Functional complementarity in a heavy oil-degrading consortium through cross-feeding, detoxification, pH stability, and transcriptional relief.

Heavy oil bioremediation is challenged by hydrocarbon recalcitrance and toxicity, requiring synergistic microbial consortia. This study formulated a tripartite biodegradation microbial consortium (BMC) comprising Micrococcus luteus BD1, Acinetobacter baumannii subsp. oleum ficedula BD54, and Ochrobactrum oleinvorans BD61, guided by genomic screening and Flux Balance Analysis (FBA). In microcosms, the BMC achieved 77% degradation of heavy oil (16° API) in 15 days, significantly outperforming monocultures. Initially, all strains attack aliphatic fractions, with BD1 operating via monooxygenases and adh, while BD54 and BD61 secrete biosurfactants (80% emulsification). In late stages, as BD54 and BD61 shift to complex aromatic cleavage (benD and pcaH), BD1 transitions into a detoxifying "metabolic sink" supporting pH stability. FBA and Flux Variability Analysis (FVA) provided a testable hypothesis of proton-associated metabolism. In vitro assays validated that the consortium maintains neutral pH, mitigates toxic phenol accumulation, and displays bidirectional exudate cross-feeding. This detoxification prevents enzyme inhibition and triggers systemic transcriptional relief, as evidenced by RT-qPCR and total oil and grease (TOG) analysis. Social coordination is supported by quorum sensing networks (LuxI/LuxR and RiPP-like). Integrating in silico modeling with in vitro validation reveals functional complementarity through cross-feeding, detoxification, pH buffering, and bioavailability enhancement, sustaining continuous heavy oil mineralization.