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

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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-L- cysteine, 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.

INTRODUCTION
Nitrification is often the rate determining step in biological nitrogen removal. In addition to the inherent slow specific growth rates and biomass yield coefficients, nitrifying microorganisms are typically more sensitive to environmental perturbations compared to heterotrophic microorganisms. The impact of heavy metals on pure and mixed nitrifying cultures has been extensively studied under different biomass concentrations, physiological states and growth modes. In addition, the inhibitory response of nitrifying bacteria to heavy metals has been quantified by several methods, including live/dead cell counts, total cell concentration, specific ammonia and oxygen uptake rates, among others (Bedard and Knowles 1989; Beg et al. 1982; Benmossa et al. 1986; Braam and Klapwijk 1981; Gernaey et al. 1997; Hooper and Terry 1973; Hyman et al. 1990; Hynes and Knowles 1983; Neufeld et al. 1980; Tomlinson 1966). Correspondingly, a wide diversity of inhibitory responses to heavy metal species has been reported in nitrifying cultures. However, the impact of such experimental factors on measured inhibitory responses has never been specifically and systematically investigated. Further, the specific mechanisms of heavy metal mediated inhibition in nitrifying bacteria have not yet been explored. In this study, we specifically examined the contribution of physiological state (exponential and stationary growth phases), growth mode (batch and continuous) and Cd(II) exposure time to the inhibitory response and recovery on monocultures of Nitrosomonas europaea. In addition, we compared different measures of inhibition determined via live/dead cell counts, total cell concentrations, specific oxygen uptake rates, and fluorescent in-situ hybridization (FISH). The relative expression of the ammonia monooxygenase gene during inhibition is under evaluation.

Based on recent reports on qualitative loss of amoA gene expression in N. europaea and Nitrosopira briensis under prolonged starvation (Bollmann et al. 2005; Stein and Arp 1998), we hypothesized that the physiological state of N. europaea during batch growth significantly influences its specific ammonia oxidation rate. Our second hypothesis was that differences in specific ammonia oxidation rates in N. europaea under different physiological states influence its susceptibility to inhibition by Cd(II). In turn, we expected that different modes of growth (batch, continuous) in N. europaea would give rise to different corresponding rates of ammonia oxidation and consequently different measured inhibitory responses to Cd(II).

Correspondingly, the objectives of this study were to:

  • Determine the inhibitory impact of Cd(II) on N. europaea measured via total cell counts, live cell fraction, specific oxygen uptake rate (sOUR) and FISH.
  • Compare the inhibitory responses of batch (exponential and stationary growth phases) and chemostat N. europaea cultures to discrete and continuous pulse Cd(II) exposure.
  • Evaluate possible mechanisms of Cd(II)-mediated inhibition in N. europaea.

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