Heavy water and (15) N labelling with NanoSIMS analysis reveals growth rate-dependent metabolic heterogeneity in chemostats.

To measure single-cell microbial activity and substrate utilization patterns in environmental systems, we employ a new technique using stable isotope labelling of microbial populations with heavy water (a passive tracer) and (15) N ammonium in combination with multi-isotope imaging mass spectrometry. We demonstrate simultaneous NanoSIMS analysis of hydrogen, carbon and nitrogen at high spatial and mass resolution, and report calibration data linking single-cell isotopic compositions to the corresponding bulk isotopic equivalents for Pseudomonas aeruginosa and Staphylococcus aureus. Our results show that heavy water is capable of quantifying in situ single-cell microbial activities ranging from generational time scales of minutes to years, with only light isotopic incorporation (∼0.1 atom % (2) H). Applying this approach to study the rates of fatty acid biosynthesis by single cells of S. aureus growing at different rates in chemostat culture (∼6 h, 1 day and 2 week generation times), we observe the greatest anabolic activity diversity in the slowest growing populations. By using heavy water to constrain cellular growth activity, we can further infer the relative contributions of ammonium versus amino acid assimilation to the cellular nitrogen pool. The approach described here can be applied to disentangle individual cell activities even in nutritionally complex environments.