Enhanced oxidation of Mn(II) and As(III) by aerobic granular sludge via ferrous citrate: Key roles of colloidal iron and extracellular superoxide radical.

Microbial manganese (Mn) oxidation plays a crucial role in shaping the fate of various elements, including arsenic (As). However, this process faces challenges in wastewater environments due to its inherent inefficiency and instability. In our initial research, a serendipitous discovery occurred: the addition of citrate to Fe(II)-containing wastewater stimulated the oxidation of Mn(II) by aerobic granular sludge (AGS). Subsequent experiments in four sequencing batch reactors (SBRs) over a 67-day period confirmed this stimulatory effect. The presence of Fe(II)-citrate led to a remarkable twofold increase in the oxidation of Mn(II) and As(III). The removal efficiency improved from 21±4 % to 87±7 % for Mn(II) and from 77.1 ± 1.8 % to 93.6 ± 0.2 % for As(III). The verification experiments demonstrated that the simultaneous addition of manganese-oxidizing bacteria (MnOB) and Fe(II)-citrate is an effective strategy for enhancing the oxidation and removal of Mn(II) and As(III) by AGS. Through a combination of genomic analysis, cell-free filtrate incubation, and bacterial batch cultivations (including monitoring the time-course changes of 17 substances and 2 free radicals), we elucidated a novel Mn(II) oxidation pathway in Pseudomonas, along with its stimulation method and mechanism. First, bacteria rapidly degrade citrate possibly via the citrate-Mg:H symporter (CitMHS) and the tricarboxylic acid (TCA) cycle, resulting in the formation of colloidal Fe(II), colloidal Fe(III), and biogenic iron (hydr)oxides (FeOx). Then, colloidal Fe(II) and colloidal Fe(III) stimulated extracellular proteins to produce superoxide radicals (·O). These radicals were responsible for oxidizing Mn(II) into Mn(III), ultimately forming biogenic manganese oxides (MnOx). Finally, MnOx effectively oxidized As(III) to the less toxic As(V). This innovative approach for bacterial Mn(II) oxidation holds promise for treating Mn(II) and As(III) in water and wastewater. Furthermore, the mechanism by which colloidal iron stimulates extracellular proteins to produce ·O, thereby facilitating Mn(II) oxidation, may widely occur across various engineering and natural ecosystems.