AbstractThe2H/1H ratio in microbial fatty acids can record information about the energy metabolism of microbes and about the isotopic composition of environmental water. However, the mechanisms involved in the fractionation of hydrogen isotopes between water and lipid are not fully resolved. We provide data aimed at understanding this fractionation in the Gram-positive obligately thermophilic anaerobe,Thermoanaerobacterium saccharolyticum, by comparing a wild-type strain to a deletion mutant in which thenfnABgenes encoding electron-bifurcating transhydrogenase have been removed. The wild-type strain showed faster growth rates and larger overall fractionationthan the mutant strain. The overall trend in growth rate and fractionation, along with the isotopic ordering of individual lipids, is consistent with results reported for the Gram-negative sulfate reducer,Desulfovibrio alaskensisG20.