Identification of iron-reducing microorganisms in anoxic rice paddy soil by 13C-acetate probing

Abstract

In anoxic rice field soil, ferric iron reduction is one of the most important terminal electron accepting processes, yet little is known about the identity of iron-reducing microorganisms. Here, we identified acetate-metabolizing bacteria by RNA-based stable isotope probing in the presence of iron(III) oxides as electron acceptors. After reduction of endogenous iron(III) for 21 days, isotope probing with 13C-labeled acetate (2 mm) and added ferric iron oxides (ferrihydrite or goethite) was performed in rice field soil slurries for 48 and 72 h. Ferrihydrite reduction coincided with a strong suppression of methanogenesis (77%). Extracted RNA from each treatment was density resolved by isopycnic centrifugation, and analyzed by terminal restriction fragment length polymorphism, followed by cloning and sequencing of 16S rRNA of bacterial and archaeal populations. In heavy, isotopically labeled RNAs of the ferrihydrite treatment, predominant 13C-assimilating populations were identified as Geobacter spp. (∼85% of all clones). In the goethite treatment, iron(II) formation was not detectable. However, Geobacter spp. (∼30%), the δ-proteobacterial Anaeromyxobacter spp. (∼30%), and novel β-Proteobacteria were predominant in heavy rRNA fractions indicating that 13C-acetate had been assimilated in the presence of goethite, whereas none were detected in the control heavy RNA. For the first time, active acetate-oxidizing iron(III)-reducing bacteria, including novel hitherto unrecognized populations, were identified as a functional guild in anoxic paddy soil.