Unbleached sulfite cellulose obtained from a mixture of 60% spruce (Picea abis) and 40% pine (Pinus sylvestris) and cooked at Domsjö mill (Sweden), was used as raw material. Prior to bleaching treatments, fibre samples were conditioned at pH 4 adjusted with H2SO4, stirred at 2% pulp consistency for 30 min and washed with de-ionized water in a glass filter funnel. This step was needed to remove contaminants and metals, and also to bring the pulp to the pH required for enzymatic treatment. The main characteristics of the starting pulp were as follows: 4.2 ± 0.2 kappa number, 61.25 ± 0.6% ISO brightness, 511 ± 11 mL/g viscosity. Carbohydrate composition, as determined by high-performance liquid chromatography (HPLC), was as follows: 92.6 ± 0.04% glucan, 6.6 ± 0.1%
mannan and xylan and 0.8 ± 0.02% glucuronic acid.
4.2.2 Enzyme and mediators
Commercial laccase from Trametes villosa was used in combination with the natural mediators syringaldehyde (SA) and p-coumaric acid (pCA), and the synthetic mediators 1-hydroxybenzotriazole (HBT) and violuric acid (VA). The enzyme was supplied by Novozymes® (Denmark) and the mediators were purchased from Sigma–Aldrich. Laccase activity was determined by monitoring the oxidation of ABTS 2,2 azinobis(3-ethylbenzthiazoline-6-sulphonate) in 0.1 M sodium acetate buffer at pH 5 at 25 ºC. One activity unit is defined as the amount of laccase required to convert 1 µmol/min of ABTS to its cation radical (ε436 = 29300 M–1cm–1).
4.2.3 Preliminary bleaching tests: selection of the mediator Unbleached sulfite cellulose was treated with a laccase–mediator system (LMS) consisting of the enzyme and either a synthetic compound (HBT or VA)
4-71 or a natural compound (SA or pCA). All treatments, at 5% pulp consistency, were conducted in an oxygen pressurized reactor (0.6 MPa), at stirring rate of 30 rpm, using 50 mM sodium tartrate buffer at pH 4, a dose of 20 U/g odp (oven dried pulp) of laccase and one of 1.5% odp of each mediator at 50 ºC for 4 h. A few drops of the surfactant Tween 20 (0.05% w/v) were also added. The enzymatic conditions were similar to those previously used by Valls et al.,( 2012) with eucalyptus pulp. The sequence was completed with a chemical bleaching stage involving alkaline peroxide bleaching procedure. Fibre samples, at 5%
consistency, were treated with 2% odp H2O2, 1.5% odp NaOH, 1% odp DTPA (diethylenetriaminepentaacetic acid) and 0.2% odp MgSO4 in a Datacolor Easydye AHIBA oscillating individual reactor at 90 ºC for 2 h (Aracri et al., 2009; Fillat et al., 2010; Andreu and Vidal, 2011). After each stage, residual liquors were collected for subsequent analysis, and pulp samples filtered and extensively washed for further processing. A laccase control treatment (KL) was conducted in parallel under the same conditions but with no presence of mediator. A conventional hydrogen peroxide bleaching treatment, i.e. a P stage applied directly to the starting pulp, was also performed in order to compare the bleaching efficiency of the laccase-mediator system in combination with a hydrogen peroxide bleaching stage.
A xylanase stage (X) was described as a pre-trial test to assess the efficiency of the enzyme on sulfite cellulose. The enzyme used was a commercial xylanase (Pulpzyme HC) supplied by Novozymes®. The X stage used 3 U/g odp xylanase at 10 % consistency adjusted with Tris-HCl buffer at pH 7 at 50 ºC for 2 h. After treatment, liquors were recovered and the resulting pulp was extensively washed as reported elsewhere for eucalyptus pulp (Valls et al., 2010e).
4.2.4 Extended biobleaching sequence: influence of the H2O2
dose and reaction time reduction
An extended TCF biobleaching sequence including a laccase-mediator treatment was applied to the initial pulp. The sequence was designated LQPO/P, where L denotes the enzymatic treatment, Q a chelating stage and PO/P a hydrogen peroxide stage lasting 6 h —the first four reinforced with pressurized oxygen and then followed by a depressurization where oxygen is removed.
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The enzymatic stage (L) was carried out with the laccase-violuric acid system (Lac-VA), using the same conditions as in the preliminary tests. The enzymatic treatment was followed by a Q stage involving the use of chelating agents to reduce the contents in metal ions (Fe2+, Cu2+, Mn2+) capable of degrading the bleaching agents and cellulose during the subsequent peroxide bleaching treatment (Heijnesson et al., 1995). The Q stage was performed with 1% odp DTPA at 5% consistency at pH 5-6 (adjusted with H2SO4 1 N) in polyethylene bags at 85 ºC for 1 h. The biobleaching sequence was completed with a chemical bleaching stage involving alkaline hydrogen peroxide bleaching procedure (PO/P). The operating conditions of PO stage differed from those of the preliminary tests —emphasising the incorporation of a multiple sequential steps. Thus, PO was carried out at 5% consistency in oxygen pressurized (0.6 MPa) reactor, using a stirring rate of 30 rpm under the following conditions:
1.5% odp NaOH, 0.3% odp DTPA and 0.2% odp MgSO4 at 90 ºC for 4 h. This stage was performed in three consecutive steps (PO1 = 1 h reaction, PO2 = 1 h reaction, PO4 = 2 h reaction) each involving the addition of 10% odp H2O2 and no interstep washing. In the last P stage, pressure was released and the temperature was maintained at 90 ºC for 2 additional hours without addition of H2O2. These last two hours of treatment were performed in order to examine the potential influence of residual hydrogen peroxide on the extent of fibre bleaching. A small amount of pulp was removed after each PO step, filtered, residual liquors collected and then extensively washed with de-ionized water in a filter funnel for subsequent analysis. A control sequence (KQPO/P) treated identically as the enzymatic sequence but in the absence of laccase and mediator was also studied. A conventional chemical hydrogen peroxide sequence (PO/P) was also performed in order to examine in terms of industrial application the effect of an enzymatic on the overall bleaching process. Thus, the enzymatic stage was omitted and the PO/P treatment was applied directly to the initial pulp using the same conditions defined for the enzymatic sequence.
4.2.5 Pulp properties
The sugar composition of the initial pulp and after xylanase treatment (X) was determined by high performance liquid chromatography (HPLC). Samples were studied on a duplicate basis using a modified version of TAPPI 249 cm-09 test method. Because the column failed to resolve xylose, mannose and
4-73 galactose, their combined content was expressed as xylose (Chapter 3, section 3.7.1).
Initial and treated pulp samples were characterized in terms of kappa number, ISO brightness and viscosity according to ISO 302:2004, ISO 2470:2009 and ISO 5351:2004, respectively. Fibre optical properties were measured in terms of the CIE L*a*b* color coordinates, namely: lightness (L*), red–green (a*) and yellow–blue (b*). Chroma (C*), which is the perpendicular distance of a point from the lightness axis [C* = (a*2 + b*2)1/2] and represents the amount of color ―saturation‖ of a sample, was also used to characterize the bleaching process. Biobleached fibre samples were subjected to accelerated thermal ageing treatment by moist heating (at 80 ºC and 65% RH) in a HC 2020 Heraeus-Vötsch climatic chamber according to ISO 5630-3:1996 (Chapter 3, section 3.7.2).
4.2.6 Effluent properties
The effluents from the xylanase treatment were collected and analysed by thin-layer chromatography (TLC) as described elsewhere (Valls et al. 2010). The effluents from L stage in combination with different mediators were collected and analysed for chemical oxygen demand (COD), residual laccase activity, colour and toxicity (Chapter 3, section 3.7.4).
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Schema 4-1 summarizes the experiments conducted and the properties analysed for each treatment.
Schema 4-1 Schema of work applied to the unbleached sulfite pulp and the respective studied properties