This study evaluated the combined effect of Cd(II), Cu(II), Ni(Il), Fe(III), Pb(II) and Zn(II) on each other removal by anaerobic biomass under sulfate reducing condition. Statistically valid Plackett-Burman design of experiments was employed to carry out this mixture study. The results obtained showed a maximum removal of Cu(II) (98.9%), followed by Ni(II) (97%), Cd(II) (94.8%), Zn(II) (94.6%), Pb(II) (94.4%) and Fe(III) (93.9%). Analysis of variance (ANOVA) of the sulfate and chemical oxygen demand (COD) reduction revealed that the effect due to copper was highly significant (P value < 0.05) on sulfate and COD removal. To establish the role of sulfate reducing bacteria (SRB) in the metal removal process, surface morphology and composition of the metal loaded biomass were analyzed. by transmission electron microscopy (TEM) equipped with energy dispersive spectroscopy (EDS) and by field emission scanning electron microscopy (FESEM) integrated with energy dispersive X-ray spectroscopy (EDX). The results obtained revealed that the metal precipitates are associated with the outer and inner cell surface of the SRB as a result of the sulfide generated by SRB. (C) 2016 Elsevier B.V. All rights reserved.