEP IL, the relative FPA (111%) was promoted. However, with increased Fe2+ concentration at 1 mM, the relative FPA (97%) was slightly inhibited. Furthermore, the addition of 0.05% PSM 60 or AEO-9 caused a 16% and 15% increase in the relative FPA, respectively. When PSM 60 increased from 0.05% to 0.1%, the relative FPA increased 29%. Moreover, in the presence of the Fe2+ at 0.5 mM, the relative CMCA (112%) was higher than the control or the other experimental groups. Similarly, 0.05% PSM 60 also promoted CMCA, which was 110% for the control. Additionally, Fe2+ improved the effect on CMCA as previously described (Hmad et al. 2017).
Fig. 5. Effect of metal ion or surfactant supplements on cellulase activity (A) and endoglucanase activity (B); the error bars indicate the standard deviation from three independent experiments
Time Course of in situ Saccharification with PSM 60 Addition
Because 0.1% PSM 60 enhanced the FPA, this concentration was selected to explore the influence on biomass hydrolysis in the reaction system with 5% [EMIM]DEP. Three kinds of biomass (MCC, BC, and bagasse) were used as the raw materials for in situ enzymatic hydrolysis. The in situ hydrolysis system of [Emim]DEP with PSM 60 exhibited a better conversion rate and efficiency as compared to that without the PSM 60 addition (Fig. 6). With the addition of 0.1% PSM 60, the amount of glucose released in the initial 12 h from MCC, BC, or bagasse was 0.30 g/L, 0.31 g/L, and 0.19 g/L, which was 107%, 119%, and 126% of the control, respectively. Therefore, this result also confirmed that the stability of PCs in the IL system was improved successfully via the addition of 0.1% PSM 60. After 108 h of enzymolysis, the conversion rates of MCC, BC, and bagasse were 77.4%, 92%, and 95.8%, respectively, in the hydrolysis system with 0.1% PSM 60 addition, while that without PSM 60 were 73.8%, 88%, and 91%, respectively, in the reaction system.
Fig. 6. The course of in situ enzymatic saccharification; the error bars indicate the standard deviation from three independent experiments
Fig. 7. FTIR patterns of MCC, BC, and bagasse
Finally, the effects of PSM 60 on the hydrolysis efficiency, FTIR, and specific surface area measurement of the three biomasses were assessed. The FTIR results in Fig. 7 show the absorption peaks of aromatic lignin rings (1510 cm-1) and the carbonyl or acetyl absorption peaks (1740 cm-1) of hemicellulose in bagasse. However, these peaks weakened or disappeared in BC, indicating the removal of lignin and hemicellulose in BC. The specific surface area (m2/g) is shown in Table 2. The order from greatest to least was bagasse (6.778) > BC (3.208) > MCC (0.921). Because all three kinds of biomass are insoluble solids, the enzymatic hydrolysis is closely related to the cellulase adsorption. The enzymatic hydrolysis of biomass is also related to the cellulose purity.
The addition of PSM 60 stimulated the efficiency of bagasse hydrolysis, and the addition of PSM 60 released the non-productive adsorption of cellulase and lignin in the biomass. Moreover, the large specific surface area of bagasse was beneficial for the adsorption of enzymes. Furthermore, the addition of nonionic surfactants can compete with the enzyme and reduce the amount of enzyme exposed in the air-liquid interface or reduce the cellulase deactivation caused by the air-liquid interface (Lou et al. 2018). Thus, this aspect might explain the promotion of PSM 60 in in situ enzymatic cellulose.
Table 2. Specific Surface Area of MCC, BC, and Bagasse
CONCLUSIONS
- In this study, the effects of various carbon sources (MCC, sucrose, CMC, cellobiose, starch, and glycerol) on the cellulase production by the strain Paenibacillus sp. LLZ1 were investigated in detail. The results showed that both filter paper activity (FPA) and CMCA induced via MCC were clearly higher than the other examined carbon sources.
- Zymography analysis revealed the existence of seven protein bands (CMC3, CMC4 and CMC6 to CMC9) with endoglucanase activity when MCC was used as the carbon source for the culture of Paenibacillus sp. LLZ1.
- The endoglucanase of Paenibacillus sp. LLZ1 that was induced by MCC showed good stability in 25% [Emim]DEP solution. Therefore, it shows biotechnological and industrial potential in the breakdown of lignocellulosic biomass.
- The addition of metal ions and surfactants improved the endoglucanase activity in the [Emim]DEP solution, where 0.5 mM of Fe2+ and 0. 05% of PSM 60 led to 12% and 10% increases in endoglucanase activity, respectively.
ACKNOWLEDGMENTS
The authors are grateful for the financial support from the National Natural Science Foundation of China (Grant Nos. 21676173, 21376156, and 21506137). This study was also supported by the Qing Lan Project of the Jiangsu Education Department. The authors are grateful for the financial support from the agricultural infrastructure project of the Suzhou Science and Technology Development plan (SNG2018046).
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Article submitted: December 15, 2018; Peer review completed: February 7, 2019; Revised version received: February 21, 2019; Accepted: February 25, 2019; Published: February 27, 2019.
DOI: 10.15376/biores.14.2.3132-3145