Enzymatic bleaching of organosolv sugarcane bagasse pulps with recombinant xylanase of the fungus Humicola grisea and with commercial cartazyme HS xylanase

Organosolv (ethanol/water and acetosolv) pulps were treated with Humicola grisea var. thermoidea and compared with Cartazyme HS xylanase-treated pulp. The ethanol/water pulps treated with H. grisea had the same viscosity as unbleached pulps (8 cP). Ethanol/water pulps treated with Cartazyme had higher viscosity than H. grisea-treated pulps (12 cP). Acetosolv pulps treated with H. grisea and Cartazyme presented a reduction in viscosity; however, the pulps treated with H. grisea had a lower reduction in viscosity than Cartazyme-treated pulps. Ethanol/water pulps treated with H. grisea had a 23% reduction in kappa number in 4 and 8 h of treatment, compared with the unbleached pulps. Cartazyme-treated pulps had a kappa number similar to that of the control pulps for 4 h of treatment. Extending the treatment time to 12 h resulted in a reduction of 33%. The acetosolv pulp treated with H. grisea had a kappa number reduced to 23% in 4 h. Cartazyme treatment resulted in a reduction of 55 and 44% in kappa number for 4 and 8 h of treatment, respectively, when compared with control pulp. Extending the treatment time to 12 h decreased the kappa number 72%. Fourier transform infrared spectra and principal component analysis showed differences among unbleached, H. grisea-treated, and Cartazyme-treated pulps.     

Effect of dose of xylanase on bleachability of sugarcane bagasse ethanol/water pulps

Pulps obtained from the ethanol/water cooking of sugarcane bagasse were bleached with the xylanase enzyme obtained from the fungus Thermomyces lanuginosus IOC-4145 and with the commercial enzyme Cartazyme HS from Sandoz. By changing the enzyme dose from 4.3 to 36 IU/g of pulp, kappa number and viscosity were maintained when the xylanase from T. lanuginosus was used. On the other hand, by using Cartazyme HS, kappa number decreased by 17%, reaching 35.5. This pulp was further extracted with NaOH without a decrease in viscosity (10 cP), and pulp with a kappa number of 13 was obtained. Xylanases had no significant effect on the ethanol/water pulps.     

Ethanol/water pulp enzymatic pretreatment: Chemical and FTIR-PCA analyses

Pulps obtained from ethanol/water cooking of sugarcane bagasse were treated at different times using xylanase enzyme obtained from Thermomyces lanuginosus IOC-4145 or commercially (Cartazyme HS, Sandoz Products Ltd.). The enzyme dosage was 18 IU per g of dry pulp and the time varied from 4 h to 12 h. When xylanase from T. lanuginosus was used, the kappa number and viscosity improved independently of the processing time used (4 h, 8 h, and 12 h). After chemical evaluation, the obtained pulps were classified using Fourier Transformed Infra-Red Spectroscopy and Principal Component Analysis. The results showed that the first three principal components explained more than 90 % of the total variance of the pulp spectra.     

Reuse of the xylanase enzyme in the biobleaching process of the sugarcane bagasse acetosolv pulp

In this work, pretreatment-enzymatic series of the bagasse-sugarcane pulp and alkaline extraction of enzyme treated pulp were carried out. In the pretreatment an enzyme dose was utilized and acetosolv pulp suspension of 3% (w/v) with different solvents (distilled water, 0.05 mol/L acetate buffer pH 5.5 and 0.05 mol/L phosphate buffer pH 7.25) stirred at 85 rpm for 2 or 4 h. The enzymes used were pulpzyme and cartazyme, both commercial. The accompaniment of the enzymatic activity was carried out through measurement in initial and finish of each enzymatic pretreatment. The xylanase-treated pulps and xylanase-alkaline-extracted pulps were analyzed regarding kappa number and viscosity. Pulpzyme recovery was better in phosphate buffered medium (84, 46, and 23% for first, second, and third enzymatic treatment, respectively) although in aqueous medium reached only 2% for every treatments. However, the improvement of pulp properties was evidenced only in aqueous medium for pulpzyme. Cartazyme recovery was similar for both solvents (water and acetate buffer), reaching values around 19% for first enzymatic treatment and 9% for second one. Nevertheless, the pulp properties increased only in acetate buffered medium.     

Bleachability and characterization by fourier transform infrared principal component analysis of acetosolv pulps obtained from sugarcane bagasse

Sugarcane bagasse Acetosolv pulps were bleached by xylanase and the pulps classified by using Fourier transform infrared (FTIR) spectroscopy and principal component analysis (PCA). Pulp was treated with xylanase for 4–8 h with stirring at 30°C. Some samples were further extracted with NaOH for 1 h at 65°C. FTIR spectra were recorded directly from the dried pulp samples by using the diffuse reflectance technique. Reduction in kappa number of 69% was obtained after sequence xylanase (4 h)-alkaline extraction. During bleaching the viscosity decreased only 12%. FTIR-PCA showed that the first three principal components (PCs) explained more than 90% of the total variance of the pulp spectra. PC2×PC1 plot showed that the points related to pulps from sequence xylanase (4 h)-alkaline extraction are different from the other. This group isenlarged by plotting PC3×PC1 or PC3×PC2 containing all pulps submitted to alkaline extraction. PC2 and PC3 are the principal factor for differentiation of the pulps. These PCs suffer influence of the ester bands (1740 and 1244 cm⁻¹). On the other hand, the pulps bleached only with xylanase could not be differentiated from the nonbleached pulps.     

Ceriporiopsis subvermispora used in delignification of sugarcane bagasse prior to soda/anthraquinone pulping

Sugarcane bagasse was pretreated with the white-rot fungus Ceriporiopsis subvermispora for 30 d of incubation. The solid-state fermentation of 800 g of bagasse was carried out in 20-L bioreactors with an inoculum charge of 250 mg of fungal mycelium/kg of bagasse. The oxidative enzymes manganese peroxidase (MnP), lignin peroxidase (LiP), and lac-case (Lac) and the hydrolytic enzyme xylanase (Xyl) were measured by standard methods and related to the fungus’s potential for delignification. Among the lignocellulolytic assayed enzymes, Xyl was detected in larger quantity (4478 IU/kg), followed by MnP (236 IU/kg). LiP and Lac were not detected. The results of chemical analysis and mass component loss showed that C. subvermispora was selective to lignin degradation. Pretreated sugarcane bagasse and control pulps were obtained by soda/anthraquinone (AQ) pulping. Pulp yields, kappa number, and viscosity of all pulps were determined by chemical analysis of the samples. Yields of soda/AQ ranged from 46 to 54%, kappa numbers were 15–25, and the viscosity ranged from 3.6 to 7 cP for pulps obtained from pretreated sugarcane bagasse.     

Bleach Enhancement of Mixed Wood Pulp by Xylanase–Laccase Concoction Derived Through Co-culture Strategy

Mixed enzyme preparation having both xylanase and laccase activity was evaluated for its bleach enhancing ability of mixed wood pulp. The enzyme was produced through co-cultivation of mutant Penicillium oxalicum SAU_E-3.510 and Pleurotus ostreatus MTCC 1804 under solid-state fermentation. Bleaching of pulp with mixed enzyme had resulted into a notable decrease in kappa number and increased brightness as compared to xylanase alone. Analysis of bleaching conditions had denoted that 8 IU g⁻¹ of mixed enzyme preparation (xylanase/laccase, 22:1) had led into maximal removal of lignin from pulp when bleaching was performed at 10% pulp consistency (55 °C, pH 9.0) for 3 h. An overall improvement of 21%, 8%, 3%, and 5% respectively in kappa number, brightness, yellowness, and viscosity of pulp was achieved under derived bleaching conditions. Process of enzymatic bleaching was further ascertained by analyzing the changes occurring in polysaccharide and lignin by HPLC and FTIR. The UV absorption spectrum of the compounds released during enzymatic treatment had denoted a characteristic peak at 280 nm, indicating the presence of lignin in released coloring matter. The changes in fiber morphology following enzymatic delignification were studied by scanning electron microscopy.     

Immobilization of Xylanase from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> Strain MK001 and its Application in Production of Xylo-oligosaccharides

Xylanase from Bacillus pumilus strain MK001 was immobilized on different matrices following varied immobilization methods. Entrapment using gelatin (GE) (40.0%), physical adsorption on chitin (CH) (35.0%), ionic binding with Q-sepharose (Q-S) (45.0%), and covalent binding with HP-20 beads (42.0%) showed the maximum xylanase immobilization efficiency. The optimum pH of immobilized xylanase shifted up to 1.0 unit (pH 7.0) as compared to free enzyme (pH 6.0). The immobilized xylanase exhibited higher pH stability (up to 28.0%) in the alkaline pH range (7.0–10.0) as compared to free enzyme. Optimum temperature of immobilized xylanase was observed to be 8 °C higher (68.0 °C) than free enzyme (60.0 °C). The free xylanase retained 50.0% activity, whereas xylanase immobilized on HP-20, Q-S, CH, and GE retained 68.0, 64.0, 58.0, and 57.0% residual activity, respectively, after 3 h of incubation at 80.0 °C. The immobilized xylanase registered marginal increase and decrease in K _m and V _max values, respectively, as compared to free enzyme. The immobilized xylanase retained up to 70.0% of its initial hydrolysis activity after seven enzyme reaction cycles. The immobilized xylanase was found to produce higher levels of high-quality xylo-oligosaccharides from birchwood xylan, indicating its potential in the nutraceutical industry.     

Effect of Microwave Irradiation on Xylanase Production from Wheat Bran and Biobleaching of Eucalyptus Kraft Pulp

Microwave irradiation (MWI) was used as pretreatment of wheat bran and eucalyptus kraft pulp to examine its effect on xylanase production by Bacillus halodurans FNP 135 using solid state fermentation and biobleaching with xylanase, respectively. Irradiation of wheat bran under optimized conditions (600?W, 6?min, and 20?% consistency) resulted in 56.8, and 31.7?% increase in xylanase yield and water absorbance of wheat bran and 17.3?% reduction in reducing sugars content. Optimized MWI of kraft pulp at 850?W, 2?min, and 20?% consistency led to 0.9?% increase in brightness, 10?% decrease in kappa number, 7.7?% increase in water absorbance, 4.6?% decrease in tear factor, 0.9?% increase in burst factor, and 7.5?% increase in viscosity. Also, MWI enhanced xylanase-mediated biobleaching by increasing brightness (1.1?%) and decreasing kappa number (14.3?%) and leading to a total of about 20?% reduction in chlorine consumption. MWI is an economical, efficient, and environment-friendly pretreatment of wheat bran and pulp for enhanced enzyme yield and rapid heating, respectively.     

A precise study on the feasibility of enzyme treatments of a kraft pulp for viscose application

The development of efficient process steps to convert paper-grade to dissolving pulps was investigated as part of the work programme to improve the process economics. The challenge of pulp refinement comprises the selective removal of hemicelluloses and the precise adjustment of the pulp viscosity, while maintaining the reactivity of the pulp as required for viscose application. The purpose of this study was to investigate the effects of various enzyme treatments on a commercial oxygen-delignified Eucalyptus globulus paper-grade kraft pulp in the course of a total chlorine free bleaching sequence in combination with refining techniques following the principle of Modified Kraft Cooking (Sixta et al. 2007). The objectives were to assess its applicability as viscose pulp besides the reduction of chemical consumption in alkaline and ozone bleaching steps by means of xylanase pre-treatment and the controlled adjustment of final pulp viscosity utilizing endoglucanase post-treatment. Xylanase pre-treatment combined with cold caustic extraction at reduced alkalinity efficiently removed the hemicelluloses from the pulp and clearly increased the pulp brightness by extensive removal of hexenuronic acid side chains. The xylanase pre-treated pulp showed increased reactivity towards xanthation and high viscose dope quality in terms of particle content. The dependence of cellulose chain scission on the applied endoglucanase concentration was analyzed in detail, and this allowed precise viscosity reduction as well as reactivity increase. The differently treated pulps, with and without xylanase pre-treatment, were of very narrow molecular weight distribution and the quality of the spun fibers were very similar to those viscose fibers from commercial dissolving pulps.     

Panus tigrinus strains used in delignification of sugarcane bagasse prior to kraft pulping

Three strains of the white-rot fungus Panus tigrinus (FTPT-4741, FTPT-4742, and FTPT-4745) were cultivated on sugarcane bagasse prior to kraft pulping. Pulp yields, kappa number, and viscosity of all pulps were determined and Fourier transform infrared (FTIR) spectra from the samples were recorded. The growth of P. tigrinus strains in plastic bags increased the manganese peroxide and xylanase activities. Lignin peroxidase was not detected in the three systems (shaken and nonshaken flasks and plastic bags). FTIR spectra were reduced to their principal components, and a clear separation between FTPT-4742 and the control was observed. Strain FTPT-4745 decayed lignin more selectively in the three systems utilized. Yields of kraft pulping were low, ranging from 20 to 45% for the plastic bag samples and from 12 to 38% for the flask samples. Kappa numbers were 1–18 and viscosity ranged from 2.3 to 6.8 cP.     

Analytical pyrolysis study of biodelignification of cloned Eucalyptus globulus (EG) clone and Pinus pinaster Aiton kraft pulp and residual lignins

This study centred on the analysis of lignin in situ of cloned eucalypt and pine kraft pulps. Trametes versicolor laccase-violuric acid system (LMS) delignifications were performed on a softwood (Pinus pinaster) and a hardwood (Eucalyptus globulus) conventional kraft pulp with an initial kappa number of 34.5 and 15.5, respectively. The LMS treated pulps were then subjected to alkaline extraction stages (E). The kappa number data show that LMS is effective at biodelignifying both softwood and hardwood kraft pulps. However, under the conditions employed in this study, a greater level of biodelignification was obtained with LMS E. globulus (hardwood) than with LMS P. pinaster (softwood), but the amount of lignin removed was higher for the softwood pulp. The original milled wood samples, kraft pulps, biodelignified kraft pulps, and isolated residual lignin and milled wood lignins from the two wood samples have been characterized by pyrolysis-gas chromatography/mass spectrometry. The analysis of the pyrograms indicates that the lignin compositions of the two wood species and corresponding pulps are very different, as expected; however, the knowledge of the chemical mechanisms of delignification is very limited and requires additional work. Analytical pyrolysis is one the few degradative methods for the analysis of biopolymers that has shown a sufficient degree of success.     

Potential of Thermo and Alkali Stable Xylanases from Thielaviopsis basicola (MTCC-1467) in Biobleaching of Wood Kraft Pulp

Thermo- and alkali-stable xylanases produced from Thielaviopsis basicola (MTCC-1467) on low-cost carbon source like rice straw were evaluated for their potential application in biobleaching of wood kraft pulp. Enzyme treatment at retention time of 240?min with 20?IU/gm of dried pulp resulted in ~85.2?% of reduction in kappa number. When compared to control, 110.8, 93, and 72.2?% of enhancement in brightness (percent International Organization of Standardization), whiteness, and fluorescence, respectively, were observed for enzyme-treated pulp. Spectroscopic analysis showed significant release of chromophoric compounds from enzyme-treated pulp. Furthermore, scanning electron microscope studies of unbleached and enzyme bleached pulp revealed the effectiveness of enzymatic treatment. The enzyme-treated pulp subjected to later stages of chemical bleaching resulted in 16?% decrease in chlorine consumption along with considerable reduction in chemical oxygen demand percentage (14.5?%) level of effluent. Various pulp properties like fiber length, fiber width, burst strength, burst index, tear strength, tear index, tensile strength, and breaking length were also significantly improved after enzyme treatment when compared to control.     

Pyrolysis-GC/MS and TG/MS study of mediated laccase biodelignification of Eucalyptus globulus kraft pulp

Biobleaching studies using laccase mediator system (LMS) were carried out, under optimized conditions, on two unbleached Eucalyptus globulus kraft pulps, one produced by conventional way, with kappa number of 16.1, and another with kappa number of 14.5, obtained by modified kraft procedure with a high liquor/wood ratio and with black liquor replacement in the middle of the cooking. The pulp properties before and after LMS and alkaline extraction were evaluated in terms of kappa number, hexeneuronic acid content, viscosity, brightness and acid insoluble lignin content.The original milled wood sample and the kraft pulps were characterized by pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) and thermogravimetry/mass spectrometry (TG/MS). Eucalypt wood lignin produces guaiacol and syringol derivatives during pyrolysis. These lignin products can be detected with high sensitivity using the selected ion chromatograms even in the bleached pulp of low lignin content (about 0.5%). Py-GC/MS revealed that the lignin moieties were similarly altered during biobleaching as during pulping, which is exemplified by the preferential removal of aldehyde groups from the alkyl side groups. Semi-quantitative analysis of the pyrograms indicates that the lignin content of the biobleached pulps is reduced by about half in comparison with the unbleached pulps. The TG/MS results show that the hemicellulose content of wood was strongly modified during pulping resulting in higher thermal stability.     

The effect of Fenton chemistry on the properties of microfibrillated cellulose

A fully bleached birch kraft pulp was treated with acidic hydrogen peroxide in the presence of ferrous ions (Fenton’s reagent) and thereafter treated mechanically in a colloid mill to produce a product containing microfibrillated cellulose (MFC). The produced MFC products were chemically and morphologically characterized and compared with MFC products produced without pretreatment as well as with enzymatic hydrolysis. Fenton treatment resulted in an increase in total charge and number of carbonyl groups while the intrinsic viscosity decreased. The Fenton treated pulps were easier to process mechanically i.e. they reached a higher specific surface area at a given mechanical treatment time and the MFC produced had a stable water-fibre suspension for at least 8 weeks compared to enzymatic pretreated pulps and pulps not subjected to any pretreatment.     

Purification and properties of a SDS-resistant xylanase from halophilic Streptomonospora sp. YIM 90494

An extracellular xylanase from halophilic Streptomonospora sp. YIM 90494 was purified to homogeneity from a fermentation broth by ammonium sulphate precipitation, gel filtration chromatography and ion exchange chromatography. The purified xylanase appeared as a single protein band on SDS-PAGE with a molecular mass of approximately 50 kDa. The xylanase had maximum activity at pH 7.5 and 55 °C. The enzyme was stable over a broad pH range (pH 4.0–10.0) and showed good thermal stability when being incubated at 60 °C for 2 h. Kinetic experiments indicated that the enzyme had K _m and V _max values of 19.24 mg/mL and 6.1 μmol/min/mg, respectively, using birch wood xylan as substrate. The inhibitory effects of various metal ions and chemical agents on the xylanase activity were investigated. It is greatly interesting to note that Ag⁺ ion and SDS, which strongly inhibited most xylanases reported previously increases the xylanase activity in this study. These characteristics suggest that the enzyme with new properties has considerable potential in industrial applications.     

Biobleaching of nonwoody pulps using xylanase of Bacillus brevis BISR-062

The efficiency of xylanase of Bacillus brevis BISR-062 as a prebleaching agent was evaluated on three nonwoody pulps at two different pH values (7.0 and 8.5). Crude xylanase was found to have an optimum temperature and pH of 65–70°C and 7.0, respectively. The stability of the enzyme was determined at two pH values (7.0 and 8.0), and it lost approx 50% of its activity at both values within 2 h at 50°C. However, the enzyme was found to be effective as a prebleaching agent only with rice straw pulp. A maximum brightness gain of 6 points was obtained with this pulp at pH 7.0. The strength properties of the rice straw pulp at pH 7.0 also improved as the result of enzyme treatment.     

Partial Characterization of Xylanase Produced by Caldicoprobacter algeriensis,a New Thermophilic Anaerobic Bacterium Isolated from an Algerian Hot Spring

To date, xylanases have expanded their use in many processing industries, such as pulp, paper, food, and textile. This study aimed the production and partial characterization of a thermostable xylanase from a novel thermophilic anaerobic bacterium Caldicoprobacter algeriensis strain TH7C1^T isolated from a northeast hot spring in Algeria. The obtained results showed that C. algeriensis xylanase seems not to be correlated with the biomass growth profile whereas the maximum enzyme production (140.0 U/ml) was recorded in stationary phase (18 h). The temperature and pH for optimal activities were 70 °C and 11.0, respectively. The enzyme was found to be stable at 50, 60, 70, and 80 °C, with a half-life of 10, 9, 8, and 4 h, respectively. Influence of metal ions on enzyme activity revealed that Ca⁺² enhances greatly the relative activity to 151.3 %; whereas Hg²⁺ inhibited significantly the enzyme. At the best of our knowledge, this is the first report on the production of xylanase by the thermophilic bacterium C. algeriensis. This thermo- and alkaline-tolerant xylanase could be used in pulp bleaching process.     

Thermostable and Alkalistable Endoxylanase of the Extremely Thermophilic Bacterium Geobacillus thermodenitrificans TSAA1: Cloning, Expression, Characteristics and Its Applicability in Generating Xylooligosaccharides and Fermentable Sugars

Xylanase encoding gene (1,224 bp) from Geobacillus thermodenitrificans was cloned in pET28a (+) vector and successfully expressed in Escherichia coli BL21 (DE3). The deduced amino acid sequence analysis revealed homology with that of glycosyl hydrolase (GH) 10 family with a high molecular mass (50 kDa). The purified recombinant xylanase is optimally active at pH 9.0 and 70 °C with T _1/2 of 10 min at 80 °C, and retains greater than 85 % activity after exposure to 70 °C for 180 min. The enzyme liberates xylose as well as xylooligosaccharides from birchwood xylan and agro-residues, and therefore, this is an endoxylanase. The xylan hydrolytic products (xylooligosaccharides, xylose, and xylobiose) find application as prebiotics and in the production of bioethanol. The xylanase being thermostable and alkalistable, it has released chromophores and phenolics from the residual lignin of pulps, suggesting its utility in mitigating chlorine requirement in pulp bleaching.     

Laccase from Trametes versicolor

The enzyme laccase was produced by the white-rot fungus Trametes versicolor in repeated batches cultures with immobilized mycelium. Two different culture conditions were used. Enzymes produced were evaluated regarding their stability a thigh temperatures (55°C and 65°C) and at alkaline conditions (pH 7.0 and pH 8.0) having in view the application of these enzymes in biobleaching of hardwood Kraft pulp. Biobleaching experiments were divided in two parts, enzymatic prebleaching followed by chemical bleaching. In the enzymatic prebleaching the enzyme laccase was used at two conditions of pH and temperature, whereas the reaction time was fixed at 1h in all pretreatments. In the chemical bleaching the DEDED and DEpDED sequences were used. The enzyme action was evaluated by Kappa number, viscosity, and brightness at the end of bleaching sequences. There were obtained values of Kappa numbers lower than control assays, viscosities compatible with industrial pulps, and brightness higher than controls, when pulps were pretreated for 1 h with laccase at pH 8.0 and 55°C.