2.3. Optimisation of Enzymatic Pre-Treatment Processes
The Kappa number is an efficient analytical technique for measuring the amount or degree of lignin in a completed or in-process pulp sample. The determination of reducing sugar (λ 540) is compulsory to support this evidence, as the reducing sugars were liberated from the polysaccharides (xylan and pectin) when the pulp was treated with xylano-pectinolytic enzymes, resulting in the high free sugar concentration (xylose and galacturonic acid) in the pulp sample. Furthermore, the reduction in kappa number, release of reducing sugars (λ 540), release of lignin (λ 280), and hydrophobic compounds (λ 465), coupled with aromatic compounds (λ 237 nm) during pulp bleaching are interrelated phenomena and reflect the dissociation of lignin-carbohydrate complex, and these can be measured using absorbance for rapid screening as suggested by many researchers [
20,
21,
22,
23].
Cellulase-free xylanase and pectinase from
B. amyloliquefaciens ADI2 were grown using inexpensive lignocellulosic material (i.e., banana peel), whereby the carbon source exhibited good xylanase-pectinase production at a ratio of 1:1.3. The same medium would extensively reduce the production cost as opposed to that of individual enzymes from different microorganisms by utilizing the bacterium to produce xylanase and pectinase concurrently within. As such, the technology would be cost-effective and commercially viable alike. The selection of
B. amyloliquefaciens ADI2 for the biobleaching pre-treatment of OPEFB pulp was supplemented by its properties, specifical stability throughout wide-ranging pH and temperature. To this end, biobleaching with the enzymatic pre-treatment revealed the optimum xylanase-pectinase dose of 15 U/g and 19.5 U/g, respectively, for the oven-dried pulp as the most effectual enzyme dosage pre-treatment (
Table 1).
In contrast, pulp treated with enzyme doses is higher than the specified values failed to yield enhanced pre-treatment effectiveness. However, reports have indicated that the enzyme dosage of 5 U/g for xylanase and pectinase alike in the process of mixed hardwood and bamboo kraft pulp biobleaching [
17,
18].
The second experimental design for the effect of retention time was carried out, whereby 180 min was deemed fit for the pre-treatment (
Table 2); beyond the aforementioned values, there was no significant improvement in biobleaching efficiency. The enzyme dosage is interrelated to the retention time. In fact, the same bleach-boosting effect can be obtained in a shorter time by increasing enzyme dosage [
24]. The same phenomenon can be observed at the third experimental design for the effect of pH on pulp biobleaching, where pH 8.5 is the best condition for biobleaching pre-treatment (
Table 3). Indicated xylano-pectinolytic enzymes work best under moderate alkaline conditions. The increased amount of positively-charged side chains due to alkaline conditions on the xylano-pectinolytic enzyme’s structure might have contributed to this finding [
25]. As for the fourth experimental design, the effect of temperature as shown in
Table 4, the optimum temperature was found suitable for pretreatment at 40 °C. The higher or lower temperature of incubation did not improve the biobleaching benefits significantly, suspected due to the thermal denaturation of enzymes structure or low kinetic energy of enzymes [
26,
27].
When subjected to the optimum conditions, the enzymatic pre-treatment could release reducing sugars as much as 66.47 ± 17.97 mg/L g of oven-dried pulp. Following the enzymatic treatment, reducing sugars found in the pulp-free filtrate were indicative of those yielded due to degraded xylan and pectin chains in the pulp fibres. Accordingly, the hydrophobic compounds (λ 465 nm) and phenolic compounds (λ 237 nm) were recorded at an absorbance of 0.22 ± 0.01 and 0.26 ± 0.04, respectively. Similarly, the characteristic peak seen post-enzymatic treatment in the pulp-free filtrate at the wavelength of 280 nm was suggestive of lignin present in the colouring matter generated. Furthermore, the enhanced filtrate absorbance observed throughout varying wavelengths post-enzymatic treatment was attributable to degraded xylan and pectin, thereby yielding maximal lignin production, reducing sugars, and chromophores from the pulp fibres.
Following enzymatic treatment, the Kappa number for the control pulp was further minimised to 110.8 units instead of its original value of 122.78 units, yielding a decrement of 9.75% reduction. The previous report indicated a reduction of 1.5 units in kappa number by xylanase from
B. licheniformis 77-2 [
28]. This could be supported by using xylanase from
B. coagulans, thus revealing a 5.45% decrease for the kappa number of non-woody pulp [
29]. Meanwhile, earlier reports detailed the synergistic action of xylanase and pectinase yielded by two bacteria,
B. pumilus and
B. subtilis reduced 1.2 units in kappa number, respectively [
18]. Similarly, post-enzymatic pre-treatment of pectinase from
B. subtilis SS on mixed hardwood and bamboo kraft pulp revealed a reduction of 5.85% for its pulp kappa number [
12]. In xylanase-treated wheat straw pulp (i.e., enzyme dose 10 U/g), its kappa number produced a decrement of 1.1 points [
30]. Therefore, these studies demonstrated a reduced kappa number following the presence of xylanase and pectinase in biobleaching pre-treatment, subsequently resulting in an improved pulp brightness.
In summary, parameters such as enzyme dosage (i.e., xylanase-pectinase dose of 15 U/g and 19.5 U/g, respectively, for optical density (OD) pulp), pH condition (i.e., pH 8.5), temperature (i.e., 40 °C), and retention time (180 min) denoted the variables leading to higher pulp solubilisation. They displayed well-correlated links to the reducing sugars yielded and kappa number reduction.
Treated and untreated OPEFB pulps with xylano-pectinolytic enzymes were both subjected to chemical bleaching, whereby treated pulp revealed slightly higher brightness compared to its untreated counterpart. This might be attributable to the selective xylan and pectin removal, thus facilitating lignin removal as per the decrease in kappa values [
31].
2.4. Biobleaching Using Xylano-Pectinolytic Enzymes on OPEFB Pulp
OPEFB pulp treated with xylano-pectinolytic enzymes isolated from
B. amyloliquefaciens ADI2 revealed an enhanced brightness by 11.25%, thereby leading to 11.25% less chlorine consumption for obtaining optical pulp attributes identical to those generated by traditional chemical bleaching (
Table 5). Consequently, the process is financially feasible and environmentally-friend alike, where they save other bleaching chemical costs and reduce toxic discharge, thus simplifying and reducing expenditure for wastewater treatment generated from mill effluent. Furthermore, the enzyme can be optimized and produced in high-yield and reproduced cost-effectively through cost-effective substrates or processes [
2]. The 11.25% decrement in chlorine or chemical consumption was due to xylanase and pectinase’s synergistic actions, which causes xylan and pectin found in the pulp fibre to be degraded. Besides, the actions amplified the bleaching chemical access to the lignin layer of the pulp.
Previous reports revealed a 3.68% increment in brightness for enzyme-treated hand sheets showing less chlorine consumption in kraft pulp upon concurrent implementation of xylanase and pectinase from
B. pumilus AJK [
9]. Meanwhile, eucalyptus kraft pulp revealed chlorine savings of 8% by utilising xylanase from
Streptomyces sp. QG-11-3 [
32] as reported earlier.
The kappa number of untreated pulp subjected to bleaching was 89.52 ± 4.33 upon the final stage of bleaching via enzymatic treatment following a reduction of 27.1% from 122.78 ± 0.24 (
Table 5). Alternatively, the enzyme-treated pulp depicted increased brightness at every bleaching phase. In the D
1EpD
2 process, approximately 35.54% of brightness increment was attained for the enzyme-treated pulp by the final stage of bleaching. This was indicative of the maximum biobleaching effect due to synergistic xylanase-pectinase action during the initial phases. Meanwhile, crude xylanase isolated from
B. licheniformis yielded 5.0 units of brightness increment upon its incorporation for eucalyptus kraft pulp bleaching [
28].
Both conventional chemical bleaching and biobleaching could improve the quality of paper compared to the control. In particular, increments were seen across various parameters for the enzyme-treated pulp, namely 30% for the tensile factor, 19.4% for the bursting factor, 20.9% for the tearing factor, and 11.25% brightness compared to conventional chemical bleaching (
Table 6). Here, its tensile factor could be related to the linking capacity between fibres. In contrast, the tear factor was better than the control, thus suggesting the lack of extensive cellulose matrix degradation in the enzymatic treatment [
33], as well as indicating the particular effect/reaction of xylanase and pectinase.
Furthermore, brightness improvements perceived following enzymatic pre-treatment could be attributed to two factors; xylanase action on xylan and pectinase capabilities. The first factor denotes xylanase action on xylan precipitate found on lignin, whereby the precipitate occurs due to lowered pH by the end of the cooking phase. Thus, xylanase action causes the precipitate removal and improves the bleaching chemical accessibility to the pulp fibres. Meanwhile, lignin’s capability for formulating complexes with polysaccharides like xylan and the alkali resistance attribute for some of the bonds may render them non-hydrolysed during the kraft process [
34,
35]. Here, xylanase causes cleavage of bonds still present linking lignin and xylan, thereby rendering the cellulose pulp structure open and xylan fragmented, following which the fragments are extracted [
36].
Secondly, pectinase can weaken the complex bond structure of lignocellulosic components found between hemicellulose-cellulose-lignin frameworks. In particular, it hydrolyses pectins present in the hemicellulose, followed by the segregation of microfibrils under alkaline conditions. This causes loosening of the framework structures, which in turn become more flexible and display higher pulp fibre porosity. The mechanism results in increased accessibility of chemical agents to the pulp fibres during chemical bleaching [
37], thus aiding in removing more lignin materials and ensuring higher brightness of the paper hand sheets.
These results indicate the enzymatic pre-bleaching’s role in facilitating increased pulp fibrillation, water retention, and bonding restoration in fibres [
8,
10], which is possibly attributable to harsh chemical additive effects on them.
Table 5 and
Table 6 display enhanced enzymatic-treated pulp physical properties compared to those subjected to chemical/conventional bleaching. Thus, this study proved the benefits of biobleaching by concurrently using enzymes such as xylanase and pectinase with chemical pre-treatment, including: enhanced pulp digestibility, improved paper quality, and the lower chemical additive amount required. The last perk would minimise the amount of polluting materials seen in the effluent.