Combined Effects of Curcumin and (-)-Epigallocatechin Gallate on Inhibition of N-Acylhomoserine Lactone-Mediated Biofilm Formation in Wastewater Bacteria from Membrane Bioreactor

This work investigated the potential of curcumin (CCM) and (-)-epigallocatechin gallate (EGCG) to inhibit N-acyl homoserine lactone (AHL)-mediated biofilm formation in gram-negative bacteria from membrane bioreactor (MBR) activated sludge. The minimum inhibitory concentrations (MICs) of CCM alone against all the tested bacteria were 200-350 μg/ml, whereas those for EGCG were 300-600 μg/ml. Biofilm formation at one-half MICs indicated that CCM and EGCG alone respectively inhibited 52-68% and 59-78% of biofilm formation among all the tested bacteria. However, their combination resulted in 95-99% of biofilm reduction. Quorum sensing inhibition (QSI) assay with known biosensor strains demonstrated that CCM inhibited the expression of C4 and C6 homoserine lactones (HSLs)-mediated phenotypes, whereas EGCG inhibited C4, C6, and C10 HSLs-based phenotypes. The Center for Disease Control biofilm reactor containing a multispecies culture of nine bacteria with one-half MIC of CCM (150 μg/ml) and EGCG (275 μg/ml) showed 17 and 14 μg/cm² of extracellular polymeric substances (EPS) on polyvinylidene fluoride membrane surface, whereas their combination (100 μg/ml of each) exhibited much lower EPS content (3 μg/cm²). Confocal laser scanning microscopy observations also illustrated that the combination of compounds tremendously reduced the biofilm thickness. The combined effect of CCM with EGCG clearly reveals for the first time the enhanced inhibition of AHL-mediated biofilm formation in bacteria from activated sludge. Thus, such combined natural QSI approach could be used for the inhibition of membrane biofouling in MBRs treating wastewaters.