Process optimization of malachite green degradation by mixed biofilm positive bacteria: Application of Box–Behnken designs, RSM and whole genome sequences analysis

Abstract

Bacterial biofilm positive strains recovered from engine oil polluted-soil was investigated for its capacity to remove malachite green (MG) in mineral salt solution. Box–Behnken designs combining four factors (glucose, yeast extract, temperature and agitation) with three levels (−1, 0, +1) was used to found the best biotic and abiotic conditions for MG removal. The biodegradation potential of the selected microbial consortium was also confirmed using analytical techniques. Then, whole genome sequencing (WGS) analysis were performed to detect genes related to biofilm formation and chemicals biodegradation. Our results showed that the selected strains (G5, G13 and G15) were able to form biofilm on congo red agar plates developing almost black, very black and almost black colonies respectively. Experimental results using Box-Behnken designs showed that decolorization rate of MG reach 89.35% in these optimized conditions: glucose (2%), yeast extract (1%), temperature (40 °C), and agitation (150 rpm). Molecular investigation revealed that the isolated strains were identified as Cronobacter sakazakii G5, Enterobacter hormaechei G13, and Klebsiella pneumoniae G15 based on the whole genome sequences . Whole genome sequences analysis demonstrated the presence of genes involved in biofilm formation as well as dyes and chemical detoxification. The findings presented here provide useful information for development an efficient microbial consortium, formed by adherent bacteria, able to remove dye from coloured wastewater.