BLEACHING OF PULP BY LACCASE FROM PLEUROTUS OSTREATUS IMI WITH ARTIFICIAL MEDIATORS

Transcription

1 BLEACHING OF PULP BY LACCASE FROM PLEUROTUS OSTREATUS IMI WITH ARTIFICIAL MEDIATORS ABSTRACT High-quality flax pulp was bleached in a totally chlorine free environment using a laccase mediator system. Two fungal laccases (Pleurotus ostreatus IMI and Trametes hirsuta MTCC 1171) were produced in a bioreactor (Chapter 3) and the produced laccase were tested for paper pulp bleaching with two well known laccase mediators like syringaldazine and 1-Hydroxybenzotriazole (HBT). Laccase from Pleurotus ostreatus IMI with HBT gave the best results in terms of high brightness (52%) and low kappa number (17.9). Xylanase (Pulpzyme) treatment was incorporated into the Pleurotus ostreatus IMI laccase (LCC2) and HBT treatment in the elemental chlorine free pulp bleaching process sequentially. The incorporation of xylanase in the treatment increased the brightness to 62% and reduced the kappa number of pulp by 12.1 respectively. The final pulp properties of the treated pulp were compared with the untreated control pulp. The combined effect of Pleurotus ostreatus IMI laccase and xylanase were found to significantly increase the brightness and decrease the kappa number with improved pulp properties. 169

2 7.1. INTRODUCTION The production and usage of paper have increased dramatically over the past several years. The forecasted annual growth of paper and board until the year 2010 was 2.7% [Diesen, 1998]. Paper is produced form woody substrate which is mainly madeup of lignocellulose. The lignocellulosic material of the wood consists of three main components, namely cellulose, hemicellulose and lignin. Quality of the paper depends upon the reduction of hemicellulose and lignin content in the finished products. The cellulose fibers have to be freed from all traces of lignin, so that no traces of color are present and finally it produces paper. The best paper is produced from long fibers of the highest brightness. Fibers in pulp may be further treated to remove residual lignin and further brighten the resultant papers. Bleaching of wood pulp is the chemical process carried out on various types of wood pulp to decrease the color of the pulp, so that it becomes whiter. Bleaching is usually done using chlorine, this process however is not an eco-friendly process. Chlorine has traditionally been used as a bleaching agent because of its strong electrophilic or oxidizing properties, but unfortunately, these properties cause adverse effects to the environment. Discharging of wastewaters containing chlorinated aromatics formed during chlorine-based bleaching into receiving waters has been proved to have cytotoxic and cytomutagenic effects on various living organisms [Kringstad and Lindstrom, 1984; Curtis and Gary, 1989; Walden and Howard, 1997]. At present there is no method to completely replace the chlorine in pulp bleaching. Enzymes are being considered as only partial replacements for the chemicals, but there is hope that in future the enzymatic treatment may completely replace the chlorine in paper pulp bleaching. The enzyme produced by a group of 170

3 white-rot fungi can break down hemicellulose and lignin; provide an environmental friendly alternative to chlorine bleaching is termed as biobleaching. Most of the countries banned the classical and elemental chlorine bleaching due to its environmental concern. Therefore, the development of environmental friendly technologies for paper manufacturing from non-woody fibers is an urgent need in both industrialized and developing countries. This should include new bleaching sequences limiting the use of chlorine-containing reagents that exert a very negative impact in the aquatic environment [Htun, 1983]. The introduction of the laccase-mediator concept based on the use of lowmolecular weight redox mediators that generate stable radicals, has enlarged the potential of laccases in different applications including the paper pulp industry [Bourbonnais and Paice, 1990]. Once the potential of the laccase mediator systems for delignifying and bleaching paper pulps has been demonstrated, a crucial aspect for their industrial implementation is to optimize the integration of the enzymatic delignification systems into Elemental Chlorine Free (ECF) and Total Chlorine free (TCF) sequences. In the present paper, we explore the potential of laccasemediator systems to remove lignin-derived products responsible for color from a high-quality flax pulp. Two fungal laccases, from Pleurotus ostreatus IMI and Trametes hirsuta MTCC 1171 combined with two mediators, syringaldazine and HBT (1-hydroxybenzotriazole) were used to treat pulp in all the possible combinitions and the two pulp properties namely kappa number and final brightness were compared. 171

4 7.2. MATERIALS AND METHODS Pulp and chemicals Flax (Linum usitatissimum) alkaline pulps with 20 25% ISO brightness and kappa number (24) were obtained from Seshayee Paper Board limited, Erode, India. Unless otherwise stated all chemicals were purchased from LOBO-Chemie (India). Xylanase (Pulpzyme) was given as a gift from Enzyme India Pvt, Tamil Nadu, India Laccase production Laccases (LCC2) from Pleurotus ostreatus IMI was produced by the optimized medium as discussed in the chapter 3. The production of laccase from Trametes hirsuta MTCC 1171 was carried out in glucose yeast extract medium [Couto et al., 2006]. The Trametes sp. was selected based on the earlier reports in the pulp bleaching by laccase [Betcheva et al., 2007; Yang et al., 2007; Frasconi et al., 2010] Experimental design Bleaching of the pulp was carried out according to the method of Camarero et al. [2007]. A control pulp was treated under the same condition without laccase. In the first experiment two different laccases, [Pleurotus ostreatus IMI (LCC2) and Trametes hirsuta MTCC 1171] were tested. The enzyme showing maximum delignification was selected and used for further studies. The treatment of the pulp was carried out in duplicate using 10 g of flax pulp, at 6-7 % consistency in phosphate buffer (100 mm; ph 6.0), 200 U of laccase. To select the best redox mediator, high potential laccase selected in the previous experiment was tested with two mediators, syringaldazine (7 mm per gram of pulp, Camarero et al. [2007]) and HBT (1.5%, Ibarra et al. [2006]) were used individually. Effect of various concentration of laccase 172

5 was determined, to optimize the quantity of laccase in the presence of suitable mediator Pulp treatment The treatments were carried out in 500 ml flasks with O 2 bubbling, placed in a thermostatic shaker at 120 rpm maintained at 50 C, for 12 h. Control pulps were treated under the same conditions but without enzyme, mediator, or both. In a subsequent step, pulps (at 5% consistency) were submitted to: (i) an alkaline extraction stage using 1.5% NaOH (referred to pulp weight) at 60 C for 1 h; or (ii) a bleaching stage using 3% H 2 O 2 and 1.5% NaOH (both referred to pulp weight) at 90 C for 2 h Incorporation of xylanase in pulp treatment The xylanase was incorporated in the pulp treatment according to the method of Bajpai et al. [2006], the pulp consistency was 10%, temperature 50 C, ph 8.0, time 2 hours and enzyme dose 0.075%. Two experiments were conducted with fixed amount of pulp, laccase and redox mediator HBT. In the first experiment, the xylanase alone was incorporated in the treatment sequence without laccase and HBT treatment. In the second experiment xylanase was incorporated in the treatment sequentially followed by laccase and HBT treatment Determination of kappa number Kappa number is determination of relative hardness, bleachability or degree of delignification of pulp. It is important parameter of unbleached pulp which is to be bleached. The method to find kappa number of pulp was done as described in Technical Association of the Pulp and Paper industry, Test methods 236 (TAPPI T 236). A term used to define the degree of delignification. The determination of kappa 173

6 number is given in appendix 3. Kappa number has the advantage of a linear relationship with lignin content over a wide range. Kappa Number x 0.15% = % lignin in pulp Determination of brightness To determine the brightness of white, near-white and naturally colored pulp, paper, and paperboard, TAPPI T 452 protocol was followed. Brightness is a commonly used industry term for the numerical value of the reflectance factor of a sample with respect to blue light of specific spectral and geometric characteristics. Elrepho 071 whiteness meter was used to measure the whiteness of the paper. This was carried out in the laboratory of Seshasayee Paper and Boards Limited, Erode, Tamil Nadu, India. This method requires an instrument employing 45 illumination and 0 viewing geometry with an illuminating and viewing beam adjusted so that translucent materials are evaluated on an arbitrary but specific scale RESULTS In the flax pulp biobleaching, two laccases were used to bleach the pulp. The best laccase was selected on basis of their performance. Most of the pulp bleaching was done by the laccase from Trametes sp. Hence to compare the potential of the Pleurotus ostreatus IMI laccase (PoL), the laccase produced from Trametes hirsuta MTCC 1171 (ThL) was also tested. Table 7.1 shows the property of the untreated pulp and the pulp treated with two laccases individually. When the pulp was treated with laccase from two different fungal organisms, there was no significant change in the pulp property. Hence the pulp treatment was carried out in the presence of redox mediator. 174

7 Figure 7.1 shows the changes of kappa number (A) and brightness (B) of pulp treated with laccases in presence of artificial mediators. Brightness and kappa number analysis showed the high effectiveness of PoL than the ThL, when HBT was used as a redox mediator. When compared with the control pulp, in pulp treated with PoL the brightness of the pulp was increased to 35% and the kappa number reduced to 20±0.8. Among the two laccases studied, the laccase produced by PoL combined with HBT shows remarkable changes in the pulp properties. The obtained data shows that PoL and ThL increase the brightness of the pulp to 47% and 35% respectively, when HBT was used as a redox mediator. Table 7.2 shows the effect of various doses of laccase along with fixed concentration of HBT as a mediator in the pulp treatment. The obtained result shows that, the reduction of kappa number and increase in brightness corresponds with the increase in laccase dose. Above 600 U of laccase no appreciable change in the pulp property was observed. Hence further increment of laccase was considered as futile for the pulp treatment. The effect of commercial xylanase (Pulpzyme) in the pulp treatment was analyzed by treating the pulp (after alkaline extraction) for 24 h simultaneously with PoL-HBT (Table7.3). In the first experiment, there was a minor change in the reduction of kappa number and brightness was observed when the pulp was treated with xylanase alone. In the second experiment, there was significant reduction of kappa number from 19.4±1.4 to 12.1±0.8 and increase in the brightness (from 62±3.2% to 49±3.1%) was observed when the pulp was treated with xylanase followed by the laccase with HBT treatment. 175

8 7.4. DISCUSSION The pulp bleaching process is regarded as one of main causes in contaminating environments since pulp and paper industries have used a lot of chlorine to remove residual lignin. The present study shows efficient bleaching of high-quality paper pulp using enzymes as an alternative to Cl 2 or ClO 2, which exert a negative environmental impact due to the release of a variety of chlorinated compounds that can be incorporated to tropic chains [Mc Kague et al., 1989]. Table 7.1 shows the treatment of paper pulp individually with laccases from Pleurous ostreatus IMI and Trametes hirsuta MTCC There was no appreciable change in the pulp property. Hence to increase the reactivity of the laccase, the pulp was treated with respective laccase along with two redox mediators viz HBT and syringaldazine separately. Kandioller and Christopher [2004] have reported that various degrees of delignificaiton depend on the pulp type, laccase and mediators used in the treatment followed by alkaline extraction. Moreover, laccase is known to oxidize phenolic units of lignin and be able to oxidize nonphenolic units in the presence of mediators [Bourbonnais et al., 1997]. While compared to other lignin oxidizing enzymes laccase was the best choice of researchers due to its dual advantage in nature. Peroxidases need peroxide to oxidize lignin, while laccase uses oxygen as an oxidizing agent to degrade lignin. There was no change in kappa number or brightness observed in the pulp, when the pulp treated with the mediators alone. This proves that individually mediators have no effect in the pulp treatment. Figure 7.1 shows the property of the pulp obtained by the various combinations of laccases and redox mediator treatment. Compared to control both the laccases reduce the kappa number when combined with HBT, nearly 44% of kappa number was reduced when the pulp was treated with 176

9 PoL-HBT. When HBT was incorporated with PoL and ThL separately, the brightness was increased to 48±2.3% and 31.4±1.8% respectively. Another selected redox mediator syringaldazine did not show any appreciable change in the pulp property with laccase, hence it was not considered for further experiments. According to Ibarra et al. [2006] if the concentration and time of introduction of HBT and pulp treatment were optimized it will certainly improve the pulp properties. Generally pulp treatment requires more quantity of laccases; hence it is necessary to optimize the laccase in the pulp treatment. Table 7.2 shows the effect of increasing the PoL units in the pulp treatment. The pulp property was significantly improved proportionally with the amount of laccase introduced (400 U). When amount of laccase was increased to 600U, there was only 7% reduction in kappa number and 3% increase in brightness of the pulp. Excess addition of laccase beyond 400 U did not have any special advantage in the reduction of kappa number and increasing of pulp brightness. To enhance the bleaching effects, xylanase was incorporated sequentially with Laccase mediator system (LMS). The incorporation of xylanase could degrade xylan, which is sandwiched between the lignin and cellulose layers, by xylanase [Suurnakki et al., 1997; Beg et al., 2000], this could eventually help in enhancing the accessibility of LMS towards lignin in the pulp fibres leading to decreased diffusion resistance to the outward movement of the degraded lignin fragments and allow the removal of the less degraded lignin fragments from the fiber wall [Bajpai, 2004]. Table 7.3 shows the incorporation of xylanase treatment in the absence and presence of the LMS. The sequential treatment of xylanase, after treating the pulp with the LMS showed increase in the reduction of kappa number to 12.1±0.8 and increase in brightness to 62±3.2% when compared to treatment with LMS alone (19.4±1.4 and 49±3.1%). The 177

10 above argument was highly correlated with Herpoel et al. [2002] report on the treatment of wheat straw chemical pulp by combining xylanases and LMS followed by alkaline extraction resulting in a final brightness of 69.0%. The reduction of 37.6% of kappa number and increase of 20.9% brightness by xylanase treatment after treating the pulp with LMS is one of the strongly recommended steps to improve the pulp property. The successive combination of the two enzymatic methods, the hydrolytic xylanase and the oxidative laccase mediator treatment has previously been shown to increase the delignification efficiency [Herpoel et al., 2002] CONCLUSION It is possible to conclude that the laccase-mediator system is especially promising for bleaching high-quality pulps from flax. Once integrated in a total chlorine free sequence, satisfactory brightness and delignification rates can be obtained. The combination of laccase with suitable mediator along with xylanase facilitate the subsequent removal of lignin in every stage and shows great improvement of the pulp in mild operational conditions. The cost of the enzymatic bleaching would be affordable because: (i) low investment for new installations would be required (ii) laccase hyperproducing strains are available or could be obtained using industrial hosts (iii) enzyme and mediator costs would be compensated by the high price of these pulps (e.g. the market price of flax pulp is around four-times that of kraft pulp) (iv) reagents would be saved due to the shortening of the bleaching sequence and (v) environmental advantages would be produced by avoiding Cl 2 and ClO 2. Once the enzyme and mediator are optimized in large scale it is possible to eliminate the hazardous chemicals in the pulp processing. 178

11 ThL-HBT ThL-Sy PoL-HBT PoL-Sy HBT Sy Control A Kappa Number ThL-HBT ThL-Sy PoL-HBT PoL-Sy HBT Sy Control B Brightness (%) Figure 7.1. Changes of kappa number (A) and brightness (B) of paper pulp treated with laccase in the presence of artificial mediators (syringaldazine, hydroxybenzotriazole) followed by hydrogen peroxide bleaching compared with control (without both laccase and mediator). Abbreviations: Sy, Syringaldazine, HBT, 1-hydroxybenzotriazole. PoL, Pleurotus ostreatus IMI laccase (LCC2), ThL, Trametes hirsuta MTCC 1711 Laccase, Variation coefficient values were below 5%. 179

12 Table 7.1. Comparison of flax pulp treated with PoL and ThL Particulars Unit of measurement Screened pulp Pulp treated with Pleurotus ostreatus LCC2 laccase isoenzyme Pulp treated with Trametes hirsute MTCC 1171 laccase Temperature ºC 65±5 60±5 60±5 ph - 8.6± ± ±0.2 consistency % 7.9± ± ±0.2 Kappa number ±2.6 24± ±2.0 Brightness % 24±2.8 29±3.2 25±3.1 Table 7.2. Effect of various concentratin of PoL (LCC2) with fixed concentration of HBT in the treatment of flax pulp Particulars Unit of measurement Test conditions and pulp properties Laccase dose Units Nil Temperature ºC ph Kappa number - 26± ± ± ±2.1 Brightness % 24±2.5 35±2.8 49±3.2 52±3.3 *HBT concentration was fixed at 1.5% 180

13 Table 7.3. Effect of incorporation of xylanase with PoL in the pulp treatment Experiment I Experiment II Particulars Unit Pulp (10 g) + laccase (LCC2) (400 U) + HBT (1.5%) xylanase treatment alone Pulp (10 g) + laccase (LCC2) (400 U) + HBT (1.5%) Followed by xylanse treatment Temperature C 60±5 50±5 60±5 50±5 ph - 6.0± ± ± ±0.2 Consistency % 6.5± ± ± ±0.2 Kappa number ± ± ± ±0.8 Brightness % 49±2.3 53±2.5 49±3.1 62±