Enhanced production of cellulase-free thermostable xylanase by Bacillus pumilus ASH and its potential application in paper industry

A very high level of cellulase-free, thermostable xylanase has been produced from newly isolated strain of Bacillus pumilus under submerged fermentation in a basal medium supplemented with wheat bran (2%, w/v) pH 8.0 and at 37 °C. After optimization of various production parameters, an increase of nearly 13-fold in xylanase production (5407 IU/ml) was achieved. The produced xylanase is stable in neutral to alkaline pH region at 70 °C. The suitability of this xylanase for use in the bioleaching of eucalyptus Kraft pulp was investigated. A xylanase dose of 5 IU/g of oven dried pulp of 10% consistency exhibited the optimum bleach boosting of the pulp at pH 7.0 and 60 °C after 180 min of treatment. An increase of 5% in brightness along with an increase of 21% and 28% in whiteness and fluorescence respectively, whereas 18% decrease in the yellowness of the biotreated pulp was observed. Enzyme treated pulp when subjected to chemical bleaching, resulted in 20% reduction in chlorine consumption and up to 10% reduction in consumption of chlorine dioxide. Also a reduction of about 16% in kappa number and 83% in permanganate number, along with a reduction in COD value and significant improvement in various pulp properties, viz. viscosity, tensile strength, breaking length, burst factor, burstness, tear factor and tearness were observed in comparison to the conventional chemical bleaching.     

Enhanced production of cellulase-free thermostable xylanase by Bacillus pumilus ASH and its potential application in paper industry

A very high level of cellulase-free, thermostable xylanase has been produced from newly isolated strain of Bacillus pumilus under submerged fermentation in a basal medium supplemented with wheat bran (2%, w/v) pH 8.0 and at 37 °C. After optimization of various production parameters, an increase of nearly 13-fold in xylanase production (5407 IU/ml) was achieved. The produced xylanase is stable in neutral to alkaline pH region at 70 °C. The suitability of this xylanase for use in the bioleaching of eucalyptus Kraft pulp was investigated. A xylanase dose of 5 IU/g of oven dried pulp of 10% consistency exhibited the optimum bleach boosting of the pulp at pH 7.0 and 60 °C after 180 min of treatment. An increase of 5% in brightness along with an increase of 21% and 28% in whiteness and fluorescence respectively, whereas 18% decrease in the yellowness of the biotreated pulp was observed. Enzyme treated pulp when subjected to chemical bleaching, resulted in 20% reduction in chlorine consumption and up to 10% reduction in consumption of chlorine dioxide. Also a reduction of about 16% in kappa number and 83% in permanganate number, along with a reduction in COD value and significant improvement in various pulp properties, viz. viscosity, tensile strength, breaking length, burst factor, burstness, tear factor and tearness were observed in comparison to the conventional chemical bleaching.     

Production of thermostable pectinase and xylanase for their potential application in bleaching of kraft pulp

A very high level of alkalophilic and thermostable pectinase and xylanase has been produced from newly isolated strains of Bacillus subtilis and Bacillus pumilus respectively. Enzyme production for pectinase was carried out under SSF using combinations of cheap agricultural residues while xylanase was produced under submerged fermentation using wheat bran as substrate to minimize the cost of production of these enzymes Among the various substrates tested, the highest yield of pectinase production was observed by using combination of WB + CW (6592 U/g of dry substrate) supplemented with 4% yeast extract when incubated at 37 °C for 72 h using deionized water of pH 7.0 as moistening agent. The biobleaching effect of these cellulase free enzymes on kraft pulp was determined. Both xylanase and pectinase showed stability over a broad range of pH from 6 to 10 and temperature from 55 to 70 °C. The bleaching efficiency of the pectinase and xylanase on kraft pulp was maximum after 150 min at 60 °C using enzyme dosage of 5 IU/ml of each enzyme at 10% pulp consistency with about 16% reduction in kappa number and 84% reduction in permanganate number. Enzyme treated pulp when subjected to CDED₁D₂ steps, 25% reduction in chlorine consumption and upto 19% reduction in consumption of chlorine dioxide was observed for obtaining the same %ISO brightness. Also an increase of 22 and 84% in whiteness and fluorescence respectively and a decrease of approximately 19% in the yellowness of the biotreated pulp were observed by pretreatment of the pulp with our enzymatic mixture.     

Application of enzymes in producing bleached pulp from wheat straw

Crude enzymes produced by different strains were used in the production of bleached wheat straw pulp. Pre-treatment with enzymes from Penicillium A10 and Aspergillus L22 at a xylanase dosage of 4 IU/g prior to pulping decreased pulp kappa number by 6.29% and 12.07% respectively as compared to the control. High cellulase activity in crude enzymes has a negative influence on pulping. Xylanase pre-bleaching reduced chlorine charge by 20-30%, or increased final brightness by approximately 4-5% ISO, and improved the pulp strength properties. Xylanase could substitute for alkali extraction in CEH sequence, and be used for treating chemical-bleached pulp, which resulted in higher strength properties for bleached pulp. Modification of bleached pulp with enzymes of 3 IU/g (on xylanase) increased pulp brightness and breaking length by 3-6% ISO and 160-790 m respectively, and decreased post color number and beating degree of pulp by 29-36% and 2.5-5.5 degrees SR respectively, as compared to the original pulp.     

Potential of xylanase from thermophilic Bacillus sp. XTR-10 in biobleaching of wood kraft pulp

An extracellular xylanase produced under optimal conditions by a thermophilic strain of Bacillus sp. XTR-10 was evaluated for its potential application in biobleaching of wood kraft pulp. Spectrophotometric analysis showed considerable release of lignin derived compounds and chromophoric material by the xylanase treated pulp samples. Xylanase was found to be effective in the liberation of reducing sugars in the pulp filtrates with increment in enzyme dose and reaction time. Eight hours pretreatment with 40 IU of xylanase/g of dry pulp resulted in 16.2% reduction of kappa number with 25.94% ISO increase in brightness as compared to the control. The same treatment slightly lowered the tensile strength and burst index, however. Enzyme pretreatment of the pulp saved 15% active chlorine charges in single step and 18.7% in multiple steps chemical bleaching with attainment of brightness at the level of the control. These results indicate the potential of enzymatic pretreatment of pulp for reduction in environmental discharge of hazardous waste from the pulp and paper industry.     

Two mannanases from Sclerotium rolfsii in total chlorine free bleaching of softwood kraft pulp

The plant pathogenic basidiomycete Sclerotium rolfsii produces a wide range of extracellular hemicellulolytic enzymes. To study the effect of β-mannanases in total chlorine free bleaching of softwood pulp, two purified β -mannanases from S. rolfsii, with molecular masses of 42 and 61 kDa, a xylanase preparation from S. rolfsii and combinations of these were tested in a O(QX)P bleaching sequence (O = oxygen delignification, X = treatment with enzymes, Q = chelation of metals, P = treatment with hydrogen peroxide in alkaline solution). A brightness increase of 1.6 and 1.9% ISO was obtained with the 42 and 61 kDa mannanase and a combination of each of these enzymes with xylanases gave a brightness increase of 2.5 and 2.8% ISO, respectively. The effect of mannanases and xylanases was nearly additive. Both mannanases alone caused a lower decrease of the kappa number as compared to xylanases. The mannanases differed in their ability to release oligosaccharides from different mannans. The 61 kDa mannanase liberated larger fragments and caused rapid depolymerisation of mannans, which seems to promote the bleaching of pulp.     

Xylanase-induced reduction of chlorine dioxide consumption during elemental chlorine-free bleaching of different pulp types

The potential of crude xylanase from Thermomyces lanuginosus and Xylanase P (a commercial xylanase) was evaluated in bleaching of various paper pulp types. Xylanases released chromophores and reducing sugars and decreased kappa number of pulps. Chlorine-bleached, alkali-extracted bagasse and post-oxygen kraft pulps, pretreated with enzymes, gained over 5 brightness points over controls. Biobleaching of soda-aq pulp with Xylanase P produced chlorine dioxide savings of up to 30% or 4.5 kg chlorine dioxide t^–1 pulp.     

Biological bleaching of chemical pulps

Use of biotechnology in pulp bleaching has attracted considerable attention and achieved interesting results in recent years. Enzymes of the hemicellulolytic type, particularly xylan-attacking enzymes, xylanases are now used commercially in the mills for pulp treatment and subsequent incorporation into bleach sequences. The aims of the enzymatic treatment depend on the actual mill conditions and may be related to environmental demands, reduction of chemical costs or maintenance or even improvement of product quality. The use of oxidative enzymes from white-rot fungi, that can directly attack lignin, is a second-generation approach, which could produce larger chemical savings than xylanase but has not yet been developed to the full scale. It is being studied in several laboratories in Canada, Japan, the U.S.A. and Europe. Certain white-rot fungi can delignify kraft pulps increasing their brightness and their responsiveness to brightening with chemicals. The fungal treatments are too slow but the enzyme manganese peroxidase and laccase can also delignify pulps and enzymatic processes are likely to be easier to optimize and apply than the fungal treatments. Development work on laccase and manganese peroxidase continues. This article presents an overview of developments in the application of hemicellulase enzymes, lignin-oxidizing enzymes and white-rot fungi in bleaching of chemical pulps. The basic enzymology involved and the present knowledge of the mechanisms of the action of enzymes as well as the practical results and advantages obtained on the laboratory and industrial scale are discussed.     

Application of crude xylanase from Bacillus licheniformis 77-2 to the bleaching of eucalyptus Kraft pulp

Alkalophilic Bacillus licheniformis 77-2 produced an extracellular alkali-tolerant xylanase with negligible cellulase activity in medium containing corn straw. The effectiveness of crude xylanase on treatment of eucalyptus Kraft pulp was evaluated. A biobleaching experiment was carried out to compare the chlorine saving with pulp treated and untreated by the enzyme. Two-stage bleaching was employed, using a ClO₂ chlorination and NaOH extraction (DE sequence). With the enzymatic treatment, in order to obtain the same value of Kappa number and brightness, respectively 28.5 and 30% less ClO₂ was required in comparison to the enzymatically untreated samples.     

The performance of fungal xylan-degrading enzyme preparations in elemental chlorine-free bleaching for Eucalyptus pulp

Cellulase-free xylan-degrading enzyme preparations from Acrophialophora nainiana, Humicola grisea var. thermoidea and two Trichoderma harzianum strains were used as bleaching agents for Eucalyptus kraft pulp, prior to a chlorine dioxide and alkaline bleaching sequence. In comparison to the control sequence (performed without xylanase pretreatment), the sequence incorporating enzyme treatment was more effective. Removal of residual lignin was indicated by a reduction in kappa number. Overall, enzyme preparations from T. harzianum were marginally more effective in reducing pulp viscosity and chlorine chemical consumption and improving the brightness of the kraft pulp. However, the highest reduction in pulp viscosity was mediated by the xylanase preparation from A. nainiana. Xylanase pretreatment compares very favorably with that of chemical pulping. Journal of Industrial Microbiology & Biotechnology (2002) 28, 204–206 DOI: 10.1038/sj/jim/7000227 Received 27 April 2001/ Accepted in revised form 03 November 2001     

Production,partial characterization and use of fungal cellulase-free xylanases in pulp bleaching

Seven fungal strains were screened for their ability to produce cellulase-free xylanases that could be used in pretreatment of sulphite pulp prior to bleaching. The potential xylanase producers were subjected to shake flask fermentations using four different carbon sources: wheat bran, corn cobs, oat spelts xylan and bleach plant effluent. When grown on corn cobs, Aspergillus foetidus (ATCC 14916) produced significant levels of xylanase (547.4 U/ml), accompanied however by 6.6 U/ml of cellulase activity. Two other strains, Aspergillus oryzae (NRRL 1808) and Gliocladium viride (CBS 658.70), produced high yields of cellulase-free xylanase on oat spelts xylan. The crude enzymes of these two isolates were characterized with respect to pH and temperature optima and stability in order to standardize the optimum conditions for their use on pulp. Although the two xylanases differed in their abilities to remove reducing sugars from pulp, their biobleaching abilities, when assessed in hydrogen peroxide delignification of pulp, were very similar: both of them increased brightness by 1.4 points and removed 7% of hemicellulose from pulp.     

Xylanase production using agro-residue in solid-state fermentation from Bacillus pumilus ASH for biodelignification of wheat straw pulp

Two stage statistical design was used to optimize xylanase production from Bacillus pumilus ASH under solid-state fermentation. Initially, Plackett-Burman designing (PB) was used for the selection of crucial production parameters. Peptone, yeast extract, incubation time, moisture level and pH were found to be the crucial factors for the xylanase production. Crucial variables were further processed through central composite designing (CCD) of response surface methodology (RSM) to maximize the xylanase yield. Each significant factor was investigated at five different levels to study their influence on enzyme production. Statistical approach resulted in 2.19-fold increase in xylanase yield over conventional strategy. The determination coefficient (R (2)) as shown by analysis of variance (ANOVA) was 0.9992, which shows the adequate credibility of the model. Potential of cellulase-free xylanase was further investigated for biobleaching of wheat straw pulp. Xylanase aided bleaching through XCDED(1)D(2) sequence resulted in 20 and 17% reduction in chlorine and chlorine dioxide consumption as compared to control. Significant increase in pulp brightness (%ISO), whiteness and improvement in various pulp properties was also observed.     

TCF mill trial on softwood pulp with Korsäs thermostable and alkaline stable xylanase T6

Abstract: Use of hemicellulases, including xylanases, for delignification in the paper industry has been slowed down by the lack of large-scale availability of enzymes which are active at a high pH (above 8) and a high temperature (above 60°C), conditions prevailing in many bleaching processes. During the past years, acidic or neutral hemicellulases, working at temperatures below 60°C, were used in most mill experiments. The Korsäs T6 xylanase from Bacillus stearothermophilus , which is active at a pH above 9.0 and at a temperature above 65°C, was produced on a large scale in collaboration with Gist-brocades and was employed on a full scale mill trial to produce a Total Chlorine chemical-Free (TCF) pulp from softwood. The bleaching sequence used was (OO)BQQPP. where O stands for oxygen delignification. B for the enzymatic treatment, Q for the chelating agent step and P for the hydrogen peroxide step. The enzyme bleaching step was performed during a period of 4 h at 63 ± 1°C and pH 8.7 ± 0.1. The results of the mill trial show that the TCF pulp produced had a brightness of 78% ISO and, at the same time, it preserved the same strength properties as chlorine dioxide-bleached pulp. The saving of hydrogen peroxide was 20%. The results on brightness, strength and chemical saving of this first full scale trial with T6 xylanase indicate that, after optimization, a TCF bleaching sequence including an enzymatic step with a xylanase working at a high pH and a high temperature, such as T6 xylanase, can be used to produce a high-strength bleached pulp. The advantages of a high pH and a high temperature enzymatic bleaching step are discussed.     

Production of multi-fiber modifying enzyme from Mamillisphaeria sp. for refining of recycled paper pulp

Enzymatic modification of pulp is receiving increasing interest for energy reduction at the refining step of the paper-making process. In this study, the production of a multi-fiber modifying enzyme from Mamillisphaeria sp. BCC8893 was optimized in submerged fermentation using a response-surface methodology. Maximal production was obtained in a complex medium comprising wheat bran, soybean, and rice bran supplemented with yeast extract at pH 6.0 and a harvest time of 7 d, resulting in 9.2 IU/mL of carboxymethyl cellulase (CMCase), 14.9 IU/mL of filter paper activity (FPase), and 242.7 IU/mL of xylanase. Treatment of old corrugated container pulp at 0.2-0.3 IU of CMCase/g of pulp led to reductions in refining energy of 8.5-14.8%. The major physical properties were retained, including tensile and compression strength. Proteomic analysis showed that the enzyme was a complex composite of endo-glucanases, cellobiohydrolases, beta-1,4-xylanases, and beta-glucanases belonging to various glycosyl hydrolase families, suggestive of cooperative enzyme action in fiber modification, providing the basis for refining efficiency.     

Application of thermoalkalophilic xylanase from Arthrobacter sp. MTCC 5214 in biobleaching of kraft pulp

A thermoalkalophilic and cellulase-free xylanase produced from Arthrobacter sp. MTCC 5214 by solid-state fermentation using wheat bran as a carbon source was evaluated for prebleaching of kraft pulp. The UV absorption spectrum of the compounds released by enzyme treatment showed a characteristic peak at 280 nm, indicating the presence of lignin in the released colouring matter. Enzymatic prebleaching of kraft pulp showed 20% reduction in kappa number of the pulp without much change in viscosity. Enzymatic treatment reduced the amount of chlorine by 29% without any decrease in brightness. The viscosity of xylanase treated pulp was 4.0 p, whereas the viscosity of the pulp treated exclusively with chlorine was 4.1 p.     

Production,purification and characterization of <Emphasis Type="Italic">Bacillus</Emphasis> sp. GRE7 xylanase and its application in eucalyptus Kraft pulp biobleaching

Production of extracellular xylanase from Bacillus sp. GRE7 using a bench-top bioreactor and solid-state fermentation (SSF) was attempted. SSF using wheat bran as substrate and submerged cultivation using oat-spelt xylan as substrate resulted in an enzyme productivity of 3,950 IU g⁻¹ bran and 180 IU ml⁻¹, respectively. The purified enzyme had an apparent molecular weight of 42 kDa and showed optimum activity at 70°C and pH 7. The enzyme was stable at 60–80°C at pH 7 and pH 5–11 at 37°C. Metal ions Mn²⁺ and Co²⁺ increased activity by twofold, while Cu²⁺ and Fe²⁺ reduced activity by fivefold as compared to the control. At 60°C and pH 6, the K _m for oat-spelt xylan was 2.23 mg ml⁻¹ and V _max was 296.8 IU mg⁻¹ protein. In the enzymatic prebleaching of eucalyptus Kraft pulp, the release of chromophores, formation of reducing sugars and brightness was higher while the Kappa number was lower than the control with increased enzyme dosage at 30% reduction of the original chlorine dioxide usage. The thermostability, alkali-tolerance, negligible presence of cellulolytic activity, ability to improve brightness and capacity to reduce chlorine dioxide usage demonstrates the high potential of the enzyme for application in the biobleaching of Kraft pulp.     

Hyper production of alkali stable xylanase in lesser duration by Bacillus pumilus SV-85S using wheat bran under solid state fermentation

High level production of an extracellular cellulase-poor alkali stable xylanase has been conceded from newly isolated Bacillus pumilus SV-85S under solid state fermentation using wheat bran as a substrate. Optimization of the fermentation conditions enhanced the enzyme production to 73,000 ± 1,000 IU/g dry substrate, which was 13.8-fold higher than unoptimized conditions (5,300 IU/g). The enzyme titre was highest after 48 h of incubation at 30°C with 1:3 ratios of substrate to moistening agent using wheat bran as a carbon source. The enzyme could be produced in significant levels by using either tap water or distilled water alone as a moistening agent. An elevated production of xylanase by B. pumilus SV-85S in the presence of wheat bran, a cheap and easily available agro-residue, in shorter duration would apparently reduce the enzyme cost substantially. The enzyme was completely stable over a broad pH (5-11) range and retained 52% of its activity at a temperature of 70°C for 30 min. The desired characteristics of this enzyme together with economic production would be important for its application in paper and pulp industry.     

Xylanases of marine fungi of potential use for biobleaching of paper pulp

Microbial xylanases that are thermostable, active at alkaline pH and cellulase-free are generally preferred for biobleaching of paper pulp. We screened obligate and facultative marine fungi for xylanase activity with these desirable traits. Several fungal isolates obtained from marine habitats showed alkaline xylanase activity. The crude enzyme from NIOCC isolate 3 (Aspergillus niger), with high xylanase activity, cellulase-free and unique properties containing 580 U l^–1 xylanase, could bring about bleaching of sugarcane bagasse pulp by a 60 min treatment at 55°C, resulting in a decrease of ten kappa numbers and a 30% reduction in consumption of chlorine during bleaching. The culture filtrate showed peaks of xylanase activity at pH 3.5 and pH 8.5. When assayed at pH 3.5, optimum activity was detected at 50°C, with a second peak of activity at 90°C. When assayed at pH 8.5, optimum activity was seen at 80°C. The crude enzyme was thermostable at 55°C for at least 4 h and retained about 60% activity. Gel filtration of the 50–80% ammonium sulphate-precipitated fraction of the crude culture filtrate separated into two peaks of xylanase with specific activities of 393 and 2,457 U (mg protein)^–1. The two peaks showing xylanase activity had molecular masses of 13 and 18 kDa. Zymogram analysis of xylanase of crude culture filtrate as well as the 50–80% ammonium sulphate-precipitated fraction showed two distinct xylanase activity bands on native PAGE. The crude culture filtrate also showed moderate activities of -xylosidase and -l-arabinofuranosidase, which could act synergistically with xylanase in attacking xylan. This is the first report showing the potential application of crude culture filtrate of a marine fungal isolate possessing thermostable, cellulase-free alkaline xylanase activity in biobleaching of paper pulp.     

Cellulase-free xylanases from Bacillus and other microorganisms

Xylanases are used mainly in the pulp and paper industries for the pretreatment of Kraft pulp prior to bleaching to minimize use of chlorine, the conventional bleaching agent. This application has great potential as an environmentally safe method. Hydrolysis by xylanases of relocated and reprecipitated xylan on the surface of cellulose fibres formed during Kraft cooking facilitates the removal of lignin by increasing permeability to oxidising agents. Most of the xylanases reported in the literature contained significant cellulolytic activity, which make them less suitable for pulp and paper industries. The need for large quantities of xylanases which would be stable at higher temperatures and pH values and free of cellulase activity has necessitated a search for novel enzymes. We have isolated and characterised several xylanase-producing cultures, one of which (an alkalophilic Bacillus SSP-34) produced more than 100 IU ml(-1) of xylanase activity. The SSP-34 xylanases have optimum activity at 50 degrees C in a pH range 6-8, with only small amounts of cellulolytic activity (CMCase (0.4 IU ml(-1), pH 7), FPase (0.2 IU ml(-1), pH 7) and no activity at pH 9).     

绿色糖单孢菌降解木质纤维胞外酶的分泌条件及其生物漂白研究

以一株耐热耐碱放线菌-绿色糖单孢菌（Saccharomonospora viridis）为研究对象,探讨其产胞外木素过氧化物酶、木聚糖酶、纤维素酶的优化发酵条件。结果表明,其最佳碳氮源分别为葡萄糖和蛋白胨,最佳接种量为1％,不同的诱导底物对三种木质纤维降解酶有不同的诱导效果,其中麦草浆的诱导效果最好。在培养基中添加0．01mol／L的Mn^2＋和0．1％的土温80能够显著促进木质纤维降解酶的产生。在pH8．0,45℃条件下,培养120h后木素过氧化物酶的酶活达到最大0．36U／mL,培养156h后木聚糖酶和纤维素酶的酶活达到最大,最高酶活分别为18．46U／mL,10．42u／mL。用含有这三种酶的粗酶液对麦草烧碱蒽醌浆进行生物漂白表明,绿色糖单孢菌所产的木质纤维降解酶具有较好的漂白效果。