A multifaceted experimental design, including factorial design, Face-centered composite design (FCCD), and mixture design, was implemented to explore competitive interaction and adsorption behavior of chromium [Cr(VI)], lead [Pb(II)], and cadmium [Cd(II)] by the immobilized extracellular polymer (EPS) based biosorbent of Pseudomonas aeruginosa OMCS-1, in single and ternary metal solution. The prepared biosorbent preferentially adsorbed Cr (47.6 mg/g), Pb (46.38 mg/g), and Cd (42.02 mg/g) in single metal system, and Pb (43.32 mg/g), Cr (40.03 mg/g) and Cd (35.9 mg/g) in multiple metal system. Adsorption behavior of all metals was successfully interpreted by the Freundlich isotherm model (R2 > 0.988), confirming multilayer sequestration. The Cr, Pb, and Cd biosorption rate followed second-order kinetics (R2 > 0.997), validating chemisorption as predominant mechanism in adsorption. The alternation in the structural morphology of EPS Ca-alginate beads and Cr, Pb, and Cd accumulation, suggesting heavy metal adsorption onto immobilized biosorbent. X-ray diffraction (XRD) pattern of multi-metal loaded biosorbent showed additional crystalline phases, indicating adsorption of metal ions. The significant (p < 0.0001; one-way ANOVA) increase in the zeta potential of Cr, Pb, and Cd loaded EPS Ca-alginate beads revealed the electrostatic interaction between biosorbent and metal ions. The hydroxyl, amine, carboxyl, and phosphate groups of formulated biosorbent contributed for metal sequestration. The adsorption-desorption efficiency retained by the biosorbent after fourth cycle was 35.41 ± 0.2% and 51.44 ± 0.98% for Cr, 51.58 ± 0.15% and 63.98 ± 0.24% for Pb, and 30.68 ± 0.13% and 60.39 ± 0.46% for Cd, respectively. The EPS Ca-alginate beads can potentially eliminate heavy metals from multi-metal contaminated water. © 2024 Elsevier Ltd