MATERIALS AND METHODS

Fungi

Trametes villosa (Fr.) Kreisel CCB176, with known ligninolytic activity (16), was obtained from the Basidiomycetes Culture Collection (CCB) of the Institute of Botany, Sao Paulo. Pycnoporus
Sanguineus (L.: Fr.) Murr. UFMGCB03 was isolated from the biological reserve of the Museum of National History, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais (20). The cultures were maintained on 2% (w/v) malt extract agar (MEA) at 4ºC.

Dyes

The reactive dyes (Table 1) were provided by Cia. Manufatora de Tecidos de Algodão (Cataguases,
MG) and are routinely used in the dyeing process of cotton fibers.

Dye decolorization on solid medium

A disc (2 mm) of fungal mycelium in MEA was inoculated into the center of Petri dishes (90 mm) containing MEA and 0.002 g L-1 of the dye, in triplicate. The plates were incubated at 28ºC in the dark until they were completely colonized with the fungus or for a maximum period of 21 days. The diameters (cm) of the decolorization and growth halos were determined in two perpendicular directions of the plate (13). Plates containing the dye but not inoculated served as control.

Decolorization of Drimaren Brilliant Blue dye in liquid medium

Three discs (2 mm) of T. villosa on MEA were transferred to 250 mL Erlenmeyer flasks containing 50 mL 2% malt extract broth (MEC) supplemented with 0.002 to 0.01 g L-1 of Drimaren Brilliant Blue, in duplicate. The flasks were incubated in the dark at 28ºC for 21 days.
Non-inoculated culture medium was used as control. The effect of shaking (130 rpm) on decolorization was evaluated at a dye concentration of 0.002 g L-1.


Influence of nitrogen on the decolorization of Drimaren Brilliant Blue


Three discs (Æ 2 mm) containing the fungus grown on 2% MEA at 28ºC were transferred to 250 mL flasks containing 50 mL modified basal medium (10) supplemented with 0.002 g L-1 Drimaren Brilliant Blue. The composition of the medium (per liter) was: 10 g glucose, 0.001 g thiamine-HCl, 1 g KH2PO4, 0.5 g MgSO4, 0.01 g CaCl2, 0.01 g FeSO4 7H2O, 0.001 g MnSO4 4H2O, 10 mL sodium acetate buffer, pH 4.5, and ammonium titrate at the concentration needed to obtain 1.2, 12 and 22.4 mm nitrogen. The flasks were incubated at 28ºC and 130 rpm. The experiments were carried out in duplicate. Non-inoculated culture medium was used as control.

Decolorization of synthetic effluent

The synthetic effluent consisted of a mixture of 10 dyes (0.001g L-1): Remazol Brilliant Orange 1, Levafix Gold Yellow 10, Procion Yellow 14, Drimaren Brilliant Blue 17, Remazol Brilliant Blue 18, Cibacron Black 55, Procion Black 59, Drimaren Turquoise Blue 62, Drimaren Brilliant Red 67, and Remazol Red 75. Two discs of fungal mycelium in MEA were inoculated into flasks containing 100 mL MEC supplemented with the synthetic effluent at a final concentration of 0.01 g L-1. For mixed cultures, one growth disc containing mycelium of each fungus was used. The flasks were incubated at 28oC and 130 rpm. The fungi were also grown in culture medium without the synthetic effluent.

Degradation of Drimaren Brilliant Blue

The content of the flasks was removed at determined time intervals and filtered (0.45μ) and the filtrate diluted 1/10 was used to determine dye degradation (4). Decolorization was analyzed by determining the absorbance at 590 nm and is expressed as relative percentage taking the non-inoculated control as 100%. The absorbance spectrum of the dye was determined in the visible range (400 to 700 nm) with a Hitachi U-3000 spectrophotometer