The Effect of Chlorine and Chloramines on the Viability and Activity of Nitrifying Bacteria

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Virginia Tech

Nitrification is a significant concern for drinking water systems employing chloramines for secondary disinfection. Utilities have implemented a range of disinfection strategies that have varying levels of effectiveness in the prevention and control of nitrification events, including optimizing the chlorine-to-ammonia ratio, maintaining chloramine residual throughout the distribution system, controlling pH, and temporal switching to free chlorination. Annual or semi-annual application of free chlorination is practiced by 23% of chloraminating systems on a temporary basis as a preventative measure, even though it has the undesirable consequences of temporarily increasing disinfection byproducts, facilitating coliform detachment, and altering water taste and odor.

Although temporal free chlorination and other nitrification control methods have been widely studied in the field and in pilot-scale systems, very little is known about the stress responses of nitrifying bacteria to different disinfection strategies and the role physiological state plays in the resistance to disinfection. It is well known that many commonly studied bacteria, such as Escherichia coli, are able to better resist disinfection by free chlorine and chloramines under nutrient limitation through regulation of stress response genes that encode for DNA protection and enzymes that mediate reactive oxygen species. We compared the genomes of E. coli and the ammonia-oxidizing bacterium Nitrosomonas europaea, and found that many of the known stress response mechanisms and genes present in E. coli are absent in N. europaea or not controlled by the same mechanisms specific to bacterial growth state. These genetic differences present a general susceptibility of N. europaea to disinfection by chlorine compounds.

Using an experimental approach, we tested the hypothesis that N. europaea does not develop increased resistance to free chlorine and monochloramine during starvation to the same degree as E. coli. In addition, N. europaea cells were challenged with sequential treatments of monochloramine and hypochlorous acid to mimic the disinfectant switch employed by drinking water utilities. Indicators of activity (specific nitrite generation rate) and viability (LIVE/DEAD® BacLight© membrane-integrity based assay) were measured to determine short-term effectiveness of disinfection and recovery of cells over a twelve day monitoring period. The results of disinfectant challenge experiments reinforce the hypothesis, indicating that the response of N. europaea to either disinfectant does not significantly change during the transition from exponential phase to stationary phase. Exponentially growing N. europaea cells showed greater susceptibility to hypochlorous acid and monochloramine than stationary phase E. coli cells, but had increased resistance compared with exponential phase E. coli cells. Following incubation with monochloramine, N. europaea showed increased sensitivity to subsequent treatment with hypochlorous acid. Complete loss of ammonia-oxidation activity was observed in cells immediately following treatment with hypochlorous acid, monochloramine, or a combination of both disinfectants. Replenishing ammonia and nutrients did not invoke recovery of cells, as detected in activity measurements during the twelve day monitoring period. The results provide evidence for the effectiveness of both free chlorine and chloramines in the inhibition of growth and ammonia-oxidation activity in N. europaea. Furthermore, comparison of viability and activity measurements suggest that the membrane integrity-based stain does not serve as a good indicator of activity. These insights into the responses of pure culture nitrifying bacteria to free chlorine and monochloramine could prove useful in designing disinfection strategies effective in the control of nitrification.

chloramines, stress response, nitrification, disinfection