Graduate Thesis Or Dissertation

 

Inhibition of the ammonia oxidizing bacteria Nitrosomonas europaea by the emerging contaminant triclosan Public Deposited

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  • Identifying the inhibition of ammonia oxidizing bacteria (AOB) by emerging organic contaminants is crucial due to the importance of AOB in wastewater treatment, the widespread use of antibacterial agents such as triclosan (TCS) in consumer products, and the sensitivity of N. europaea to inhibitors. Triclosan inhibition of nitrification by AOB N. europaea cells was determined via suspended cell batch reactor experiments using cells grown in batch and in a chemostat. Specific oxygen uptake rate tests (SOURs) were performed on both the ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO) enzymes to determine the enzyme inhibition mechanism of TCS. Inhibition of long term growth of N. europaea cells and development of antibacterial resistance when exposed to low concentrations of TCS were also studied via suspended cell batch growth experiments. A colorimetric nitrite assay was used to quantify nitrite production and UHPLC-MS analysis was used to analyze for the cometabolic transformation of TCS. Three hour inhibition tests showed that N. europaea cells are inhibited by TCS at concentrations ranging from 0.1 to 8 ppm TCS, but activity partially recovered when washed and re-suspended in fresh media. Triclosan inhibition results show a non-linear increase in nitrification inhibition with increasing TCS concentration with 40% inhibition occurring at 1 ppm TCS and 90% inhibition at 8 ppm TCS. The recovery of previously inhibited cells, relative to control cells, fit a semi-log plot, with cell inactivation being dependent on TCS concentration and the time of exposure, similar to models used for disinfection. Results of the exposure of chemostat-grown N. europaea cells to 1 ppm TCS showed less inhibition than observed with batch-grown cells. SOURs results indicate that the AMO enzyme is directly inhibited by TCS, either through competition for its active site or interactions of TCS with AMO, and that the HAO enzyme is not inhibited by TCS. Results of suspended cell growth experiments indicate that growth of N. europaea is inhibited by approximately 30% at 0.01 ppm TCS; where concentrations at or exceeding 0.05 ppm TCS significantly inhibit cell growth. Resistance tests showed no development in resistance to 0.01 ppm TCS over three growth cycles or approximately 67 generations of growth. UHPLC-MS analyses indicate that TCS is not transformed in the presence of N. europaea over a three hour direct exposure period or during long term growth of several days.
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