Azo dyes impact the environment and deserve attention due to their widespread use in textile and tanning industries and challenging degradation. The high temperature, pH, and salinity used in these industries render industrial effluent decolorization and detoxification a challenging process. An enrichment technique was employed to screen for cost-effective biodegraders of Direct Red 81 (DR81) as a model for diazo dye recalcitrant to degradation. Our results showed that three mixed bacterial cultures achieved ≥80% decolorization within 8 h of 40 mg/L dye in a minimal salt medium with 0.1% yeast extract (MSM-Y) and real wastewater. Moreover, these mixed cultures showed ≥70% decolorization within 24 h when challenged with dye up to 600 mg/L in real wastewater and tolerated temperatures up to 60 °C, pH 10, and 5% salinity in MSM-Y. Azoreductase was the main contributor to DR81 decolorization based on crude oxidative and reductive enzymatic activity of cell-free supernatants and was stable at a wide range of pH and temperatures. Molecular identification of azoreductase genes suggested multiple AzoR genes per mixed culture with a possible novel azoreductase gene. Metabolite analysis using hyphenated techniques suggested two reductive pathways for DR81 biodegradation involving symmetric and asymmetric azo-bond cleavage. The DR81 metabolites were non-toxic to Artemia salina nauplii and Lepidium sativum seeds. This study provided evidence for DR81 degradation using robust stress-tolerant mixed cultures with potential use in azo dye wastewater treatment.
Keywords: Direct Red 81 (DR81); azo dye; azoreductase; biodegradation; biodegradation genes; biodegradative pathways; detoxification; metabolite identification; mixed bacterial cultures; wastewater.