Transcriptional and physiological response of Nitrosomonas europaea to inhibition by chlorinated aromatics and heavy metals Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/g732dd534

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  • This research investigates the physiological and transcriptional responses of Nitrosomonas europaea when exposed to chlorinated aromatic compounds and heavy metals under varying environmental conditions. It was found that transcriptional responses of identified sentinel genes correlate well with nitrification inhibition. Sorption of metals to biomass was also investigated and found not to correlate well with N. europaea inhibition. Whole genome microarray experiments were performed to define the transcriptional response of N. europaea when exposed to chlorobenzene. 13 out of 2460 N. europaea genes were significantly up-regulated after a 1-hour exposure to 4 μM chlorobenzene. HPLC analysis revealed that chlorobenzene was being oxidized primarily into 4-chlorophenol, and further physiological studies revealed that the presence of 4-chlorophenol could account for the inhibitory responses observed. RT-qPCR analysis of several differentially regulated genes verified that similar transcriptional responses were occurring for both chlorobenzene and 4-chlorophenol. 50% inhibitory concentrations of chlorobenzene and 4-chlorophenol resulted in moderate up-regulation of studied genes, however, increasing the concentration of 4-chlorophenol to achieve nitrification inhibition of 93% or more dramatically increased the fold regulation of several of the identified up- and down- regulated genes of interest. Increasing the 4-chlorophenol exposure time to 3 hours at the higher inhibition levels led to a general decrease in amplitude of transcriptional response for all genes tested. Cultures of N. europaea were exposed to various amounts of cadmium in aqueous solution containing EDTA, a strong metal-chelating organic, to control free ionic cadmium²⁺ (Cd²⁺) concentrations. Inhibition of ammonia oxidation as well as transcriptional up-regulation of merA, an identified sentinel gene for exposure to cadmium was found to correlate well with the concentration of Cd²⁺. The concentration of Cd²⁺ required to significantly affect N. europaea cells was found to be in the nanomolar range, which is several orders of magnitude lower than values reported in the literature for cadmium inhibition to mixed-culture activated sludge systems. The sorption of cadmium to the cells was found to be proportional to both the concentration of total cadmium and the concentration of Cd²⁺. At the concentration of metals required to cause approximately 50% nitrification inhibition, specific oxygen uptake results indicate the inhibition is specific to AMO with HAO and downstream energy-generation processes intact. To investigate more closely the inhibitory interactions between heavy metals and AMO, N. europaea inhibition by cadmium, zinc and silver was studied under substrate-limiting conditions. Unlike incubation in oxic environments, 1 hour incubations of N. europaea with cadmium and silver under anoxic conditions did not cause inhibition of nitrification activity after re-suspension in oxic media. In contrast, zinc, which is normally considered an analogue of cadmium in terms of toxic effect and transport mechanisms, was non-inhibitory to N. europaea when exposed in media lacking ammonia. Transcriptional response of merA closely followed the inhibition patterns, with samples which were inhibited after the removal of the metal having significant up-regulation of the gene, and those samples which were uninhibited showing no significant change in merA transcript levels compared to controls. Although sorbed metal concentrations were not found to be predictive of either extent of inhibition or transcriptional response, significantly more cadmium, zinc and silver were sorbed to biomass when incubated in aerobic media compared to anoxic media. Sorption in oxic media was found to be independent of AMO activity and similar results were obtained using Deinococcus radiodurans, a non-nitrifying gram-positive extremophile. The results indicate that greater heavy metal sorption to biomass in oxic environments may be due to general membrane chemistry effects.
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