Effects of short- and long-term exposure of silver nanoparticles and silver ions to Nitrosomonas europaea biofilms and planktonic cells.

The increasing use of silver nanoparticles (AgNPs) in consumer products, and their resulting influx into wastewater, may pose a threat to biological nutrient removal in wastewater treatment plants. Planktonic ammonia-oxidizing bacteria (AOB), which convert ammonia to nitrite in the first step of nitrification, are highly sensitive to AgNPs and their released silver ions (Ag+), but the sensitivity of AOB biofilms to AgNPs and Ag+ is less clear. This study demonstrated that biofilms of Nitrosomonas europaea, a model AOB, were more resistant to both short-term and long-term exposure to AgNP and Ag+ than planktonic cells. The increased resistance of N. europaea biofilms was attributed primarily to the increased biomass and slower growth rates present in the biofilm. Similar inhibition mechanisms were observed for AgNPs and Ag+ in both planktonic cells and biofilms with enzymatic inhibition observed at lower concentrations and cell lysis observed at higher concentrations. Long-term continuous exposure to AgNPs lowered the inhibitory concentration by 1-2 orders of magnitude below that required by short-term exposures. Although the total AgNP load was similar between the short and long-term exposure scenarios, the long-term exposure resulted in an order of magnitude more silver being associated in the biofilms and is the primary reason for the increased sensitivity observed. This suggests that short-term batch toxicity assays may greatly underestimate the sensitivity of biofilm treatment systems to long-term exposures of low concentrations of AgNPs and Ag+.

Zinc chloride inhibition of Nitrosococcus mobilis.

Nitrosococcus mobilis, a halophilic nitrifier, plays an important role in global nitrogen cycling and in the removal of nitrogen from wastewater treatment plants. However, ammonia oxidation is sensitive to a wide variety of inhibitors, including the heavy metal, zinc. Using a metabolic-steady-state reactor, shotgun DNA microarrays, and quantitative polymerase chain reaction (qPCR), this research looked at the dynamic physiological and transcriptional responses of N. mobilis to 1 and 10 microM ZnCl2. By oxygen uptake rate measurements, zinc was determined to act directly on the ammonia monooxygenase (AMO) enzyme. The addition of excess copper prevented the inhibition of AMO by ZnCl2 suggesting that zinc and copper compete for placement in the metal active site in AMO. Shotgun DNA microarrays identified four previously unsequenced genes that were up- or down-regulated in response to 10 microM ZnCl2. Genes up-regulated in response to zinc inhibition include methionine synthase I, UbiA prenyltransferase and a recG-like helicase. RuBisCO was the lone down-regulated gene identified. qPCR was used to track the gene expression of the identified genes over the course of the 4-h experiment for both ZnCl2 concentrations. Because of their physiological importance, the expressions of AMO and hydroxylamine oxidoreductase (HAO) were also monitored via qPCR. The qPCR results showed general agreement with the shotgun DNA microarray results for metH, UbiA, recG and RuBisCO, and revealed that AMO and HAO expression levels were maintained or modestly up-regulated during ZnCl2 inhibition.