Biologically active, passive treatment systems are commonly employed for removing high concentrations of dissolved Mn(II) from coal mine drainage (CMD). Studies of microbial communities contributing to Mn attenuation through the oxidation of Mn(II) to sparingly soluble Mn(III/IV) oxide minerals, however, have been sparse to date. This study reveals a diverse community of Mn(II)-oxidizing fungi and bacteria existing in several CMD treatment systems.Acidic, metal-laden mine drainage is a significant problem for many regions in the United States and throughout the world. In Appalachia, centuries of coal mining has left thousands of abandoned mines that are discharging waters containing elevated levels of metals-particularly Mn, with concentrations as high as 150 mg liter Ϫ1 (see reference 9 and references therein and reference 23). In the eastern United States, one of the most common methods to remediate coal mine drainage (CMD) is the use of biologically active limestone treatment beds. In essence, dissolved metals, such as Mn(II), are immobilized in the treatment bed via precipitation of sparingly soluble oxide minerals (23, 24) that effectively remove other metal contaminants (e.g., Ni, Co, and Zn) through coprecipitation and surface adsorption reactions (26,31,47).The importance of microbial activity in the remediation of Mn-contaminated waters has frequently been observed (6,20,21,24,25). Several strains of Mn(II)-oxidizing bacteria have even been used for treating manganiferous mine waters (49). Recently Mariner et al. (32) identified Mn(II)-oxidizing fungi, in addition to bacteria, successfully growing in a Mn-attenuating bioreactor for treatment of mine waters. We also observed that the addition of fungicides inhibited Mn(II) oxidation in laboratory-based CMD treatment simulations (W. D. Burgos, H. Tan, C. M. Santelli, and C. M. Hansel, presented at the National Meeting of the American Society of Mining and Reclamation, Pittsburgh, PA, 5 to 11 June 2010), suggesting a role for fungal activity in Mn remediation. The identities, growth characteristics, and oxidation mechanisms of the microbial community contributing to CMD remediation, however, remain largely unresolved. The objective of this study was to define the Mn(II)-oxidizing microbial community existing in passive treatment systems designed to remove dissolved Mn(II) from CMD. Because the mechanisms of microbial Mn(II) oxidation are not fully elucidated and are not genetically tractable (13, 18), we initiated an extensive culture survey to identify microorganisms that catalyze Mn(II) oxidation and precipitate Mn(III/IV) oxide minerals. These results provide the foundation for future explorations identifying the key players in CMD remediation and factors impacting their activity.In October 2007, we sampled four Mn attenuation bedsSaxman Run (SRC1) and DeSale phases I, II, and III (DS1 to DS3)-in central Pennsylvania that are currently treating exceptionally high Mn concentrations (up to 119 mg liter Ϫ1 ; 2.2 mM) generated from abandoned coal mines. Ea...