Biodegradation of azo dyes by bacterial cells and azoreductase

Abstract

Azo dyes are widely used by different industries including the textile industry. In Pakistan, dye-containing industrial wastewater is commonly used to irrigate crops, which leads to the contamination of agricultural soils. These azo dyes may influence soil microbes adversely. Hence, a study was conducted to assess the persistence of azo dyes in soil and their impact on soil microbial community structural changes. Furthermore, biodegradation of these azo dyes by bacterial cells and enzyme azoreductase was examined in the liquid medium. Three azo dyes such as Direct Red 81, Reactive Black 5 and Acid Yellow 19 were added into 10 g soil at concentration of 160 mg kg-1 soil. Azo dyes were found to be quite stable and degraded slowly in the soil. A substantial amount of Direct Red 81 (63.5%), Reactive Black 5 (17.3%) and Acid Yellow 19 (24.6%) was recovered from soil upon treatment with a mixture of four solvents (water, methanol, acetone, chloroform, 1:1:1:1 v/v) after 14 days of spiking. Phospholipid fatty acid (PLFA) analysis showed significant changes in the soil microbial community structure after treatment of the soil with azo dyes compared to untreated soil. To prevent contamination of soil, dye-contaminated wastewater discharged by dyeing units requires treatment prior to its release into water streams and soil. For this purpose, thirty bacterial strains capable of degrading azo dyes were isolated from wastewater of textile industry. Isolate IFN4 was identified by 16S rRNA gene sequencing. It belonged to genus Shewanella and was named as Shewanella sp. strain IFN4. This bacterium was highly efficient in decolorizing four, structurally different azo dyes (200 mg L-1) individually as well as in mixture, and 72-99% decolorization was achieved just in 4 h under static incubation. Optimum pH and temperature for the decolorization of dye mixtures were 8.5 and 35 °C, respectively. Decolorization of the dyes was dependent on the presence of co-substrate in medium, and yeast extract was used preferably by the strain IFN4 as a co-substrate for the decolorization of dye mixtures compared to other co-substrates. Maximum decolorization occurred when the dye solution was supplemented with 6 g L-1 yeast extract. Moreover, azoreductase activity of strain IFN4 was significantly higher in the dye solution containing 2 g L-1 yeast extract (4.19 U/mg proteins) than that observed without yeast extract (1.32 U/mg proteins). Michaelis-Menten kinetics was employed to calculate Km and Vmax values for crude proteins of strain IFN4, and were 0.062 g L-1 yeast extract and 4.44 U/mg proteins, respectively. Among the components (riboflavin, pyridoxine and thiamine) of yeast extract, only riboflavin enhanced the decolorization of azo dyes by bacterial cells and azoreductase. Textile wastewater contains toxic heavy metals and salts, thus decolorizing activity of strain IFN4 and its azoreductase was evaluated in the presence of various metals and salts. The decolorization efficiency of Shewanella sp. strain IFN4 was not affected by the addition of Ni2+, Cr2+, Pb2+, Fe3+ and Mn2+ in liquid medium containing 200 mg L-1 Reactive Black 5. However, addition of Cu2+, Zn2+, Co2+ and Cd2+ substantially reduced the decolorization rate. Cd2+ was highly toxic as no decolorization was observed at concentration of 10 mg L-1. In contrast, strain IFN4 was able to decolorize Reactive Black 5 dye efficiently in the presence of metal ion mixture with concentration up to 15 mg L-1 dye solution. The results also revealed that Zn2+, Co2+, Cd2+ and Cu2+ inhibited bacterial growth while Fe3+ and Mn2+ enhanced it. The metal ions did not cause a significant inhibition in the azoreductase activity except Cu2+. Furthermore, strain IFN4 was able to decolorize Reactive Black 5 at salt concentration of 50 g NaCl L-1 and 60 g Na2SO4 L-1. However, this strain was unable to decolorize Reactive Black 5 in the presence of even 2 g NaNO3 L-1 medium. Azoreductase activity of strain IFN4 was not significantly decreased at salt concentration of 30 and 60 g L-1 Na2SO4 and NaCl, respectively. However, a significant inhibition in the enzyme activity was observed above these concentrations. Moreover, azoreductase of strain IFN4 showed broad substrate specificity and maximum decolorization of azo dyes was observed at pH 8.0 and 45 °C. Azoreductase activity was dependent on coenzymes (NADH or NADPH), flavin and quinone compounds as enzyme activity increased by their presence in the assay. The azoreductase of Shewanella sp. strain IFN4 had a molecular mass of 33±0.5 kDa and was identified as Na (+)-translocating NADH-quinone reductase subunit F. This study suggested that Shewanella sp. strain IFN4 and its azoreductase are the potential tools to treat textile wastewater.