Functional characterization of bacterial isolates from dye decolorizing consortia and a step-up metabolic engineering based on NADH-regeneration

Azo dye decolorizing acclimatized decolorizing consortia are enriched microbial sources of potential azoreductase-efficient bioremediation strains. Here, we characterized eight selected consortial members for their azo decolorization and azoreductase profiling. These efficient dye decolorizing bacterial isolates were affiliated to two major phyla viz. Firmicute (genus-Enterococcus) and Proteobacteria ({gamma}-group). Redox-mediators such as AQDS and AQS were found to significantly increase decolorization except for menadione, and IR functional group signatures highlighted the azo bond reduction and degraded metabolites profiles of each strain. Among isolates, Enterococcus sp. L2 was found to be the most effective strain as it could reduce >90mg/L Reactive violet 5R (RV5R) dye in 3h of incubation. Furthermore, strain L2 possesses profound high NADH and NADPH-dependent azoreductase activity which also corroborated with its superior azo decolorization. As per physicochemical parameters, strain L2 showed an optimum decolorization at pH 8, 40 {degrees}C and up to 2% w/v salinity. To channelize reducing equivalence (NADH) to further enhance the dye decolorization in NADH-azoreductase efficient Enterococcus sp. L2, we augmented an NADH co-factor regeneration system. Using pMGS100, a Gram-positive expression vector a constitutive heterologous expression of Mycobacterium vaccae encoded NAD+-dependent formate dehydrogenase enhanced NADH pool which led to a significant 3.2 fold increased dye decolorization in Enterococcus sp. L2 harboring pMGS100 fdh along with a positive effect on growth. Ultimately, an augmentation of formate utilization step could further accelerate azo dye decolorization by fulfilling the co-factor (NADH) requirement of azoreductase along with a growth advantage in the non-model azoreductase-efficient environmentally important strain L2.