Reactive black 5

This method was established by using Phanerochaete velutina (FBCC 941, previous number 244i) and can be applied to other white rot fungi. We present here the results of four persistent textile dyes. However, the method can be applied to on any dye and any white rot fungus. The investigated dyes were Reactive Orange 16 (Sigma-Aldrich, Germany, CAS, no. 12225-83-1, referred as “RO16”), Reactive Black 5 (Sigma-Aldrich, Germany, CAS, no. 17095-24-8, referred as “RB5”), Reactive Blue 4 (Sigma-Aldrich, Germany, CAS, no. 13324-20-4, referred as “RB4”), and Reactive Green 19 (Sigma-Aldrich, Germany, CAS, no. 61931-49-5, referred as “RG19”).
We present and discuss the method step by step providing methodological steps that are necessary to obtain kinetic data on the overall decolorization, and the decolorization caused by non-enzymatic degradation, as well as the decolorization caused by enzymatic degradation.

2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and 1-aminobeznotriazole (ABT) were obtained from Sigma-Aldrich (Germany). Stock solutions of ABT were prepared in ethanol at a concentration of 50 mM.
o-tolidine was purchased from Chempur (Poland). A stock solution of o-tolidine (10 mg mL^-1) was prepared in ethanol. Liquid chromatography solvents were obtained from Avantor Performance Materials (Poland).
The azo dyes Reactive Orange 16 (CAS 12225–83-1), Acid Red 27 (CAS 915–67-3), Acid Orange 7 (CAS 633–96-5), Reactive Red 120 (CAS 61951–82-4), and Reactive Black 5 (CAS 17095–24-8) were purchased from Sigma-Aldrich (Germany). Stock solutions were prepared in water at a concentration of 50 mg mL^-1.
Cd(NO₃)₂ × 4 H₂O, K₂Cr₂O₇, and ZnSO₄ × 7 H₂O were purchased from Sigma-Aldrich (Germany). Metal stock solutions were prepared by dissolving in deionized water at a concentration of 1 M.
Samples of landfill leachate (L1 and L2) collected from the landfill of the former “Boruta” Dye Industry Plant in Zgierz, Poland (Fig. 1) were kindly supported by the Voivodeship Inspectorate of Environmental Protection in Łódź, Poland.

Saccharomonospora viridis DSM 43017 was obtained from the China General Microbiological Culture Collection Center, Beijing (reference number CGMCC 4.1324). The strain was cultivated in a shaker flask at 45°C in STS medium (1.0% (w/v) soy peptone, 1.0% (w/v) glucose, 0.2% (w/v) yeast extract, 0.2% (w/v) NaCl, and 0.2% casein enzymatic hydrolysate, all from Biodee, Beijing, China), adjusted pH to 8.0 with NaOH prior to autoclaving.
Escherichia coli DH5α and E. coli BL21 (Tiangen, Beijing, China) (were cultivated in Luria Bertani (LB) medium at 37°C for gene cloning, sequencing, and expression. The pEASY-T3 vector (TransGen, Beijing, China) was used for plasmid gene cloning and sequencing. The plasmid pET-28a (+) (Takara Bio, Otsu, Japan) was used as an expression vector. Manganese peroxidase (MnP), azure B, brilliant green, reactive blue 19, reactive green 19, reactive yellow 2, reactive black 5, reactive red 120, malachite green and crystal violet were purchased from Sigma (St. Louis, MO, USA). All other reagents used were of analytical grade unless otherwise stated.

Polyethylene glycol with average molecular weight 200 (PEG) (Sigma-Aldrich, Burlington, MA, USA, P3015), copper(II) nitrate (Merck, Darmstadt, Germany, 0140630), L-cysteine (Sigma-Aldrich, W326305), ammonium citrate dibasic (Fluka Analytical, 09831), ascorbic acid (Merck, F971227), zinc nitrate (Merck, CC396733), titanium(IV) isopropoxide (Aldrich Chemistry, 87560), isopropyl alcohol (Merck, 1.09634) and sodium chloride (Fluka, S9888), reagents of analytical grade, were acquired from Sigma-Aldrich. The dye references were: Methyl Orange (Fluka, 68250), Orange II sodium salt (Aldrich, 195235), Reactive Black 5 (Sigma-Aldrich, 306452) and Remazol Brilliant Blue R (Sigma, R8001), acquired from Sigma-Aldrich. The chemical structures of the dyes are shown in Figure 1.
A typical real textile effluent was obtained from a textile dying and printing industry located in Barcelos (Portugal), with the following features: COD, 339 ± 9 mg O₂/L; turbidimetry, 57.7 ± 0.3 NTU; pH, 8.66 ± 0.01; conductivity, 4.61 ± 0.01 mS/cm; chloride anion, 100 mg/L; carbonates, >356 mg/L. Moreover, optical microscopy analysis of the raw textile effluent showed the presence of a considerable number of textile fibers, microplastics and microorganisms (raw effluent is a mixture of industrial effluent and sanitary sewage).

All analytical grade chemicals were used in the present study. The substrates: 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and Reactive Black 5 (RB5) were procured from Sigma-Aldrich (USA). Azure B and manganese chloride (MnCl₂) were purchased from Hi-Media.