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Abstract： Enzymatic pretreatment of pulp is demonstrated to be potentially effective for decreasing the energy consumption in the refining process. Herein， a neutral cellulase was utilized for the pretreatment of bleached softwood pulp in order to improve the refining performance. Cellulase pretreatment effectively improved the drainability of the pulp and could thus reduce the energy consumption in the refining process. The beating degree of the pulp was significantly improved at 6000 PFI revolutions， at which a maximum increase of 70% could be obtained. The water retention value （WRV） of the pulp increased by 17% after treatment with cellulase at a dosage of 5 IU/g， and the fibers could be easily torn apart after enzymatic treatment. To achieve the same beating degree， the refining time could be shortened by 80% when the pulp was treated with cellulase. Using a low dosage of cellulase， the freeness of the pulp increased rapidly without deterioration of the mechanical properties.
Keywords： cellulase pretreatment； refining； physical properties； energy consumption
1 Introduction
Refining is an important step in the pulping process， wherein cellulosic fibers are mechanically treated in water， resulting in morphological and structural changes. The main effects of refining on the fibers are internal and external fibrillation， fines formation， fiber shortening or cutting， and fiber curling or straightening[1]. Achieving the desired properties of paper involves subjecting the pulp to the refining process， which accounts for 15%～18% of the total energy consumption for producing paper from wood[2]. Due to regional policy and environmental regulations， energy consumption reduction is a requisite， especially for the pulp and paper industry， which is an energy intensive industry[3]. Any treatment of pulp or equipment improvement that significantly decreases the energy requirement of the refining process will have a significant beneficial effect on the overall energy input of the pulp and paper industry.
Over the past decades， biotechnology has undergone major developments for the manufacture of a variety of industrial products， including pulp and paper[4]. The application of enzymes in the pulp and paper industry has been attempted in order to provide green catalysts and an environmentally friendly process. Cellulolytic enzymes have been intensively studied and applied in many fields， although they are still under development in several areas. Cellulases have been the focus of several studies for use in the bioconversion of agricultural wastes[5]. These enzymes are being introduced into the paper industry to improve the manufacture of recycled paper[6-7]. It has been shown that controlled treatment with cellulases can improve the mechanical properties of paper. Although some reports indicated that endoglucanases are more effective than exoglucanases for modifying the properties of fibers[8]， the desirable characteristics of endoglucanases for improving the strength properties of pulp and paper have yet to be established. Enzymatic pretreatment to improve the refining performance of pulp fibers has been proven efficient[9]. Almost all commercial pulp-related refining-assisted enzyme products utilize the activity of cellulase and hemicellulase， in which cellulase plays the central role in altering the freeness of bleached pulp. Cellulase treatment of pulp can be performed before refining or between two cycles of refining to increase the fibrillation and flexibility of fiber to promote the drainage of pulp[10]. When cellulase treatment was applied before the refining process， some studies reported enhanced pulp drainage for recycled fibers. There is great interest in improving the paper machine runnability[11]. Enzymatic treatment of old corrugated containers （OCCs） can lead to significant energy savings[12]， but in some cases， these treatments may cause an obvious decline in the paper resistance. Simao pine bleached kraft pulp[13]， hardwood kraft pulp[14]， and bamboo pulp[15] have been subjected to enzymatic treatment， leading to energy savings benefits in the refining process. When cellulase treatment is applied before the refining process， the content of fines can be reduced so as to enhance the dewatering rate and increase the maximum speed of the paper machine. For the purpose of enhancing pulp drainability， a neutral monocomponent cellulase endoglucanase （such as Novozym 476） is generally applied[16]. Pala et al suggested that in this process， the enzymes modified the surface properties of the fibers， and increased the water affinity[17].
In this study， bleached softwood kraft pulp was treated with neutral cellulase in different dosages. The subsequent refining performance （specifically the freeness and physical properties） of the fibers was evaluated， which may provide a scenario for energy saving in the refining process.
2 Experimental
2.1 Pulp and enzyme
Bleached softwood pulp was provided by Guangzhou Paper Group Co.， Ltd. （Guangdong province， China）. The brightness of the pulp was 89%ISO. Neutral cellulase was supplied by Hu’nan Youtell Biochemical Co.， Ltd. （Yueyang， Hu’nan province， China）. The initial activity of the neutral cellulase was 8000 IU/mL.
2.2 Cellulase treatment of pulp
For each pretreatment experiment， 30 g （oven dried pulp） of original pulp and cellulase were placed into a polyethylene plastic bags. After mixing， the bags were placed into a thermostatic water bath at a constant temperature of 50℃. The pretreatment conditions for neutral cellulase were as follows： pulp consistency 5%， cellulase dosage 1.0， 2.5， 5.0， 7.5 and 10.0 IU/g （based on oven dried pulp）， pH value 7.0， pretreatment time 60 min. The control samples were pretreated with 0.1 mol/L phosphate buffer under similar conditions. Finally， the pretreated pulps were placed into 90℃ water to inactivate the cellulase and then washed thoroughly for beating. 2.3 Water retention value and carbohydrate analysis
The fiber hydration was determined using the water retention value （WRV）， according to the method described by Silvy. This method involves soaking the pulp samples in water with further centrifugation （at 3000g for 15 min）； the WRV is calculated using the following equation：
Where， Mw is the mass of the wet sample after centrifugation， and the Md is the mass after drying the initially wet sample to constant weight at 105℃.
The carbohydrates hydrolysis in the cellulase treatment process were analyzed as total reducing sugars by the colorimetric method using 3，5-dinitrosalicylic （DNS） acid.
2.4 Pulp refining and physical properties of pulp
The pulp samples were beaten in a PFI refiner to determine the refining responses. The samples were refined using 3000， 6000， 9000 and 12000 revolutions， respectively， according to the ISO 5264-2 standard method. Handsheets were prepared according to the ISO 5269-2 standard method. The basic weight of the paper was 60 g/m2. The tensile index， tear index， and burst index were measured according to ISO standard methods.
2.5 Fiber characterization during refining process
The fiber size distribution was measured using a Kajaani FS300 analyzer （Metso， Finland）. Scanning electron microscopy （ZEISS EVO18， Germany） was used to observe the surface morphology of the refined fibers. After refining， the fibers were dispersed in ethanol for dehydration， and then freeze-dried in order to reveal the external fibrillation.
3 Results and discussion
3.1 Effect of cellulase pretreatment on pulp
Cellulosic fibers may be damaged or hydrolyzed by cellulase components. However， if these components are controlled， the fiber features may be changed without damage. Cellulases are a group of enzymes mainly consisting of different isozymes， including endoglucanases （EGI and EGII）， exoglucanases （CBHI and CBHII）， and b-glucosidases， which work synergistically to hydrolyze cellulose into reducing sugars. The generation of reducing sugars via cellulose degradation in the cellulase pretreatment process should be considered when cellulase is used for enzyme-assisted refining. The pulp yield is significantly affected by the generation of reducing sugars. A lower pulp yield increases the production cost， which is the main factor that limits enzyme application， especially on the mill-scale.
The effluent was filtered from the substrate after cellulase treatment and the amount of reducing sugars released was determined. As shown in Fig.1， the content of reducing sugars increased with increasing cellulase dosage. At low cellulase dosages， such as 1.0 IU/g and 2.5 IU/g， the reducing sugar contents were 1.8% and 2.7%， respectively， i.e.， less than 3%. The reducing sugar contents were 4.8% and 7.5% （oven dried pulp） with cellulase dosages of 5.0 IU/g and 10.0 IU/g （oven dried pulp）， respectively. The reducing sugar content was lower than that reported in the literature for a mixture of cellulase produced by a selected strain of the fungus T. reesei used to improve the refining performance of bleached eucalyptus globulus kraft pulp[14]. The present results show that the relationship between the reducing sugar content and cellulase dosage is not linear. This is associated with the composition of the enzyme. Soluble oligosaccharides were formed from the cellulose fragments hydrolyzed by exoglucanase， but these oligosaccharides were not converted into glucose. A similar phenomenon was reported by Liu[18]. The pulp yield calculation is also affected by the presence of oligosaccharides. The pulp yield calculated by applying the colorimetric method was significantly different from that obtained by the dry weight method. The dry weight loss ratio was greater than the reducing sugar content， but the general trends in the changes were almost the same. Some cellulose degradation during pretreatment with cellulase is inevitable. In general， this is acceptable that pulp hydrolysis is controlled within 3% during the pretreatment process with cellulase[19]. Thus， a high cellulase dosage may not be suitable for pretreatment on the mill-scale in consideration of the production cost.
The WRV is an important parameter for analyzing the water absorption inside the fiber wall and has been used to characterize the swelling behavior and hydration of cellulosic materials[20]. The WRV of pulp fiber is mainly affected by the fine structure of the pulp， associated with the fibrils during mechanical treatment or the parenchyma of the cell. Hydration and swelling are easily achieved for samples with a high specific surface area and loose structure. However， a high WRV has adverse effects on sheet formation. First， dehydration of the pulp is difficult with a high WRV， and second， a high content of fine deteriorates the mechanical properties of the paper， making it more prone to breaks.
The WRV was determined after pretreatment with cellulase to evaluate the fiber swelling and fiber cell wall hydration. The results presented in Fig.2 show that the WRV increased slightly with increasing cellulase dosage. Compared with the control sample， the maximum increase in the WRV of 17% was obtained with a cellulase dosage of 7.5 IU/g. This indicates that swelling of the fibers was increased by cellulase treatment. This result is similar to that presented by other authors[14， 21]， where it was found that a smaller increase in the WRV was observed after cellulase treatment. This index is mainly associated with internal fibrillation of the fibers[14， 22]. At the highest cellulase dosage （10.0 IU/g）， the WRV decreased compared to that at a cellulase dosage of 7.5 IU/g. This is caused by degradation of the fibrils or fines during the cellulase treatment process with a high cellulase dosage. The results indicate that the surface and internal structure of the fibers can be modified through cellulase treatment. This effect mainly depends on the cellulase component.
Thus， the pulp underwent some changes after treatment with cellulase， which leads to some degradation of the fibers， simultaneously enhancing swelling of the fibers and making them easier to refine.
3.2 Effect of cellulase pretreatment on pulp refining performance
The evolution of the pulp freeness （beating degree） during the refining process was observed after cellulase pretreatment. The data in Fig.3（a） shows an increase in the beating degree of the pulp sample subjected to the cellulase treatment compared to that of the control. Pulp refining was promoted by different dosages of cellulase with a visible improvement in the degree of pulp beating. This result is especially significant for 6000 PFI revolutions， where a maximum increase of 300% could be obtained. On the other hand， fewer PFI revolutions were needed to achieve the same beating degree after cellulase pretreatment. For example， to achieve the target of 40°SR， 4800 PFI revolutions and 6000 PFI revolutions were respectively applied with cellulase dosages of 5.0 IU/g and 2.5 IU/g， whereas 7500 PFI revolutions were performed for the control experiment. This result is better than that reported in the literature. The drainage property of pulp is mainly influenced by the fibers fibrillation and the amount of fines. The increase in the beating degree， which is related to the external fibrillation， supports the hypothesis that cellulase treatment modifies the surface structure of the fibers. This increase in the external fibrillation is probably related to the action of endoglucanases in the cellulase mixture， which apparently cut the cellulose chains on the fiber surface without liberating them from the surface at low dosage levels[14， 23]. The flexibility of the fibers and the improvement of the fiber bonding capacity are related to the beating degree； thus， the beating degree is an indicator of the maintenance of the pulp strength. When the data in Fig.3（a） were processed appropriately by plotting the beating degree （ordinate） against the cellulase dosage （abscissa）， Fig.3（b） was obtained. Obviously， for the same PFI revolutions， when a higher dosage of cellulase was used in the pretreatment process， a higher beating degree was obtained. The beating degree increased disproportionally with the cellulase dosage. There was an inflection point in the correlation curve. At low PFI revolution （6000 r）， the cellulase dosage corresponding to the inflection point was 7.5 IU/g， whereas at high PFI revolutions （9000～12000 r）， the cellulase dosages were 2.5 IU/g. This indicates that at high PFI revolution， low cellulase dosage can lead to a greater improvement in the beating degree. However， it was difficult to gain further benefits by increasing the cellulase dosage. This result also shows that there is a theoretical limit on the pulp refining performance after pretreatment with natural cellulase. Nevertheless， enzyme-assisted refining can furnish a higher final beating degree compared with the control. For example， at high PFI revolutions， the beating degree of the pulp subjected to cellulase pretreatment could exceed 80°SR， whereas the beating degree of the control pulp was less than 60°SR. This means that enzyme-assisted refining is more suitable for pulp that demands a high beating degree， such as the pulp for special wrapping paper production.
Cellulase treatment modified the fiber surface structure and enhanced the refining performance of the pulp. However， this increment was not unlimited. The pulp treated with cellulase had a higher final beating degree compared with the control.
3.3 Fiber characteristics during enzyme-assisted refining
When the pulp was treated with the neutral cellulase before refining， the average fiber length of the pulp increased to some extent， and the amount of fines decreased， as shown in Table 1. The loss of fines is attributed to their high specific surface area and tiny size， which promotes rapid hydrolysis by the enzymes. The original or long fibers are not easily degraded by enzymes. The cellulolytic enzymes attack the pulp fibers weakly when the dosage is not high； thus， the fibers were not cut off. However， the enzyme-treated pulp was refined and fibrillation of the pulp became easier because the strength of the bonds between the microfibers was lessened after enzyme attack. On the other hand， after cellulase treatment， the length of the fiber declined sharply during the refining process； accordingly， the fine content increased significantly. This trend was more obvious with a high cellulase dosage. Compared with the data in Fig.3， the decrease in the fiber length and increase in the beating degree were consistent. This also indicates that one aspect of the enzyme-assisted refining mechanism is that cellulase treatment makes the pulp fibers easier to cut off in the refining process. Although cutting of the fiber is inevitable， it is better to reduce fiber cutting for a given beating degree. Some studies have shown that the lumen of the fibers collapses and the fiber shape changes from tubular to flat after refining[24]. In this study， scanning electron microscopic （SEM） micrographs of the untreated pulp and pulp subjected to cellulase pretreatment， refined for 6000 PFI revolutions， were obtained to demonstrate the morphological change of the fibers after cellulase pretreatment compared to the control， as shown in Fig.4. The observations suggest that obvious changes in the fiber took place after cellulase pretreatment followed by 6000 PFI revolutions. Fiber cutting and fragmentation mainly occurred with 2.5 IU/g of cellulase compared to the control. Cellulase affects the outer fiber surface as a result of increased external fibrillation[14]. Fibrils were peeled off from the fiber surface through mechanical refining and formed fine or fragments. During the fibrillation process， gel-like layers were formed due to the release of some hydrophilic compounds from the fiber cell wall[1]. This is clearly observed in Fig.4（b）. Fiber-fiber bonding was improved in the films with these gelatinous layers during the drying process[24]. This result is in agreement with the fiber analysis data， where the fiber length dropped abruptly and the fine content increased significantly during the cellulase-assisted refining process.
3.4 Effect of cellulase pretreatment on mechanical properties of pulp
As previously mentioned， cellulose degradation occurs during cellulase pretreatment. In order to investigate the impact of cellulase pretreatment on the mechanical properties of the pulp， handsheets were prepared. The mechanical properties are greatly influenced by the beating degree. Thus， the beating degree of all pulps was controlled to around （32±2）°SR. The physical strength parameters， such as the tensile index， tear index， and burst index， were determined.
As shown in Fig.5， the physical strength parameters increased slightly at low cellulase dosage and declined at high cellulase dosage. In fact， the fibers were well swollen， fibrillated， and maintained a good length after cellulase pretreatment with an appropriate enzyme dosage， resulting in enhanced adhesion strength of the fibers. Therefore， the physical parameters increased slightly.
The tear index declined after pretreatment with a high dosage of cellulase （Fig.5）， where major degradation was observed （i.e.， compared with the control sample， around 20% decrease in the tear index when the cellulase dosage was 5.0 IU/g）. Generally， the tear index is strongly affected by the fiber length. However， in this study， modification of the fiber length induced by cellulase treatment was not as important as the effect on the tear index at the same drainability. This could be attributed to weakening of the fibers at the ultra-structural level by enzymatic pretreatment[25]. The cellulase used in the pretreatment process probably preferentially attacked specific zones （amorphous cellulose） and created weak points. These weak points became fracture zones during refining[21]. Additionally， the fiber degradation increased when the fibers were subjected to drying and pressing processes during sheet manufacture. The fibers are resistant to mechanical treatment when they are wet and uncollapsed. However， their intrinsic strength decreases during drying due to the severity of the cellulase treatment[26]. This could explain why the tear index decline was more significant than expected. The tensile strength of the paper is affected by the fiber bonding strength and fiber length. The tensile index could be maintained at an adequate level at low cellulase dosage （1.0 IU/g）， but decreased at high cellulase dosage （higher than 2.5 IU/g） during the enzyme-assisted refining process. As discussed previously， the fiber length was not the main factor affecting the tensile index for pulps with the same freeness level. The decrease in the fiber bonding strength， reduced by the greater number of fiber weakening points with a high cellulase dosage for pretreatment， may also play a role. The same principle could also explain the lower burst index with high cellulase dosage during the pretreatment. This result also indicates that a low cellulase dosage during pretreatment could enhance the mechanical properties of paper.
At low cellulase dosages， the pulp freeness increases as soon as contacting with the enzymes， without any reduction of the mechanical properties of the paper. This can improve the paper machine speed and allow more dilution in the headbox， which leads to better sheet formation and better physical properties of the paper.
3.5 Energy saving in enzyme-assisted refining process
The main purpose of enzyme-assisted refining is to reduce the energy consumption of the refining process. The energy consumed during refining can be represented well by PFI revolutions in the laboratory. In order to investigate the impact of cellulase pretreatment on the energy consumption， we calculated the PFI revolution savings for the same beating degree compared to that of the control. The selected beating degrees were 30， 45， 60， and 70°SR.
As shown in Fig.6， for the four different beating degrees， there was a saving of more than 30% PFI revolutions after cellulase pretreatment with different enzyme dosages， where the maximum was 86.7%. This indicates that cellulase pretreatment can significantly reduce the energy consumption in the refining process. These results are better than those reported in other studies[27]. At low beating degrees （30°SR and 45°SR）， the difference in the PFI revolution savings was not significant when the cellulase dosage was more than 5.0 IU/g， whereas at high beating degrees （60°SR and 70°SR）， the PFI revolution savings were similar for all cellulase dosages. The increase in the PFI revolution savings at low beating degree was more evident than at high beating degree after cellulase pretreatment. These results are similar to the trends in Fig.3（a）. In the early period of the enzyme-assisted refining process， the beating degree increased rapidly， but plateaued at high PFI revolutions. Otherwise， the data in Fig.6 showed that the PFI revolution savings at a high beating degree of 30°SR were much greater than that at a low beating degree when the cellulase loading was less than 2.5 IU/g. When the cellulase loading was more than 5.0 IU/g， the PFI revolution savings at low beating degree were higher. This indicated that low dosages of cellulase were more beneficial for pulp requiring a high beating degree， and a high cellulase dosage was more suitable for low-beating-degree pulp. 4 Conclusions
Cellulase treatment improved the swelling of cellulose fibers， making them more easily refined. The pulp refining performance was promoted by cellulase pretreatment at various dosages. The improvement in the pulp beating degree was especially significant when the pulp was beaten at 6000 PFI revolutions. The final beating degree of the pulp could surpass 80°SR after treatment with cellulase， whereas that of the control pulp （without enzyme assistance） did not exceed 60°SR， even with beating for a longer time. Cellulase treatment increased the WRV of the pulp， making the pulp fibers easier to tear apart in the refining process. Even at low cellulase dosage， there was a significant increase in the pulp freeness without any reduction of the mechanical properties of the paper. Thus， enzymatic pretreatment is beneficial for reducing the energy consumption in the refining process； 86.7% PFI revolutions were saved with cellulase treatment to obtain 30°SR pulp. Interestingly， with regard to reducing energy consumption during the refining process， a low cellulase loading （less than 2.5 IU/g） is more beneficial for high-beating-degree pulp， whereas a high cellulase dosage is more suitable for low-beating-degree pulp.
Acknowledgments
The authors are grateful for financial support from the National Natural Science Foundation of China （31570569）.
References
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Keywords： cellulase pretreatment； refining； physical properties； energy consumption
1 Introduction
Refining is an important step in the pulping process， wherein cellulosic fibers are mechanically treated in water， resulting in morphological and structural changes. The main effects of refining on the fibers are internal and external fibrillation， fines formation， fiber shortening or cutting， and fiber curling or straightening[1]. Achieving the desired properties of paper involves subjecting the pulp to the refining process， which accounts for 15%～18% of the total energy consumption for producing paper from wood[2]. Due to regional policy and environmental regulations， energy consumption reduction is a requisite， especially for the pulp and paper industry， which is an energy intensive industry[3]. Any treatment of pulp or equipment improvement that significantly decreases the energy requirement of the refining process will have a significant beneficial effect on the overall energy input of the pulp and paper industry.
Over the past decades， biotechnology has undergone major developments for the manufacture of a variety of industrial products， including pulp and paper[4]. The application of enzymes in the pulp and paper industry has been attempted in order to provide green catalysts and an environmentally friendly process. Cellulolytic enzymes have been intensively studied and applied in many fields， although they are still under development in several areas. Cellulases have been the focus of several studies for use in the bioconversion of agricultural wastes[5]. These enzymes are being introduced into the paper industry to improve the manufacture of recycled paper[6-7]. It has been shown that controlled treatment with cellulases can improve the mechanical properties of paper. Although some reports indicated that endoglucanases are more effective than exoglucanases for modifying the properties of fibers[8]， the desirable characteristics of endoglucanases for improving the strength properties of pulp and paper have yet to be established. Enzymatic pretreatment to improve the refining performance of pulp fibers has been proven efficient[9]. Almost all commercial pulp-related refining-assisted enzyme products utilize the activity of cellulase and hemicellulase， in which cellulase plays the central role in altering the freeness of bleached pulp. Cellulase treatment of pulp can be performed before refining or between two cycles of refining to increase the fibrillation and flexibility of fiber to promote the drainage of pulp[10]. When cellulase treatment was applied before the refining process， some studies reported enhanced pulp drainage for recycled fibers. There is great interest in improving the paper machine runnability[11]. Enzymatic treatment of old corrugated containers （OCCs） can lead to significant energy savings[12]， but in some cases， these treatments may cause an obvious decline in the paper resistance. Simao pine bleached kraft pulp[13]， hardwood kraft pulp[14]， and bamboo pulp[15] have been subjected to enzymatic treatment， leading to energy savings benefits in the refining process. When cellulase treatment is applied before the refining process， the content of fines can be reduced so as to enhance the dewatering rate and increase the maximum speed of the paper machine. For the purpose of enhancing pulp drainability， a neutral monocomponent cellulase endoglucanase （such as Novozym 476） is generally applied[16]. Pala et al suggested that in this process， the enzymes modified the surface properties of the fibers， and increased the water affinity[17].
In this study， bleached softwood kraft pulp was treated with neutral cellulase in different dosages. The subsequent refining performance （specifically the freeness and physical properties） of the fibers was evaluated， which may provide a scenario for energy saving in the refining process.
2 Experimental
2.1 Pulp and enzyme
Bleached softwood pulp was provided by Guangzhou Paper Group Co.， Ltd. （Guangdong province， China）. The brightness of the pulp was 89%ISO. Neutral cellulase was supplied by Hu’nan Youtell Biochemical Co.， Ltd. （Yueyang， Hu’nan province， China）. The initial activity of the neutral cellulase was 8000 IU/mL.
2.2 Cellulase treatment of pulp
For each pretreatment experiment， 30 g （oven dried pulp） of original pulp and cellulase were placed into a polyethylene plastic bags. After mixing， the bags were placed into a thermostatic water bath at a constant temperature of 50℃. The pretreatment conditions for neutral cellulase were as follows： pulp consistency 5%， cellulase dosage 1.0， 2.5， 5.0， 7.5 and 10.0 IU/g （based on oven dried pulp）， pH value 7.0， pretreatment time 60 min. The control samples were pretreated with 0.1 mol/L phosphate buffer under similar conditions. Finally， the pretreated pulps were placed into 90℃ water to inactivate the cellulase and then washed thoroughly for beating. 2.3 Water retention value and carbohydrate analysis
The fiber hydration was determined using the water retention value （WRV）， according to the method described by Silvy. This method involves soaking the pulp samples in water with further centrifugation （at 3000g for 15 min）； the WRV is calculated using the following equation：
Where， Mw is the mass of the wet sample after centrifugation， and the Md is the mass after drying the initially wet sample to constant weight at 105℃.
The carbohydrates hydrolysis in the cellulase treatment process were analyzed as total reducing sugars by the colorimetric method using 3，5-dinitrosalicylic （DNS） acid.
2.4 Pulp refining and physical properties of pulp
The pulp samples were beaten in a PFI refiner to determine the refining responses. The samples were refined using 3000， 6000， 9000 and 12000 revolutions， respectively， according to the ISO 5264-2 standard method. Handsheets were prepared according to the ISO 5269-2 standard method. The basic weight of the paper was 60 g/m2. The tensile index， tear index， and burst index were measured according to ISO standard methods.
2.5 Fiber characterization during refining process
The fiber size distribution was measured using a Kajaani FS300 analyzer （Metso， Finland）. Scanning electron microscopy （ZEISS EVO18， Germany） was used to observe the surface morphology of the refined fibers. After refining， the fibers were dispersed in ethanol for dehydration， and then freeze-dried in order to reveal the external fibrillation.
3 Results and discussion
3.1 Effect of cellulase pretreatment on pulp
Cellulosic fibers may be damaged or hydrolyzed by cellulase components. However， if these components are controlled， the fiber features may be changed without damage. Cellulases are a group of enzymes mainly consisting of different isozymes， including endoglucanases （EGI and EGII）， exoglucanases （CBHI and CBHII）， and b-glucosidases， which work synergistically to hydrolyze cellulose into reducing sugars. The generation of reducing sugars via cellulose degradation in the cellulase pretreatment process should be considered when cellulase is used for enzyme-assisted refining. The pulp yield is significantly affected by the generation of reducing sugars. A lower pulp yield increases the production cost， which is the main factor that limits enzyme application， especially on the mill-scale.
The effluent was filtered from the substrate after cellulase treatment and the amount of reducing sugars released was determined. As shown in Fig.1， the content of reducing sugars increased with increasing cellulase dosage. At low cellulase dosages， such as 1.0 IU/g and 2.5 IU/g， the reducing sugar contents were 1.8% and 2.7%， respectively， i.e.， less than 3%. The reducing sugar contents were 4.8% and 7.5% （oven dried pulp） with cellulase dosages of 5.0 IU/g and 10.0 IU/g （oven dried pulp）， respectively. The reducing sugar content was lower than that reported in the literature for a mixture of cellulase produced by a selected strain of the fungus T. reesei used to improve the refining performance of bleached eucalyptus globulus kraft pulp[14]. The present results show that the relationship between the reducing sugar content and cellulase dosage is not linear. This is associated with the composition of the enzyme. Soluble oligosaccharides were formed from the cellulose fragments hydrolyzed by exoglucanase， but these oligosaccharides were not converted into glucose. A similar phenomenon was reported by Liu[18]. The pulp yield calculation is also affected by the presence of oligosaccharides. The pulp yield calculated by applying the colorimetric method was significantly different from that obtained by the dry weight method. The dry weight loss ratio was greater than the reducing sugar content， but the general trends in the changes were almost the same. Some cellulose degradation during pretreatment with cellulase is inevitable. In general， this is acceptable that pulp hydrolysis is controlled within 3% during the pretreatment process with cellulase[19]. Thus， a high cellulase dosage may not be suitable for pretreatment on the mill-scale in consideration of the production cost.
The WRV is an important parameter for analyzing the water absorption inside the fiber wall and has been used to characterize the swelling behavior and hydration of cellulosic materials[20]. The WRV of pulp fiber is mainly affected by the fine structure of the pulp， associated with the fibrils during mechanical treatment or the parenchyma of the cell. Hydration and swelling are easily achieved for samples with a high specific surface area and loose structure. However， a high WRV has adverse effects on sheet formation. First， dehydration of the pulp is difficult with a high WRV， and second， a high content of fine deteriorates the mechanical properties of the paper， making it more prone to breaks.
The WRV was determined after pretreatment with cellulase to evaluate the fiber swelling and fiber cell wall hydration. The results presented in Fig.2 show that the WRV increased slightly with increasing cellulase dosage. Compared with the control sample， the maximum increase in the WRV of 17% was obtained with a cellulase dosage of 7.5 IU/g. This indicates that swelling of the fibers was increased by cellulase treatment. This result is similar to that presented by other authors[14， 21]， where it was found that a smaller increase in the WRV was observed after cellulase treatment. This index is mainly associated with internal fibrillation of the fibers[14， 22]. At the highest cellulase dosage （10.0 IU/g）， the WRV decreased compared to that at a cellulase dosage of 7.5 IU/g. This is caused by degradation of the fibrils or fines during the cellulase treatment process with a high cellulase dosage. The results indicate that the surface and internal structure of the fibers can be modified through cellulase treatment. This effect mainly depends on the cellulase component.
Thus， the pulp underwent some changes after treatment with cellulase， which leads to some degradation of the fibers， simultaneously enhancing swelling of the fibers and making them easier to refine.
3.2 Effect of cellulase pretreatment on pulp refining performance
The evolution of the pulp freeness （beating degree） during the refining process was observed after cellulase pretreatment. The data in Fig.3（a） shows an increase in the beating degree of the pulp sample subjected to the cellulase treatment compared to that of the control. Pulp refining was promoted by different dosages of cellulase with a visible improvement in the degree of pulp beating. This result is especially significant for 6000 PFI revolutions， where a maximum increase of 300% could be obtained. On the other hand， fewer PFI revolutions were needed to achieve the same beating degree after cellulase pretreatment. For example， to achieve the target of 40°SR， 4800 PFI revolutions and 6000 PFI revolutions were respectively applied with cellulase dosages of 5.0 IU/g and 2.5 IU/g， whereas 7500 PFI revolutions were performed for the control experiment. This result is better than that reported in the literature. The drainage property of pulp is mainly influenced by the fibers fibrillation and the amount of fines. The increase in the beating degree， which is related to the external fibrillation， supports the hypothesis that cellulase treatment modifies the surface structure of the fibers. This increase in the external fibrillation is probably related to the action of endoglucanases in the cellulase mixture， which apparently cut the cellulose chains on the fiber surface without liberating them from the surface at low dosage levels[14， 23]. The flexibility of the fibers and the improvement of the fiber bonding capacity are related to the beating degree； thus， the beating degree is an indicator of the maintenance of the pulp strength. When the data in Fig.3（a） were processed appropriately by plotting the beating degree （ordinate） against the cellulase dosage （abscissa）， Fig.3（b） was obtained. Obviously， for the same PFI revolutions， when a higher dosage of cellulase was used in the pretreatment process， a higher beating degree was obtained. The beating degree increased disproportionally with the cellulase dosage. There was an inflection point in the correlation curve. At low PFI revolution （6000 r）， the cellulase dosage corresponding to the inflection point was 7.5 IU/g， whereas at high PFI revolutions （9000～12000 r）， the cellulase dosages were 2.5 IU/g. This indicates that at high PFI revolution， low cellulase dosage can lead to a greater improvement in the beating degree. However， it was difficult to gain further benefits by increasing the cellulase dosage. This result also shows that there is a theoretical limit on the pulp refining performance after pretreatment with natural cellulase. Nevertheless， enzyme-assisted refining can furnish a higher final beating degree compared with the control. For example， at high PFI revolutions， the beating degree of the pulp subjected to cellulase pretreatment could exceed 80°SR， whereas the beating degree of the control pulp was less than 60°SR. This means that enzyme-assisted refining is more suitable for pulp that demands a high beating degree， such as the pulp for special wrapping paper production.
Cellulase treatment modified the fiber surface structure and enhanced the refining performance of the pulp. However， this increment was not unlimited. The pulp treated with cellulase had a higher final beating degree compared with the control.
3.3 Fiber characteristics during enzyme-assisted refining
When the pulp was treated with the neutral cellulase before refining， the average fiber length of the pulp increased to some extent， and the amount of fines decreased， as shown in Table 1. The loss of fines is attributed to their high specific surface area and tiny size， which promotes rapid hydrolysis by the enzymes. The original or long fibers are not easily degraded by enzymes. The cellulolytic enzymes attack the pulp fibers weakly when the dosage is not high； thus， the fibers were not cut off. However， the enzyme-treated pulp was refined and fibrillation of the pulp became easier because the strength of the bonds between the microfibers was lessened after enzyme attack. On the other hand， after cellulase treatment， the length of the fiber declined sharply during the refining process； accordingly， the fine content increased significantly. This trend was more obvious with a high cellulase dosage. Compared with the data in Fig.3， the decrease in the fiber length and increase in the beating degree were consistent. This also indicates that one aspect of the enzyme-assisted refining mechanism is that cellulase treatment makes the pulp fibers easier to cut off in the refining process. Although cutting of the fiber is inevitable， it is better to reduce fiber cutting for a given beating degree. Some studies have shown that the lumen of the fibers collapses and the fiber shape changes from tubular to flat after refining[24]. In this study， scanning electron microscopic （SEM） micrographs of the untreated pulp and pulp subjected to cellulase pretreatment， refined for 6000 PFI revolutions， were obtained to demonstrate the morphological change of the fibers after cellulase pretreatment compared to the control， as shown in Fig.4. The observations suggest that obvious changes in the fiber took place after cellulase pretreatment followed by 6000 PFI revolutions. Fiber cutting and fragmentation mainly occurred with 2.5 IU/g of cellulase compared to the control. Cellulase affects the outer fiber surface as a result of increased external fibrillation[14]. Fibrils were peeled off from the fiber surface through mechanical refining and formed fine or fragments. During the fibrillation process， gel-like layers were formed due to the release of some hydrophilic compounds from the fiber cell wall[1]. This is clearly observed in Fig.4（b）. Fiber-fiber bonding was improved in the films with these gelatinous layers during the drying process[24]. This result is in agreement with the fiber analysis data， where the fiber length dropped abruptly and the fine content increased significantly during the cellulase-assisted refining process.
3.4 Effect of cellulase pretreatment on mechanical properties of pulp
As previously mentioned， cellulose degradation occurs during cellulase pretreatment. In order to investigate the impact of cellulase pretreatment on the mechanical properties of the pulp， handsheets were prepared. The mechanical properties are greatly influenced by the beating degree. Thus， the beating degree of all pulps was controlled to around （32±2）°SR. The physical strength parameters， such as the tensile index， tear index， and burst index， were determined.
As shown in Fig.5， the physical strength parameters increased slightly at low cellulase dosage and declined at high cellulase dosage. In fact， the fibers were well swollen， fibrillated， and maintained a good length after cellulase pretreatment with an appropriate enzyme dosage， resulting in enhanced adhesion strength of the fibers. Therefore， the physical parameters increased slightly.
The tear index declined after pretreatment with a high dosage of cellulase （Fig.5）， where major degradation was observed （i.e.， compared with the control sample， around 20% decrease in the tear index when the cellulase dosage was 5.0 IU/g）. Generally， the tear index is strongly affected by the fiber length. However， in this study， modification of the fiber length induced by cellulase treatment was not as important as the effect on the tear index at the same drainability. This could be attributed to weakening of the fibers at the ultra-structural level by enzymatic pretreatment[25]. The cellulase used in the pretreatment process probably preferentially attacked specific zones （amorphous cellulose） and created weak points. These weak points became fracture zones during refining[21]. Additionally， the fiber degradation increased when the fibers were subjected to drying and pressing processes during sheet manufacture. The fibers are resistant to mechanical treatment when they are wet and uncollapsed. However， their intrinsic strength decreases during drying due to the severity of the cellulase treatment[26]. This could explain why the tear index decline was more significant than expected. The tensile strength of the paper is affected by the fiber bonding strength and fiber length. The tensile index could be maintained at an adequate level at low cellulase dosage （1.0 IU/g）， but decreased at high cellulase dosage （higher than 2.5 IU/g） during the enzyme-assisted refining process. As discussed previously， the fiber length was not the main factor affecting the tensile index for pulps with the same freeness level. The decrease in the fiber bonding strength， reduced by the greater number of fiber weakening points with a high cellulase dosage for pretreatment， may also play a role. The same principle could also explain the lower burst index with high cellulase dosage during the pretreatment. This result also indicates that a low cellulase dosage during pretreatment could enhance the mechanical properties of paper.
At low cellulase dosages， the pulp freeness increases as soon as contacting with the enzymes， without any reduction of the mechanical properties of the paper. This can improve the paper machine speed and allow more dilution in the headbox， which leads to better sheet formation and better physical properties of the paper.
3.5 Energy saving in enzyme-assisted refining process
The main purpose of enzyme-assisted refining is to reduce the energy consumption of the refining process. The energy consumed during refining can be represented well by PFI revolutions in the laboratory. In order to investigate the impact of cellulase pretreatment on the energy consumption， we calculated the PFI revolution savings for the same beating degree compared to that of the control. The selected beating degrees were 30， 45， 60， and 70°SR.
As shown in Fig.6， for the four different beating degrees， there was a saving of more than 30% PFI revolutions after cellulase pretreatment with different enzyme dosages， where the maximum was 86.7%. This indicates that cellulase pretreatment can significantly reduce the energy consumption in the refining process. These results are better than those reported in other studies[27]. At low beating degrees （30°SR and 45°SR）， the difference in the PFI revolution savings was not significant when the cellulase dosage was more than 5.0 IU/g， whereas at high beating degrees （60°SR and 70°SR）， the PFI revolution savings were similar for all cellulase dosages. The increase in the PFI revolution savings at low beating degree was more evident than at high beating degree after cellulase pretreatment. These results are similar to the trends in Fig.3（a）. In the early period of the enzyme-assisted refining process， the beating degree increased rapidly， but plateaued at high PFI revolutions. Otherwise， the data in Fig.6 showed that the PFI revolution savings at a high beating degree of 30°SR were much greater than that at a low beating degree when the cellulase loading was less than 2.5 IU/g. When the cellulase loading was more than 5.0 IU/g， the PFI revolution savings at low beating degree were higher. This indicated that low dosages of cellulase were more beneficial for pulp requiring a high beating degree， and a high cellulase dosage was more suitable for low-beating-degree pulp. 4 Conclusions
Cellulase treatment improved the swelling of cellulose fibers， making them more easily refined. The pulp refining performance was promoted by cellulase pretreatment at various dosages. The improvement in the pulp beating degree was especially significant when the pulp was beaten at 6000 PFI revolutions. The final beating degree of the pulp could surpass 80°SR after treatment with cellulase， whereas that of the control pulp （without enzyme assistance） did not exceed 60°SR， even with beating for a longer time. Cellulase treatment increased the WRV of the pulp， making the pulp fibers easier to tear apart in the refining process. Even at low cellulase dosage， there was a significant increase in the pulp freeness without any reduction of the mechanical properties of the paper. Thus， enzymatic pretreatment is beneficial for reducing the energy consumption in the refining process； 86.7% PFI revolutions were saved with cellulase treatment to obtain 30°SR pulp. Interestingly， with regard to reducing energy consumption during the refining process， a low cellulase loading （less than 2.5 IU/g） is more beneficial for high-beating-degree pulp， whereas a high cellulase dosage is more suitable for low-beating-degree pulp.
Acknowledgments
The authors are grateful for financial support from the National Natural Science Foundation of China （31570569）.
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