Cd(II) Mediated Inhibition of Nitrosomonas europaea is Linked to Oxidative Stress and is Impacted by Physiological State and Growth Mode

Abstract

In this study, we systematically investigated the impact of physiological state and growth mode on the biokinetics and cadmium mediated inhibition of monocultures of Nitrosomonas europaea strain 19718. During batch growth and in the absence of an inhibitor, specific ammonia oxidation activity, measured as specific oxygen uptake rate, (sOUR), increased during exponential phase and then decreased after transition into stationary phase. On the other hand total cell counts increased during exponential growth phase and stabilized in stationary phase, as expected. Cd(II) mediated inhibition of N. europaea was studied based on three experimental designs; (1) exposure of stationary and exponential phase N. europaea cultures to three discrete concentrations, 0.1, 0.5 and 1 mM Cd(II) for 1, 4 and 7 h, (2) exposure of continuous flow (chemostat) cultures to a 20 h Cd(II) pulse at an influent Cd(II) concentration of 2 mM and peak reactor concentration of 0.6 mM and (3) exposure of batch stationary and exponential cultures to a similar 20 h Cd(II) pulse. Among the different inhibitory responses studied, irrespective of physiological state, growth mode or mode of Cd(II) exposure, sOUR was the most sensitive and rapid indicator of inhibition compared to total cell abundance, membrane integrity, and intracellular 16S rRNA:DNA content, as determined by fluorescent in situ hybridization (FISH) and DAPI staining. Notably, intracellular 16S rRNA:DNA content did not correlate with sOUR under non-inhibited batch growth or with differing severity of inhibition. Based on sOUR measurements, a strong time-dependent exacerbation of inhibition (in terms of a computed inhibition coefficient, KI), was observed in exponentially growing N. europaea cells. Long-term inhibition was also especially severe in actively growing chemostat cultures and was comparable with long-term inhibition of exponentially growing cells in batch culture. In contrast, inhibition was ten-fold lower and invariant with time of exposure in stationary phase cultures. Additionally, N-acetyl-Lcysteine, a scavenger of reactive oxygen species and source of intracellular glutathione, reversed indicators of inhibition in exponential phase N. europaea cultures subject to short-term (1-5 h) exposure of Cd(II) or hydrogen peroxide. Therefore, we also describe for the first time, that Cd(II) mediated inhibition in N. europaea could be attributed to oxidative stress.