Effects of Oxyanions, Natural Organic Matter, and Bacterial Cell Numbers on the Bioreduction of Lepidocrocite ( γ-FeOOH) and the Formation of Secondary Mineralization Products EDWARD J. O’LOUGHLIN,* ,† CHRISTOPHER A. GORSKI, ‡ MICHELLE M. SCHERER, ‡ MAXIM I. BOYANOV, † AND KENNETH M. KEMNER † Biosciences Division, Argonne National Laboratory, Argonne, Illinois 60439-4843, and Department of Civil and Environmental Engineering, University of Iowa, Iowa City, Iowa 52242-1527 Received January 27, 2010. Revised manuscript received May 5, 2010. Accepted May 7, 2010. Microbial reduction of Fe(III) oxides results in the production of Fe(II) and may lead to the subsequent formation of Fe(II)- bearing secondary mineralization products including magnetite, siderite, vivianite, chukanovite (ferrous hydroxy carbonate (FHC)), and green rust; however, the factors controlling the formation of specific Fe(II) phases are often not well-defined. This study examined effects of (i) a range of inorganic oxyanions (arsenate, borate, molybdate, phosphate, silicate, and tungstate), (ii) natural organic matter (citrate, oxalate, microbial extracellular polymeric substances [EPS], and humic substances), and (iii) the type and number of dissimilatory iron-reducing bacteria on the bioreduction of lepidocrocite and formation of Fe(II)- bearing secondary mineralization products. The bioreduction kinetics clustered into two distinct Fe(II) production profiles. “Fast” Fe(II) production kinetics [19-24 mM Fe(II) d -1 ] were accompanied by formation of magnetite and FHC in the unamended control and in systems amended with borate, oxalate, gellan EPS, or Pony Lake fulvic acid or having “low” cell numbers. Systems amended with arsenate, citrate, molybdate, phosphate, silicate, tungstate, EPS from Shewanella putrefaciens CN32, or humic substances derived from terrestrial plant material or with “high” cell numbers exhibited comparatively slow Fe(II) production kinetics [1.8-4.0 mM Fe(II) d -1 ] and the formation of green rust. The results are consistent with a conceptual model whereby competitive sorption of more strongly bound anions blocks access of bacterial cells and reduced electron-shuttling compounds to sites on the iron oxide surface, thereby limiting the rate of bioreduction. Introduction The biogeochemical cycling of Fe in aquatic and terrestrial environments is complex, involving a suite of highly inter- dependent biotic and abiotic processes. For example, microbial reduction of Fe(III) oxides results in the production of Fe(II) and may lead to formation of Fe(II)-bearing secondary mineralization products including magnetite, siderite, vivianite, chukanovite (ferrous hydroxy carbonate (FHC)), and green rust. Green rustssmixed Fe(II)/Fe(III) layered double hydroxidesshave been reported as products of the bioreduction of Fe(III) oxides in laboratory-based studies (1-13), as well as in Fe(III)/Fe(II) transition zones in natural systems (14-18). The factors controlling formation of specific Fe(II) phases as a consequence of microbial Fe(III) reduction are complex and not fully understood; however, the rate and magnitude of Fe(II) production and its reaction with residual Fe(III) phases and other ligands (e.g., phosphate, carbonate) are often cited as primary factors (2, 19-23). In laboratory experiments with single Fe(III) oxide phases, the formation of green rust as a secondary mineralization product is typically linked to phosphate concentration or the number of dis- similatory iron-reducing bacteria (IRB) present (1, 2, 5, 11, 13). In natural systems, the factors contributing to green rust formation have yet to be identified. Soils and sediments contain a range of organic and inorganic ligands (e.g., inorganic oxyanions such as phosphate and silicate, as well as natural organic matter [NOM] ranging from low-molec- ular-mass aliphatic acids to high-molecular-mass biopoly- mers and humic substances) that are known to affect Fe(II)/ Fe(III) redox transformations and accompanying changes in Fe speciation (24); however, their potential role in the formation of green rusts in these environments is uncertain. For example, a green rust phase was identified by X-ray absorption spectroscopy analysis of Fe-rich lacustrine sedi- ments containing high As levels (18), although what role, if any, As played in formation of green rust in these sediments remains unclear. To better understand the factors contributing to the formation of green rust as a secondary mineralization product of the bioreduction of Fe(III) oxides, we examined the effects of (i) the oxyanions arsenate, borate, molybdate, phosphate, silicate, and tungstate; (ii) NOM including aliphatic acids (citrate and oxalate), humic substances (Elliott soil humic acid [ESHA], leonardite humic acid [LHA], Pony Lake fulvic acid [PLFA], Suwannee River fulvic acid [SRFA], and Su- wannee River humic acid [SRHA]), and microbially produced extracellular polymeric substances (EPS); and (iii) the type and number of bacterial cells on the bioreduction of lepidocrocite (γ-FeOOH) and the accompanying formation of Fe(II)-bearing secondary mineralization products. Experimental Section Details on the sources of chemicals, synthesis and charac- terization of lepidocrocite, and isolation of EPS produced by Shewanella putrefaciens CN32 (hereafter designated CN32 EPS) are in the Supporting Information. The experimental systems consisted of sterile 160 mL serum vials containing 100 mL of sterile defined mineral medium (DMM) (6) with Fe(III) as lepidocrocite (80 mM), formate (75 mM), and anthraquinone-2,6-disulfonate (AQDS) (100 µM); the levels of lepidocrocite and formate were chosen to provide sufficient material for frequent sampling over the course of the experiments and to ensure that electron donor limiting conditions did not develop. DMM was prepared by com- bining all components except formate, AQDS, and oxyanions/ NOM. The pH was adjusted to 7.5, and the medium was autoclaved. After the medium cooled to ambient temperature, * Corresponding author phone: 630-252-9902; fax: 630-252-9793; e-mail: oloughlin@anl.gov. † Argonne National Laboratory. ‡ University of Iowa. Environ. Sci. Technol. 2010, 44, 4570–4576 4570 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 12, 2010 10.1021/es100294w  2010 American Chemical Society Published on Web 05/17/2010