Values of Δ34S (= δ34SHS - δ34SSO4, where δ34SHS and δ34SSO4 indicate the differences in the isotopic compositions of the HS- and SO42- in the eluent, respectively) for many modern marine sediments are in the range of -55 to -75‰, much greater than the -2 to -46‰ ε34S (kinetic isotope enrichment) values commonly observed for microbial sulfate reduction in laboratory batch culture and chemostat experiments. It has been proposed that at extremely low sulfate reduction rates under hypersulfidic conditions with a nonlimited supply of sulfate, isotopic enrichment in laboratory culture experiments should increase to the levels recorded in nature. We examined the effect of extremely low sulfate reduction rates and electron donor limitation on S isotope fractionation by culturing a thermophilic, sulfate-reducing bacterium, Desulfotomaculum putei, in a biomass-recycling culture vessel, or "retentostat." The cell-specific rate of sulfate reduction and the specific growth rate decreased progressively from the exponential phase to the maintenance phase, yielding average maintenance coefficients of 10-16 to 10-18 mol of SO4 cell-1 h-1 toward the end of the experiments. Overall. S mass and isotopic balance were conserved during the experiment. The differences in the δ34S values of the sulfate and sulfide eluting from the retentostat were significantly larger, attaining a maximum Δ34S of -20.9‰, than the -9.7‰ observed during the batch culture experiment, but differences did not attain the values observed in marine sediments. Copyright © 2009, American Society for Microbiology. All Rights Reserved.