A wide diversity of inhibitory responses to heavy metal species has been reported in nitrifying bacteria. Part of the variability could be attributed to the different conditions under which heavy-metal inhibition tests have been performed. In this study, we specifically and systematically examined the contribution of physiological state (exponential and stationary growth phases) and growth mode (batch and continuous) to several inhibitory responses in pure monocultures of Nitrosomonas europaea. The inhibitory responses measured were total cell abundance, live cell fraction, specific oxygen uptake rate (sOUR) and intra-cellular 16S ribosomal RNA (rRNA) content. Based on our experiments, the specific rate of ammonia oxidation was strongly impacted by physiological state. During uninhibited batch growth, specific ammonia oxidation rates during exponential growth phase were much higher than those during stationary phase. Further, sOUR values were rapidly and strongly inhibited (~ 85% inhibition in exponential phase) at even the lowest Cd(II) concentrations and exposure times tested (0.1 mM Cd(II) and 1h, respectively). The relative inhibition was more marked in actively growing cultures because of their higher un-inhibited sOUR values. In contrast, at all physiological states and growth modes, Cd(II) concentrations or exposure times, the total cell count and live cell fraction were nearly constant, whether inhibited or not (data not shown), indicating that cell lysis and membrane disruption did not contribute to observed respirometric inhibition. Notably, relative FISH signal intensity, a measure of intra-cellular 16S rRNA content was a poor indicator of activity in inhibited N. europaea. Finally, inhibition of N. europaea by Cd(II) involved oxidative stress, and not mechanisms such as membrane disruption.

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