Most microorganisms live in environments where nutrients are limited and fluctuate over time. Cells respond to nutrient fluctuations by sensing and adapting their physiological state. Recent studies suggest phenotypic heterogeneity(1) in isogenic populations as an alternative strategy in fluctuating environments, where a subpopulation of cells express a function that allows growth under conditions that might arise in the future(2-9). It is unknown how environmental factors such as nutrient limitation shape phenotypic heterogeneity in metabolism and whether this allows cells to respond to nutrient fluctuations. Here, we show that substrate limitation increases phenotypic heterogeneity in metabolism, and this heterogeneity allows cells to cope with substrate fluctuations. We subjected the N-2-fixing bacterium Klebsiella oxytoca to different levels of substrate limitation and substrate shifts, and obtained time-resolved single-cell measurements of metabolic activities using nanometre-scale secondary ion mass spectrometry (NanoSIMS). We found that the level of NH4+ limitation shapes phenotypic heterogeneity in N-2 fixation. In turn, the N-2 fixation rate of single cells during NH4+ limitation correlates positively with their growth rate after a shift to NH4+ depletion, experimentally demonstrating the benefit of heterogeneity. The results indicate that phenotypic heterogeneity is a general solution to two important ecological challenges-nutrient limitation and fluctuations-that many microorganisms face.