Materials and Methods
3.7 Cellulosic fiber characterization
3.7.4 Effluent characterization
Chemical Oxygen Demand (COD) and Color
COD and color were measured according to ASTM 1252-06 and 1209-05, respectively, on duplicate samples. Absorbance data were recorded at 600 nm for COD and 465 nm for color. A Thermo Scientific Evolution 600 spectrophotometer was used in both cases.
Toxicity
Effluent toxicity was determined with the Microtox method, using the marine luminescent bacterium Vibrio fischeri in accordance with UNE-EN ISO 11348-3: 1999 on a Microtox M500 Analyzer (Strategic Diagnostic Inc., Azur Environmental). The difference between the amount of light emitted before and after addition of the sample was used to determine its toxicity. In order to prevent pH effects, samples were adjusted to pH 6–8 with NaOH/HCl solution.
Ecotoxicity was quantified as EC50, which is defined as the effective concentration of sample reducing the light emission intensity by 50% after 15 min of contact. EC50 is inversely proportional to biological toxicity, expressed in toxicity units (TU). The reference toxicant ZnSO4∙7H2O was used to control Vibrio fischeri batch quality in accordance with the Basic Test procedure.
Toxicity measurements were color-corrected as per the recommendations of the equipment manufacturer. Toxicity tests were conducted in duplicate or triplicate.
Residual laccase activity
Residual laccase activity in the effluents was measured after filtration of each pulp slurry treated in the reactor and according to the procedure described in section 3.2.2. Residual laccase activity values were corrected for the dilution factor and expressed as percentages of the initial laccase rate.
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Released reducing sugars
Dissolved carbohydrates present in the liquors released from the cellulase treatments of biobleached dissolving pulps were quantified by HPLC (Agilent 1200 HPLC instrument), using a Biorad Aminex HPX-87H ion-exchange resin column that affords the separation of glucose, xylose and arabinose. Prior to analysis, the liquors were subjected to acid hydrolysis with 4% H2SO4 and the flasks placed in an autoclave at 103 ± 7 kPa for 20 min in order to convert oligomers into monomers for easier on-column separation. Then, the hydrolysed solutions were filtered through Whatman membranes of 0.45 µm pore size and the chromatographic determination was performed under the following conditions: mobile phase, 6 mmol/L sulphuric acid; flow rate, 0.7 mL/min;
column temperature, 60 ºC.
The concentration of oligosaccharides present in the treatment liquors was also determined by HPLC, using a Biorad Aminex HPX-42A ion-exchange column (Garcia-Ubasart et al., 2013a). Effluents were filtered using a 0.45 µm pore size Whatman membrane and neutralized to pH 7 using HCl or NaOH solution. The determination was performed under the following conditions:
mobile phase, ultrapure water; flow rate, 0.35 mL/min; column temperature, 65 ºC. Identification and quantification of compounds was done by interpolation into calibration curves run from glucose-oligosaccharides (DP 1 to 6) and xylose oligosaccharides (DP 1 to 4).
3-63 The methods used in each chapter are summarized in Schema 3-2.
Schema 3-2 Summary of the different characterization methods used in the thesis.
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