Effects of tea saponin on glucan conversion and bonding behaviour of cellulolytic enzymes during enzymatic hydrolysis of corncob residue with high lignin content
© Feng et al.; licensee BioMed Central Ltd. 2013
Received: 5 August 2013
Accepted: 5 November 2013
Published: 14 November 2013
Recently, interest in the utilization of corncob residue (CCR, with high lignin of 45.1%) as a feedstock for bioethanol has been growing. Surfactants have been one of the most popular additives intended to prevent the inhibitory effect of lignin on cellulolytic enzymes, thereby improving hydrolysis. In this study, the effects of biosurfactant tea saponin (TS) on the enzymatic hydrolysis of CCR and the bonding behavior of cellulolytic enzymes to the substrate were investigated. The surface tension in the supernatant was also detected to obtain information about the characteristics and stability of TS.
The glucose concentration was 17.15 mg/mL at 120 hours of hydrolysis with the low loading of cellulolytic enzymes (7.0 FPU/g cellulose and 10.5 BGU/g cellulose) and 5% CCR. The optimal dosage of TS was its critical micelle concentration (cmc, 1.80 mg/mL). The glucose yield was enhanced from 34.29 to 46.28 g/100 g dry matter by TS. The results indicate that TS can promote the adsorption of cellulolytic enzymes on the substrate and mediate the release of adsorbed enzymes. Meanwhile, TS improves the recovery of the cellulolytic enzymes after a hydrolysis cycle and prevents deactivation of the enzymes during the intense shaking process. The surface tension in supernatants of digested CCR with TS remained at 50.00 mN/m during the course of hydrolysis. It is interesting to note that biosurfactant TS can maintain the surface tension in supernatants, despite its digestibility by cellulolytic enzymes.
Serving as an accelerant of lignocellulose hydrolysis, TS can also be degraded by the cellulolytic enzymes and release glucose while retaining stability, which reduces the cost of both the cellulolytic enzymes and the additive. As the glucose from the TS could be utilized by yeast, further efforts will investigate the mechanism of function and the application of TS in the production of ethanol by simultaneous saccharification and fermentation (SSF).
One of the major limitations of cellulosic ethanol production is the release of fermentable sugars from lignocellulose using cellulolytic enzymes[1, 2]. Recently, interest in the utilization of corncob residue (CCR) as a feedstock for the production of bioethanol has been growing[3–6]. CCR is an industrial byproduct of furfural manufacture from corncobs, in which hemicelluloses are acid-hydrolyzed to produce furfural. The cellulose and lignin present in corncobs are relatively stable during the acid hydrolyzation of hemicelluloses. Therefore, the lignocellulosic residues from furfural production are mainly composed of cellulose and lignin, the components of which in the residues were on average about 43% and 45%, respectively[9–11]. It has been reported that 12 to 15 tons of CCR can be gained from the production of 1 ton of furfural, while 23 million tons of CCR have been available each year on average for alternative usage in China. The advantages of using CCR in ethanol bioconversion have been reported. As these residues are byproducts of hemicelluloses extraction, CCR is rich in cellulose. When acid treatment was applied during the manufacture procedure, the lignin in the CCR was less polymerized, and the cellulose was more accessible[14, 15].
However, despite intensive research efforts, an efficient hydrolysis of CCR by cellulolytic enzymes is still difficult to accomplish. Lignocellulose conversion to sugar monomers on a commercial scale is hampered by the inhibitory effect of lignin[16, 17]. Lignin provides a physical barrier limiting the accessibility of cellulolytic enzymes to the substrate, and the residual lignin could block the removal of the cellulase from the cellulose chain. In addition, the non-productive adsorption of lignin on cellulolytic enzymes reduces the productive hydrolysis of the substrate. Lignin may also directly inhibit the activities of cellulolytic enzymes. Therefore, studies are focusing on additives that improve the conversion of lignocellulosic feedstock.
In recent years, surfactants have been one of the most popular additives intended to prevent the inhibitory effect of lignin on cellulolytic enzymes, thereby improving hydrolysis. A large number of reports have stated that surfactants, especially non-ionic surfactants, were the most suitable additives for improving the saccharification of lignocellulose and the recovery of cellulolytic enzymes[21–25].
However, most of the surfactants that have been studied recently were chemicals. The application of natural biosurfactants in lignocellulose hydrolysis has been less extensively investigated. Biosurfactants were more popular for their high efficiency and avirulence. It was found that biosurfactant monorhamnolipid may promote hydrolysis of NaOH-pretreated rice straw by 23.15%, and increase the stability of cellulase by 24% to 36%. The improvement of the production of cellulases and xylanase from Penicillium expansum via the addition of biosurfactant rhamnolipid was also confirmed by Wang et al.. The rhamnolipid increased the activity of cellulase by 25.5% to 102.9%, and protected cellulase from degradation and inactivation. However, the reducing sugars by hydrolyzing wheat straw were not visibly increased by the rhamnolipid. Zhang et al. also found the rhamnolipid prevented unproductive binding of enzymes to lignin. The increment of 20% was found by Menon et al., who investigated the positive effect of sophorolipid on the hydrolysis of oat spelt xylan and wheat bran hemicelluloses with Thermomonospora xylanase.
Tea seed is an agricultural byproduct of Camellia oleifera Abel, which is commonly used for the production of cooking oil. On average, the production of 15 million tons of tea seed oil will obtain 50 million tons of residues annually in China. The defatted tea seed residues contain 11% to 17% saponin, which is usually used for detergents or organic fertilizers with low economic value. Tea saponin (TS) is a type of tea seed-derived natural non-ionic biosurfactant. The TS had a weight-average molecular weight of 809.12 g/mol and contained four aglycones of L-rhamnose, D-galactose, D-glucose, and D-glucuronic acid. A critical micelle concentration (cmc) of 1.80 mg/mL and a minimum surface tension (γcmc) of 43.5 mN/m were determined for the TS. The cmc is the threshold value that limits the formation of micelles. Micelles will form in the solution, combining enzymes and hindering the exchange of materials, when the surfactant concentration is higher than its cmc. Nevertheless, few studies seem to have been conducted regarding the effects of biosurfactant TS on lignocellulose conversion, especially in the case of CCR saccharification.
One of the typical characteristics of a surfactant is that it can stabilize the surface tension in a solution. However, the function of surfactants in lignocellulose hydrolysis has mostly been investigated by the determination of the cellulose conversion, the enzymatic protein content, and the stability and activity of cellulolytic enzymes[27, 28]. The impact of the surfactant on the surface tension in the supernatant during the course of hydrolysis has not been extensively considered.
Biosurfactant TS was investigated for its ability to improve the enzymatic hydrolysis of CCR in this study. The components of glucan and lignin in the prepared CCR were 48.3% and 45.1%, respectively. The substrate was hydrolyzed with Trichoderma reesei cellulase in commercial mixtures. The influence of TS on the efficiency of CCR hydrolysis and on the adsorption and recovery of cellulolytic enzymes was detected by considering the alteration of the glucose yield, protein concentrations, and enzymatic activity in the hydrolysate. To obtain the information about characteristics and stability of biosurfactant TS, the surface tension in the supernatant was also detected during the process of CCR hydrolysis.
Results and discussion
Effect of TS on CCR hydrolysis
It is interesting to note that glucose could be released in the control group without the addition of CCR (Figure 1). Glucose production in the solution of TS with cellulolytic-enzyme loading levels of 7.0 FPU/g cellulose and 10.5 BGU/g cellulose increased when more TS was added into the mixture. The glucose concentration reached 2.31 mg/mL when the dosage of TS was 7.20 mg/mL. This result indicates that cellulolytic enzymes can release glucose from the biosurfactant TS. However, the glucose released from the TS was eliminated from the calculation of the substrate saccharification yield in the above CCR enzymatic hydrolysis process involving TS to ensure the applicability and accuracy of the experimental data.
Effect of TS on protein concentrations in the supernatants of digested CCR
Effect of TS on filter paper activity in the supernatants of digested CCR
Effect of TS on surface tension in the supernatants of digested CCR
Process of CCR hydrolysis with the participation of TS
Therefore, the positive effect of TS on the bonding and recovery of cellulolytic enzymes, accessibility of lignocelluloses, and homogenization of hydrolysates may provide the explanation for the improved conversion of CCR in the presence of TS. However, more experimental studies are needed to confirm the comments.
Biosurfactant TS can promote CCR saccharification by 34.97%. The functions of TS in CCR hydrolysis were investigated through the detection of the protein concentrations, FPAs, and surface tensions in supernatants. The results indicate that TS can improve the combination of the enzymes and the substrates, and the recovery and stability of the enzymes for recycling, thereby enhancing the hydrolysis of the lignocellulose substrate. Serving as an accelerant of lignocellulose hydrolysis, TS can also be degraded by the cellulolytic enzymes and release glucose while retaining stability, which reduces the cost of both cellulases and additives. As the glucose from the TS could be utilized by yeast, further efforts will investigate the mechanism of TS action and application of TS in the production of ethanol by simultaneous saccharification and fermentation (SSF).
Materials and methods
The CCR was kindly supplied by the Chunlei Furfural Corporation (Hebei, China). The residues, which had a pH of 2 to 3 initially, were immersed in a 1% NaOH solution for half an hour and then washed with fresh tap water until neutral. The samples were dried at 50°C for 12 hours and milled to a size of below 40 to 60 mesh. According to the National Renewable Energy Laboratory (NREL) methods that were employed to determine and calculate the constituent contents of the samples, the proportions of glucan, xylan, and lignin in the CCR were 48.3%, 3.6%, and 45.1%, respectively. Whatman No 1 filter paper was purchased from the Sigma-Aldrich (Beijing, China).
Celluclast 1.5 L, a cellulase preparation from Trichoderma reesei, and Novozyme 188, a β-glucosidase preparation from Aspergillus niger, were purchased from Novozymes investment Co, Ltd (Beijing, China). The activity of Celluclast 1.5 L was detected to be 74 FPU/mL. The activity of Novozyme 188 was detected to be 175 BGU/mL.
Biosurfactant TS was isolated and purified from the defatted seed of Camellia oleifera Abel in the laboratory. The TS had a weight-average molecular weight of 809.12 g/mol and contained four aglycones of L-rhamnose, D-galactose, D-glucose, and D-glucuronic acid. A cmc of 1.80 mg/mL and γcmc of 43.5 mN/m were determined for the TS.
Hydrolysis of CCR with cellulolytic enzymes
Enzymatic hydrolysis was accomplished with an enzyme loading of 7.0 FPU/g cellulose to evaluate the hydrolytic potential of the CCR by the commercial cellulase preparations with the addition of biosurfactant TS. The efficiency of the hydrolysis was improved by β-glucosidase supplementation with Novozyme 188 at an enzyme loading of 10.5 BGU/g cellulose. The cellulosic substrates were diluted with the addition of 0.1 mol/L sodium acetate buffer (pH 4.8) to 50 g substrate/L in a total reaction volume of 100 mL. Saccharification was performed at 45°C on a rotary shaker at 180 rpm for 120 hours. The samples were withdrawn and centrifuged at 10,000 × g for 10 minutes. The supernatants were withdrawn for the evaluations of FPAs, protein concentrations, and surface tension. The supernatants were also filtered through 0.2 μm filters and diluted as indicated for neutral sugar analysis. Control hydrolysis without any substrate was performed to avoid the release of sugars from the TS and cellulolytic enzymes.
The FPA was evaluated using the standard method of the International Union of Pure and Applied Chemistry (IUPAC). The β-glucosidase activity (BGA) was determined using the modified Berghem’s method. The enzymatic protein concentration was determined by the Bradford method using BSA as a standard. The above experiments were repeated three times, and the data presented are the mean values.
The samples were filtered through a 0.22 μm filter and diluted appropriately by distilled water. Glucose and cellobiose were analyzed by HPLC (Waters 2695e, Waters, Milford, MA, USA) with an Aminex HPX-87P column (300 × 7.8 mm, Bio-Rad, Hercules, CA, USA) at 85°C and by a refractive index detector at 35°C. The injection volume of the sample was 10 μL, and distilled water was used as the eluent at a flow rate of 0.6 mL/min. The glucose yield was expressed as the weight of the glucose released in the supernatant to the 100 g dry matter of the loading substrate. The sugar determination was performed in duplicate under the same conditions, and the average values were computed. The standard deviations were less than 3.6%.
The surface tensions of the supernatant samples were determined according to Jian’s method on the automatic tension meter (model JK99B, Zhongchen digital technology equipment Co, Ltd, Shanghai, China) at 20°C in an aqueous medium.
Bovine serum albumin
Critical micelle concentration
Filter paper activity
Filter paper unit
High performance liquid chromatography
International union of pure and applied chemistry
National renewable energy laboratory
Simultaneous saccharification and fermentation
Minimum surface tension.
The authors acknowledge the support of the National Basic Research Program of China (2011CB403202), National Science Foundation of China (31070510, 40930107), and the Strategic Priority Research Program of Chinese Academy of Sciences (XDA05050201, XDA05020300).
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