The effect of endo-1, 4-β-xylanase from Thermomyces dupontii KKU−CLD−E2−3 on eucalyptus pulp bleaching and effluent treatment

The effect of endo-1, 4-β-xylanase from Thermomyces dupontii KKU−CLD−E2−3 on eucalyptus pulp bleaching and effluent treatment | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The effect of endo-1, 4-β-xylanase from Thermomyces dupontii KKU−CLD−E2−3 on eucalyptus pulp bleaching and effluent treatment Wasan Seemakram, Hiroyuki Harada, Jindarat Ekprasert, Sophon Boonlue This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-33851/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract Endo-1, 4-β-xylanase-chlorine dioxide bleaching of eucalyptus pulp and analysis of effluent was investigated. The eucalyptus pulp bleaching in D0 stage was prepared with enzyme dose 100 Unit of 10% consistency and incubated at 70°C for 30 min. The brightness level was achieved up to 72.50 (% ISO). The kappa number and viscosity of eucalyptus pulp was found to be 1.70 and 8.90 (cp), respectively. The paper from pulp bleaching with enzyme has shown an increase in brightness, breaking length, bursting strength, and tearing resistance to be 89.60% IOS, 37.27 Nm/g, 138.81 kPa and 218.02 mN, respectively. The SEM and FTIR analysis of pulp fibers revealed a significance of morphological and structural changes. The analysis of the effluent also showed statistically-significant differences in TOC, BOD, TDS and TSS. The FTIR analysis of the effluent showed the organic compounds and chloride dioxin in cases of those treated by chemicals. Biobleaching Chlorine dioxin Effluent Environmental Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Xylanases (EC 3.2.1.8) are a group of glycoside hydrolase enzymes that can degrade the linear polysaccharide β-1, 4-xylan resulting in xylose 1 . The xylanase cleaves the glycosidic bonds in the xylan backbone, leading to a reduction in the degree of polymerization of the pulp fiber. The mechanism how the enzyme attacks the molecule of xylan depends on the nature of the pulp fiber molecule, such as on the chain length of branching, and the presence of substituents 2 . Recently, Orozco-Colonia et al. 3 reported that xylanase could attack the xylan of hemicellulose by inserting between cellulose and lignin resulting in removal of lignin associated hemicelluloses is facilitated with minimal damage of pulp fiber. In addition, pretreatment of pulp with xylanase can decrease the partial disruption of lignin and carbohydrate bonds in pulp fiber, as the result of enhancing the accessibility of the subsequent bleaching chemicals to the pulp 4 . In general, during the process of pulping, bleaching and washing in pulp and paper industry produces effluent that contains with high amount of organic and inorganic compounds including lignin, hemicellulose, cellulose fragments chlorophenols, fatty acids, inorganic salts and the chlorinated dioxins 5 . Thus, the application of xylanase in the pulp and paper industry focuses to reduce the use of chlorine dioxide in pulp bleaching process that decreases the toxicity in effluents. Besides, there was also a report that the effluent produced from paper bleaching by xylanase after pulp washing could reduce the TSS, TDS, TOC, BOD, colour, and pH 4 . Currently, the pre-treatment of pulp using xylanase has been adopted to be alternate approach that plays an environmental friendly and economically viable method for paper industries. However, in order to use xylanase in the pulp bleaching process, several properties are needed to be considered. The enzyme should be free of cellulase and active at high temperatures. Thermo-alkali-stable xylanase has attracted to use for pulp bleaching which can work well at relatively higher temperatures without additional cooling process requirement. Thus, there is a continuous need to find novel xylanase which is active in alkaline pH and also thermostable during the pulping process. This work presents the application of purified endo-1, 4-β-xylanase from Thermomyces dupontii KKU−CLD−E2−3 in pulp bleaching step under routinely performed by Phoenix Pulp and Paper Public Company Limited in SCG Packaging, Khon Kaen, Thailand, which is tested at alkaline pH and high temperature. Characterization of pulp, paper and effluent after being treated with enzyme and chlorine dioxide bleaching was presented. Results Eucalyptus pulp bleaching by the enzyme and chlorine dioxide were analyzed for their brightness, kappa number, and viscosity. The brightness was increased from 53.60 to 72.50% ISO, when bleaching with xylanase at 100 Unit and chlorine dioxide at 82.80% ISO (Fig. 1 ). In cases of bleaching with xylanase and chlorine dioxide, the Kappa number decreased from 3.20 to 1.70 and 0.20, respectively (Table 1 ). On the other hand, the viscosity decreased from 9.40 to 9.10 and 7.80 cp, respectively. The effluents after bleaching with xylanase and chlorine dioxide were also characterized. The sample treated with xylanase revealed the maximum free reducing sugars released from pulp compared to the untreated sample (Table 1 ). Chromophoric and hydrophobic compounds were observed in xylanase and chlorine treated pulp samples, as reported in Table 1 . The surface states of fibers in pulp samples treated with endo-1,4-β-xylanase and chloride were analyzed by SEM. The SEM analysis revealed noticeable changes in fiber disintegration on pulp in treated enzyme and chloride, compared to the untreated case (Fig. 2 ). The surface of untreated eucalyptus pulp was smooth and tight (Fig. 2 A). When the eucalyptus pulp was attacked by purified xylanase, its fiber surface became rough, loose and was in a peeling state and loss in compactness (Fig. 2 B). In contrast, the eucalyptus pulp which was attacked by chlorine dioxide became lost in compactness of pulp fibers and more disintegrating (Fig. 2 C). FTIR spectra of untreated, enzyme-treated and chlorine dioxide-treated eucalyptus pulp were compared. The functional group levels were changes in the peaks of modified in pulp samples (Fig. 3 ). The peaks of FTIR spectra in the pulp fiber were assigned as shown in Table 2 . Eucalyptus pulp samples after passing through the D1 step were used to prepare papermaking of 20 gram. The quality of paper was analyzed for its brightness, breaking length, bursting strength, and tearing resistance. The examination of the brightness from the enzyme-treated and chlorine-treated samples were significantly increased at 89.6% and 92.8% ISO, respectively. In addition, the paper samples from pulp bleached with enzymes treatment showed a maximum value of breaking length, bursting strength, and tearing resistance of 52.25 Nm/g, 138.81 kPa and 218.00 mN, respectively, when compared to the untreated control and the chlorine-treated sample (Table 3 ). The present work revealed that the wastewater obtained after pulp bleaching by endo-1,4-β-xylanase significantly showed lower values of the TSS and TDS of effluents of pulp bleaching for chloride compared to xylanase treatment reduced from 102 to 57 mg/L, and 2183 to 1139 mg/L, respectively. There was also a reduction of TOC and BOD from 60.15 to 6.81 mg/L and 538.00 to 61.33 mg/L, respectively (Table 4 ). These values are in the range of industrial effluent standards assigned in Thailand (IEST). The TSS, TDS, TOC and BOD in effluent significantly decreased when pulp was bleached by the enzyme compared to using chlorine dioxide are shown in Table 4 . The FTIR spectra of effluents obtained from untreated, enzyme-treated and chlorine-treated samples was compared. The band assignment of the FTIR spectrum of effluent was listed in Table 5 . The functional groups remarkable profiles were changed in the wavenumbers of 500 to 3900 cm −1 as shown in Figure 4 . Discussion We observed effects of the ability of endo-1, 4-β-xylanase from T. dupontii KKU−CLD−E2−3 in the bleaching of eucalyptus pulp at D0 stage under pulp quality assessment of SCG company was investigated. The results revealed that kappa number significantly decreased up to 46.87% and the brightness increased up to 35.26% after being treated by endo-1, 4-β-xylanase dose 100 U, as compared to the untreated control. The chemical pulp bleach can reduce kappa number of 93.75%, resulting in no lignin in the pulp fiber and an increase in brightness of 82.80% ISO. The kappa number is an indication of the residual lignin content in pulp fiber which can be used as an indicator of the efficiency of bleaching process. A reduction of kappa number and an increase in the percentage of brightness in our experiment was higher than those reported in previous studies. Kumar et al. 6 reported that the brightness of hardwood pulp pre-treated with xylanase increased 21.56% while the kappa number decreased 20% compared to the untreated samples. According to Wu et al. 7 who reported that the brightness of eucalyptus kraft was significantly increased about 14.5%, while 24.5% of kappa number was reduced after bleaching by endoxylanase from Streptomyces griseorubens LH–3. The viscosity of pulp after bleaching with endo-1, 4-β-xylanase dose 100 U was found comparatively higher (9.10 cp) than chlorine dioxide bleached pulp (7.80 cp) (Table 1 ), which was not significantly different when compared to the untreated case. Higher viscosity means higher cellulose content which is directly proportional to the greater physical strength of pulp 8 . In addition, the release of reducing sugar, phenolic compounds and hydrophobic compounds also increased when the enzyme concentration increased (Table 1 ). The amount of sugar in the effluent of eucalyptus pulp bleaching for xylanase also significantly increased, which shows that the final product from the enzyme treated pulp was released to sugar. The release of reducing sugars, phenolics, and hydrophobic compounds are a result of the reaction of disintegration of lignin and carbohydrate complex from the pulp fibers 9 . The surface states of fibers have exhibited noticeable changes on the surface fiber was treated with endo-1, 4-β-xylanase as compared to the untreated case. This indicated that the enzymatic reaction was more specific to xylan in the pulp fiber more than chemical one. The enzyme treatments may expose cellulose fibers to the surface 11 cause surface alteration of the enzyme-treated pulp was due to the activity of xylanase 10 . Also, the xylanase activity in the digestion of xylan causes the release of lignin. The pretreatment of eucalyptus pulp using chlorine dioxide caused severe damage to the pulp, in which the strength of fibers was reduced and the cracks and peeling of pulp surface were appeared. FTIR spectra showed functional group alteration in the eucalyptus pulp samples after bleaching in D0 stage. The peak at 1614 cm −1 which corresponds to the C=O stretching vibration in conjugated carbonyl of lignin was shown in the untreated and the enzyme-treated pulp samples, but not in the chemical-treated one 11 . The peaks at 1430 cm −1 corresponds to the symmetric CH 2 bending vibration 12 . The presence of peaks at 1372 cm −1 and 1318 cm −1 were due to the aliphatic C–H stretching in methyl and the CH in-plane bending vibration in cellulose I and cellulose II 13 . Peak at 1163 cm −1 and 1113 cm −1 indicated the C–O–C asymmetric stretching in cellulose I and cellulose II and the C–O–C stretching of hemicellulose 11 . The peaks at 1060 cm −1 and 1035 cm −1 were attributed to the C–O stretching vibration of acetyl cellulose and hemicellulose and the C–O stretching vibration of cellulose, hemicellulose and lignin, respectively 10 , 14 . Eucalyptus pulp samples after passing through the D1 step were used to prepare papermaking of 20 grams. The quality of paper was analyzed for its brightness, breaking length, bursting strength, and tearing resistance. The examination of the brightness of papers treated by enzyme showed an increase of 29.20% compared to the control, but < 3.45% compared to chlorine dioxide. However, the paper from pulp bleached with enzymes treatment are shown the value of breaking length, bursting strength, and tearing resistance at 52.25 Nm/g, 138.81 kPa and 218.00 mN, respectively. The fiber properties of enzyme pre-bleached pulp had a higher value of tear index compared to that of the control, but no significant difference was observed. The tearing index of enzyme-treated pulp increased up to 15.71% which was higher than those from the untreated pulp 15 . Obviously, pretreatments by enzyme could improve value tensile, tearing and bursting index better than the use only chemical process. The paper industry worldwide usually causes serious environmental burdens due to its 40% global industry wastewater discharge leading to environmental pollution problems 4 , 16 . The present work revealed that the wastewater obtained after pulp bleaching by endo-1,4-β-xylanase significantly showed lower values of the TSS and TDS as compared to chloride treatment reduced 44.12% and 45.73%, respectively. There was also a reduction of TOC and BOD of 80.68% and 88.60%, respectively (Table 3 ). These values are in the range of industrial effluent standards assigned in Thailand (IEST). The TSS, TDS, TOC and BOD in effluent significantly decreased when pulp was bleached by the enzyme compared to using chlorine dioxide. Similarly, Sridevi et al. 4 reported that the use of xylanase in pulp bleaching process resulted in the reduction of BOD (89.83%), TSS (72.14%) and TDS (53.32%) in the wastewater discharge. The use of our xylanase for pulp bleaching could bring about not only higher quality of effluent which is even better than the IEST, but also reduce the amount of organic/inorganic materials in the effluent. Therefore, the quality of the effluent from xylanase-bleaching process is ready to be discharged without passing through the company’s wastewater treatment processes. The FTIR spectra of effluents obtained from untreated, enzyme-treated and chlorine-treated samples was compared. The functional groups remarkable profiles were changed in the wave numbers of 500 to 3900 cm −1 as shown in Figure 3 . Interestingly, there were some significant changes at peak around 941.24 cm −1 corresponding to C–O stretching of chloride dioxin caused by the combination of Cl and lignin rings which was seen in case of pulp bleaching by chloride only 17 , while pulp bleached by endo-1, 4-β-xylanase could not be observed. Nevertheless, the chlorine dioxin was shown between 935 and 950 cm −1 18 which caused by holocellulose of pulp from eucalyptus wood with bleached by chlorine dioxide pretreated in pulp bleaching 19 . This provided evidence that endo-1, 4-β-xylanase has good exceeding for pulp bleaching compared to commercial chemical treatment. Because in the pulp bleaching process by endo-1, 4-β-xylanase can help to improve the quality of pulp and paper. Also, using enzyme has efficiency for economic improvement of effluents and reduces the risk of cancer cause by dioxin for the staffs in working area. Additionally, the odor pollution from the use of chemicals in the factory can be reduced as well. Conclusions An endo-1, 4-β-xylanase bleaching of eucalyptus pulp in D0 stage could increase the paper brightness. The physical properties of the paper including breaking length, bursting strength, and tearing resistance were more improved than those using chemicals bleaching works well in realistic situations. The variety of contaminants present in the effluent from the pulp bleaching process after enzyme bleaching was degraded. Therefore, this bio-bleaching process is cost-effective and has environmental benefits than the chemical bleaching. Materials And Methods Pulp and enzyme Eucalyptus pulp used in the experiment was obtained from press 3 stage of Phoenix Pulp and Paper Public Co., Ltd. in SCG Packaging, Khon Kaen, Thailand. The characteristics of the pulp were as followed: 3.2 kappa number, 53.6% ISO of brightness, 9.4 cp of viscosity and initial pH 9.7. The purified endo-1, 4-β-xylanase was produced from Thermomyces dupontii KKU−CLD−E2−3 (Accession number LC428093) 20 . The eucalyptus pulp bleaching was carried out by following the method which routinely performed by Phoenix Pulp and Paper Public Company Limited in SCG Packaging. Effect of endo - 1, 4 - β - xylanase on eucalyptus pulp in Chlorine dioxide stage (D0) Eucalyptus pulp was prepared by treating with an enzyme dose 20–100 (U/50 g pulp) in sealed plastic bags with intermittent kneading and 10% (w/v) pulp consistency for a time period of 30 min at 70 °C. Control sample was untreated by enzyme and treated by chlorine dioxide (ClO 2 ) at a concentration of 15 kg/T pulp under the same conditions with an activated enzyme. Effect of endo - 1, 4 - β - xylanase on eucalyptus pulp in Extraction - Peroxide stage ( EOP ) and Chlorine dioxide stage (D1) After passing the procedure D0, excess enzyme and buffer in eucalyptus pulp were removed by pressing and draining from the pretreated pulp. The eucalyptus pulp samples prepared were treated with 10% (w/v) pulp consistency. The eucalyptus pulp samples were bleached in EOP staged with a NaOH (14 kg/T pulp) and H 2 O 2 (5 kg/T pulp) incubated at 65 ºC for 120 min. After that, the chemicals in eucalyptus pulp samples were removed by pressing and draining with tap water, and were treated with a ClO 2 (5 kg/T pulp) of time period for 240 min at 70 °C. Finally, the eucalyptus pulp samples were washed by pressing and draining with tap water again. Analyses of eucalyptus pulp properties The eucalyptus pulp bleaching obtained from D0 stage was prepared for hand sheets and analyzed for brightness using a brightness machine (Technidyne Corporation, Color Touch™ 2). The efficiency of the bleaching treatment process was observed for the reduction of kappa number 21 and viscosity. The eucalyptus pulp samples were dehydrated in acetone, critical point dried and mounted on sample stubs. The feature surfaces of the xylanase-treated and the control samples were observed under scanning electron microscope (SEM) (SEC, SNE–4500M). Characterization of papers Eucalyptus pulp samples after passing the procedure D1 were used for papermaking at 20 gram to study the properties of the paper. The paper was obtained from pulp bleaching with purified xylanase. Thereafter, they were massaged pulp fibers by basket centrifuge at 4500 rpm and then were prepared wet sheets forming by hand sheets with 10% (w/v) pulp consistency. The eucalyptus pulp samples were pressed for consolidation of wet. The paper was baked to remove moisture so that the remaining moisture was about 4–6%, which dried overnight in a temperature-controlled room where it operated at temperature of 25 ºC, and the moisture was kept at 55%. The quality of paper was analyzed for their brightness (Technidyne Corporation, Color Touch™ 2), breaking length (REGMED, Horizontal tensile−D−21), bursting tester (ABB AB/Lorentzen & Wettre, RS232C), and tearing tester (Labthink, SLY−S1), respectively. Effluent characterization of eucalyptus pulp bleaching The effluent was characterized before disposal by following the standard method described by Sridevi et al. 4 . The enzyme-mediated release of hydrophobic and chromophoric compounds from pulp bleaching process were measured the absorbance at 280 nm and 465 nm, respectively 22 . Total amount of reducing sugars released from pulp bleaching was determined by the DNS method 23 . The FTIR analysis of effluent after treated eucalyptus pulp by enzyme was carried out. We aim to investigate the functional groups of the contaminant particles obtained during enzymatic treatment into the effluent. The characterization properties of effluent were determined for several parameters including pH, TSS, TDS, BOD (VELP; Scientifica), and TOC (SHIMADZU, SA24). Statistical analysis Experimental designs were statistically analyzed by a Completely Randomized Design (CRD) and pair comparisons in the least significant difference (LSD) test to determine variations and the significance of differences between treatments. All experiments were performed in triplicate, and the results were expressed as the mean. Effects of the variables and the significance of regression coefficients were determined at F-test (P ≤ 0.05). Declarations Acknowledgements This study was financially supported by Protein and Proteomics Research Centre for Commercial and Industrial Purposes in FY 2016 in ongoing project no ProCCI60001, and Student Exchange Program 2019 from Faculty of Science, Khon Kaen University, Thailand and Student Exchange Program 2019 from Hiroshima Prefectural of University, Japan. The authors thank the Phoenix Pulp and Paper Public Company Limited in SCG Packaging for supporting the equipment and facilities on laboratory analysis. Author information Affiliations Department of Microbiology, Faculty of Science, Khon Kaen University, Khon Kaen, 40002, Thailand Wasan seemakram, Jindarat Ekprasert and Sophon Boonlue Department of Environmental Sciences, Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, Shobara, Japan Hiroyuki Harada Corresponding author Correspondence to Sophon Boonlue, Email: [email protected] Author Contributions W.S. and S.B. planned the experiment, contributed to design and selection of methodology of the experiment. H.H. and J.E. provided critical feedback and helped to shape the manuscript. All authors (W.S., S.B., H.H., and J.E.) discussed the results and contributed to the final manuscript. Ethics declarations Competing interests The authors declare no competing interests Additional Information Competing Interests: The authors declare no competing interests. Additional information Correspondence and requests for materials should be addressed to S.B. Data Availability/Availability of Data and Materials All data generated and analyzed during this study are included in this published article References Beg, Q.K., Kapoor, M., Mahajan, L. & Hoondal, G.S. Microbial Xylanases and Their Industrial Applications: A Review. Appl. Microbiol. Biotechnol. 56 , 326–338 (2001). Walia, A., Guleria, S., Mehta, P., Chauhan, A. & Parkash, J. Microbial xylanases and their industrial application in pulp and paper biobleaching: a review. 3 Biotech. 7 , 11–23 (2017). Orozco Colonia, B.S., Woiciechowski, A.L., Malanski, R., Letti, L.A.J. & Soccol, C.R. 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Conditions Brightness (%ISO) Kappa Viscosity (cp) Reducing sugar (µ mol/g pulp) Chromophoric compounds Hydrophobic compounds Un 53.60 f 3.20 a 9.40 a 0.00 d 1.54 d 0.12 cd ClO 2 82.80 a 0.20 c 7.80 b 0.32 c 3.56 a 0.09 d 20 U 55.60 e 2.90 a 9.10 ab 1.29 bc 2.25 c 0.20 bc 40 U 57.70 d 2.70 a 9.10 ab 1.29 bc 3.35 b 0.25 ab 80 U 63.60 c 1.90 b 9.10 ab 2.38 b 3.43 b 0.26 ab 100 U 72.50 b 1.70 b 9.10 ab 13.10 a 3.60 a 0.33 a %CV. 1.30 16.27 9.48 6.41 2.82 2.08 F-Test ** ** * ** ** ** Numbers followed by the same letter in the same column are not significantly different according to LSD test (P≤0.05) (Un = untreated pulp; ClO 2 = chlorine dioxide-treated pulp similar to the factory condition; 20U-100U = dose of enzyme used to treat pulp, * Significant difference at P≤0.05, ** Significant difference at P≤0.01) Table 2 The signals assignment in FTIR spectra of the pulp fiber. Wave number (cm −1 ) Assignment 3355–3345 –OH stretching of acid and methanol groups in cellulose 2900–2892 C=H stretching of aliphatic group 1642–1640 C=O stretching vibration in conjugated carbonyl of lignin 1431–1429 CH 2 bending vibration 1373–1371 C–H stretching of aliphatic in methyl group 1318.01 –CH in˗plane bending vibration in cellulose 1164–1161 C–O–C asymmetric stretching vibration 1115–1112 C–O–C stretching of hemicellulose 1061–1057 C–O stretching vibration of cellulose and hemicellulose 1035.11 C–O stretching vibration of lignocellulose 898.19 C–H stretching of deformation in carbohydrates 665.71 =C–H bending vibration of alkenes group 617 ≡C–H bending vibration of alkynes group Table 3 Properties of paper of acquired pulp bleaching with endo-1,4-β-xylanase and chlorine dioxide. Conditions Brightness (%ISO) Breaking length (Nm/g) Bursting Strength (kPa) Tearing Resistance (mN) Un 69.35 e 45.79 c 119.13 b 210.26 ab ClO 2 92.80 a 48.99 b 112.25 c 182.66 c 20 U 79.70 d 46.97 bc 119.38 b 188.75 bc 40 U 80.80 d 47.01 bc 123.50 b 191.00 bc 80 U 86.00 c 47.74 bc 124.19 b 207.50 ab 100 U 89.60 b 52.25 a 138.81 a 218.02 a %CV. 1.12 5.66 6.40 7.78 F-Test ** ** ** ** Numbers followed by the same letter in the same column are not significantly different according to LSD test (P≤0.05) (Un = untreated pulp; ClO 2 = chlorine dioxide-treated pulp similar to the factory condition; 20U-100U = dose of enzyme used to treat pulp, * Significant difference at P≤0.05, ** Significant difference at P≤0.01) Table 4 Characteristics of the effluent with IEST standards. Conditions pH TOC mg/L BOD mg/L TDS mg/L TSS mg/L IEST limits 5.5-9.0 - <60 2,000 50 Un 9.50 c 0.05 e 24.00 d 410 d 35 c ClO 2 1.72 a 60.15 a 538.00 a 2183 a 102 a 20 8.50 b 2.68 b 26.67 d 586 d 44 bc 40 8.60 b 3.53 cd 36.67 cd 729 c 47 bc 80 8.50 b 4.84 c 53.33 bc 774 c 53 b 100 8.40 b 6.81 b 61.33 b 1139 b 57 b %CV 1.93 33.21 7.76 52.13 16.73 F-Test ** ** ** ** ** Numbers followed by the same letter in the same column are not significantly different according to LSD test (P≤0.05) (Un = untreated pulp; ClO 2 = chlorine dioxide-treated pulp similar to the factory condition; 20U-100U = dose of enzyme used to treat pulp, * Significant difference at P≤0.05, ** Significant difference at P≤0.01) Table 5 The band assignment of the FTIR spectrum of effluent. Wavenumber (cm −1 ) Assignment 3800–3557 OH stretching vibration of silanol group 3310–2900 C–H stretching vibration of alkyl group in aliphatic 2358.47 C–O stretching vibration of phosphine 1729.45 C=O stretching of ketone group 1593–1582 C=C stretching, aromatic ring of lignin 1414–1409 OH bending vibration 1356–1396 C–H bending vibration of methyl group 1138.06 C–O stretching of alcohols group 1111.61 C–O–C vibration of ethylene glycol 1045–1039 C–O stretching of ester group 968.74 C=C bending vibration of carboxylic acid group 941.24 C–O stretching of chloride dioxin 849–842 C–H bending of aromatic ring in lignin 877–769 C–H out-of-plane bending of aromatic compounds 699.50 C–Cl stretching 680.21 C–Br stretching of alkyl halides 626–621 C=C bending, out of plane ring Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-33851","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":86887121,"identity":"d7eeadb5-5460-48d0-b00b-84650631a1c5","order_by":0,"name":"Wasan Seemakram","email":"","orcid":"","institution":"Khon Kaen University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wasan","middleName":"","lastName":"Seemakram","suffix":""},{"id":86887122,"identity":"1c747f06-8f3a-4158-8cbd-3527cb3354b2","order_by":1,"name":"Hiroyuki Harada","email":"","orcid":"","institution":"Prefectural University of Hiroshima","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hiroyuki","middleName":"","lastName":"Harada","suffix":""},{"id":86887123,"identity":"6e6cc631-90a4-4f20-9792-9b9385cdb840","order_by":2,"name":"Jindarat Ekprasert","email":"","orcid":"","institution":"Khon Kaen University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jindarat","middleName":"","lastName":"Ekprasert","suffix":""},{"id":86887124,"identity":"54864385-6e3e-42fb-915a-2831c692295b","order_by":3,"name":"Sophon Boonlue","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsklEQVRIiWNgGAWjYBACAyBmZmCwMZCACiQQqyUNpsWAaC2HSdBizn/42OOCivPGkjMSGD/8YPiTR1CL5Yy0dOMZZ26bSUskMEv2MBgUE3bYDR4zad622zZyEgkM0kB+YgNBLefPgLScA2lh/k2clgM5IC0HQA5jI84WsF94ziQbS/Y8bLPsMTAmrAUcYjwVdoYzjicfvvGjQo6wFiBgg9KMDZBoIkHLKBgFo2AUjAIcAADxqzUnmOCi+AAAAABJRU5ErkJggg==","orcid":"","institution":"Khon Kaen University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Sophon","middleName":"","lastName":"Boonlue","suffix":""}],"badges":[],"createdAt":"2020-06-05 15:28:24","currentVersionCode":2,"declarations":"","doi":"10.21203/rs.3.rs-33851/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-33851/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":18700979,"identity":"03f94ad1-0c25-4d2b-bcce-e966f7709ed3","added_by":"auto","created_at":"2022-02-28 19:38:21","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":263260,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of eucalyptus pulp bleaching in D0 stage. A; Untreated, B; Chlorine dioxide (ClO2) treatment, C; Endo-1,4-β-xylanase (100 U) treatment.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figure1..jpg","url":"https://assets-eu.researchsquare.com/files/rs-33851/v2/7050c7fad035dbc197082c06.jpg"},{"id":18700981,"identity":"b258641d-9a80-4a4f-b970-eb9c095b07b4","added_by":"auto","created_at":"2022-02-28 19:38:21","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":407250,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of eucalyptus pulp bleaching treated by endo-1,4-β-xylanase A), untreated pulp; B), enzyme-treated pulp; C), chlorine dioxide-treated pulp. Features of surface pulp fiber are presented by arrow head.\u003c/p\u003e","description":"","filename":"Figure2..jpg","url":"https://assets-eu.researchsquare.com/files/rs-33851/v2/8daa3a9d86f26f9d4170d84f.jpg"},{"id":18700980,"identity":"9de33da4-9de7-4c7e-8889-c774ad03c369","added_by":"auto","created_at":"2022-02-28 19:38:21","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":95164,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra of eucalyptus pulp bleaching in D0 stage; A), untreated; B), enzyme treated and C), chemical treated.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-33851/v2/c484afcedc0c0b9e395893d2.jpg"},{"id":18700982,"identity":"90569b8b-5b7f-4da3-992e-369b6b0c261b","added_by":"auto","created_at":"2022-02-28 19:38:21","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":103208,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra of effluent after eucalyptus pulp bleaching; A), untreated; B), endo-1,4-β-xylanase-treated and C), chlorine dioxide-treated.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-33851/v2/ecea48ff496a69e1b430dfcf.jpg"},{"id":19803890,"identity":"58858c79-f7ae-4d2a-81c6-ac0ec3f2a416","added_by":"auto","created_at":"2022-03-31 04:14:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":669370,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-33851/v2/64d9fc73-0c62-48d9-a70c-6b4040c5b742.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The effect of endo-1, 4-β-xylanase from Thermomyces dupontii KKU−CLD−E2−3 on eucalyptus pulp bleaching and effluent treatment","fulltext":[{"header":"Introduction","content":"\u003cp\u003eXylanases (EC 3.2.1.8) are a group of glycoside hydrolase enzymes that can degrade the linear polysaccharide β-1, 4-xylan resulting in xylose \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The xylanase cleaves the glycosidic bonds in the xylan backbone, leading to a reduction in the degree of polymerization of the pulp fiber. The mechanism how the enzyme attacks the molecule of xylan depends on the nature of the pulp fiber molecule, such as on the chain length of branching, and the presence of substituents \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Recently, Orozco-Colonia et al. \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e reported that xylanase could attack the xylan of hemicellulose by inserting between cellulose and lignin resulting in removal of lignin associated hemicelluloses is facilitated with minimal damage of pulp fiber. In addition, pretreatment of pulp with xylanase can decrease the partial disruption of lignin and carbohydrate bonds in pulp fiber, as the result of enhancing the accessibility of the subsequent bleaching chemicals to the pulp \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. In general, during the process of pulping, bleaching and washing in pulp and paper industry produces effluent that contains with high amount of organic and inorganic compounds including lignin, hemicellulose, cellulose fragments chlorophenols, fatty acids, inorganic salts and the chlorinated dioxins \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Thus, the application of xylanase in the pulp and paper industry focuses to reduce the use of chlorine dioxide in pulp bleaching process that decreases the toxicity in effluents. Besides, there was also a report that the effluent produced from paper bleaching by xylanase after pulp washing could reduce the TSS, TDS, TOC, BOD, colour, and pH \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCurrently, the pre-treatment of pulp using xylanase has been adopted to be alternate approach that plays an environmental friendly and economically viable method for paper industries. However, in order to use xylanase in the pulp bleaching process, several properties are needed to be considered. The enzyme should be free of cellulase and active at high temperatures. Thermo-alkali-stable xylanase has attracted to use for pulp bleaching which can work well at relatively higher temperatures without additional cooling process requirement. Thus, there is a continuous need to find novel xylanase which is active in alkaline pH and also thermostable during the pulping process. This work presents the application of purified endo-1, 4-β-xylanase from \u003cem\u003eThermomyces dupontii\u003c/em\u003e KKU\u0026minus;CLD\u0026minus;E2\u0026minus;3 in pulp bleaching step under routinely performed by Phoenix Pulp and Paper Public Company Limited in SCG Packaging, Khon Kaen, Thailand, which is tested at alkaline pH and high temperature. Characterization of pulp, paper and effluent after being treated with enzyme and chlorine dioxide bleaching was presented.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eEucalyptus pulp bleaching by the enzyme and chlorine dioxide were analyzed for their brightness, kappa number, and viscosity. The brightness was increased from 53.60 to 72.50% ISO, when bleaching with xylanase at 100 Unit and chlorine dioxide at 82.80% ISO (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn cases of bleaching with xylanase and chlorine dioxide, the Kappa number decreased from 3.20 to 1.70 and 0.20, respectively (Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). On the other hand, the viscosity decreased from 9.40 to 9.10 and 7.80 cp, respectively. The effluents after bleaching with xylanase and chlorine dioxide were also characterized. The sample treated with xylanase revealed the maximum free reducing sugars released from pulp compared to the untreated sample (Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Chromophoric and hydrophobic compounds were observed in xylanase and chlorine treated pulp samples, as reported in Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe surface states of fibers in pulp samples treated with endo-1,4-β-xylanase and chloride were analyzed by SEM. The SEM analysis revealed noticeable changes in fiber disintegration on pulp in treated enzyme and chloride, compared to the untreated case (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The surface of untreated eucalyptus pulp was smooth and tight (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). When the eucalyptus pulp was attacked by purified xylanase, its fiber surface became rough, loose and was in a peeling state and loss in compactness (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). In contrast, the eucalyptus pulp which was attacked by chlorine dioxide became lost in compactness of pulp fibers and more disintegrating (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). FTIR spectra of untreated, enzyme-treated and chlorine dioxide-treated eucalyptus pulp were compared. The functional group levels were changes in the peaks of modified in pulp samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The peaks of FTIR spectra in the pulp fiber were assigned as shown in Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eEucalyptus pulp samples after passing through the D1 step were used to prepare papermaking of 20 gram. The quality of paper was analyzed for its brightness, breaking length, bursting strength, and tearing resistance. The examination of the brightness from the enzyme-treated and chlorine-treated samples were significantly increased at 89.6% and 92.8% ISO, respectively. In addition, the paper samples from pulp bleached with enzymes treatment showed a maximum value of breaking length, bursting strength, and tearing resistance of 52.25 Nm/g, 138.81 kPa and 218.00 mN, respectively, when compared to the untreated control and the chlorine-treated sample (Table \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe present work revealed that the wastewater obtained after pulp bleaching by endo-1,4-β-xylanase significantly showed lower values of the TSS and TDS of effluents of pulp bleaching for chloride compared to xylanase treatment reduced from 102 to 57 mg/L, and 2183 to 1139 mg/L, respectively. There was also a reduction of TOC and BOD from 60.15 to 6.81 mg/L and 538.00 to 61.33 mg/L, respectively (Table \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These values are in the range of industrial effluent standards assigned in Thailand (IEST). The TSS, TDS, TOC and BOD in effluent significantly decreased when pulp was bleached by the enzyme compared to using chlorine dioxide are shown in Table \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe FTIR spectra of effluents obtained from untreated, enzyme-treated and chlorine-treated samples was compared. The band assignment of the FTIR spectrum of effluent was listed in Table \u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The functional groups remarkable profiles were changed in the wavenumbers of 500 to 3900 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e as shown in Figure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe observed effects of the ability of endo-1, 4-β-xylanase from \u003cem\u003eT. dupontii\u003c/em\u003e KKU\u0026minus;CLD\u0026minus;E2\u0026minus;3 in the bleaching of eucalyptus pulp at D0 stage under pulp quality assessment of SCG company was investigated. The results revealed that kappa number significantly decreased up to 46.87% and the brightness increased up to 35.26% after being treated by endo-1, 4-β-xylanase dose 100 U, as compared to the untreated control. The chemical pulp bleach can reduce kappa number of 93.75%, resulting in no lignin in the pulp fiber and an increase in brightness of 82.80% ISO. The kappa number is an indication of the residual lignin content in pulp fiber which can be used as an indicator of the efficiency of bleaching process. A reduction of kappa number and an increase in the percentage of brightness in our experiment was higher than those reported in previous studies. Kumar et al. \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e reported that the brightness of hardwood pulp pre-treated with xylanase increased 21.56% while the kappa number decreased 20% compared to the untreated samples. According to Wu et al. \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e who reported that the brightness of eucalyptus kraft was significantly increased about 14.5%, while 24.5% of kappa number was reduced after bleaching by endoxylanase from \u003cem\u003eStreptomyces griseorubens\u003c/em\u003e LH\u0026ndash;3. The viscosity of pulp after bleaching with endo-1, 4-β-xylanase dose 100 U was found comparatively higher (9.10 cp) than chlorine dioxide bleached pulp (7.80 cp) (Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), which was not significantly different when compared to the untreated case. Higher viscosity means higher cellulose content which is directly proportional to the greater physical strength of pulp \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn addition, the release of reducing sugar, phenolic compounds and hydrophobic compounds also increased when the enzyme concentration increased (Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The amount of sugar in the effluent of eucalyptus pulp bleaching for xylanase also significantly increased, which shows that the final product from the enzyme treated pulp was released to sugar. The release of reducing sugars, phenolics, and hydrophobic compounds are a result of the reaction of disintegration of lignin and carbohydrate complex from the pulp fibers \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe surface states of fibers have exhibited noticeable changes on the surface fiber was treated with endo-1, 4-β-xylanase as compared to the untreated case. This indicated that the enzymatic reaction was more specific to xylan in the pulp fiber more than chemical one. The enzyme treatments may expose cellulose fibers to the surface \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e cause surface alteration of the enzyme-treated pulp was due to the activity of xylanase \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Also, the xylanase activity in the digestion of xylan causes the release of lignin. The pretreatment of eucalyptus pulp using chlorine dioxide caused severe damage to the pulp, in which the strength of fibers was reduced and the cracks and peeling of pulp surface were appeared.\u003c/p\u003e \u003cp\u003eFTIR spectra showed functional group alteration in the eucalyptus pulp samples after bleaching in D0 stage. The peak at 1614 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e which corresponds to the C=O stretching vibration in conjugated carbonyl of lignin was shown in the untreated and the enzyme-treated pulp samples, but not in the chemical-treated one \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The peaks at 1430 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e corresponds to the symmetric CH\u003csub\u003e2\u003c/sub\u003e bending vibration \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The presence of peaks at 1372 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e and 1318 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e were due to the aliphatic C\u0026ndash;H stretching in methyl and the CH in-plane bending vibration in cellulose I and cellulose II \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Peak at 1163 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e and 1113 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e indicated the C\u0026ndash;O\u0026ndash;C asymmetric stretching in cellulose I and cellulose II and the C\u0026ndash;O\u0026ndash;C stretching of hemicellulose \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The peaks at 1060 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e and 1035 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e were attributed to the C\u0026ndash;O stretching vibration of acetyl cellulose and hemicellulose and the C\u0026ndash;O stretching vibration of cellulose, hemicellulose and lignin, respectively \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eEucalyptus pulp samples after passing through the D1 step were used to prepare papermaking of 20 grams. The quality of paper was analyzed for its brightness, breaking length, bursting strength, and tearing resistance. The examination of the brightness of papers treated by enzyme showed an increase of 29.20% compared to the control, but \u0026lt; 3.45% compared to chlorine dioxide. However, the paper from pulp bleached with enzymes treatment are shown the value of breaking length, bursting strength, and tearing resistance at 52.25 Nm/g, 138.81 kPa and 218.00 mN, respectively. The fiber properties of enzyme pre-bleached pulp had a higher value of tear index compared to that of the control, but no significant difference was observed. The tearing index of enzyme-treated pulp increased up to 15.71% which was higher than those from the untreated pulp \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Obviously, pretreatments by enzyme could improve value tensile, tearing and bursting index better than the use only chemical process.\u003c/p\u003e \u003cp\u003eThe paper industry worldwide usually causes serious environmental burdens due to its 40% global industry wastewater discharge leading to environmental pollution problems \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. The present work revealed that the wastewater obtained after pulp bleaching by endo-1,4-β-xylanase significantly showed lower values of the TSS and TDS as compared to chloride treatment reduced 44.12% and 45.73%, respectively. There was also a reduction of TOC and BOD of 80.68% and 88.60%, respectively (Table \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These values are in the range of industrial effluent standards assigned in Thailand (IEST). The TSS, TDS, TOC and BOD in effluent significantly decreased when pulp was bleached by the enzyme compared to using chlorine dioxide. Similarly, Sridevi et al. \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e reported that the use of xylanase in pulp bleaching process resulted in the reduction of BOD (89.83%), TSS (72.14%) and TDS (53.32%) in the wastewater discharge. The use of our xylanase for pulp bleaching could bring about not only higher quality of effluent which is even better than the IEST, but also reduce the amount of organic/inorganic materials in the effluent. Therefore, the quality of the effluent from xylanase-bleaching process is ready to be discharged without passing through the company\u0026rsquo;s wastewater treatment processes.\u003c/p\u003e \u003cp\u003eThe FTIR spectra of effluents obtained from untreated, enzyme-treated and chlorine-treated samples was compared. The functional groups remarkable profiles were changed in the wave numbers of 500 to 3900 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e as shown in Figure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Interestingly, there were some significant changes at peak around 941.24 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e corresponding to C\u0026ndash;O stretching of chloride dioxin caused by the combination of Cl and lignin rings which was seen in case of pulp bleaching by chloride only \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, while pulp bleached by endo-1, 4-β-xylanase could not be observed. Nevertheless, the chlorine dioxin was shown between 935 and 950 cm\u003csup\u003e\u0026minus;1 18\u003c/sup\u003e which caused by holocellulose of pulp from eucalyptus wood with bleached by chlorine dioxide pretreated in pulp bleaching \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. This provided evidence that endo-1, 4-β-xylanase has good exceeding for pulp bleaching compared to commercial chemical treatment. Because in the pulp bleaching process by endo-1, 4-β-xylanase can help to improve the quality of pulp and paper. Also, using enzyme has efficiency for economic improvement of effluents and reduces the risk of cancer cause by dioxin for the staffs in working area. Additionally, the odor pollution from the use of chemicals in the factory can be reduced as well.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eAn endo-1, 4-β-xylanase bleaching of eucalyptus pulp in D0 stage could increase the paper brightness. The physical properties of the paper including breaking length, bursting strength, and tearing resistance were more improved than those using chemicals bleaching works well in realistic situations. The variety of contaminants present in the effluent from the pulp bleaching process after enzyme bleaching was degraded. Therefore, this bio-bleaching process is cost-effective and has environmental benefits than the chemical bleaching.\u003c/p\u003e "},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003ePulp and enzyme\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEucalyptus pulp used in the experiment was obtained from press 3 stage of Phoenix Pulp and Paper Public Co., Ltd. in SCG Packaging, Khon Kaen, Thailand. The characteristics of the pulp were as followed: 3.2 kappa number, 53.6% ISO of brightness, 9.4 cp of viscosity and initial pH 9.7. The purified endo-1, 4-\u0026beta;-xylanase was produced from \u003cem\u003eThermomyces dupontii\u003c/em\u003e KKU\u0026minus;CLD\u0026minus;E2\u0026minus;3 (Accession number LC428093) \u003csup\u003e20\u003c/sup\u003e. The eucalyptus pulp bleaching was carried out by following the method which routinely performed by Phoenix Pulp and Paper Public Company Limited in SCG Packaging.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of endo\u003cem\u003e-\u003c/em\u003e1, 4\u003cem\u003e-\u003c/em\u003e\u0026beta;\u003cem\u003e-\u003c/em\u003exylanase on eucalyptus pulp in Chlorine dioxide stage (D0)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEucalyptus pulp was prepared by treating with an enzyme dose 20\u0026ndash;100 (U/50 g pulp) in sealed plastic bags with intermittent kneading and 10% (w/v) pulp consistency for a time period of 30 min at 70 \u0026deg;C. Control sample was untreated by enzyme and treated by chlorine dioxide (ClO\u003csub\u003e2\u003c/sub\u003e) at a concentration of 15 kg/T pulp under the same conditions with an activated enzyme.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of endo\u003cem\u003e-\u003c/em\u003e1, 4\u003cem\u003e-\u003c/em\u003e\u0026beta;\u003cem\u003e-\u003c/em\u003exylanase on eucalyptus pulp in Extraction\u003cem\u003e-\u003c/em\u003ePeroxide stage \u003cem\u003e(\u003c/em\u003eEOP\u003cem\u003e) \u003c/em\u003eand Chlorine dioxide stage (D1)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter passing the procedure D0, excess enzyme and buffer in eucalyptus pulp were removed by pressing and draining from the pretreated pulp. The eucalyptus pulp samples prepared were treated with 10% (w/v) pulp consistency. The eucalyptus pulp samples were bleached in EOP staged with a NaOH (14 kg/T pulp) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (5 kg/T pulp) incubated at 65 \u0026ordm;C for 120 min. After that, the chemicals in eucalyptus pulp samples were removed by pressing and draining with tap water, and were treated with a ClO\u003csub\u003e2 \u003c/sub\u003e(5 kg/T pulp) of time period for 240 min at 70 \u0026deg;C. Finally, the eucalyptus pulp samples were washed by pressing and draining with tap water again.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalyses of eucalyptus pulp properties\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe eucalyptus pulp bleaching obtained from D0 stage was prepared for hand sheets and analyzed for brightness using a brightness machine (Technidyne Corporation, Color Touch\u0026trade; 2). The efficiency of the bleaching treatment process was observed for the reduction of kappa number \u003csup\u003e21\u003c/sup\u003e and viscosity. The eucalyptus pulp samples were dehydrated in acetone, critical point dried and mounted on sample stubs. The feature surfaces of the xylanase-treated and the control samples were observed under scanning electron microscope (SEM) (SEC, SNE\u0026ndash;4500M). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterization of papers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEucalyptus pulp samples after passing the procedure D1 were used for papermaking at 20 gram to study the properties of the paper. The paper was obtained from pulp bleaching with purified xylanase. Thereafter, they were massaged pulp fibers by basket centrifuge at 4500 rpm and then were prepared wet sheets forming by hand sheets with 10% (w/v) pulp consistency. The eucalyptus pulp samples were pressed for consolidation of wet. The paper was baked to remove moisture so that the remaining moisture was about 4\u0026ndash;6%, which dried overnight in a temperature-controlled room where it operated at temperature of 25 \u0026ordm;C, and the moisture was kept at 55%. The quality of paper was analyzed for their brightness (Technidyne Corporation, Color Touch\u0026trade; 2), breaking length (REGMED, Horizontal tensile\u0026minus;D\u0026minus;21), bursting tester (ABB AB/Lorentzen \u0026amp; Wettre, RS232C), and tearing tester (Labthink, SLY\u0026minus;S1), respectively. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffluent characterization of eucalyptus pulp bleaching\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effluent was characterized before disposal by following the standard method described by Sridevi et al. \u003csup\u003e4\u003c/sup\u003e. The enzyme-mediated release of hydrophobic and chromophoric compounds from pulp bleaching process were measured the absorbance at 280 nm and 465 nm, respectively \u003csup\u003e22\u003c/sup\u003e. Total amount of reducing sugars released from pulp bleaching was determined by the DNS method \u003csup\u003e23\u003c/sup\u003e. The FTIR analysis of effluent after treated eucalyptus pulp by enzyme was carried out. We aim to investigate the functional groups of the contaminant particles obtained during enzymatic treatment into the effluent. The characterization properties of effluent were determined for several parameters including pH, TSS, TDS, BOD (VELP; Scientifica), and TOC (SHIMADZU, SA24).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperimental designs were statistically analyzed by a Completely Randomized Design (CRD) and pair comparisons in the least significant difference (LSD) test to determine variations and the significance of differences between treatments. All experiments were performed in triplicate, and the results were expressed as the mean. Effects of the variables and the significance of regression coefficients were determined at F-test (P \u0026le; 0.05).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financially supported by Protein and Proteomics Research Centre for Commercial and Industrial Purposes in FY 2016 in ongoing project no ProCCI60001, and Student Exchange Program 2019 from Faculty of Science, Khon Kaen University, Thailand and Student Exchange Program 2019 from Hiroshima Prefectural of University, Japan. The authors thank the Phoenix Pulp and Paper Public Company Limited in SCG Packaging for supporting the equipment and facilities on laboratory analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAffiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDepartment of Microbiology, Faculty of Science, Khon Kaen University, Khon Kaen, 40002, Thailand\u003c/p\u003e\n\u003cp\u003eWasan seemakram, Jindarat Ekprasert and Sophon Boonlue\u003c/p\u003e\n\u003cp\u003eDepartment of Environmental Sciences, Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, Shobara, Japan\u003c/p\u003e\n\u003cp\u003eHiroyuki Harada\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Sophon Boonlue, Email: [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eW.S. and S.B. planned the experiment, contributed to design and selection of methodology of the experiment. H.H. and J.E. provided critical feedback and helped to shape the manuscript. All authors (W.S., S.B., H.H., and J.E.) discussed the results and contributed to the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompeting Interests: The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence and requests for materials should be addressed to S.B.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability/Availability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated and analyzed during this study are included in this published article\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBeg, Q.K., Kapoor, M., Mahajan, L. \u0026amp; Hoondal, G.S. 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Purification, characterization and partial amino acid sequences of thermo-alkali-stable and mercury ion-tolerant xylanase from \u003cem\u003eThermomyces dupontii\u003c/em\u003e KKU\u0026ndash;CLD\u0026ndash;E2\u0026ndash;3. \u003cem\u003eScientific Reports\u003c/em\u003e, \u003cstrong\u003e10\u003c/strong\u003e, 21663 (2020).\u003c/li\u003e\n\u003cli\u003eTAPPI.Technical association of the pulp and paper industry, standard T230. TAPPI Press, Atlanta. (1990).\u003c/li\u003e\n\u003cli\u003eManimaran, A. \u0026amp; Vatsala, T.M. Biobleaching of banana Wbre pulp \u003cem\u003eusing Bacillus subtilis\u003c/em\u003e C O1 xylanase produced from wheat bran under solid-state cultivation. \u003cem\u003eJ. Ind. Microbiol. Biotechnol\u003c/em\u003e. \u003cstrong\u003e34\u003c/strong\u003e, 745\u0026ndash;749 (2007). \u003c/li\u003e\n\u003cli\u003eMiller, G.L. Use of dinitrosalicylic acid reagent for determination of reducing sugar. \u003cem\u003eAnalytic. Chem.\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, 426\u0026ndash;428 (1959).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of enzyme dose on eucalyptus pulp bleaching in D0 stage.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConditions\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrightness (%ISO)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKappa\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eViscosity (cp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eReducing sugar\u003c/p\u003e \u003cp\u003e(\u0026micro; mol/g pulp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChromophoric compounds\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHydrophobic compounds\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUn\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e53.60\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.20\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.40\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.00\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.54\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.12\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e82.80\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.20\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.80\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.32\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.56\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.09\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20 U\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e55.60\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.90\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.10\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.29\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.25\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.20\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e40 U\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e57.70\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.70\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.10\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.29\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.35\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.25\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e80 U\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e63.60\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.90\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.10\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.38\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.43\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.26\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e100 U\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e72.50\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.70\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.10\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13.10\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.60\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.33\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e%CV.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF-Test\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eNumbers followed by the same letter in the same column are not significantly different according to LSD test (P\u0026le;0.05) (Un = untreated pulp; ClO\u003csub\u003e2\u003c/sub\u003e = chlorine dioxide-treated pulp similar to the factory condition; 20U-100U = dose of enzyme used to treat pulp, * Significant difference at P\u0026le;0.05, ** Significant difference at P\u0026le;0.01)\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe signals assignment in FTIR spectra of the pulp fiber.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWave number (cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAssignment\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3355\u0026ndash;3345\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ndash;OH stretching of acid and methanol groups in cellulose\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2900\u0026ndash;2892\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC=H stretching of aliphatic group\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1642\u0026ndash;1640\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC=O stretching vibration in conjugated carbonyl of lignin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1431\u0026ndash;1429\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCH\u003csub\u003e2\u003c/sub\u003e bending vibration\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1373\u0026ndash;1371\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u0026ndash;H stretching of aliphatic in methyl group\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1318.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ndash;CH in˗plane bending vibration in cellulose\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1164\u0026ndash;1161\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u0026ndash;O\u0026ndash;C asymmetric stretching vibration\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1115\u0026ndash;1112\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u0026ndash;O\u0026ndash;C stretching of hemicellulose\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1061\u0026ndash;1057\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u0026ndash;O stretching vibration of cellulose and hemicellulose\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1035.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u0026ndash;O stretching vibration of lignocellulose\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e898.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u0026ndash;H stretching of deformation in carbohydrates\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e665.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e=C\u0026ndash;H bending vibration of alkenes group\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e617\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026equiv;C\u0026ndash;H bending vibration of alkynes group\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eProperties of paper of acquired pulp bleaching with endo-1,4-β-xylanase and chlorine dioxide.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConditions\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrightness (%ISO)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBreaking length (Nm/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBursting Strength (kPa)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTearing Resistance (mN)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUn\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e69.35\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e45.79\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e119.13\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e210.26\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e92.80\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48.99\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e112.25\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e182.66\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20 U\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e79.70\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46.97\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e119.38\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e188.75\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e40 U\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e80.80\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e47.01\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e123.50\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e191.00\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e80 U\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e86.00\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e47.74\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e124.19\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e207.50\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e100 U\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e89.60\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e52.25\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e138.81\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e218.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e%CV.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.78\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF-Test\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eNumbers followed by the same letter in the same column are not significantly different according to LSD test (P\u0026le;0.05) (Un = untreated pulp; ClO\u003csub\u003e2\u003c/sub\u003e = chlorine dioxide-treated pulp similar to the factory condition; 20U-100U = dose of enzyme used to treat pulp, * Significant difference at P\u0026le;0.05, ** Significant difference at P\u0026le;0.01)\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacteristics of the effluent with IEST standards.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConditions\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTOC mg/L\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBOD mg/L\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTDS mg/L\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTSS mg/L\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIEST limits\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.5-9.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUn\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.50\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.05\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24.00\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e410\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e35\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.72\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e538.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2183\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e102\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.50\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.68\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26.67\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e586\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e44\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.60\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.53\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e36.67\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e729\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e47\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.50\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.84\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e53.33\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e774\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e53\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.40\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.81\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e61.33\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1139\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e57\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e%CV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e52.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16.73\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF-Test\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eNumbers followed by the same letter in the same column are not significantly different according to LSD test (P\u0026le;0.05) (Un = untreated pulp; ClO\u003csub\u003e2\u003c/sub\u003e = chlorine dioxide-treated pulp similar to the factory condition; 20U-100U = dose of enzyme used to treat pulp, * Significant difference at P\u0026le;0.05, ** Significant difference at P\u0026le;0.01)\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe band assignment of the FTIR spectrum of effluent.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWavenumber (cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAssignment\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3800\u0026ndash;3557\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOH stretching vibration of silanol group\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3310\u0026ndash;2900\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u0026ndash;H stretching vibration of alkyl group in aliphatic\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2358.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u0026ndash;O stretching vibration of phosphine\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1729.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC=O stretching of ketone group\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1593\u0026ndash;1582\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC=C stretching, aromatic ring of lignin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1414\u0026ndash;1409\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOH bending vibration\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1356\u0026ndash;1396\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u0026ndash;H bending vibration of methyl group\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1138.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u0026ndash;O stretching of alcohols group\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1111.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u0026ndash;O\u0026ndash;C vibration of ethylene glycol\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1045\u0026ndash;1039\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u0026ndash;O stretching of ester group\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e968.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC=C bending vibration of carboxylic acid group\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e941.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u0026ndash;O stretching of chloride dioxin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e849\u0026ndash;842\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u0026ndash;H bending of aromatic ring in lignin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e877\u0026ndash;769\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u0026ndash;H out-of-plane bending of aromatic compounds\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e699.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u0026ndash;Cl stretching\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e680.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u0026ndash;Br stretching of alkyl halides\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e626\u0026ndash;621\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC=C bending, out of plane ring\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Biobleaching, Chlorine dioxin, Effluent, Environmental","lastPublishedDoi":"10.21203/rs.3.rs-33851/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-33851/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEndo-1, 4-β-xylanase-chlorine dioxide bleaching of eucalyptus pulp and analysis of effluent was investigated. The eucalyptus pulp bleaching in D0 stage was prepared with enzyme dose 100 Unit of 10% consistency and incubated at 70\u0026deg;C for 30 min. The brightness level was achieved up to 72.50 (% ISO). The kappa number and viscosity of eucalyptus pulp was found to be 1.70 and 8.90 (cp), respectively. The paper from pulp bleaching with enzyme has shown an increase in brightness, breaking length, bursting strength, and tearing resistance to be 89.60% IOS, 37.27 Nm/g, 138.81 kPa and 218.02 mN, respectively. The SEM and FTIR analysis of pulp fibers revealed a significance of morphological and structural changes. The analysis of the effluent also showed statistically-significant differences in TOC, BOD, TDS and TSS. The FTIR analysis of the effluent showed the organic compounds and chloride dioxin in cases of those treated by chemicals.\u003c/p\u003e","manuscriptTitle":"The effect of endo-1, 4-β-xylanase from Thermomyces dupontii KKU−CLD−E2−3 on eucalyptus pulp bleaching and effluent treatment","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2022-02-28 19:38:19","doi":"10.21203/rs.3.rs-33851/v2","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2020-06-29 23:40:29","doi":"10.21203/rs.3.rs-33851/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f9f3c686-3b38-4f89-9dbc-1254f77f4530","owner":[],"postedDate":"February 28th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-03-31T04:14:18+00:00","versionOfRecord":[],"versionCreatedAt":"2022-02-28 19:38:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-33851","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-33851","identity":"rs-33851","version":["v2"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}