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Bacterial Mn(II) oxidation is believed to play a dominant role in accelerating the rate of Mn biomineralization in nature. Commonly, bacteria employ two ways involving Mn(II) oxidases and reactive oxygen species to oxidize Mn(II). In this study, a new strategy for bacterial Mn(II) oxidation involving the pyocyanin, a greenish blue phenazine pigment from Pseudomonas aeruginosa, was discovered. To begin with, a bacterial strain L3 was isolated from soils and identified as Pseudomonas aeruginosa, which exhibited the ability of Mn(II) oxidation. Next, the pyocyanin was purified from strain L3 cultures and proven to be involved in Mn(II) oxidation. Particularly, the oxidation of Mn(II) by pyocyanin was dependent on its ambient pH. In comparison with pH of 5 and 7, pyocyanin (the initial value of OD387 was 0.56 at pH of 2) showed a stronger capability of oxidizing Mn(II) at pH of 9, reaching 144.03 μg L−1 of Mn oxides after 108 h of Mn(II) oxidation, while pyocyanin ultimately produced 43.81 μg L−1 at pH of 7 and 3.32 μg L−1 at pH of 5, respectively. Further, strain L3 cultures were fractionated into three parts, i.e., the cell culture solution, fermentation supernatant, and cell suspension, and the Mn(II)-oxidizing activity was found to be distributed in the cell culture solution and fermentation supernatant, as evidenced by the formation of blackish glossy Mn oxides. Specifically, in the first half, the rate of Mn(II) oxidation by the fermentation supernatant was higher than that by the cell culture solution, whereas in the second half, the cell culture solution showed the much higher Mn(II)-oxidizing activity compared to the fermentation supernatant. Last but not least, the collective results from mineral characterization demonstrated that, the Mn oxides produced by P. aeruginosa strain L3, either by the cell culture solution or by the fermentation supernatant, were bixbyite-type Mn2O3 with poor crystallinity. [ABSTRACT FROM AUTHOR]