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Fractionation and cellulase treatment for enhancing the properties of kraft-based dissolving pulp
Bioresource Technology xxx (2016) xxx–xxx
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Fractionation and cellulase treatment for enhancing the properties of kraft-based dissolving pulp Chao Duan a,b,⇑, Xinqi Wang a, YanLing Zhang a, Yongjian Xu a, Yonghao Ni a,b a b
College of Bioresources Chemical and Materials Engineering, Shaanxi University of Science and Technology, Xi’an 710021, China Limerick Pulp and Paper Centre, University of New Brunswick, Fredericton, New Brunswick E3B 5A3, Canada
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
A combined process to improve MWD
and reactivity of dissolving pulp was proposed. Pulp fractionation and cellulase treatment of each fraction were involved in the combined process. The SF had the highest accessibility and highest cellulase adsorption capacity. The proposed process led to a narrower MWD and a higher reactivity due to more homogenous reactions.
a r t i c l e
i n f o
Article history: Received 3 October 2016 Received in revised form 22 October 2016 Accepted 25 October 2016 Available online xxxx Keywords: Dissolving pulp Kraft-based process Fractionation Cellulase treatment Molecular weight distribution Fock reactivity
a b s t r a c t The aim of this study was to investigate a combined process involving pulp fractionation and cellulase treatment of each fraction for improving the molecular weight distribution (MWD) and reactivity of a kraft-based dissolving pulp. Three pulp fractions, namely long-fiber, mid-fiber and short-fiber fractions (LF, MF and SF, respectively), were used as the substrates. The results showed that the SF had the highest accessibility, lowest viscosity, and highest cellulase adsorption capacity, while the opposite was true for the LF. At a given viscosity, the combined process led to a lower polydispersity index (3.71 vs 4.98) and a higher Fock reactivity (85.6% vs 76.3%), in comparison to the conventional single-stage cellulase treatment. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Dissolving pulp (i.e., lignocellulose-derived pulp with a high cellulose content) has attracted intensive interest in recent years due to its green and sustainable nature for many applications, including cellulose rayon, cellulose esters, cellulose ethers, and ⇑ Corresponding author at: College of Bioresources Chemical and Materials Engineering, Shaanxi University of Science and Technology, Xi’an 710021, China. E-mail address: [email protected] (C. Duan).
other cellulose-based new products (e.g. cellulose nano-crystals, cellulose filaments) (Miao et al., 2014; Sixta et al., 2013; Wang et al., 2015a). As a dominant consumer, viscose rayon manufacturers always have a high demand for the quality parameters of dissolving pulp (e.g. a narrow molecular weight distribution and a high reactivity), to ensure homogenous and efficient reactions in downstream processes, such as mercerization and xanthation (Ibarra et al., 2010b; Strunk et al., 2011; Tian et al., 2014). However, in practice, in order to mitigate the material shortage and save the cost, mills often manufacture the dissolving pulp using mixed
http://dx.doi.org/10.1016/j.biortech.2016.10.077 0960-8524/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Duan, C., et al. Fractionation and cellulase treatment for enhancing the properties of kraft-based dissolving pulp. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.10.077
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A commercial endoglucanase-rich cellulase (FiberCare D, EG) was supplied by Novozymes A/S (Denmark) and its cellulase activity of 460 U/mL was determined as sodium carboxymethyl cellulose (CMC-Na) activity. The protein concentration of FiberCare D solution was 68 mg protein/mL cellulase, as determined by following the Bradford method using a coomassie protein assay kit that was purchased from Sigma-Aldrich.
wood chips as their feedstock, which results in products with poor homogeneity (Chen et al., 2016; Duan et al., 2015a). Wood-derived pulp fibers can be fractionated based on fiber sizes to minimize the size-induced differentiation, and the fractionation treatment has been practiced or demonstrated in terms of pulp and paper production (Abubakr et al., 1995; Lei et al., 2013; Li et al., 2015). For the paper industry, Abubakr et al. (1995) studied the fractionation of mixed office waste fibers to upgrade fiber quality, and found that fractionation was effective in upgrading the long-fiber component, thus increasing the strength indices of paper. In the area of dissolving pulp, Li et al. (2015) developed a sequential process consisting of fiber fractionation and followed by cold alkali extraction (CCE), and the results showed that hemicelluloses removal for the long-fiber fraction was more pronounced than the short-fiber fraction. Recently, cellulase treatment has shown to be effective in improving the critical properties of kraft-based dissolving pulp because it can not only adjust pulp viscosity, but also enhance pulp reactivity (Duan et al., 2016; Engstrom et al., 2006; Gehmayr and Sixta, 2012; Wang et al., 2015b). The efficiency of cellulase treatment is closely related to substrate-related factors: the intimate contact between enzyme and cellulose is the prerequisite for efficient enzymatic treatment (Arantes and Saddler, 2011; Meng and Ragauskas, 2014). For dissolving pulp, cellulose accessibility is mainly governed by fiber morphology, crystallinity and degree of polymerization (DP), etc. (Duan et al., 2015a; Gehmayr and Sixta, 2012; Leu and Zhu, 2012). Generally, the cellulase preferentially adsorbs onto substrates with a high cellulose accessibility, and these substrates can be characteristic of smaller size, high specific surface area, large porosity, and low crystallinity (Ju et al., 2013; Ko et al., 2011). The objective of this study was to develop a sequential process consisting of pulp fractionation and cellulase treatment to improve the molecular weight distribution and reactivity of kraft-based dissolving pulp. The effect of pulp fractionation on cellulase treatment efficiency was investigated. Three fiber fractions, namely long-fiber fraction (LF), mid-fiber fraction (MF), and short-fiber fraction (SF), fractioned from a kraft-based dissolving pulp, were used for this purpose. Cellulose accessibility, cellulase adsorption, and viscosity reduction (chain scission) kinetics of each fiber fraction were also discussed.
2.2. Methods 2.2.1. Pulp fractionation Fractionation was carried out with a Bauer-McNett fiber classifier (MC Tec Co., Ltd, Giessen, Netherlands) by following Tappi T 233 cm-95. Pulp fibers that were retained on the 50-mesh screen were the LF, those fibers passed through 50-mesh screen but retained on the 200-mesh screen were the MF, while the SF was defined as those fibers that passed through the 200-mesh screen. 2.2.2. Enzyme adsorption kinetics and protein content measurements The cellulase adsorption experiments were conducted using a buffer (pH 4.8) in a 500 mL beaker placed in a water bath shaker (25 °C, 200 rpm). Approximately 5 g of the slurry was taken out at intervals and filtered through a 0.45 mL syringe membrane to collect the fiber-free permeate. The amount of cellulase adsorbed to pulps was determined as the difference between the initial amount of cellulase added to the beaker and the amount of free cellulase in the aqueous medium. The amount of free cellulase (protein) was determined by following the Bradford method using bovine serum albumin (BSA) as the standard (Duan et al., 2015b). 2.2.3. Cellulase treatment Cellulase treatment was conducted using 5 g (oven dry weight) of pulp (whole pulp or each pulp fraction, 10% pulp consistency) at varying treatment times in a citrate buffer system (pH 4.8), and a polyethylene bag placed in a water bath (55 °C) was used. For a homogeneous distribution, cellulase was added to the buffer, and the mixture was then added to the pulp. The cellulase e dosage was 0.23 U/g odp or 0.5 mg cellulase/g odp unless otherwise stated. The samples were periodically taken out of the bath and kneaded for 10 to 15 s, in particular within the initial 10 min. After the completion of the cellulase treatment, the samples were placed in hot water (90 °C) for 15 min to denature the enzymes, and subsequently filtered and washed. For the conventional process, namely a single-stage cellulase treatment of the whole pulp, 0.5 g of the pulp was treated with 0.5 mg cellulase/g odp for 60 min to obtain a cellulase-treated sample with a viscosity of 460 ml/g. In the case of the combined process, namely an initial pulp fractionation followed by a cellulase treatment on each fraction, 0.5 g of the LF, MF and SF were treated with 0.5 mg cellulase/g odp for 20, 40 and 120 min to obtain three samples with viscosities of 458, 463 and 465 mL/g, respectively. After that, the three cellulase-treated samples were mixed together based on their weighted percent (shown in Table 1) to re-make the pulp sample with a final viscosity of 462 mL/g. All the cellulase treatment times
2. Experimental 2.1. Materials A commercial hardwood-derived dissolving pulp from the prehydrolysis kraft-based process was provided by a pulp mill in Eastern Canada. The whole pulp (WP) was subjected to rewetting and dispersion, and then used for the preparation of the three fiber fractions, namely the long-fiber fraction (LF), mid-fiber fraction (MF), and short-fiber fraction (SF), respectively. All the samples were stored in sealed plastic bags and refrigerated prior to subsequent treatments and analyses. The properties of pulp samples with respect to yield, purity, viscosity and Fock reactivity are listed in Table 1.
Table 1 Characteristics of pulp samples. Sample
WP
LF
MF
SF
Yield, % Glucose, % Hemicellulose, % Intrinsic viscosity, g/mL Fock reactivity, %
– 94.76 ± 0.25 4.43 ± 0.08 560 ± 4 48.9 ± 0.4
62.5 ± 0.5 95.39 ± 0.22 4.02 ± 0.10 591 ± 5 45.5 ± 0.2
26.1 ± 0.3 94.02 ± 0.32 5.21 ± 0.12 548 ± 4 42.3 ± 0.3
11.4 ± 0.2 93.03 ± 0.28 6.45 ± 0.13 500 ± 3 56.4 ± 0.4
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during the conventional and sequential processes were determined according to the parameters relevant to viscosity decrease kinetics.
samples were air-dried to constant moisture content in a constant temperature/humidity room.
2.3. Analyses
3. Results and discussion
2.3.1. Cellulose accessibility Cellulose accessibility can be determined on the basis of morphological changes, which involves several parameters: fiber length, crystallinity, pore structure, and specific surface area. The fiber length of each fraction was measured using the Fiber Quality Analyzer (OpTest Equipment Inc., Canada). The crystallinity of each fraction was determined based on the X-ray diffraction (XRD) method (Duan et al., 2015a; Park et al., 2010). The XRD scattering analysis of the samples was carried out on a Bruker D8 XRD system using Cu-K as a source (k = 0.154 nm) in the 2h range 5°–40°, with scanning speed of 1.2°/min. The pore structure (pore volume and diameter) and specific surface area of each fiber fraction were measured based on the Brunauer-Emmett-Teller (BET) analysis of nitrogen absorption isotherms using a Belsorp-Max volumetric gas adsorption instrument (Bel Japan, Inc., Osaka, Japan).
3.1. Proposed process concept involving pulp fractionation and cellulase treatment for improving the critical properties of dissolving pulp
Chain scission ¼ 1=DP t 1=DP 0
ð1Þ
The hypothesis of the present study is that a sequential treatment consisting of pulp fractionation and cellulase treatment may facilitate the production of purified cellulose products (dissolving pulp) with a narrow molecular weight distribution. Such a treatment is expected to more efficient than a single-stage cellulase treatment (the conventional process) even at a similar cellulose viscosity. In accordance with this hypothesis, different fiber fractions, with varying chemical compositions, viscosities, and physical/chemical characteristics, may have different behaviors in the subsequent cellulase treatment. For the conventional process (Left in S-Fig. 1), the cellulase would be preferentially adsorbed onto the SF due to its highest cellulose accessibility. Consequently, upon cellulase treatment, the viscosity of the SF (the lowest among the three fiber fractions, as shown in Table 1) would be more readily decreased than that of the LF, which would lead to inhomogeneous reactions in the whole pulp, thus resulting in a broader MWD of the resultant dissolving pulp. By contrast, for the sequential treatment (Right in S-Fig. 1), the introduction of pulp fractionation prior to cellulase treatment tends to facilitate homogeneous reactions in each fiber fraction, and hence the resultant dissolving pulp can have a narrower MWD and an even higher reactivity. Dasari and Berson (2007) had studied the effect of initial size of sawdust on enzymatic hydrolysis. The results revealed that the sawdust with a smaller size had a higher substrate accessibility, thus facilitating more efficient reactions between substrate and cellulase, Lv et al. (2011) investigated the adsorption behavior of cellulase on bleached softwood fibers with varying fiber lengths, and found that the short-fiber fraction (48–100 mesh) had higher cellulase adsorption than the other two fractions (larger than 28 mesh and 28 48 mesh). In a recent study relevant to the interaction of cellulase with dissolving pulp, it was found that when compared to the air-dried and oven-dried kraft-based dissolving pulps, the never-dried one had the highest cellulase adsorption and lowest viscosity due to its highest accessibility to cellulase (Duan et al., 2015b).
DP0:905 ¼ 0:75 ½g
ð2Þ
3.2. Fiber characteristics and accessibilities of different pulp fractions
2.3.2. Carbohydrates analyses The carbohydrates composition of the pulp samples was determined using an Ion Chromatography (IC) unit equipped with CarboPac TM PA1 column (Dionex-300, Dionex Cooperation, Canada) and a pulsed amperometric detector (PAD). The detailed sample preparation procedure was based on an NREL protocol (Sluiter et al., 2008). 2.3.3. Intrinsic viscosity and chain scission The intrinsic viscosity of all samples was measured according to the T 230 om-99 standard method using copperethylenediamine (CED) solution as solvent. All measurements were carried out in duplicate and the average results were presented. The chain scission (cellulose depolymerization), which represents the number of chain cleavage steps per initial cellulose, can be calculated from the degree of polymerization (DP), as shown in Eq. (1) (Duan et al., 2015b). The conversion between cellulose DP and intrinsic viscosity was based on the SCAN C15:62 as expressed in Eq. (2).
where DP0 is the initial DP of cellulose, DPt is the DP at a random time (t) and the [g] is intrinsic viscosity. 2.3.4. Molecular weight distribution The determination of molecular weight distribution (MWD) was conducted on a GPC device (Waters 600E), equipped with a differential refractometer detector (Waters 410) and a Waters Styragel HR 5E column. Prior to determination, the pulp sample was dissolved in an 8% DMAc/LiCl solution. The mobile phase of 0.5% DMAc/LiCl was pumped into the system at a flow rate of 0.3 mL/ min. The system was operated at a column temperature of 50 °C, with the injection volume of 10 lL. 2.3.5. Fock reactivity The Fock reactivity can usually be used as an indicator of the reactivity of pulp, which was conducted according to the modified method reported in the literature (Tian et al., 2013). The cellulose xanthation was performed at 19 °C in a water bath, and the pulp
The fiber morphologies of each pulp fraction, with respect to fiber length, crystallinity, pore diameter, specific surface area (SSA), and water retention value (WRV) are shown in Table 2. After pulp fractionation, the mean fiber length for the LF fraction was 0.80 mm, while the mean lengths for the MF and SF fractions were 0.44 cm and 0.27 cm, respectively. Correspondingly, the crystallinity for the LF fraction was the highest (66.79%), followed by the MF fraction (64.14%), while the SF fraction had the lowest crystallinity (62.42%). Thus, a larger size of the fiber fraction was associated with a higher crystallinity. By contrast, the specific surface area (SSA) for the SF was the highest (2.28 m2/g), followed by 1.62 m2/g for the MF, and 1.15 m2/g for the LF. In addition, the mean pore diameter was 7.04, 5.81 and 4.35 nm for the LF, MF, and SF, respectively. Although the mean pore diameter for the SF was the lowest, the WRV of the SF was the highest (118%), which can be explained by the fact that the SF contained the highest fines content with the highest SSA and free hydroxyl groups, thus exhibiting the highest swelling efficiency (Chen et al., 2009).
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Table 2 Fiber lengths, crystallinities, pore diameters, specific surface areas (SSA) and water retention values (WRV) of different fiber fractions. Sample
WP
LF
MF
SF
Fiber length, mm Crystallinity, % SSA, m2/g Pore diameter, nm WRV, %
0.64 ± 0.06 64.9 ± 0.5 1.43 ± 0.03 6.42 ± 0.22 101 ± 4
0.80 ± 0.08 66.8 ± 0.4 1.15 ± 0.02 7.04 ± 0.28 95 ± 3
0.44 ± 0.03 64.2 ± 0.3 1.62 ± 0.03 5.81 ± 0.18 106 ± 3
0.27 ± 0.02 62.4 ± 0.4 2.28 ± 0.04 4.35 ± 0.15 118 ± 4
Based on the crystallinity, SSA and WRV of each fraction, it can be concluded that the SF had the highest cellulose accessibility among the three fiber fractions. In a previous study, Yeh et al. (2010) investigated the effect of milling pretreatment on enzymatic hydrolysis of cotton cellulose, and found that the milling pretreatment decreased the cotton particle size to submicron scale, the specific surface area (SSA) increased, and the crystallinity decreased; as a result, the rate of enzymatic hydrolysis increased at least 5 folds at a 3% substrate concentration.
3.3. Cellulase adsorption The results on the cellulase adsorption for each fraction are shown in Fig. 1. The SF sample had the highest amount of adsorbed cellulase in comparison with the MF and LF samples, for example, 94.4% (of the added cellulase) for the SF, while 88.8% and 82.4% for the MF and LF sample respectively after 120 min. Ko et al. (2011) investigated the effect of fiber morphology on the interaction between EG and eucalypt kraft pulp and found that a high accessibility of the pulp lead to a higher maximum enzyme adsorption capacity. The adsorption of cellulase onto the three pulp fractions and original pulp nearly reached equilibrium within 60 min and displayed a significant binding rate during the first 20 min, especially for the SF (approx. 75% bound fraction). In a study, Tu et al. (2007) reported that the cellulase binding on both the steam exploded and ethanol pretreated wood chips of Lodgepole pine reached a plateau at about 60 min. These results suggested that the cellulase adsorption onto the different fiber fractions was highly correlated to their accessibilities, thus, to some extent, determining the enzymatic treatment efficiency, such as the viscosity decrease and chain scission during the cellulase treatment, both of which will be discussed in the following section.
Fig. 1. Adsorption of cellulase on different fiber fractions (5% pulp consistency, 25 °C, pH 4.8, total initial cellulase loading of 340 lg protein/g odp).
3.4. Kinetics of viscosity decrease and chain scission of pulp samples during the cellulase treatment Shown in Fig. 2 are the kinetics of the initial whole pulp (WP) and three pulp fractions (SF, MF and LF) with respect to viscosity and chain scission during the cellulase treatment. The viscosity of the SF sample decreased most significantly in comparison with those of the MF and LF samples during the cellulase treatment process, and particularly in the first 2 h, the viscosity decreased by 162 units for SF sample as compared to 143, 132 and 125 units for the MF, LF and WP samples respectively (Fig. 2a). In addition, a rapid reduction in viscosity was observed in the initial few hours of the cellulase treatments, but slowed down as the process continued, which is consistent with the behavior of cellulase-treated dissolving pulp (without pulp fractionation) as shown in a previous study (Duan et al., 2015b). These results were further evaluated
Fig. 2. Kinetics of (a) viscosity and (b) chain scission of different samples during the cellulase treatment (cellulase dosage of 0.5 mg/g odp, 10% pulp consistency, pH 4.8, and 55 °C).
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C. Duan et al. / Bioresource Technology xxx (2016) xxx–xxx Table 3 Molecular weight, polydispersity index and Fock reactivity of different samples under different treatment conditions. Sample 1
Sample A Sample B2 Sample C3 1 2 3
Mw, kg/mol
Mn, kg/mol
PDI
Fock reactivity, %
326.5 ± 5.0 289.3 ± 4.2 270.1 ± 3.9
79.2 ± 1.4 58.1 ± 0.8 72.8 ± 1.2
4.12 ± 0.15 4.98 ± 0.14 3.71 ± 0.12
48.9 ± 0.4 76.3 ± 0.2 85.6 ± 0.3
Sample A: the original untreated dissolving pulp. Sample B: cellulase- treated pulp based on the conventional process (treatment time of 60 min for the WP, pulp viscosity of 460 mL/g). Sample C: cellulase- treated pulp based on the combined sequential process (20, 40, 120 min for SF, MF and LF, respectively, and pulp viscosity of 462 mL/g).
in terms of the cellulose chain scission. As shown in Fig. 2b, the SF sample showed the highest depolymerization rate in the first four hours and the most robust continuing chain scission during the following cellulase treatment process among the four samples studied. Therefore, it is evident that the enzymatic treatment efficiency was highly dependent on the cellulose accessibility and cellulase adsorption: a substrate with higher cellulose accessibility and cellulase adsorption will have higher enzymatic treatment efficiency. Ibarra et al. (2010a) reported that upon enzymatic treatment (N476, an EG enzyme), the never-dried acid sulfite dissolving pulps (derived from hardwood and softwood) with less pulp hornification and thus better cellulose accessibility exhibited more pronounced viscosity drops in comparison with those dried pulps due to the fact that never-dried pulps. In another study, Luo et al. (2011) investigated the enzymatic hydrolysis of two types of substrates: pretreated lignocellulosic material and bleached pulp sample, and the results indicated that the substrate enzymatic digestibility (SEDs) was proportional to the adsorbed amount of a commercial endoglucanase. Moreover, apart from more available surface/mass ratio, the more remarkable viscosity reduction and chain scission in the case of SF fraction may be also attributed to its better mass transfer between the solid (cellulose fibers) and liquid (enzyme solution) phases (Wojtusik et al., 2016). On the basis of these results, it was concluded that the cellulase was preferentially adsorbed onto the SF due to its highest cellulose accessibility, and the viscosity decrease and chain scission of the SF were more remarkable than those of other fiber fractions.
3.5. Molecular weight distribution and reactivity As shown in Fig. 3, Sample A, i.e., the original untreated dissolving pulp, had the highest molecular weight (Mw) of cellulose fraction among the three samples. After cellulase treatment, the highMw peaks for both Sample B and C shifted to the low- Mw region. However, at a similar pulp viscosity (460 mg/L vs 462 mg/L), Sample C (produced based on the sequential treatment process), showed a narrower molecular weight distribution in comparison to Sample B (treated with the conventional process). The above results are consistent with those, of the polydispersity index (PDI) (Table 3). For example, the PDI of Sample A was 4.12, and that of Sample B (the conventional cellulase treated sample) was 4.98, due to the heterogenous nature of the treatment. In contrast, the PDI of Sample C (after the combined treatment process) was 3.71, significantly lower than that of Sample B. Included in Table 3 are the reactivity data. As shown, the Fock reactivity of Sample A was 48.9%, and that of Sample B (the conventional cellulase treated sample) was 76.2%. Importantly, the Fock reactivity of Sample C (after the sequential treatment process) was 85.6%. The higher Fock reactivity in Sample C as compared to Sample B, can be attributed to more homogeneous reactions for these fibers. Therefore, these results further support the hypothesis that the sequential/combined process can facilitate homogeneous reactions for each fiber fraction, resulting in a cellulose product with a narrow molecular weight distribution and a high reactivity. In terms of the commercial use of cellulase for the improvement of the properties of dissolving pulp with respect to molecular weight distribution and reactivity, it would be conceivable that the cellulase treatment can be applied to the fractionated cellulose fibers due to homogeneous, efficient reaction for each pulp fraction. The pulp fractionation can be readily conducted using the screening systems in the pulp mill, such as the pressure screen, and the adjustment of cellulase addition level as well as the reaction time for each pulp fraction in the combined process can be easily practiced by the industry. 4. Conclusions
Fig. 3. Molecular weight distribution (MWD) of untreated pulp and cellulasetreated pulps (cellulase dosage of 0.5 mg/g odp, 10% pulp consistency, pH 4.8, and 55 °C). Sample A: the original untreated dissolving pulp; Sample B: the cellulasetreated pulp based on the conventional process (treatment time of 60 min for the WP, and of pulp viscosity of 460 mL/g); Sample C: cellulase- treated pulp based on the sequential process (20, 40 and 120 min treatment times for SF, MF and LF, respectively, and pulp viscosity of 462 mL/g).
A sequential/combined process, consisting of pulp fractionation and cellulase treatment of each fraction, was investigated to improve the properties of a kraft-based dissolving pulp. Results showed that the SF had the lowest viscosity, highest accessibility, and cellulase adsorption capacity among the three fiber fractions. At a given viscosity (approx. 460 mL/g), the proposed combined process led to a narrower MWD (PDI, 3.71 vs 4.98) and a higher Fock reactivity (85.6% vs 76.3%) in comparison with the conventional process (single-stage cellulase treatment). The enhancement in MWD and reactivity was associated with homogeneous cellulase actions on each pulp fraction. Acknowledgement The authors would like to acknowledge the Doctoral Scientific Fund Project of Shaanxi University of Science and Technology
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(SUST), China and Atlantic Innovation Fund (AIF), Canada, for supporting this research. The authors would also like to thank Xiaojuan Ma of Fujian Agriculture and Forestry University (FAFU) for her assistance in the molecular weight distribution analysis. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2016.10. 077. References Abubakr, S.M., Scott, G.M., Klungness, J.H., 1995. Fiber fractionation as a method of improving handsheet properties after repeated recycling. Tappi J. 78 (5), 123– 126. Arantes, V., Saddler, J.N., 2011. Cellulose accessibility limits the effectiveness of minimum cellulase loading on the efficient hydrolysis of pretreated lignocellulosic substrates. Biotechnol. Biofuel 4 (1), 3–18. Chen, C., Duan, C., Li, J., Liu, Y., Ma, X., Zheng, L., Stavik, J., Ni, Y., 2016. Cellulose (Dissolving Pulp) manufacturing processes and properties: a mini-review. Bioresources 11 (2), 5553–5564. Chen, H., Park, A., Heitmann, J.A., Hubbe, M.A., 2009. Importance of cellulosic fines relative to the dewatering rates of fiber suspensions. Ind. Eng. Chem. Res. 48 (20), 9106–9112. Dasari, R.K., Berson, R.E., 2007. The effect of particle size on hydrolysis reaction rates and rheological properties in cellulosic slurries. Appl. Biochem. Biotechnol. 137 (1–12), 289–299. Duan, C., Li, J.G., Ma, X.J., Chen, C.X., Liu, Y.S., Stavik, J., Ni, Y.H., 2015a. Comparison of acid sulfite (AS)- and prehydrolysis kraft (PHK)-based dissolving pulps. Cellulose 22 (6), 4017–4026. Duan, C., Long, Y.D., Li, J.G., Ma, X.J., Ni, Y.H., 2015b. Changes of cellulose accessibility to cellulase due to fiber hornification and its impact on enzymatic viscosity control of dissolving pulp. Cellulose 22 (4), 2729–2736. Duan, C., Verma, S.K., Li, J., Ma, X., Ni, Y., 2016. Viscosity control and reactivity improvements of cellulose fibers by cellulase treatment. Cellulose 23 (1), 269– 276. Engstrom, A.C., Ek, M., Henriksson, G., 2006. Improved accessibility and reactivity of dissolving pulp for the viscose process: pretreatment with monocomponent endoglucanase. Biomacromolecules 7 (6), 2027–2031. Gehmayr, V., Sixta, H., 2012. Pulp properties and their influence on enzymatic degradability. Biomacromolecules 13 (3), 645–651. Ibarra, D., Köpcke, V., Ek, M., 2010a. Behavior of different monocomponent endoglucanases on the accessibility and reactivity of dissolving-grade pulps for viscose process. Enzyme Microb. Technol. 47 (7), 355–362. Ibarra, D., Kopcke, V., Larsson, P.T., Jaaskelainen, A.S., Ek, M., 2010b. Combination of alkaline and enzymatic treatments as a process for upgrading sisal paper-grade pulp to dissolving-grade pulp. Bioresour. Technol. 101 (19), 7416–7423. Ju, X., Grego, C., Zhang, X., 2013. Specific effects of fiber size and fiber swelling on biomass substrate surface area and enzymatic digestibility. Bioresour. Technol. 144, 232–239.
Ko, C., Chen, F., Lee, J., Tzou, D.M., 2011. Effects of fiber physical and chemical characteristics on the interaction between endoglucanase and eucalypt fibers. Cellulose 18 (4), 1043–1054. Lei, M., Zhang, H., Li, J., Duan, J., 2013. Characteristics of poplar preconditioning followed by refining chemical treatment alkaline peroxide mechanical pulp fiber fractions and their effects on formation and properties of high-yield pulp containing paper. Ind. Eng. Chem. Res. 52 (11), 4083–4088. Leu, S., Zhu, J., 2012. Substrate-related factors affecting enzymatic saccharification of lignocelluloses: our recent understanding. Bioenergy Res. 6 (2), 405–415. Li, J., Zhang, H., Duan, C., Liu, Y., Ni, Y., 2015. Enhancing hemicelluloses removal from a softwood sulfite pulp. Bioresour. Technol. 192, 11–16. Luo, X., Zhu, J., Gleisner, R., Zhan, H., 2011. Effects of wet-pressing-induced fiber hornification on enzymatic saccharification of lignocelluloses. Cellulose 18 (4), 1055–1062. Lv, J., Zhang, H., Fu, S., 2011. Adsorption behaviors of cellulase on cellulose fiber fractions with different fiber length. Trans. China Pulp Pap. 2, 37–41. Meng, X., Ragauskas, A.J., 2014. Recent advances in understanding the role of cellulose accessibility in enzymatic hydrolysis of lignocellulosic substrates. Curr. Opin. Biotechnol. 27, 150–158. Miao, Q., Chen, L., Huang, L., Tian, C., Zheng, L., Ni, Y., 2014. A process for enhancing the accessibility and reactivity of hardwood kraft-based dissolving pulp for viscose rayon production by cellulase treatment. Bioresour. Technol. 154, 109– 113. Park, S., Baker, J.O., Himmel, M.E., Parilla, P.A., Johnson, D.K., 2010. Research cellulose crystallinity index: measurement techniques and their impact on interpreting cellulase performance. Biotechnol. Biofuels 3 (10), 1–10. Sixta, H., Iakovlev, M., Testova, L., Roselli, A., Hummel, M., Borrega, M., van Heiningen, A., Froschauer, C., Schottenberger, H., 2013. Novel concepts of dissolving pulp production. Cellulose 20 (4), 1547–1561. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker, D. 2008. Determination of Structural Carbohydrates and Lignin in Biomass. LAP-002 NREL Analytical Procedure National Renewable Energy Laboratory Golden CO. Strunk, P., Eliasson, B., Hägglund, C., Agnemo, R., 2011. The influence of properties in cellulose pulps on the reactivity in viscose manufacturing. Nord. Pulp Pap. Res. J. 26 (1), 81–89. Tian, C., Zheng, L., Miao, Q., Nash, C., Cao, C., Ni, Y., 2013. Improvement in the Fock test for determining the reactivity of dissolving pulp. Tappi J. 12 (11), 21–26. Tian, C., Zheng, L.Q., Miao, Q.X., Cao, C.Y., Ni, Y.H., 2014. Improving the reactivity of kraft-based dissolving pulp for viscose rayon production by mechanical treatments. Cellulose 21 (5), 3647–3654. Tu, M., Chandra, R.P., Saddler, J.N., 2007. Recycling cellulases during the hydrolysis of steam exploded and ethanol pretreated Lodgepole pine. Biotechnol. Prog. 23 (5), 1130–1137. Wang, Q., Liu, S., Yang, G., Chen, J., Ni, Y., 2015a. Cationic polyacrylamide enhancing cellulase treatment efficiency of hardwood kraft-based dissolving pulp. Bioresour. Technol. 183, 42–46. Wang, Q., Liu, S., Yang, G., Chen, J., Ni, Y., 2015b. High consistency cellulase treatment of hardwood prehydrolysis kraft based dissolving pulp. Bioresour. Technol. 189, 413–416. Wojtusik, M., Zurita, M., Villar, J.C., Ladero, M., Garcia-Ochoa, F., 2016. Influence of fluid dynamic conditions on enzymatic hydrolysis of lignocellulosic biomass: effect of mass transfer rate. Bioresour. Technol. 216, 28–35. Yeh, A., Huang, Y., Chen, S.H., 2010. Effect of particle size on the rate of enzymatic hydrolysis of cellulose. Carbohydr. Polym. 79 (1), 192–199.
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Fractionation and cellulase treatment for enhancing the properties of kraft-based dissolving pulp Chao Duan a,b,⇑, Xinqi Wang a, YanLing Zhang a, Yongjian Xu a, Yonghao Ni a,b a b
College of Bioresources Chemical and Materials Engineering, Shaanxi University of Science and Technology, Xi’an 710021, China Limerick Pulp and Paper Centre, University of New Brunswick, Fredericton, New Brunswick E3B 5A3, Canada
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
A combined process to improve MWD
and reactivity of dissolving pulp was proposed. Pulp fractionation and cellulase treatment of each fraction were involved in the combined process. The SF had the highest accessibility and highest cellulase adsorption capacity. The proposed process led to a narrower MWD and a higher reactivity due to more homogenous reactions.
a r t i c l e
i n f o
Article history: Received 3 October 2016 Received in revised form 22 October 2016 Accepted 25 October 2016 Available online xxxx Keywords: Dissolving pulp Kraft-based process Fractionation Cellulase treatment Molecular weight distribution Fock reactivity
a b s t r a c t The aim of this study was to investigate a combined process involving pulp fractionation and cellulase treatment of each fraction for improving the molecular weight distribution (MWD) and reactivity of a kraft-based dissolving pulp. Three pulp fractions, namely long-fiber, mid-fiber and short-fiber fractions (LF, MF and SF, respectively), were used as the substrates. The results showed that the SF had the highest accessibility, lowest viscosity, and highest cellulase adsorption capacity, while the opposite was true for the LF. At a given viscosity, the combined process led to a lower polydispersity index (3.71 vs 4.98) and a higher Fock reactivity (85.6% vs 76.3%), in comparison to the conventional single-stage cellulase treatment. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Dissolving pulp (i.e., lignocellulose-derived pulp with a high cellulose content) has attracted intensive interest in recent years due to its green and sustainable nature for many applications, including cellulose rayon, cellulose esters, cellulose ethers, and ⇑ Corresponding author at: College of Bioresources Chemical and Materials Engineering, Shaanxi University of Science and Technology, Xi’an 710021, China. E-mail address: [email protected] (C. Duan).
other cellulose-based new products (e.g. cellulose nano-crystals, cellulose filaments) (Miao et al., 2014; Sixta et al., 2013; Wang et al., 2015a). As a dominant consumer, viscose rayon manufacturers always have a high demand for the quality parameters of dissolving pulp (e.g. a narrow molecular weight distribution and a high reactivity), to ensure homogenous and efficient reactions in downstream processes, such as mercerization and xanthation (Ibarra et al., 2010b; Strunk et al., 2011; Tian et al., 2014). However, in practice, in order to mitigate the material shortage and save the cost, mills often manufacture the dissolving pulp using mixed
http://dx.doi.org/10.1016/j.biortech.2016.10.077 0960-8524/Ó 2016 Elsevier Ltd. All rights reserved.
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A commercial endoglucanase-rich cellulase (FiberCare D, EG) was supplied by Novozymes A/S (Denmark) and its cellulase activity of 460 U/mL was determined as sodium carboxymethyl cellulose (CMC-Na) activity. The protein concentration of FiberCare D solution was 68 mg protein/mL cellulase, as determined by following the Bradford method using a coomassie protein assay kit that was purchased from Sigma-Aldrich.
wood chips as their feedstock, which results in products with poor homogeneity (Chen et al., 2016; Duan et al., 2015a). Wood-derived pulp fibers can be fractionated based on fiber sizes to minimize the size-induced differentiation, and the fractionation treatment has been practiced or demonstrated in terms of pulp and paper production (Abubakr et al., 1995; Lei et al., 2013; Li et al., 2015). For the paper industry, Abubakr et al. (1995) studied the fractionation of mixed office waste fibers to upgrade fiber quality, and found that fractionation was effective in upgrading the long-fiber component, thus increasing the strength indices of paper. In the area of dissolving pulp, Li et al. (2015) developed a sequential process consisting of fiber fractionation and followed by cold alkali extraction (CCE), and the results showed that hemicelluloses removal for the long-fiber fraction was more pronounced than the short-fiber fraction. Recently, cellulase treatment has shown to be effective in improving the critical properties of kraft-based dissolving pulp because it can not only adjust pulp viscosity, but also enhance pulp reactivity (Duan et al., 2016; Engstrom et al., 2006; Gehmayr and Sixta, 2012; Wang et al., 2015b). The efficiency of cellulase treatment is closely related to substrate-related factors: the intimate contact between enzyme and cellulose is the prerequisite for efficient enzymatic treatment (Arantes and Saddler, 2011; Meng and Ragauskas, 2014). For dissolving pulp, cellulose accessibility is mainly governed by fiber morphology, crystallinity and degree of polymerization (DP), etc. (Duan et al., 2015a; Gehmayr and Sixta, 2012; Leu and Zhu, 2012). Generally, the cellulase preferentially adsorbs onto substrates with a high cellulose accessibility, and these substrates can be characteristic of smaller size, high specific surface area, large porosity, and low crystallinity (Ju et al., 2013; Ko et al., 2011). The objective of this study was to develop a sequential process consisting of pulp fractionation and cellulase treatment to improve the molecular weight distribution and reactivity of kraft-based dissolving pulp. The effect of pulp fractionation on cellulase treatment efficiency was investigated. Three fiber fractions, namely long-fiber fraction (LF), mid-fiber fraction (MF), and short-fiber fraction (SF), fractioned from a kraft-based dissolving pulp, were used for this purpose. Cellulose accessibility, cellulase adsorption, and viscosity reduction (chain scission) kinetics of each fiber fraction were also discussed.
2.2. Methods 2.2.1. Pulp fractionation Fractionation was carried out with a Bauer-McNett fiber classifier (MC Tec Co., Ltd, Giessen, Netherlands) by following Tappi T 233 cm-95. Pulp fibers that were retained on the 50-mesh screen were the LF, those fibers passed through 50-mesh screen but retained on the 200-mesh screen were the MF, while the SF was defined as those fibers that passed through the 200-mesh screen. 2.2.2. Enzyme adsorption kinetics and protein content measurements The cellulase adsorption experiments were conducted using a buffer (pH 4.8) in a 500 mL beaker placed in a water bath shaker (25 °C, 200 rpm). Approximately 5 g of the slurry was taken out at intervals and filtered through a 0.45 mL syringe membrane to collect the fiber-free permeate. The amount of cellulase adsorbed to pulps was determined as the difference between the initial amount of cellulase added to the beaker and the amount of free cellulase in the aqueous medium. The amount of free cellulase (protein) was determined by following the Bradford method using bovine serum albumin (BSA) as the standard (Duan et al., 2015b). 2.2.3. Cellulase treatment Cellulase treatment was conducted using 5 g (oven dry weight) of pulp (whole pulp or each pulp fraction, 10% pulp consistency) at varying treatment times in a citrate buffer system (pH 4.8), and a polyethylene bag placed in a water bath (55 °C) was used. For a homogeneous distribution, cellulase was added to the buffer, and the mixture was then added to the pulp. The cellulase e dosage was 0.23 U/g odp or 0.5 mg cellulase/g odp unless otherwise stated. The samples were periodically taken out of the bath and kneaded for 10 to 15 s, in particular within the initial 10 min. After the completion of the cellulase treatment, the samples were placed in hot water (90 °C) for 15 min to denature the enzymes, and subsequently filtered and washed. For the conventional process, namely a single-stage cellulase treatment of the whole pulp, 0.5 g of the pulp was treated with 0.5 mg cellulase/g odp for 60 min to obtain a cellulase-treated sample with a viscosity of 460 ml/g. In the case of the combined process, namely an initial pulp fractionation followed by a cellulase treatment on each fraction, 0.5 g of the LF, MF and SF were treated with 0.5 mg cellulase/g odp for 20, 40 and 120 min to obtain three samples with viscosities of 458, 463 and 465 mL/g, respectively. After that, the three cellulase-treated samples were mixed together based on their weighted percent (shown in Table 1) to re-make the pulp sample with a final viscosity of 462 mL/g. All the cellulase treatment times
2. Experimental 2.1. Materials A commercial hardwood-derived dissolving pulp from the prehydrolysis kraft-based process was provided by a pulp mill in Eastern Canada. The whole pulp (WP) was subjected to rewetting and dispersion, and then used for the preparation of the three fiber fractions, namely the long-fiber fraction (LF), mid-fiber fraction (MF), and short-fiber fraction (SF), respectively. All the samples were stored in sealed plastic bags and refrigerated prior to subsequent treatments and analyses. The properties of pulp samples with respect to yield, purity, viscosity and Fock reactivity are listed in Table 1.
Table 1 Characteristics of pulp samples. Sample
WP
LF
MF
SF
Yield, % Glucose, % Hemicellulose, % Intrinsic viscosity, g/mL Fock reactivity, %
– 94.76 ± 0.25 4.43 ± 0.08 560 ± 4 48.9 ± 0.4
62.5 ± 0.5 95.39 ± 0.22 4.02 ± 0.10 591 ± 5 45.5 ± 0.2
26.1 ± 0.3 94.02 ± 0.32 5.21 ± 0.12 548 ± 4 42.3 ± 0.3
11.4 ± 0.2 93.03 ± 0.28 6.45 ± 0.13 500 ± 3 56.4 ± 0.4
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during the conventional and sequential processes were determined according to the parameters relevant to viscosity decrease kinetics.
samples were air-dried to constant moisture content in a constant temperature/humidity room.
2.3. Analyses
3. Results and discussion
2.3.1. Cellulose accessibility Cellulose accessibility can be determined on the basis of morphological changes, which involves several parameters: fiber length, crystallinity, pore structure, and specific surface area. The fiber length of each fraction was measured using the Fiber Quality Analyzer (OpTest Equipment Inc., Canada). The crystallinity of each fraction was determined based on the X-ray diffraction (XRD) method (Duan et al., 2015a; Park et al., 2010). The XRD scattering analysis of the samples was carried out on a Bruker D8 XRD system using Cu-K as a source (k = 0.154 nm) in the 2h range 5°–40°, with scanning speed of 1.2°/min. The pore structure (pore volume and diameter) and specific surface area of each fiber fraction were measured based on the Brunauer-Emmett-Teller (BET) analysis of nitrogen absorption isotherms using a Belsorp-Max volumetric gas adsorption instrument (Bel Japan, Inc., Osaka, Japan).
3.1. Proposed process concept involving pulp fractionation and cellulase treatment for improving the critical properties of dissolving pulp
Chain scission ¼ 1=DP t 1=DP 0
ð1Þ
The hypothesis of the present study is that a sequential treatment consisting of pulp fractionation and cellulase treatment may facilitate the production of purified cellulose products (dissolving pulp) with a narrow molecular weight distribution. Such a treatment is expected to more efficient than a single-stage cellulase treatment (the conventional process) even at a similar cellulose viscosity. In accordance with this hypothesis, different fiber fractions, with varying chemical compositions, viscosities, and physical/chemical characteristics, may have different behaviors in the subsequent cellulase treatment. For the conventional process (Left in S-Fig. 1), the cellulase would be preferentially adsorbed onto the SF due to its highest cellulose accessibility. Consequently, upon cellulase treatment, the viscosity of the SF (the lowest among the three fiber fractions, as shown in Table 1) would be more readily decreased than that of the LF, which would lead to inhomogeneous reactions in the whole pulp, thus resulting in a broader MWD of the resultant dissolving pulp. By contrast, for the sequential treatment (Right in S-Fig. 1), the introduction of pulp fractionation prior to cellulase treatment tends to facilitate homogeneous reactions in each fiber fraction, and hence the resultant dissolving pulp can have a narrower MWD and an even higher reactivity. Dasari and Berson (2007) had studied the effect of initial size of sawdust on enzymatic hydrolysis. The results revealed that the sawdust with a smaller size had a higher substrate accessibility, thus facilitating more efficient reactions between substrate and cellulase, Lv et al. (2011) investigated the adsorption behavior of cellulase on bleached softwood fibers with varying fiber lengths, and found that the short-fiber fraction (48–100 mesh) had higher cellulase adsorption than the other two fractions (larger than 28 mesh and 28 48 mesh). In a recent study relevant to the interaction of cellulase with dissolving pulp, it was found that when compared to the air-dried and oven-dried kraft-based dissolving pulps, the never-dried one had the highest cellulase adsorption and lowest viscosity due to its highest accessibility to cellulase (Duan et al., 2015b).
DP0:905 ¼ 0:75 ½g
ð2Þ
3.2. Fiber characteristics and accessibilities of different pulp fractions
2.3.2. Carbohydrates analyses The carbohydrates composition of the pulp samples was determined using an Ion Chromatography (IC) unit equipped with CarboPac TM PA1 column (Dionex-300, Dionex Cooperation, Canada) and a pulsed amperometric detector (PAD). The detailed sample preparation procedure was based on an NREL protocol (Sluiter et al., 2008). 2.3.3. Intrinsic viscosity and chain scission The intrinsic viscosity of all samples was measured according to the T 230 om-99 standard method using copperethylenediamine (CED) solution as solvent. All measurements were carried out in duplicate and the average results were presented. The chain scission (cellulose depolymerization), which represents the number of chain cleavage steps per initial cellulose, can be calculated from the degree of polymerization (DP), as shown in Eq. (1) (Duan et al., 2015b). The conversion between cellulose DP and intrinsic viscosity was based on the SCAN C15:62 as expressed in Eq. (2).
where DP0 is the initial DP of cellulose, DPt is the DP at a random time (t) and the [g] is intrinsic viscosity. 2.3.4. Molecular weight distribution The determination of molecular weight distribution (MWD) was conducted on a GPC device (Waters 600E), equipped with a differential refractometer detector (Waters 410) and a Waters Styragel HR 5E column. Prior to determination, the pulp sample was dissolved in an 8% DMAc/LiCl solution. The mobile phase of 0.5% DMAc/LiCl was pumped into the system at a flow rate of 0.3 mL/ min. The system was operated at a column temperature of 50 °C, with the injection volume of 10 lL. 2.3.5. Fock reactivity The Fock reactivity can usually be used as an indicator of the reactivity of pulp, which was conducted according to the modified method reported in the literature (Tian et al., 2013). The cellulose xanthation was performed at 19 °C in a water bath, and the pulp
The fiber morphologies of each pulp fraction, with respect to fiber length, crystallinity, pore diameter, specific surface area (SSA), and water retention value (WRV) are shown in Table 2. After pulp fractionation, the mean fiber length for the LF fraction was 0.80 mm, while the mean lengths for the MF and SF fractions were 0.44 cm and 0.27 cm, respectively. Correspondingly, the crystallinity for the LF fraction was the highest (66.79%), followed by the MF fraction (64.14%), while the SF fraction had the lowest crystallinity (62.42%). Thus, a larger size of the fiber fraction was associated with a higher crystallinity. By contrast, the specific surface area (SSA) for the SF was the highest (2.28 m2/g), followed by 1.62 m2/g for the MF, and 1.15 m2/g for the LF. In addition, the mean pore diameter was 7.04, 5.81 and 4.35 nm for the LF, MF, and SF, respectively. Although the mean pore diameter for the SF was the lowest, the WRV of the SF was the highest (118%), which can be explained by the fact that the SF contained the highest fines content with the highest SSA and free hydroxyl groups, thus exhibiting the highest swelling efficiency (Chen et al., 2009).
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Table 2 Fiber lengths, crystallinities, pore diameters, specific surface areas (SSA) and water retention values (WRV) of different fiber fractions. Sample
WP
LF
MF
SF
Fiber length, mm Crystallinity, % SSA, m2/g Pore diameter, nm WRV, %
0.64 ± 0.06 64.9 ± 0.5 1.43 ± 0.03 6.42 ± 0.22 101 ± 4
0.80 ± 0.08 66.8 ± 0.4 1.15 ± 0.02 7.04 ± 0.28 95 ± 3
0.44 ± 0.03 64.2 ± 0.3 1.62 ± 0.03 5.81 ± 0.18 106 ± 3
0.27 ± 0.02 62.4 ± 0.4 2.28 ± 0.04 4.35 ± 0.15 118 ± 4
Based on the crystallinity, SSA and WRV of each fraction, it can be concluded that the SF had the highest cellulose accessibility among the three fiber fractions. In a previous study, Yeh et al. (2010) investigated the effect of milling pretreatment on enzymatic hydrolysis of cotton cellulose, and found that the milling pretreatment decreased the cotton particle size to submicron scale, the specific surface area (SSA) increased, and the crystallinity decreased; as a result, the rate of enzymatic hydrolysis increased at least 5 folds at a 3% substrate concentration.
3.3. Cellulase adsorption The results on the cellulase adsorption for each fraction are shown in Fig. 1. The SF sample had the highest amount of adsorbed cellulase in comparison with the MF and LF samples, for example, 94.4% (of the added cellulase) for the SF, while 88.8% and 82.4% for the MF and LF sample respectively after 120 min. Ko et al. (2011) investigated the effect of fiber morphology on the interaction between EG and eucalypt kraft pulp and found that a high accessibility of the pulp lead to a higher maximum enzyme adsorption capacity. The adsorption of cellulase onto the three pulp fractions and original pulp nearly reached equilibrium within 60 min and displayed a significant binding rate during the first 20 min, especially for the SF (approx. 75% bound fraction). In a study, Tu et al. (2007) reported that the cellulase binding on both the steam exploded and ethanol pretreated wood chips of Lodgepole pine reached a plateau at about 60 min. These results suggested that the cellulase adsorption onto the different fiber fractions was highly correlated to their accessibilities, thus, to some extent, determining the enzymatic treatment efficiency, such as the viscosity decrease and chain scission during the cellulase treatment, both of which will be discussed in the following section.
Fig. 1. Adsorption of cellulase on different fiber fractions (5% pulp consistency, 25 °C, pH 4.8, total initial cellulase loading of 340 lg protein/g odp).
3.4. Kinetics of viscosity decrease and chain scission of pulp samples during the cellulase treatment Shown in Fig. 2 are the kinetics of the initial whole pulp (WP) and three pulp fractions (SF, MF and LF) with respect to viscosity and chain scission during the cellulase treatment. The viscosity of the SF sample decreased most significantly in comparison with those of the MF and LF samples during the cellulase treatment process, and particularly in the first 2 h, the viscosity decreased by 162 units for SF sample as compared to 143, 132 and 125 units for the MF, LF and WP samples respectively (Fig. 2a). In addition, a rapid reduction in viscosity was observed in the initial few hours of the cellulase treatments, but slowed down as the process continued, which is consistent with the behavior of cellulase-treated dissolving pulp (without pulp fractionation) as shown in a previous study (Duan et al., 2015b). These results were further evaluated
Fig. 2. Kinetics of (a) viscosity and (b) chain scission of different samples during the cellulase treatment (cellulase dosage of 0.5 mg/g odp, 10% pulp consistency, pH 4.8, and 55 °C).
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C. Duan et al. / Bioresource Technology xxx (2016) xxx–xxx Table 3 Molecular weight, polydispersity index and Fock reactivity of different samples under different treatment conditions. Sample 1
Sample A Sample B2 Sample C3 1 2 3
Mw, kg/mol
Mn, kg/mol
PDI
Fock reactivity, %
326.5 ± 5.0 289.3 ± 4.2 270.1 ± 3.9
79.2 ± 1.4 58.1 ± 0.8 72.8 ± 1.2
4.12 ± 0.15 4.98 ± 0.14 3.71 ± 0.12
48.9 ± 0.4 76.3 ± 0.2 85.6 ± 0.3
Sample A: the original untreated dissolving pulp. Sample B: cellulase- treated pulp based on the conventional process (treatment time of 60 min for the WP, pulp viscosity of 460 mL/g). Sample C: cellulase- treated pulp based on the combined sequential process (20, 40, 120 min for SF, MF and LF, respectively, and pulp viscosity of 462 mL/g).
in terms of the cellulose chain scission. As shown in Fig. 2b, the SF sample showed the highest depolymerization rate in the first four hours and the most robust continuing chain scission during the following cellulase treatment process among the four samples studied. Therefore, it is evident that the enzymatic treatment efficiency was highly dependent on the cellulose accessibility and cellulase adsorption: a substrate with higher cellulose accessibility and cellulase adsorption will have higher enzymatic treatment efficiency. Ibarra et al. (2010a) reported that upon enzymatic treatment (N476, an EG enzyme), the never-dried acid sulfite dissolving pulps (derived from hardwood and softwood) with less pulp hornification and thus better cellulose accessibility exhibited more pronounced viscosity drops in comparison with those dried pulps due to the fact that never-dried pulps. In another study, Luo et al. (2011) investigated the enzymatic hydrolysis of two types of substrates: pretreated lignocellulosic material and bleached pulp sample, and the results indicated that the substrate enzymatic digestibility (SEDs) was proportional to the adsorbed amount of a commercial endoglucanase. Moreover, apart from more available surface/mass ratio, the more remarkable viscosity reduction and chain scission in the case of SF fraction may be also attributed to its better mass transfer between the solid (cellulose fibers) and liquid (enzyme solution) phases (Wojtusik et al., 2016). On the basis of these results, it was concluded that the cellulase was preferentially adsorbed onto the SF due to its highest cellulose accessibility, and the viscosity decrease and chain scission of the SF were more remarkable than those of other fiber fractions.
3.5. Molecular weight distribution and reactivity As shown in Fig. 3, Sample A, i.e., the original untreated dissolving pulp, had the highest molecular weight (Mw) of cellulose fraction among the three samples. After cellulase treatment, the highMw peaks for both Sample B and C shifted to the low- Mw region. However, at a similar pulp viscosity (460 mg/L vs 462 mg/L), Sample C (produced based on the sequential treatment process), showed a narrower molecular weight distribution in comparison to Sample B (treated with the conventional process). The above results are consistent with those, of the polydispersity index (PDI) (Table 3). For example, the PDI of Sample A was 4.12, and that of Sample B (the conventional cellulase treated sample) was 4.98, due to the heterogenous nature of the treatment. In contrast, the PDI of Sample C (after the combined treatment process) was 3.71, significantly lower than that of Sample B. Included in Table 3 are the reactivity data. As shown, the Fock reactivity of Sample A was 48.9%, and that of Sample B (the conventional cellulase treated sample) was 76.2%. Importantly, the Fock reactivity of Sample C (after the sequential treatment process) was 85.6%. The higher Fock reactivity in Sample C as compared to Sample B, can be attributed to more homogeneous reactions for these fibers. Therefore, these results further support the hypothesis that the sequential/combined process can facilitate homogeneous reactions for each fiber fraction, resulting in a cellulose product with a narrow molecular weight distribution and a high reactivity. In terms of the commercial use of cellulase for the improvement of the properties of dissolving pulp with respect to molecular weight distribution and reactivity, it would be conceivable that the cellulase treatment can be applied to the fractionated cellulose fibers due to homogeneous, efficient reaction for each pulp fraction. The pulp fractionation can be readily conducted using the screening systems in the pulp mill, such as the pressure screen, and the adjustment of cellulase addition level as well as the reaction time for each pulp fraction in the combined process can be easily practiced by the industry. 4. Conclusions
Fig. 3. Molecular weight distribution (MWD) of untreated pulp and cellulasetreated pulps (cellulase dosage of 0.5 mg/g odp, 10% pulp consistency, pH 4.8, and 55 °C). Sample A: the original untreated dissolving pulp; Sample B: the cellulasetreated pulp based on the conventional process (treatment time of 60 min for the WP, and of pulp viscosity of 460 mL/g); Sample C: cellulase- treated pulp based on the sequential process (20, 40 and 120 min treatment times for SF, MF and LF, respectively, and pulp viscosity of 462 mL/g).
A sequential/combined process, consisting of pulp fractionation and cellulase treatment of each fraction, was investigated to improve the properties of a kraft-based dissolving pulp. Results showed that the SF had the lowest viscosity, highest accessibility, and cellulase adsorption capacity among the three fiber fractions. At a given viscosity (approx. 460 mL/g), the proposed combined process led to a narrower MWD (PDI, 3.71 vs 4.98) and a higher Fock reactivity (85.6% vs 76.3%) in comparison with the conventional process (single-stage cellulase treatment). The enhancement in MWD and reactivity was associated with homogeneous cellulase actions on each pulp fraction. Acknowledgement The authors would like to acknowledge the Doctoral Scientific Fund Project of Shaanxi University of Science and Technology
Please cite this article in press as: Duan, C., et al. Fractionation and cellulase treatment for enhancing the properties of kraft-based dissolving pulp. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.10.077
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Please cite this article in press as: Duan, C., et al. Fractionation and cellulase treatment for enhancing the properties of kraft-based dissolving pulp. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.10.077