432 JOURNAL OF BIOSCIENCE AND BIOENGINEERING © 2007, The Society for Biotechnology, Japan Vol. 103, No. 5, 432–439. 2007 DOI: 10.1263/jbb.103.432 Production of Biogenic Manganese Oxides by Repeated-Batch Cultures of Laboratory Microcosms Naoyuki Miyata, 1 * Daisuke Sugiyama, 2 Yukinori Tani, 1 Hiroshi Tsuno, 3§ Haruhiko Seyama, 4 Masahiro Sakata, 1 and Keisuke Iwahori 1 Institute for Environmental Sciences, University of Shizuoka, 52-1 Yada, Suruga, Shizuoka 422-8526, Japan, 1 Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, 52-1 Yada, Suruga, Shizuoka 422-8526, Japan, 2 National Institute of Advanced Industrial Science and Technology, 16-1 Onogawa, Tsukuba 305-8569, Japan, 3 and National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba 305-8506, Japan 4 Received 11 December 2006/Accepted 14 February 2007 We investigated the production of manganese (Mn) oxides using repeated-batch bioreactors maintained over long periods under laboratory conditions. Freshwater epilithic biofilms were used as the initial inocula. The bioreactors yielded suspended solids that could remove 0.1 mM dissolved Mn(II) within a few days. Chemical titration, X-ray absorption near-edge structure spectroscopy, and X-ray diffraction analysis revealed that the Mn(II) had been converted to poorly crystallized layer-type Mn(IV) oxides, which were similar to known biogenic Mn oxides from pure bacterial cultures. Spherical or rod-shaped Mn microconcretions occurred in the sus- pended solids; transmission electron microscopy showed that these structures likely resulted from the microbial activity but not represent living cells. Instead, the presence of encapsulated, sheathed, and hyphal budding cells in the suspended solids indicated that a range of Mn-deposit- ing bacteria contributed to the Mn oxide formation. To our knowledge, our data represent the first observation of production of such Mn oxides in a laboratory microcosm wherein a range of Mn-depositing bacteria coexist. The fact that sorption of trace Zn(II) and Ni(II) ions onto the sus- pended solids co-occurred with the removal of dissolved Mn(II) emphasizes the important role of Mn-oxidizing microorganisms in the fates of trace or contaminant metals in the aquatic environ- ment. [Key words: biogenic manganese oxides, X-ray absorption near-edge structure, X-ray diffraction, laboratory microcosm, trace metal sorption] In freshwater environments, the oxic compartments and oxic-anoxic interfaces contain poorly crystallized Mn(III, IV) (hydr)oxides as micro- and macro-concretions and crusts, usually in conjunction with Fe (hydr)oxides. These Mn oxide phases serve as primary adsorbents for trace or contaminant metal cations, such as Co, Ni, Zn, Cu, Mn, Pb, and Cd, in lakes and stream sediments (1–4). However, the degrees of distribution and enrichment of these metal cations upon Mn oxides are different in different locations, due to complex environmental factors (e.g., pH, Eh, the presence of compet- ing ligands, and biological oxidation and sorption activities) that control interactions between metals and metal oxides (5). Mn oxides also oxidize numerous inorganic and organic compounds because of their highly reactive surfaces (5). Reproducing the occurrence of Mn oxide minerals and their interactions with trace elements and compounds under con- trolled laboratory conditions could help to understand the role of Mn oxide minerals in the environment. Mn(II)-oxidizing microorganisms (bacteria and fungi) are ubiquitous in freshwater environments and are thought to participate in the deposition of Mn oxides (6). In bacteria, such as a marine Bacillus sp., Leptothrix discophora, and Pseudomonas putida, the enzymatic oxidation of Mn(II) yields nanocrystallized Mn oxides with Mn valences close to +4 (5). These biogenic oxides resemble the layer-type Mn oxides vernadite (δ-MnO 2 ) and birnessite, which are common in natural environments (7). Cultures of the L. discophora and P. putida have been used as efficient models for study- ing the occurrence and roles of Mn oxide minerals in fresh- water (8–14). These Mn oxides exhibit high capacities for Pb(II) sorption due to their high specific surface area (100– 250 m 2 g –1 ) and the negatively charged layer structures (8, 11). While detailed studies on biological Mn oxidation have been conducted with pure microbial cultures, mixed cul- tures capable of producing analogues of natural Mn oxides have not been examined in detail. If such cultures can be obtained under laboratory conditions, they may also be use- * Corresponding author. e-mail: miyatan@smail.u-shizuoka-ken.ac.jp phone: +81-(0)54-264-5649 fax: +81-(0)54-264-5594 § Present address: Faculty of Education and Human Sciences, Yokohama National University, 79-2 Tokiwadai, Hodogaya, Yokohama 240-8501, Japan.