Shewanella putrefaciens strain CN-32 and Shewanella algae strain BrY were grown in laboratory cultures at 30 degreesC to characterize carbon and hydrogen isotope fractionation patterns related to the growth of iron-reducing bacteria. Ferric citrate or hydrous ferric oxide (14170) was provided as the electron acceptor and lactate or H-2 (balanced with CO2) was used as the electron donor. Because these bacteria are not known to grow chemoautotrophically, yeast extract was provided as a carbon source when cultures were grown on H-2/CO2. Siderite formed only when HFO was used as the electron acceptor, possibly because of chelation of ferrous iron with dissolved citrate when ferric citrate was used as the electron acceptor. Carbon isotope enrichment factors for the siderite-CO2 System (epsilon(sid-CO2)) ranged from 13.3 parts per thousand to 14.5 parts per thousand when lactate was used as the carbon and energy source, which were consistent with theoretical calculations of equilibrium isotope fractionation (alpha(sid-CO2)) for the siderite-CO2 system [Geochim. Int. 18 (1981) 85]. In experiments using H2/CO2 as the energy source and yeast extract as the carbon source, carbon isotope enrichment factors were relatively low (0.5 parts per thousand to 7.4 parts per thousand). The potential exists that a kinetic effect related to siderite precipitation rate influenced isotope partitioning or a dynamic balance was established between carbon sinks (i.e. biomass and solid carbonate) of diverging carbon isotope composition. A more quantitative estimate of epsilon(sid-CO2) for biological systems that contain ambient dissolved inorganic carbon (DIC) requires a deeper understanding of carbon flow dynamics in these compartmentalized closed systems. Finally, in experiments using H-2/CO2 as an energy source, the hydrogen isotope composition of head gas H2 and water were analyzed for D/H ratio. The results indicate that bacterial metabolism potentially facilitates isotope exchange between water and H-2. (C) 2002 Elsevier Science B.V. All rights reserved.