Additives are additional materials that do not belong to the original cellulosic-hydrolysis system. The mechanism of additives, including bovine serum albumin (BSA) (Yang and Wyman, 2006), Tween 20 (Zheng et al., 2008), various polyethylene glycols (PEG) (Börjesson et al., 2007; Zhang et al., 2010) and yeast extract, improves cellulases hydrolysis efficiency of lignocellulose.
One of the vital additives of enzymatic hydrolysis is bovine serum albumin (BSA). A capacity for BSA could adsorb even more cellulase, which found adsorption of a substantial amount of cellulase on both lignin and cellulose. Adding BSA with cellulase
resulted in some promise in both rates and yields, and after hydrolysis began also enhanced rates whether pretreated fiber was treated with BSA prior to hydrolysis (Yang and Wyman, 2006). In addition, Tween 20 is also a common additive. The effect of the surfactant Tween 20 on the hydrolysis of different cellulosic fibers was investigated and related to the cellulose fiber structure. It was found that this non-ionic surfactant enhanced the enzymatic saccharification of highly crystalline cellulose (Mizutani et al., 2002). Moreover, polyethylene glycols can raise the hydrolysis yield due to the following two effects, hydrophobic interactions and hydrogen bonds between PEG and lignin. The phenolic hydroxyl group of lignin can interact with the ether oxygen bond (-O-) to form a hydrogen bond (Börjesson et al., 2007; Zhang et al., 2010).
Since yeast extract is commonly present in the simultaneous saccharification and fermentation processes (SSF) (Ballesteros et al,. 2004), its effect on the accessibility and degradability of the crude endoglucanase was also examined. Molecular weight reduction and reducing sugar were monitored for the extents of hydrolysis (Ouyang et al., 2010). The cellulase hydrolysis was related to the degree to which this saccharification was enhanced by the presence by additives which block the cellulases from binding to the lignin. Therefore, more cellulases could participate in lignocellulosic hydrolysis (Ander et al., 2008; Ramírez et al., 2008; Mamma et al., 2009).
(III) Refinig of Pulp Stock 1. PFI
Cellulases were the most efficient enzymes as refining helpers (Gil et al., 2009).
During papermaking, the bleached pulp must be mechanically produced to obtain a certain reduced paper web drainage (defined as freeness) to improve paper web
formation and flexibility (Kamaya, 1996; Mutjé et al., 2005), and the cellulase pretreatment of the pulp could result in noticeable savings in electricity of power, while maintaining the strength properties of the pulp (Bajpai et al., 2006).
The cellulases have effective ability to alter fiber morphology, and could increase drainability (Schopper-Riegler method) and water retention value (WRV) of bleached pulp. They could improve inter-fiber bonding in the refining process. However, the drainability and paper strength results have not always been consistent; also, cellulases are often thought to have a harmful impact on paper properties (Oksanen et al., 2000;
García et al., 2002; Pala et al., 2002; Cadena et al., 2010).
2. Scanning Electron Microscope (SEM)
A scanning electron microscope (SEM) is a type of electron microscope that images a sample by scanning it with a high-energy beam of electrons in a raster scan pattern. The objectives of using SEM were to explore the morphological change on the surface of fiber, to compare at the microscopic level. Visual observation revealed differences in fiber fracture between treated and control samples (Garmaroody et al., 2011; Shi et al., 2011). High resolution SEM analysis of the fiber surface was the methods of choice.
The images should be interpreted in view of possible differences in the fiber fracture mechanism caused by the cellulase treatment (Suchy et al., 2009). In this study, the morphology will be examined by SEM which showed that differences of refined fibers between control and enzyme pretreated.
III Materials and Methods
(I) Microorganism and Growth Measurements
The thermo-alkaline Paenibacillus campinanesis BL 11 used in this study was isolated at 90℃, pH 9 environment in a kraft pulp mill (Ko et al., 2007). Chemicals were obtained from Sigma (St. Louis, USA) or Merck (Darmstadt, Germany). This strain was routinely plated on Luria-Bertani (LB) plate and incubated at 37℃ for 1 day.
(II) Expression of the Recombinant Endoglucanase
One colony of the expression strain transformed by pETCMC2 insertion is inoculated into 2 ml of Luria-Bertani medium containing 100 μg of ampicillin/ml and allowed to grow overnight at 37°C in a rotary shaker. The overnight culture is then transferred to 30 ml of the same medium and grown to an A600 value of 0.4-0.5. Protein productions are induced by the addition of isopropyl-β-D-thio-galactopyranoside (IPTG) to a final concentration of 0.1 mM and grown overnight at 28°C, after which time the cells are harvested by centrifugation, washed, and disrupted by sonication in a phosphate buffered saline (PBS) buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.4). Clear lysate of the extracts is loaded on a Ni-NTA agarose (QIAgen) volume. The resin is then washed twice with wash buffer (300 mM NaCl, 50 mM NaH2PO4, 25-30 mM imidazole, pH 7.0) and the protein is eluted by adding 200 µl elution buffer (300 mM NaCl, 50 mM NaH2PO4, 100 mM imidazole, pH 7.0).
(III) Fiber Preparation and Chemical Analysis
Norway spruce (Picea abies) wood chips were obtained from the Heiberg Experimental Forest, State University of New York, College of Environmental Science
and Forestry, Tully, New York, USA (Ko et al., 2010a). The kraft spruce pulps obtained by different chemical treatments. The delignification of spruce chips was carried out according to the procedure described by kraft method with 21% and 25% NaOH and sodium sulfite mixed liquor (w/w with respect to O.D. material, sulfide 25 ± 1) and 700 g spruce chips (O.D.), then cooking at constant temperature of 150 min. The liquor to solid ratio was changed and fixed at 4 due to the experimental device used and temperatures 165℃were tested. All the experiments were conducted in a 1 L reactor, in which the heating time to reach the constant temperature was 150 min. Then, the pulp made in the chemical condition with 21% NaOH and sodium sulfite mixed liquor was treated by oxygen delignification. Oxygen delignification was conducted with 1.2%
(w/w) NaOH (O.D.) pulp under 4 kg/cm2 oxygen pressure raised to 99℃ within 60 min, then maintained isothermally for next 15 min (O15). Fully bleached pulps were prepared from oxygen bleached pulps by using a commercial DEDD bleaching sequence.
(IV) DEDD Bleaching
DEDD bleaching (Wong et al., 2001) was carried out using chlorine dioxide (ClO2) that obtained from an industrial source contained 10% active chlorine as ClO2. The first chlorine dioxide stage (D0) was conducted at 10% pulp consistency, 50°C and for 1 h.
The kappa factor is defined as active chlorine multiple (A.C.M.) (Reeve, 1996), which is a predetermined quantity representing the percentage of active chlorine divided by the kappa number of the pulp (Reeve, 1996). The reason for defining the kappa factor is to normalize bleaching agent usage with respect to pulp lignin contents in industrial practice. The reason for defining the active chlorine values is to normalize weight-based usage for various bleaching agents with respect to chlorine molecular weight in
industrial practice. Alkaline extraction stage (E) were performed at 10% consistency, 70°C for 1 h, and with the charge of NaOH equivalent to one half of the initial active chlorine charge. The second chlorine dioxide stages (D1) were carried out at 10%
consistency with 1% ClO2 solution, at pH 4, 70°C for 3 h. The third chlorine dioxide stages (D2) were carried out at 10% consistency with 1% ClO2 solution, at pH 4, 70°C for 3 h.
(V) Characterization of Pulps
After pulp making, the obtained pulps were washed several times through a wire until obtaining a clear filtrate and characterized in terms of yield, kappa number, residual lignin, holocellulose and pentosan. The cooking yield was calculated as the ratio of the weight of O.D. material after washing to that of initial raw material. The residual lignin was determined from both the Klason lignin and the soluble lignin measured by UV absorption of a filtrate specimen at 280 nm (TAPPI method UM 250). The viscosity of pulp (g in mPa．s) dissolved in a cupriethylene-diamine solution was determined according to TAPPI standard (T230 om-99). These values were then converted into degrees of polymerization (DP) thanks to the following relation proposed by Sihtola et al. (1963):
DP= [0.75 (954Log10η – 325)1.105] (1)
The values of both rate constant (k) and LODP have been calculated with the aid of a non-linear curve fitting software (Calvini, 2005):
(1/DP - 1/DP0) = (1/LODP-1/DP0) × (1-e-kt) (2)
(VI) Cellulase Activity
The cellulase was tested in a fixed volume, containing diluted enzyme dosage, 4%
CMC (Carboxymethyl cellulose, pH 7) in the Tris buffer (0.5 M, pH 7) contained 100 mM Tris buffer, 20 mM CaCl2 and 0.04% (v/v) Tween 20 (Bio Basic). The tubes were incubated for 20 min at 40◦C, and the reaction was terminatedly added to dinitrosalicylic acid (DNSA) including 1% (w/v) dinitrosalicylic acid (Sigma), and 0.4 M sodium hydroxide solution (10%, v/v) and 30% (w/v) potassium sodium tartrate tetrahydrate (Sigma), and the tubes were boiled for 10 min, then cooling. The absorbance then was measured at 540 nm (Miller, 1959; König et al., 2002).
(VII) Enzymatic Hydrolysis of Pulp
The crude endoglucanase from P. campinasensis BL 11 were used to hydrolyze cellulose in spruce kraft pulp. Enzymes were added to make 5, 10, 20 and 50 U/g pulp.
Experiments were carried out in heat-resisting plastic bags containing 1 g of kraft pulp (O.D.) and a total liquid volume of 10 mL, with pulp consistence 10% (i.e., cellulase diluted in Tris buffer of pH 7) for 0, 1, 2, 4, 6, 8 and 24 h at 40◦C in a thermostatic water bath. At periodic time intervals, glucose and DP were measured.
(VIII) Additive of Yeast Extract
0.25 g yeast extract (Fluka) was added to 5 mL 50 mM tris buffer, pH 7, to make a 5
% solution, and then kraft pulps (12.43% and 1.24% lignin content) at 20% (w/v) were added. After mixing completely, the final pulps consistence was 10% and yeast extract concentration was 25% to the pulps (w/w). The hydrolysis steps were same to foregoing method “Enzymatic hydrolysis of pulp”.
(IX) Endoglucanase Accessibility and Digestibility
To determine enzyme accessibility, adsorption on three pulps was performed in 2 mL 0.1 M sodium acetate, 20 mM CaCl2, 0.04 % (w/v) Tween 20, pH 6 at 4℃ to avoid hydrolysis. The substrate concentration was 0.1% (w/v), with the dosages for 5, 10, 15, 20, 30, 40, 50 mg enzyme per gram O.D. pulp. The mixtures were loaded in 2.5 mL centrifuge tubes; then turned end-over-end on a home-made rotator. Tubes in triplicates were removed over 1 h and then centrifuged at 5,000 rpm. The adsorbed enzymes were determined by the difference between the amounts of initially added protein and free protein in the supernatant assayed by the Bradford method.
Kumar and Wyman (2009) indicated that adsorption parameters (maximum adsorption capacity [σ] and equilibrium constant [Kd]) were determined by non-linear regression of the adsorption data to the Langmuir expression, using Sigma Plot software (ver 10.0, SPSS Inc., Chicago):
[CE] = (σ × [St] × [Ef]) / (Kd + [Ef]) (3)
where [CE] is the amount of adsorbed enzyme in mg/mL, [Ef] is the free enzyme concentration in mg/mL, σ is the maximum adsorption capacity in mg/mg substrate, [St] is the substrate concentration in mg/mL, and Kd is the equilibrium constant = [C][E]/[CE] in mg of enzyme/mL (Kumar and Wyman, 2009).
To determine enzyme adsorption-desorption kinetics during hydrolysis, the dosage at 6 mg enzyme per gram O.D. pulp was chosen with the above solution at 40℃. Tubes in triplicates were removed over 1 h to 48 h to quantify the adsorbed enzymes. The digestibility was measured by the change of intrinsic viscosity and the release of reducing sugars. Intrinsic viscosities were analyzed following ISO 5351: 2004 standard
method. The released reducing sugars were measured by the dinitrosalicylic acid (DNSA) method.
(X) Enzyme Treatment
The pulp was treated with 1 IU, 2.5 IU and 5 IU of cellulases and endoglucanases per gram of oven-dry (O.D.) pulp. This treatment was carried out with a pulp suspension having a consistency of 10% at 40℃, in beakers, with continuous mechanical agitation.
The following reaction times were tested for 1 h. The pH was adjusted to 7 with Tris buffer for cellulases treatment.
(XI) Pulp Refining
The fully bleached spruce pulp was place in a polyethylene bag at 10% consistency at pH 7.0, 40℃ for 1 h, simulating actual mill operating conditions. Constant pH values were monitored for the filtrates of the pulp-enzyme mixtures before and after enzyme treatment. Crude 38-kDa Cel-BL11 cellulase directly from cell lysate after the disruption of E. coli was applied at levels of 2.5 IU and 5 IU per gram of oven-dried pulp (O.D.). After treatment, the reaction mixtures were washed, diluted up to 400 mL with cold water and subjected to freeness tests. The recovered pulp was beaten at 10%
consistency in a PFI (Papir-og fiberinstituttet) mill, following ISO 5264-2 (ISO, 2002), homogenized in a disintegrator at 1.2% consistency for 2 min and subjected to freeness measurements.
Freeness values of pulp were measured by TAPPI method T227-om04 (TAPPI, 2004) and expressed as Canadian Standard Freeness (CSF) values. The fiber hydratation was determined using the water retention value (WRV), according to the method described by Silvy et al. (1968). This method consists of the soaking of the pulp samples in water
with further centrifugation, and the WRV was calculated from the following equation:
WRV [%] = [(Ww–Wd)/Wd]×100, where Ww is the mass of the wet sample after centrifugation, and the Wd is that after wet sample drying at 105℃ to constant weight.
Handsheets of 75 ± 2 g/m2 grammage were prepared on Rapid-Köhten equipment according to ISO 5331 and tested mechanically in accordance with the following standards: tensile index, ISO 1924; burst index, ISO 2758; tear index, ISO 1974; folding endurance, ISO 5626. Experimental errors were calculated as prescribed by the respective standards.
(XII) Morfi
Morfi (TECHPAP, 10 rue de Mayencin 38400 Saint Martin d’Hères) was used in this experiment to analyzed the morphology of fibers. 30 mg pulp samples were put in 1 L plastic beacker with water to 0.3% consistency, and to analyze using Morfi. Morfi provides the distribution of fines area, fines length, fiber distribution, and fiber width.
Fiber length was 200-10,000 μm, fiber width was 5-75 μm, fines length was defined shorter than 200 μm, and width of fines was defined as shorter than 5 μm.
(XIII) Scanning Electron Microscope (SEM)
The sample of spruce fibers before tested would be formed very thin papers; then, the fibers were coated with gold to provide electrical conductivity. SEM was used to analyze fiber morphology using and acceleration voltage of 20 kV. The tested fibers were randomly chosen, and the better image has a whole fiber in the picture center.
Their dimensions were measured using software (Shi et al., 2011).
IV Results and Discussion
(I) Impacts of Lignin Contents and Yeast Extract Addition on the Interaction between Spruce Pulps and Crude Recombinant Paenibacillus Endoglucanase
Chemical compositions of the pulps were listed in Table 1. Pulp samples were coded with their total lignin contents as follows: LIG 12.4, LIG 8.29, LIG 6.22, and LIG 1.24.
The result of statistics showed the chemical properties of four pulps have some difference, analyzed by Scheffé's method.
Table 1. Chemical properties of four spruce pulps.
Chemical properties
Sample code
LIG 12.4 LIG 8.3 LIG 6.2 LIG 1.2 Pulp lignin content (%) 12.43 ± 0.13 8.29 ± 0.14 6.22 ± 0.12 1.24 ± 0.03
Holocellulose (%) 83.69 ± 0.10 89.50 ± 0.08 93.45 ± 0.05 98.72 ± 0.01 α-cellulose (%) 69.81 ± 0.09 70.54 ± 0.09 78.71 ± 0.02 83.73 ± 0.04 Pentosan (%) 8.83 ± 0.11 8.53 ± 0.13 6.65 ± 0.09 5.98 ± 0.08
DP 3876 ± 85 3226 ± 63 2462 ±37 2135± 26
1. Effects on the Accessibility of Proteins
The effect of lignin contents and added yeast extract addition on the accessibility of proteins (crude endoglucanase) onto to the four spruce pulps at 4oC was shown in Fig. 1.
The results at 4oC demonstrated that the pulp with more lignin contents accommodated more proteins as shown Fig. 1A. The above observation contradicts the findings that biomass with more cellulose favored cellulase adsorption, according to several studies using purified cellulases (Chernoglazov et al., 1988), mixed cellulase (Ooshima et al.,
1990; Boussaid and Saddler, 1999), purified CBH (Ishizawa et al., 2009), and commercial endoglucanase (Ko et al., 2011). The above discrepancy could be explained:
CMCase activity of the purified and crude recombinant Paenibacillus endoglucanase was measured at 250 IU/mg (Ko et al., 2010b) and 35.2 IU/mg (this study) at pH 7 and 40oC. So it could be assumed that the mass of irrelevant proteins were 6.1 times more than the purified endoglucanase. In contrast to the behaviors of mostly pure cellulases used in the above studies, Fig. 1A described the adsorption behavior of the mixture of less endoglucanase and much more irrelevant proteins derived from cell rupture. Figure 1A showed that the interaction between irrelevant protein and overall pulp surfaces dominated those between pure endoglucanase and cellulose surface.
The addition of yeast extract facilitated even more protein adsorption onto pulps with more lignin contents, as shown in Fig. 1B (Yang and Wyman, 2006). Yeast extract, composted of amino acid and peptides, also could be regarded as of a mixture of electrolyte and polyelectrolyte. Hence, the above finding was consistent to the effect on increasing protein adsorption by the addition of bovine serum albumin (BSA, also a polylelectolyte) (Yang and Wyman, 2006) and polyethylene glycol (PEG) (Ouyang et al., 2010).
0.00 0.02 0.04 0.06 0
2 4 6 8 10 12
0.00 0.02 0.04 0.06 Adsorbed protein (mg g-1 substrate)
Free protein in the supernatant (mg mL-1)
A B
Fig. 1. Effect of yeast extract addition on the accessibility of protein (crude recombinant Paenibacillus endoglucanase) on four spruce pulps at 4oC. Panel A: Controls with closed symbols. Panel B: Yeast extract with open symbols. Legends of pulps with different lignin contents: LIG 12.4 (★), LIG 8.3 (■), LIG 6.2 (▲), LIG 1.2 (●).
The adsorption parameters were also estimated by nonlinear regression of adsorption data for the protein (very crude endoglucanase) and four spruce pulps with or without yeast extract addition action, using the Langmuir equation. As illustrated in Table 2, the adsorption data of the protein mostly followed the Langmuir relationship. The results of Table 2 showed that the addition of yeast extract significantly increased the maximum adsorption capacities of the protein onto all four pulps. The maximum adsorption capacities [σ] in Table 3 were much lower than published values of treated biomass by leading treatments (Kumar and Wyman, 2009), although in same order of magnitude.
The above observation might be due to much smaller specific surface areas of complete softwood fibers used in the study, when compared with much smaller fibers obtained by leading treatments (Kumar and Wyman, 2009). In general, the maximum adsorption capacities [σ] decreased with pulps of decreasing lignin contents. The above trend matched increasing equilibrium constants [Kd] with pulps of decreasing lignin contents.
The discrepancy between parameters of LIG 8.3 and LIG 6.2 might due to two competing interactions for irrelevant protein/overall pulp surfaces and pure endoglucanase/cellulose surface. Addition of yeast extract increased the maximum adsorption capacities [σ] and equilibrium constants [Kd] of all four pulps; the interaction between amino acid/peptides and pulp surface might create more available binding sites for the proteins.
Table 2. Effects of pulp lignin contents and yeast extract addition on the maximum adsorption capacity (σ) and equilibrium constants (Kd) of the protein onto four spruce pulps.
Sample code
σ Kd R2
Control Y. E. Control Y. E. Control Y. E.
LIG 12.4 7.14 13.93 0.0129 0.0128 0.9002 0.9624 LIG 8.3 6.19 14.87 0.0195 0.0325 0.9790 0.9760 LIG 6.2 4.29 11.20 0.0150 0.0265 0.9720 0.9922 LIG 1.2 2.02 3.82 0.0317 0.0519 0.8275 0.9712
Table 3. Effects of pulp lignin contents and dosages of endoglucanase applied on degradation rate constants (k) and leveling-off degree of polymerization (LODP) during hydrolysis of four spruce pulps.
Sample code
Enzyme dosage (IU/g. o. d. p.)
5 10 20 50
k LODP k LODP k LODP k LODP
LIG 12.4 0.2132 3192 0.2514 2783 0.2958 2624 0.3250 2410 LIG 8.3 0.3141 2147 0.3287 2148 0.3736 2069 0.3856 1982 LIG 6.2 0.2518 1996 0.2913 1949 0.3179 1810 0.3352 1550 LIG 1.2 0.1815 1489 0.1841 1441 0.1863 1392 0.1940 1297
2. Effects on the Preferential Adsorption of Irrelevant Proteins
To further verify the different adsorption of endoglucanase and of irrelevant proteins, the specific endoglucanase (CMCase) activities of supernatant portion during adsorption were separately measured at pH 7 and 40oC. Yeast extract couldn’t interfere with the Bradford assay, or with the DNSA assay. Since the CMCase activity of crude recombinant Paenibacillus endoglucanase was measured at 35.2 IU/mg at pH 7 and 40oC, the preferential adsorption of irrelevant proteins could be indicated by the more than 35.2 IU/mg activity values of proteins. The effect of yeast extract addition on specific endoglucanase activity of proteins in supernatants is shown in Fig. 2. Figure 2A showed specific endoglucanase activity of proteins in supernatants without yeast extract addition. With sample LIG 12.4, the specific endoglucanase activities of proteins in supernatants were higher than 35.2 IU/mg. The above trend was more so at lower total proteins loading. However, an opposite trend was shown for LIG 1.2: the specific endoglucanase activity of proteins in supernatants was slightly lower than 35.2 IU/mg at low protein loading. Interestingly, the specific endoglucanase activities of proteins in supernatants for all pulps more or less converged at around 35.2 IU/mg. It could be deduced that the interaction between cellulose and endoglucanase dominated for the low lignin pulp (LIG 1.2) at low protein loading. On the other hand, the interaction between pulp surface and irrelevant proteins dominated for the high lignin pulps at low protein loadings. Protein concentrations were quite dilute at low protein loadings, and the collisions between endoglucanase/irrelevant proteins and pulp surface were fewer. In the above case, not every collision led to successful adsorption. However, the discretion between endoglucanase/irrelevant proteins was diminished at higher protein loading, since there were too many collisions between both endoglucanase/irrelevant proteins and pulp surfaces.
0.01 0.02 0.03 0.04 0.05 0.06 30
35 40 45 50 55 60 65
0.01 0.02 0.03 0.04 0.05 0.06
A
Specific endoglucanase activity of protein in supernatant(IU/mg)
Free protein content in the supernatant (mg/mL)
B
Fig. 2. Effect of yeast extract addition on specific endoglucanase activity of proteins in supernatants. Panel A: Controls with closed symbols. Panel B: Yeast extract with open symbols. Legends of pulps with different lignin contents: LIG 12.4 (★), LIG 8.3 (■), LIG 6.2 (▲), LIG 1.2 (●).
Figure 2B shows the specific endoglucanase activity of proteins in supernatants with yeast extract addition. Since a lot of amino acid and peptide (with much smaller molecular weights) was brought by yeast extract addition, the surfaces of all pulps were further attached by yeast extract to facilitate the adsorption by irrelevant proteins. Again,
Figure 2B shows the specific endoglucanase activity of proteins in supernatants with yeast extract addition. Since a lot of amino acid and peptide (with much smaller molecular weights) was brought by yeast extract addition, the surfaces of all pulps were further attached by yeast extract to facilitate the adsorption by irrelevant proteins. Again,