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• The combination of natural microbe species enhanced both direct and indirect electron transport mechanisms. • The alliance system expands the utilization spectra of the carbon source. • Riboflavin and Phenazine two electron shuttles work in coordination. Microbial fuel cells with mixed bacteria always show better power output capacity than those with single bacteria due to synergetic effects between species. Here, a rational-designed synthetic three-species alliance system based on Shewanella oneidensis MR-1 (the model exoelectrogen), Pseudomonas aeruginosa, and Lactobacillus plantarum was constructed upon process optimization, the three-species alliance system in MFC delivered a maximum power density of ∼ 207 mW/m2 by using the glucose as the substrate, which was 2 ∼ 58 times higher than that of MFC inoculated with single species. In addition, the synthetic alliance system could generate electricity from broad substrate spectrum (lactose, saccharose, maltose, arabinose, cellobiose) with enhanced performance. Electrochemical and metabolism analysis indicated inter-species synergetic enhancement on charge transfer, substrate utilization, and biofilm formation was the underlying mechanism for performance improvement in MFC by this synthetic alliance system. In this system, L. plantarum uses glucose to produce lactic acid as the electrogenic substrate and electron donor of S. oneidensis MR-1, and P. aeruginosa uses glucose to produce another electron shuttle phenazine, which can also promote the growth of S. oneidensis MR-1 biofilm and help the alliance system to transfer electrons better. This work demonstrated a new strategy to enhance the MFC performance with a rational designed synthetic bacterial community, which would further renew the toolbox for MFC optimization. [ABSTRACT FROM AUTHOR]