Production of Paper Using Biopulping of Pineapple Leaves Fibers (PALF) Followed by Chemical and Xylanase-Enzymatic Processing

ABSTRACT Pineapple leaf fibers (PALF) were biopulped using Trametes versicolor, and the resulting biopulp was bleached with xylanase-enzymatic treatments. The biopulping was extensively described using determinations of fiber morphology, color, chemical composition, extractive content, and thermal stability using the structural characteristics determined by XRD and paper properties. The results showed that the chemical and enzymatic treatments shortened the fiber, almost to 50%, and the Kappa index decreased from 27 to 13. Cellulose and holocellulose contents increased from 65% to 74% and from 86% to 91%, respectively, but extractives, lignin (from 12% to 4%), pentosans (from 25% to 14%) and the crystallinity decreased from 58% to 67% in both chemical bleaching and further xylanase-enzymatic processing. Xylanase-enzymatic processing allowed us to obtain whiter (increased lightness color and decreased redness and yellowness tonality) and heavier paper, even though it presented decreased mechanical properties (decreased stress resistance, rupture length, tear resistance and index longitudinal tearing). The xylanase-enzymatic treatment with the best pulping and paper properties is when the biopulp is treated with a xylanase enzyme concentration of 0.04% (w/w). GRAPHICAL ABSTRACT


Introduction
Pineapple crops are the third most economically important tropical fruit in the world (Lobo and Siddiq 2016). However, pineapple production generates more agro-industrial wastes than any other agricultural crop (Roda and Lambri 2019). A potential use for pineapple wastes, particularly for the leaves, is to produce fibers (Sibaly and Jeetah 2017). Recent reports have demonstrated interesting characteristics of pineapple leaf fibers (PALF), including physical, mechanical and chemical properties from various pineapple species (Padzil et al. 2020). These PALF are useful for different purposes, including pulp and paper production (Padzil et al. 2020).
Biopulping is an alternative method involving biochemical conversions. It uses fungal pretreatment of lignocellulosic materials to produce pulps (Laftah and Rahman 2016). Biopulping is based on the ability of a restricted number of white-rot fungi to colonize and selectively degrade lignin, thereby leaving cellulose relatively intact (Singh et al. 2013). Advanced stages of delignification are characterized by middle lamella degradation, while the cellulose-rich secondary walls separate (Singh et al. 2013). Fungi, such as Trametes versicolor, are used in pulp production by biopulping (Moya et al. 2016). Trametes versicolor is a white-rot fungus showing a simultaneous degradation of lignin and holocellulose, leaving the cells either pierced with bore holes or with thinned secondary walls (Singh et al. 2013). It is easily available and exhibits fast growth and has the ability to use various lignocellulosic materials as substrates (Singh et al. 2013).
The production of better-quality pulps can be assisted by enzymatic methods, which have the advantage of being environmentally benign and mitigate the use of costly chemical methods (Janardhnan and Sain 2011). Enzymes could enhance or restore fiber strength, reduce beating times in the refining processes, assist in the bleaching process and increase interfiber bonding through fibrillation (Hassan et al. 2014). Xylanase enzymes are reported to partially hydrolyze the hemicellulose portion of pulps (Hassan et al. 2014). It is presumed that the enzyme hydrolyzes xylenes into smaller fragments, allowing lignin that is associated with these short hemicellulose chains to be more easily removed (Laftah and Rahman 2016). Xylanases have saved chemical costs for the industry without interfering with the existing process (Kenealy and Jeffries 2003). These enzymes have been used on a wide variety of pulp fibers (Ruzene and Gonçalves 2003). However, the application or use of xylanase enzymes is limited in PALF, despite the number of treatments applied for pulping production (Padzil et al. 2020).
Xylanases have saved chemical costs for the industry without interfering with the existing process (Kenealy and Jeffries 2003). These enzymes have been used on a wide variety of pulp fibers. Pulps from softwood, hardwoods, bagasse, eucalyptus and bamboo have all been treated with xylanases, and the treatment appears useful in assisting the bleaching of the corresponding pulp (Ruzene and Gonçalves 2003).
Different studies have been carried out on the use of PALF in paper production (Laftah and Rahman 2016). However, the use of enzymatic treatments to help bleaching is scarce (Laftah and Rahman 2016). This study aimed to improve paper produced from PALF biopulped with Trametes versicolor and further processed by chemical bleaching followed by enzymatic treatments with xylanase enzymes from Thermomyces lanuginosus at concentrations of 0.01%, 0.02% and 0.04%, and the biopulp was extensively analyzed for its color, chemical composition and structure characterized by its crystallinity obtained using X-ray diffraction (XRD) and its physical and mechanical paper properties. Therefore, the use of enzymatic treatments to help bleaching could dissociate the fiber bundles to increase their reinforcement of paper from this crop residue in countries where pineapple is planted.

Extraction and processing of fibers
Pineapple leaves were obtained from 18-month-old Ananas comosus plantations located in the dry tropical zone of the Central Pacific Coast of Costa Rica. Details about the source of the materials and the procedure used for extraction of the fibers were previously described elsewhere (Moya et al. 2014). With the leaf tips facing forward, approximately 4 to 6 pineapple leaves were introduced into the machine. Once the leaves were inserted up to approximately half of their length, they were pulled backward. At this moment, the pineapple leaf fibers were stripped of their parenchymal tissue. The operator then held the leaf by the fiber-free end and again introduced it from the base-side until reaching the exposed fibers. At this point, the leaves were removed, thus completely extracting the PALF. The fibers obtained were washed with water to remove any remaining chlorophyll, which produced a green tonality, and then stored in humid conditions until fungal inoculation. This material was named PALF.

Fungal inoculation
Pineapple leaf fibers (PALF) were first cut into small pieces 1 cm in length. The resulting material was separated into 30 samples of 100 g each and stored inside autoclave-resistant plastic bags. A small sample of ca. 1 g was used to determine the moisture content (MC). The samples were slightly moistened to reach 50% MC, followed by sterilization in an autoclave for 1 h at 120°C. Subsequently, 15 samples were inoculated with Trametes versicolor (L.) Lloyd. This fungus was selected due to its aggressiveness in lignin degradation (Moya et al. 2016). T. versicolor fungus was previously cultivated in a Petri dish in malt agar for 10 days until the mycelium reached three quarters of the Petri dish diameter. Then, 2.5 cm diameter mycelium segments were extracted and located on PALF. These segments were left to grow for 14 days in 250 mL flasks with 50 mL of a malt extractbased liquid medium.

Biopulping process
The flasks containing the inoculated fungi were kept intact to avoid the formation of mycelial lumps. Once enough mycelial mass was obtained, the samples were placed in 2000 mL beakers and blended at 3000 rpm with an electric manual whisk previously sterilized with alcohol. This enabled the rupture of the mycelia to attain a greater contact surface. The liquid medium containing the fungus was then poured into each plastic bag containing PALF, which were then stored in a dark environment at 26°C with 80% relative humidity during the fungal colonization period of Trametes versicolor. The colonization period was 4 weeks, according to Moya et al. (2016).
At the end of the fungal colonization period, the bags of pulp were washed to eliminate the mycelia. Subsequently, 500 mL of water was blended with fiber-mycelia for 2 min to completely separate the pulp. Finally, the mixture was washed until the fungal mycelium disappeared. The pulp was then placed on a 0.40 mm mesh sieve and manually pressed to eliminate the excess water, after which it was stored at 3°C. Before storage, three samples of 3 g each were taken to determine MC. This pulp was called biopulp from PALF or BP-PALF.

Bleached biopulp
The biopulp (BP-PALF) was bleached using the methods detailed by Cherian et al. (2010) where it was treated with 2% sodium hydroxide (NaOH) with a fiber to liquor ratio of 1:10, and a 1:3 mixture acetic acid (27%) and sodium hypochlorite (78.8%) solutions. The bleaching was repeated six times. After bleaching, the resulting material was thoroughly washed with distilled water and dried. The bleached BP-PALF was named bleached biopulp or BBP-PALF.

Enzymatic treatments of bleached biopulp
Analytical grade sodium citrate and citric acid were used for preparation of a citrate buffer (pH = 5.3). Commercial xylanases from Thermomyces lanuginosus were purchased from Sigma Aldrich (Sigma-Aldrich, St. Louis, MO) and used as received. Poly(diallyldimethylammonium chloride) (PolyDADMAC) with an average molecular weight of 400-500 kDa as a 20% (w/w) solution in water was purchased from Sigma-Aldrich. Enzymatic treatments of bleached pulp (BBP-PALF) with xylanase enzymes were carried out according to Hassan et al. (2010), as follows: 20 g of BBP-PALF was treated with xylanase enzymes in citrate buffer prepared (pH = 5.3) in a 500 mL conical flask at 10% (w/w). The concentrations of xylanase enzymes in pulp were 0.01%, 0.02% and 0.04% (w/w). Each reaction mixture was kept under gentle stirring at 65°C for 4 h. At the end of the corresponding reaction, the temperature was raised to 90°C to deactivate the enzymes. Then, the corresponding pulp was filtered and thoroughly washed with distilled water. The resulting pulps were named BBP-PALF -1X, BBP-PALF-2X and BBP-PALF-4X for treatments with xylanase concentrations in pulp of 0.01%, 0.02% and 0.04% (w/w), respectively.

Scanning electron microscopy
One sample of each material was analyzed by scanning electron microscopy (SEM) with a Hitachi Tabletop Microscope TM3000 (Hitachi High-Tech Corporation, Tokyo, Japan). This equipment was used for the visual determination of the degree of polymerization of fiber bundles. The samples were placed onto conductive carbon tape and directly analyzed using a working distance (WD) of 3.8-5.8 mm, an accelerating voltage of 7.5 kV and a magnification of 200 × .

Fiber morphology
The different materials were analyzed using macerated samples of each treatment, previously dissociated using customary methods proposed by Franklin (Moya et al. 2016). For the maceration procedures, small samples of each material were mixed with glacial acetic acid and hydrogen peroxide (1:1 v/v) and heated for 4 h. Then, 30 replicates of each treatment were stained with safranin and analyzed to determine the fiber dimensions of the sample (fiber length, fiber diameter, lumen diameter and cell wall thickness) using a digital camera on an optical microscope.

X-ray diffraction (XRD)
Air-dried samples of the different materials were cut into 2 mm pieces and then analyzed using XRD. XRD diffractograms were acquired using a PANalytical Empyrean diffractometer (Malvern Panalytical, Malvern, United Kingdom) with a Cukα1 radiation source at 40 mA and 45 kV. Each pattern was obtained in the 2θ range of 5.0085-44.9925° with steps of 0.0070° and step times of 13.7700 s. The Segal method-modified crystallinity index (CrI) was calculated from the XRD data based on the formula presented in the literature (Segal et al. 1959), as described in Equation 1: where I 002 is the intensity for the crystalline portion of the sample (i.e., cellulose) at 22° and I am the intensity related to the amorphous fraction at 16° (i.e., cellulose, hemicellulose and lignin).

Paper properties
The different pulps obtained were used for paper fabrication, and the resulting products were analyzed in detail, as described below.

Paper fabrication
First, the kappa index of each pulp was determined according to APPI/ANSI T 236 om-13. Paper production was developed following the TAPPI T 205 sp-02 standard (TAPPI 2002). In a typical procedure, 100 g of the dry pulp of interest (BP-PALF, BBP-PALF, BBP-PALF-1X, BBP-PALF-2X and BBP-PALF-4X) was used to obtain 20 sheets of paper, each with a diameter of 20 cm, using a sheet former Ogawa Seiki model OSK-2444 (Ogawa Seiki Co., Ltd., Japan). Each pulp was placed in a pile within a container of water for 30 min to refine the fibers. Then, the mixture was placed in the sheet former and drained, and the sheets were pressed for 5 min on each side using a pressure of 5 kg cm −2 . Finally, the sheets were dried in a ventilation chamber for 24 hours. The approximate grammage of the paper was 60 g m −2 .

Paper characteristics
Some physical and mechanical properties of the paper fabricated with each pulp were determined (BP-PALF, BBP-PALF, BBP-PALF-1X, BBP-PALF-2X and BBP-PALF-4X). For physical properties, the TAPPI T 410 om-13 standard was used for grammage determination, TAPPI T 412 mo-02 standard for moisture content, TAPPI T 411 om-10 standard for thickness, and the apparent density of each paper was determined by calculating the corresponding ratio between weight and volume (with volume calculated as thickness * area of sheet). For mechanical properties, the TAPPI T-494-mo-13 standard was used to determine stress resistance, TAPPI T 409 sp-15 standard for rupture length and TAPPI T 414 om-12 standard for tear resistance and index of longitudinal tearing determination.

Statistical analysis
A variance analysis (ANOVA) was verified to establish differences among the pulp treatments and paper properties (fiber morphology, chemical composition, paper characteristics and paper color). Tukey's test was carried out to determine the significant differences among means. Statistical analysis was conducted using SAS 8.1 software (SAS Institute, Cary, NC, USA).

Scanning electron microscopy
When fibers were extracted from the pineapple leaves, it can be observed that fiber bundles are free of the fundamental parenchyma tissue (Figure 1a), which is small in diameter and rounded in transversal sections (Moya et al. 2016). In the case of PALF subjected to colonization with T. versicolor (BP-PALF), fiber bundles remained joined to one another (Figure 1b).
In the case of BBP-PALF, as well as in BBP-PALF-1X, BBP-PALF-2X and BBP-PALF-4X, it was observed that fiber bundles were no longer frequently present but that the fibers that composed this type of structure were already separated or dissociated (Figure 1d-f). The amount of fiber bundles was lower in BBP-PALF (Figure 1c), and high dissociation of fiber bundles was observed for xylanases from Thermomyces lanuginosus in the enzymatic treatments (Figure 1d-f). A slightly higher separation of fibers in fiber bundles was observed in the BBP-PALF-4X treatment than in the other two concentrations (Figure 1f).
When extracted from leaves, PALF still possess approximately 15.34% of the total chemical components and forms a network within the fiber bundles to hold together the cellulose chains of these bundles (Moya et al. 2016). The white-rot fungus used for biopulping (T. versicolor) affects the complex structure of lignin (Laftah and Rahman 2016), penetrating at the extracellular level of lignin via enzymes with a complex known as the ligninolytic system (Laftah and Rahman 2016). Then, the fiber bundles of BBP-PALF and different concentrations of enzymes (BBP-PALF-1X, BBP-PALF-2X and BBP-PALF-4X treatment) produced fiber bundles with more dissociation than fiber bundles from PALF (Figure 1d-f).
The utilization of xylanases from Thermomyces lanuginosus is known in the paper industry for its bleaching characteristics, reduction in the Kappa index, increase in brightness (Walia et al. 2017), and reduction in chemical agents in pulp production processes (Walia et al. 2017). The dissociation of fibers by an increased enzyme concentration (Figure 1d-f) is due to the xylanases depolymerizing the hemicellulose on the surface of the fibers or the removal of chromophoric groups, in this case those that compose the bundler fiber, producing openings between the fibers (Walia et al. 2017). The openings between fibers and on the surface produce a dissociation of cellulose or show a slight decrease in the interfiber bonding strength (Valls, Vidal, and Roncero 2010).

Fiber morphology
The fiber dimensions of different samples are detailed in Table 1. Fungus treatment of PALF (generating BP-PALF) and bleaching of this fiber (producing BBP-PALF) first produced a decrease in length, fiber diameter and lumen diameter (Table 1). Enzymatic treatments (producing BBP-PALF -1X, BBP-PALF-2X and BBP-PALF-4X) also reduced fiber dimensions, but exposure to 3 concentrations of enzyme showed no difference in fiber dimensions (Table 1).
In the fiber treatments during the biopulping process with T. versicolor, the fungal hyphae have the ability to penetrate and degrade the complex structure of hemicellulose and cellulose in the fibers in bundles (Singh et al. 2013), producing a fractionation of the fibers compared to PALF (Table 1). However, another important aspect to note is that different concentrations of xylanase enzymes produced uniform fiber fractionation as the fiber length was maintained. This is because this type of enzyme was intended to improve surface quality and remove lignin residues in the fiber after biopulping (Kenealy and Jeffries 2003).
The Kappa index was reduced after treatment with BBP-PALF, BBP-PALF-1X, BBP-PALF-2X and BBP-PALF-4X, especially when PALF was treated with 0.04% xylanase or BBP-PALF-4× (Table 1). The decrease in these parameters can be attributed to the reduction of fiber length (Table 1) and better delignification for the xylanase treatment stage, which is the result of bleaching PALF (Kenealy and Jeffries 2003).

Chemical analyses
The chemical analysis of the BBP-PALF, BBP-PALF-1X, BBP-PALF-2X and BBP-PALF-4X samples showed that the amount of cellulose and holocellulose increased with respect to the initial material (PALF), while lignin and pentosans decreased (Table 2). However, despite the increase in cellulose content in those samples, they showed lower α-cellulose content ( Table 2).
As expected, treatment with xylanase enzymes removes lignin residue and bleaching in biopulps, specifically lignin associated with hemicellulose (Walia et al. 2017). According to 2017), xylanase treatment helps in the removal of chromophoric groups from the pulp as well as partial hydrolysis of the reprecipitated xylan or lignin-carbohydrate complexes, thus increasing the porosity of the pulp to allow the free diffusion of bleaching chemicals or splitting of the linkage between the residual lignin and carbohydrates. It is proposed that the released xylan contains carbohydrate complexes, and both mechanisms may allow enhanced diffusion of entrapped lignin from the fiber wall.
The cellulose and holocellulose contents were higher and the amount of lignin was lower in BBP-PALF, BBP-PALF-1X, BBP-PALF-2X and BBP-PALF-4X compared to PALF ( Table 2). The decrease in pentosans in BBP-PALF-1X, BBP-PALF-2X and BBP-PALF-4X (with respect to PALF) indicated that possibly the pectin bound in hemicellulose was partly reduced, which led to the disintegration and rupture of the fiber bundle and the dissolution of part of the hemicellulose. The changes mentioned, as shown below, produce an improvement in paper properties, resulting from the breakdown of hemicellulose and lignin (Liu et al. 2017). Regarding α-cellulose and pentosans, these types of cellulose and hemicellulose (respectively) decreased with enzyme treatments in relation to PALF, indicating a degree of degradation of α-cellulose from pentosans in such treatments (Liu et al. 2017).
The comparison of the chemical properties of these biopulps with other types of biomasses, including trees (Kapun et al. 2022), plants (Ferhi et al. 2014), grass (Ferdous et al. 2021) and nonwoody cellulosic (Mannai et al. 2016), showed that pulling from PALF and the treatment tested produced pulp with the highest quantity of cellulose, hemicellulose and pentosans.

X-ray diffraction (XRD) analysis
XDR diffractograms of all the samples are presented in Figure 2(a-f). Signals observed at a 2θ value of 16.8° represent amorphous regions of the materials, while at approximately 22°, the sharp and intense peaks show their highly crystalline portions (Nam et al. 2016). The modified Segal index was calculated (Equation 1) because of its ease of use and is widely employed to determine relative changes in crystallinity after physicochemical and biological treatments of cellulose materials, in which the cellulose changes are observed at 16.8° and 22° (Nam et al. 2016). The diffraction patterns were similar in all 5 treatments (Figure 2a-f), with a slight increase in intensity at 22.15°, indicating an increase in the crystallinity of the cellulose (Nam et al. 2016). This increase was verified by calculating the crystallinity index of the different materials (Figure 2g). The lowest crystallinity index was observed in PALF, followed by BP-PALF and then by BBP-PALF. The highest values were obtained after enzymatic treatments (Figure 2g). This result confirms the higher cellulose crystallinity when PALF was biopulped, bleached and treated enzymatically. The crystallinity index may be increased by the dissolution of noncrystalline materials, such as hemicellulose, and the amorphous areas of cellulose were attacked simultaneously, and the proportion of the crystalline areas increased (Ling et al. 2019). However, cellulose crystallinity xylanase enzymes are introduced into the fiber molecules during the process of lignin degradation and may produce swelling of the crystalline area (Ling et al. 2019) and alter paper properties, such as the mechanical properties, as described below.

Paper properties
The physical characteristics of paper fabricated with different pulps are shown in Table 3. The moisture content was different in papers fabricated with BP-PALF and paper fabricated with BBP-PALF and BBP-PALF-1X. The thickness and apparent density of paper were significantly different in all treatments, except in BBP-PALF and BBP-PALF-1X for thickness and in BBP-PALF-1X and BBP-PALF-4X for density. The highest values of grammage were found in BBP-PALF-4X, while the other papers showed lower values, all of them with no significant difference. In relation to the mechanical properties of the papers, there were important differences between the pulps used to fabricate them (Figure 3). Enzymatic treatments deteriorate mechanical properties (Figure 3). Tensile stress resistance and rupture length were higher in BP-PALF, and the lowest was found in BBP-PALF-2X and BBP-PALF-4X (Figure 3a-b). The tear resistance and index of longitudinal tearing decreased significantly in paper made from biopulp treated with the three enzyme concentrations (Figure 3c-d).
The variation in paper properties indicates the differences found in fiber dimensions (Table 1). It was observed that the treatments with xylanase enzymes, mainly BBP-PALF-2X and BBP-PALF-4X, produced shorter fiber lengths in relation to the other treatments (Table 1), so this effect allowed a better arrangement of the fibers in the sheet, resulting in a higher bulk density and a higher grammage (Table 3). Although these physical properties were improved, in these two treatments, the mechanical properties (stress and tear resistance, rupture length and index of longitudinal tearing) decrease because the fibers suffer a degradation of the surface (Gangwar, Prakash, and Prakash 2014), affecting the strength properties of the papers produced ( Figure 3). However, despite the results, the biopulping process is technologically feasible and cost-effective (Walia et al. 2017). This process reduces electrical energy consumption, increases mill through-put for mechanical pulping and reduces its environmental impact (Walia et al. 2017).
A comparison of the properties of these paper samples with trees (Kapun et al. 2022), plants (Ferhi et al. 2014), grass (Ferdous et al. 2021) and nonwoody cellulosic (Mannai et al. 2016) demonstrated that biopulp from PALF and treatment produced pulping with some properties that were higher and other properties that were lower, for example, tensile force and stress and tear resistance, respectively.

Color properties
The color parameters (L*, a* and b*) of different samples are detailed in Table 4 and Figure 4. The three treatments BBP-PALF-1X, BBP-PALF-2X and BBP-PALF-4X exhibited the greatest change, as measured by the ∆E* parameter shown in Table 4. The BP-PALF treatment exhibited the lowest ∆E*, while BBP-PALF exhibited values close to the enzymatically treated pulps (BBP-PALF-1X, BBP-PALF -2X and BBP-PALF-4X), indicating the importance of bleaching in the increase in lightness (Perng, Wang, and Chang 2015). The cleavage of the carbohydrate portion of the lignin-carbohydrate complex to smaller residual lignin molecules, which were easier to remove, is also a possible mechanism of xylanase prebleaching (Perng, Wang, and Chang 2015).

Conclusions
Biopulping using Trametes versicolor and further processing by enzymatic treatments using xylanase enzymes from Thermomyces lanuginosus produces changes in the morphology (fiber bundles were shortened and dissociated), chemical characteristics (increased cellulose and holocellulose), crystallinity (decreased), color (increased lightness) and properties of the paper (some mechanical properties decreased). Generally, during the process of biopulping, chemical bleaching and processing with xylanase enzymes, the amount of lignin in the pulp decreased, and therefore, the amount of cellulose with a higher  crystallinity increased. These changes were advantageous in that the treatment with enzymes decreases the Kappa index and crystallinity index and increases the lightness of the paper, but were disadvantageous in that the mechanical properties of the paper decreased. According to the results, the biopulp of PALF treated with 0.04% (w/w) xylanase enzymes produced paper with the best properties.

Highlight
• Pineapple residues represent an alternative raw material for pulp and paper • Biopulping is an attractive method involving biochemical conversions • Pineapple leaves fibers were non-conventionally treated to produce pulps and papers • A comprehensive characterization of the materials involved is presented • Chemical and enzymatic processing act together to provide whiter and heavier paper