Production of xylanase by Aspergilli using alternative carbon sources: application of the crude extract on cellulose pulp biobleaching

The ability of xylanolytic enzymes produced by Aspergillus fumigatus RP04 and Aspergillus niveus RP05 to promote the biobleaching of cellulose pulp was investigated. Both fungi grew for 4-5 days in liquid medium at 40A degrees C, under static conditions. Xylanase production was tested using different carbon sources, including some types of xylans. A. fumigatus produced high levels of xylanase on agricultural residues (corncob or wheat bran), whereas A. niveus produced more xylanase on birchwood xylan. The optimum temperature of the xylanases from A. fumigatus and A. niveus was around 60-70A degrees C. The enzymes were stable for 30 min at 60A degrees C, maintaining 95-98% of the initial activity. After 1 h at this temperature, the xylanase from A. niveus still retained 85% of initial activity, while the xylanase from A. fumigatus was only 40% active. The pH optimum of the xylanases was acidic (4.5-5.5). The pH stability for the xylanase from A. fumigatus was higher at pH 6.0-8.0, while the enzyme from A. niveus was more stable at pH 4.5-6.5. Crude enzymatic extracts were used to clarify cellulose pulp and the best result was obtained with the A. niveus preparation, showing kappa efficiency around 39.6% as compared to only 11.7% for that of A. fumigatus.

Characterization of a cellulase-free xylanase producing Bacillus sp for biobleaching of kraft pulp

Application of the xylanase in the pulp bleaching process has been shown to be effective in decreasing the amount of chlorinating agents in the process and improving the brightness of the pulp. The use of thermostable cellulase-free xylanase might enhance both the technical and economic feasibility of the process. In this work an alkalophylic strain of Bacillus sp 77-2, was isolated which showed a high production of xylanase and free cellulases. The xylanase of Bacillus sp displayed an optimum pH of 6.0 (with 70% activity at pH 9.0), all optimum temperature of 60 degrees C, pH stability in the range 5-10 and thermal stability of 50 degrees C. These characteristics are important to the kraft pulp bleaching because they are similar to those found in the industrial paper environment.

Effect of Bacillus circulans D1 thermostable xylanase on biobleaching of eucalyptus kraft pulp

The alkalophilic Bacillus circulans D1 was isolated from decayed wood. It produced high levels of extracellular cellulase-free xylanase. The enzyme was thermally stable up to 60°C, with an optimal hydrolysis temperature of 70°C. It was stable over a wide pH range (5.5-10.5), with an optimum pH at 5.5 and 80% of its activity at pH 9.0. This cellulase-free xylanase preparation was used to biobleach kraft pulp. Enzymatic treatment of kraft pulp decreased chlorine dioxide use by 23 and 37% to obtain the same kappa number (κ number) and brightness, respectively. Separation on Sephadex G-50 isolated three fractions with xylanase activity with distinct molecular weights.

Xylanase and β-Xylosidase from Penicillium janczewskii: Production, Physico-Chemical properties, and application of the crude extract to pulp biobleaching

Extracellular xylanase and β-xylosidase production by a Penicillium janczewskii strain were investigated in liquid cultures with xylan from oat spelts under different physical and chemical conditions. The selected conditions for optimized production of xylanase and β-xylosidase were 7 days, pH 6.5, at 30 °C and 8 days, pH 5.0, at 25 °C, respectively. The xylanase exhibited optimal activity in pH 5.0 at 50 °C and the β- xylosidase in pH 4.0 at 75 °C. The xylanase was more stable at pH 6.0 to 9.5, while the β-xylosidase remained stable at pH ranging from 1.6 to 5.5. The xylanase half-life (T50) at 40, 50, and 60 °C was 183, 15, and 3 min, respectively. β-xylosidase half-life was 144, 8, and 4 min at 50, 65, and 75 °C, respectively. When applied to the biobleaching of Eucalyptus kraft pulp, xylanase dosages of 2 and 4 U/g dried pulp reduced, respectively, kappa number by 3.0 and 3.3 units after 1 h treatment, demonstrating that the use of P. janczewskii xylanases in this process is quite promising. The pulp viscosity was not altered, confirming the absence of cellulolytic enzymes in the fungal extract.

Xylanase and beta-Xylosidase from Penicillium janczewskii: Production, Physico-chemical Properties, and Application of the Crude Extract to Pulp Biobleaching

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Bioprocess and biotecnology: effect of xylanase from Aspergillus niger and Aspergillus flavus on pulp biobleaching and enzyme production using agroindustrial residues as substract

This study compares two xylanases produced by filamentous fungi such as A. niger and A. flavus using agroindustrial residues as substract and evaluated the effect of these enzymes on cellulose pulp biobleaching process. Wheat bran was the best carbon source for xylanase production by A. niger and A. flavus. The production of xylanase was 18 and 21% higher on wheat bran when we compare the xylanase production with xylan. At 50°C, the xylanase of A. niger retained over 85% activity with 2 h of incubation, and A. flavus had a half-life of more than 75 minutes. At 55°C, the xylanase produced by A. niger showed more stable than from A. flavus showing a half-life of more than 45 minutes. The xylanase activity of A. niger and A. flavus were somehow protected in the presence of glycerol 5% when compared to the control (without additives). On the biobleaching assay it was observed that the xylanase from A. flavus was more effective in comparison to A. niger. The kappa efficiency corresponded to 36.32 and 25.93, respectively. That is important to emphasize that the cellulase activity was either analyzed and significant levels were not detected, which explain why the viscosity was not significantly modified.

Production and properties of xylanases from Aspergillus terricola Marchal and Aspergillus ochraceus and their use in cellulose pulp bleaching

Aspergillus terricola and Aspergillus ochraceus, isolated from Brazilian soil, were cultivated in Vogel and Adams media supplemented with 20 different carbon sources, at 30 A degrees C, under static conditions, for 120 and 144 h, respectively. High levels of cellulase-free xylanase were produced in birchwood or oat spelt xylan-media. Wheat bran was the most favorable agricultural residue for xylanase production. Maximum activity was obtained at 60 A degrees C and pH 6.5 for A. terricola, and 65 A degrees C and pH 5.0 for A. ochraceus. A. terricola xylanase was stable for 1 h at 60 A degrees C and retained 50% activity after 80 min, while A. ochraceus xylanase presented a t (50) of 10 min. The xylanases were stable in an alkali pH range. Biobleaching of 10 U/g dry cellulose pulp resulted in 14.3% delignification (A. terricola) and 36.4% (A. ochraceus). The brightness was 2.4-3.4% ISO higher than the control. Analysis in SEM showed defibrillation of the microfibrils. Arabinase traces and beta-xylosidase were detected which might act synergistically with xylanase.

Xylanases from Aspergillus niger, Aspergillus niveus and Aspergillus ochraceus produced under solid-state fermentation and their application in cellulose pulp bleaching

This study describes the production of xylanases from Aspergillus niveus, A. niger, and A. ochraceus under solid-state fermentation using agro-industrial residues as substrates. Enzyme production was improved using a mixture of wheat bran and yeast extract or peptone. When a mixture of corncob and wheat bran was used, xylanase production from A. niger and A. ochraceus increased by 18%. All cultures were incubated at 30 A degrees C at 70-80% relative humidity for 96 h. For biobleaching assays, 10 or 35 U of xylanase/g dry cellulose pulp were incubated at pH 5.5 for 1 or 2 h, at 55 A degrees C. The delignification efficiency was 20%, the brightness (percentage of ISO) increased two to three points and the viscosity was maintained confirming the absence of cellulolytic activity. These results indicated that the use of xylanases could help to reduce the amount of chlorine compounds used in cellulose pulp treatment.

Aproximación al diseño de sistemas de bio-reactores para la delignificación de materiales lignocelulósicos y para la decontaminación de suelos y efluentes. Approximation to the design of bio-reactors systems for the delignificación of lignocellulosic materials and for the decontamination of soil and effluents

Los materiales lignocelulósicos residuales de las actividades agroindustriales pueden ser aprovechados como fuente de lignina, hemicelulosa y celulosa. El tratamiento químico del material lignocelulósico se debe enfrentar al hecho de que dicho material es bastante recalcitrante a tal ataque, fundamentalmente debido a la presencia del polímero lignina. Esto se puede lograr también utilizando hongos de la podredumbre blanca de la madera. Estos producen enzimas lignolíticas extracelulares fundamentalmente Lacasa, que oxida la lignina a CO2. Tambien oxida un amplio rango de sustratos ( fenoles, polifenoles, anilinas, aril-diaminas, fenoles metoxi-sustituídos, y otros), lo cual es una buena razón de su atracción para aplicaciones biotecnológicas. La enzima tiene potencial aplicación en procesos tales como en la delignificación de materiales lignocelulósicos y en el bioblanqueado de pulpas para papel, en el tratamiento de aguas residuales de plantas industriales, en la modificación de fibras y decoloración en industrias textiles y de colorantes, en el mejoramiento de alimentos para animales, en la detoxificación de polutantes y en bioremediación de suelos contaminados. También se la ha utilizado en Q.Orgánica para la oxidación de grupos funcionales, en la formación de enlaces carbono- nitrógeno y en la síntesis de productos naturales complejos. HIPOTESIS: Los hongos de podredumbre blanca, y en condiciones óptimas de cultivo producen distintos tipos de enzimas oxidasas, siendo las lacasas las más adecuadas para explorarlas como catalizadores en los siguientes procesos:  Delignificación de residuos de la industria forestal con el fin de aprovechar tales desechos en la alimentación animal.  Decontaminación/remediación de suelos y/o efluentes industriales. Se realizarán los estudios para el diseño de bio-reactores que permitan responder a las dos cuestiones planteadas en la hipótesis. Para el proceso de delignificación de material lignocelulósico se proponen dos estrategias: 1- tratar el material con el micelio del hongo adecuando la provisión de nutrientes para un desarrollo sostenido y favorecer la liberación de la enzima. 2- Utilizar la enzima lacasa parcialmente purificada acoplada a un sistema mediador para oxidar los compuestos polifenólicos. Para el proceso de decontaminación/remediación de suelos y/o efluentes industriales se trabajará también en dos frentes: 3) por un lado, se ha descripto que existe una correlación positiva entre la actividad de algunas enzimas presentes en el suelo y la fertilidad. En este sentido se conoce que un sistema enzimático, tentativamente identificado como una lacasa de origen microbiano es responsable de la transformación de compuestos orgánicos en el suelo. La enzima protege al suelo de la acumulación de compuestos orgánicos peligrosos catalizando reacciones que involucran degradación, polimerización e incorporación a complejos del ácido húmico. Se utilizarán suelos incorporados con distintos polutantes(por ej. policlorofenoles ó cloroanilinas.) 4) Se trabajará con efluentes industriales contaminantes (alpechínes y/o el efluente líquido del proceso de desamargado de las aceitunas). The lignocellulosic raw materials of the agroindustrial activities can be taken advantage as source of lignin, hemicellulose and cellulose. The chemical treatment of this material is not easy because the above mentioned material is recalcitrant enough to such an assault, due to the presence of the lignin. This can be achieved also using the white-rot fungi of the wood. It produces extracellular ligninolitic enzymes, fundamentally Laccase, which oxidizes the lignin to CO2. The enzyme has application in such processes as in the delignification of lignocellulosic materials and in the biobleaching of fibers for paper industry, in the treatment of waste water of industrial plants, in the discoloration in textile industries, in the improvement of food for ruminants, in the detoxification of polutants and in bioremediation of contaminated soils. HYPOTHESIS: The white-rot fungi produce different types of enzymes, being the laccases the most adapted to explore them as catalysts in the following processes:  Delignification of residues of the forest industry in order to take advantage of such waste in the animal feed.  Decontamination of soils and / or waste waters. The studies will be conducted for the design of bio reactors that allow to answer to both questions raised in the hypothesis. For the delignification process of lignocellulosic material they propose two strategies: 1- to treat the material with the fungi 2-to use the partially purified enzyme to oxidize the polyphenolic compounds. For the soil and/or waste water decontamination process, we have: 3- Is know that the enzyme protects to the soil of the accumulation of organic dangerous compounds catalyzing reactions that involve degradation, polymerization and incorporation to complexes of the humic acid. There will be use soils incorporated into different pollutants. 4- We will work with waste waters (alpechins or the green olive debittering effluents.

Mecanismos envolvidos na biodegradação de materiais lignocelulósicos e aplicações tecnológicas correlatas

The biodegradation of lignocellulosic materials is an important natural process because it is responsible for the carbon recycling. When induced under controlled conditions, this process can be used for technological applications such as biopulping, biobleaching of cellulosic pulps, pre-treatment for subsequent saccharification and cellulosic-ethanol production, and increase of the digestibility in agroindustrial residues used for animal feed. In the present work, the enzymatic and non-enzymatic mechanisms involved in the biodegradation of lignocellulosic materials by fungi were reviewed. Furthermore, the technological applications of these extracellular metabolites are presented and discussed.

Production and Characterization of Endocylanase from Bacillus Pumilus Using Solidstate Fermentation and its Application in 'Paper Pulp Processing

In the present studies it is clear that Bacillus pumilus xylanase is having the characteristic suited for an industrial enzyme (xylanases that are active and stable at elevated temperatures and alkaline pH are needed). SSF production of xylanases and its application appears to be an innovative technology where the fermented substrate is the enzyme source that is used directly in the bleaching process without a prior downstream processing. The direct use of SSF enzymes in bleaching is a relatively new biobleaching approach. This can certainly benefit the bleaching process to lower the xylanase production costs and improve the economics and viability of the biobleaching technology. The application of enzymes to the bleaching process has been considered as an environmentally friendly approach that can reduce the negative impact on the environment exerted by the use of chlorine-based bleaching agents. It has been demonstrated that pretreatment of kraft pulp with xylanase prior to bleaching (biobleaching) can facilitate subsequent removal of lignin by bleaching chemicals, thereby, reducing the demand for elemental chlorine or improving final paper brightness. Using this xylanase pre-treatment, has resulted in an increased of brightness (8.5 Unit) when compared to non-enzymatic treated bleached pulp prepared using identical conditions. Reduction of the consumption of active chlorine can be achieved which results in a decrease in the toxicity, colour, chloride and absorbable organic halogen (AOX) levels of bleaching effluents. The xylanase treatment improves drainage, strength properties and the fragility of pulps, and also increases the brightness of pulps. This positive result shows that enzyme pre-treatment facilitates the removal of chromophore fragments of pulp there by making the process more environment friendly

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.

Xilanases, ß-xilosidases de Penicillium janczewskii: purificação, caracterização e aplicação no branqueamento da polpa celulósica e para ração animal

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Production of a xylose-stimulated beta-glucosidase and a cellulase-free thermostable xylanase by the thermophilic fungus Humicola brevis var. thermoidea under solid state fermentation

Humicola brevis var. thermoidea cultivated under solid state fermentation in wheat bran and water (1:2 w/v) was a good producer of beta-glucosidase and xylanase. After optimization using response surface methodology the level of xylanase reached 5,791.2 +/- A 411.2 U g(-1), while beta-glucosidase production was increased about 2.6-fold, reaching 20.7 +/- A 1.5 U g(-1). Cellulase levels were negligible. Biochemical characterization of H. brevis beta-glucosidase and xylanase activities showed that they were stable in a wide pH range. Optimum pH for beta-glucosidase and xylanase activities were 5.0 and 5.5, respectively, but the xylanase showed 80 % of maximal activity when assayed at pH 8.0. Both enzymes presented high thermal stability. The beta-glucosidase maintained about 95 % of its activity after 26 h in water at 55 A degrees C, with half-lives of 15.7 h at 60 A degrees C and 5.1 h at 65 A degrees C. The presence of xylose during heat treatment at 65 A degrees C protected beta-glucosidase against thermal inactivation. Xylanase maintained about 80 % of its activity after 200 h in water at 60 A degrees C. Xylose stimulated beta-glucosidase activity up to 1.7-fold, at 200 mmol L-1. The notable features of both xylanase and beta-glucosidase suggest that H. brevis crude culture extract may be useful to compose efficient enzymatic cocktails for lignocellulosic materials treatment or paper pulp biobleaching.