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The objectives of this research were to: i) develop a mechanistic understanding of the synergy between microbial co-inoculation, nitrogen (N) fertilizer, and maize plants on biological 15 N fixation, and 15 N-recovery from applied fertilizers; and ii) explore the mechanist effects of microbial co-inoculation on N fractionations and derivation (fertilizer, atmosphere and soil), physiological responses on water use and carboxylation efficiencies and growth by using two different isotopic techniques under field conditions. Treatments included four seed inoculations (Control, B. subtilis, A. brasilense, and the combination of B. subtilis and A. brasilense), along with five levels of N application (0-240 kg N ha -1 ). Overall, the results indicate that maize co-inoculation with the above-mentioned bacteria enhanced photosynthetic efficiency leading to improved carboxylation efficiency and instantaneous water use efficiency in maize plants, likely due to an increase in net photosynthetic rate. This effect was more evident under low N availability. The findings also suggest that co-inoculation enhanced the ability of maize plants to absorb CO 2 , adjust to different soil N levels, and carry out photosynthesis, which resulted in higher carbon fixation and better maize growth. The N obtained from the atmosphere resulting from inoculation ranged from 25 to 50 kg N ha -1 . Nonetheless, N application rates exceeding 186 kg N ha -1 substantially diminished the ability of these bacteria to fix N 2 . The combination of inoculation with the application of 120-180 kg N ha -1 led to a synergistic effect resulting in the greatest N-use efficiency, -recovery and grain yield., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)