Commercial carriers Kaldnes™ K1 (Anoxkaldnes™, Sweden), made of h

Commercial carriers Kaldnes™ K1 (Anoxkaldnes™, Sweden), made of high density polyethylene (density 950 kg/m3; surface area 500 m2/m3), were used as inert supports. The carriers were autoclaved at 121 °C for 20 min until used. Sunflower (Helianthus annuus) seeds were obtained from a local market Afatinib and the shells were collected after normal human consumption of the seeds. The sunflower seed shells (SS) were autoclaved at 121 °C for 20 min before use. The chemical

composition of the SS according to Gullón et al. [6] is 23 ± 0.15% glucan, 29.4 ± 0.0016% klason lignin, 25.8 ± 0.07% hemicelluloses, 5.40 ± 0.03% extractives and 4 ± 0.15% ash. Cultivation was carried out in cotton-plugged Erlenmeyer flasks (250 mL) containing 3 g of Kaldnes™ K1 carriers or 1.5 g SS, according to the experiment, and 20 mL of culture medium. The culture medium composition was the same as that of the medium M1 described in Rodriguez-Couto [16] and consisted of 10 g/L glucose, 20 g/L yeast extract, 0.9 g/L

(NH4)2SO4, 2 g/L KH2PO4, 0.5 g/L MgSO4·7H2O, 0.1 g/L CaCl2·2H2O, 0.5 g/L KCl and 0.5 g/L thiamine hydrochloride in 0.05 M citrate–phosphate buffer (pH 4.5). To boost laccase production 0.5 mM Cu+2 was added to the cultures on the 3rd cultivation day [18]. Inoculation was carried out directly in the Erlenmeyer flasks. Three agar plugs (diameter, 7 mm), from a 7-day grown fungus on PDA, per Erlenmeyer were used as inoculum. The Erlenmeyer flasks were incubated statically under an air atmosphere at 30 °C and in complete darkness. Glucose consumption, Bak apoptosis measured as reducing sugars, was determined with the dinitrosalicylic acid reagent (DNS) using d-glucose as a standard according to the method described by Miller [11]. Laccase activity

was spectrophotometrically determined as described by Niku-Paavola et al. [12] with 2,2′-azino-di-[3-ethyl-benzo-thiazolin-sulphonate] (ABTS) as a substrate. One activity unit (U) was defined as the amount of enzyme very that oxidised 1 μmol of ABTS per min. The activities were expressed in U/L. Manganese-dependent peroxidase activity was spectrophotometrically assayed at 468 nm by the method of Kuwahara et al. [9]. The reaction was started by adding 0.4 mM H2O2. One activity unit (U) was defined as 1 μmol of 2,6-dimethoxyphenol oxidised per minute and the activities were expressed in U/L. Lignin peroxidase activity was spectrophotometrically determined at 310 nm according to Tien and Kirk [22]. The reaction was starting by adding 0.4 mM H2O2. One activity unit (U) was defined as 1 μmol of veratryl alcohol oxidised in 1 min and the activities were reported as U/L. The dyes used were the textile dyes Bemaplex Navy M-T (CI Acid Blue 193), an acid chromium-complex dye and Bezaktiv Blue BA (CI Reactive Blue 235), a reactive copper-complex dye. They were a kind gift of CTH R. Beilich GmbH (Barcelona, Spain). They were used as received, without further purification.

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