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Transcriptional and physiological responses of nitrifying bacteria to heavy metal inhibition
Kapoor, V., X. Li, M. Elk, K. Chandran, C. Impellitteri, AND J. Santodomingo. Transcriptional and physiological responses of nitrifying bacteria to heavy metal inhibition. ENVIRONMENTAL SCIENCE & TECHNOLOGY. American Chemical Society, Washington, DC, 49:13454-13462, (2015).
Inhibition of nitrification by heavy metals has more traditionally been studied using analytical procedures, such as those based on ammonia-dependent specific oxygen uptake rate . A wide range of nitrifying bacteria inhibition rates have been reported in the scientific literature in the presence of specific metals, variability that may be explained by a number of factors. For example, the reported values for concentration of Cu that can cause a 50 % decrease in nitrification activity extends from 61 to 173 mg/L. Few studies have attempted to understand this variability by characterizing the physiological, transcriptional and proteomic responses of nitrifying bacteria in response to nitrification inhibition by heavy metals. Most of these studies have used pure cultures (i.e., Nitrosomonas europaea), and therefore limited data is available on how complex nitrifying microbial communities (such as those from activated sludge) respond to the presence of various metals. Moreover, there has been minimal data on the correspondence between results obtained from biochemical and molecular assays for same samples of nitrifying populations in response to heavy metal inhibition. To address this research gap, we examined the physiological and transcriptional responses of nitrifying bacteria upon exposure to several heavy metals, specifically, Ni, Zn, Cd and Pb. Specifically, we compared the transcriptional responses to ammonia-dependent sOUR measurements for the same set of samples to evaluate the efficacy of using functional-gene transcript levels as indicators of nitrification inhibition in cells exposed to heavy metals. The bacterial community composition of the nitrifying bioreactor was also examined to confirm the presence and determine diversity of nitrifying bacteria in the enrichment cultures.
Heavy metals have been shown to inhibit nitrification, a key process in the removal of nitrogen in wastewater treatment plants. In the present study, the effects of nickel, zinc, lead and cadmium on nitrifying enrichment cultures were studied in batch reactors. The transcriptional responses of amoA, hao, nirK and norB were measured in conjunction with the specific oxygen uptake rate (sOUR) for nitrifying bacteria exposed to different concentrations of each metal. For Ni (0.03 – 3 mg/L), Zn (0.1 – 10 mg/L) and Cd (0.03 – 1 mg/L), there was a noticeable decrease in sOUR with increasing metal concentration. However, there were no considerable changes in sOUR during Pb exposure (1 -100 mg/L), except when concentration reached 1000 mg/L. Based on RT-qPCR data, the transcript levels of amoA and hao decreased when exposed to increasing Ni concentrations. Slight up-regulation of amoA, hao, and nirK (i.e., 0.5 to 1.5-fold) occurred after exposure to 0.3, 1 and 3 mg/L Zn, although, their expression decreased by more than half-fold when exposed to 10 mg/L Zn. Stimulation of all four genes occurred when exposed to Cd, with amoA highly up-regulated at 0.3 mg/L Cd and hao at 1 mg/L Cd. During Pb exposure, the transcript levels of all genes increased by more than half-fold for 100 mg/L Pb, but amoA and hao decreased 2-fold and 1-fold respectively, for 1000 mg/L Pb. While overall the results show that RNA-based function specific assays can be used as potential surrogates for measuring nitrification inhibition by heavy metals, transcription levels may not always be a direct indicator of the responses at the enzymatic activity level (as determined by the sOUR data). We suggest that the fluctuations in the transcription of specific functional genes involved in nitrification could serve as an early warning indicator of nitrification inhibition in biological nitrogen removal.