Spectroscopy reveals cooperation in bacterial biofilm resistance: Cross-Fed metabolites enable less-resistant species to dominate Cd immobilization

Interspecies interactions within microbial biofilms play a critical role in shaping community responses to environmental stressors such as heavy metal exposure, yet the underlying mechanisms remain poorly understood. This study investigated the cooperative behavior and cadmium (Cd) resistance mechanisms in dual-species biofilms of Pseudomonas putida and Shewanella oneidensis which contain contrasting resistance strategies against heavy metals. Cadmium exposure reduced bacterial biomass by 67% in mono-species cultures but only by 38% in dual-species cultures throughout cultivation in triangular flask, demonstrating enhanced resistance to Cd in the bacterial consortium. Unexpectedly, P. putida dominated the dual-species biofilms (70% of total biomass) despite its six-fold lower Cd resistance than S. oneidensis , and it contributed more effectively to Cd immobilization than S. oneidensis by secreting extracellular polymeric substances (EPS). The combination of genome-scale metabolic models (GEMs) and X-ray absorption spectroscopy (XAS) further demonstrated that bacterial cells immobilized Cd via Cd-S coordination, with S. oneidensis likely supplying sulfhydryl amino acids to P. putida , which enhanced Cd resistance and sequestration by dual-species biofilms. Concurrently, fluorescence and atomic force microscopy analysis revealed that S. oneidensis cells were encapsulated by EPS within the biofilm, facilitating survival of both species. These findings elucidate interspecies interactions in biofilms, paving the way for engineering microbial communities to enhance heavy metal bioremediation, although verification in natural environments remains essential. • Dual-species bacterial biofilms exhibited higher Cd resistance than mono-species biofilms • Cd localized primarily within extracellular polymeric substances of dual-species biofilms • Pseudomonas putida dominated biofilms and encapsulated Shewanella oneidensis by EPS • Sulfhydryl-binding accounted for 89-98% of Cd immobilization in bacterial cells • Genome-scale metabolic models revealed sulfhydryl amino acid transfer between species