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