Tyler S. Radniecki

Influence of liberated silver from silver nanoparticles on nitrification inhibition of Nitrosomonas europaea

The ecotoxicity of silver nanoparticles (Ag-NPs) to wastewater biota, including ammonia oxidizing bacteria (AOB), is gaining increasing interest as the number of products containing Ag-NPs continues to rise exponentially and they are expected to accumulate in wastewater treatment plants. This research demonstrated that the addition order of Ag-NP and the media constituents had a profound influence on the stability of the Ag-NP suspension and the corresponding repeatability of results and sensitivity of Nitrosomonas europaea. N. europaea, a model AOB, was found to be extremely sensitive to ionic silver (Ag(+)) and two sizes of Ag-NPs (20 and 80 nm). Ag(+) exposures resulted in the highest level of toxicity with smaller Ag-NPs (20 nm) being more toxic than larger Ag-NPs (80 nm). The increased sensitivity of N. europaea to smaller Ag-NPs was caused by their higher rates of dissolved silver (dAg) release, via dissolution, due to a greater surface area to volume ratio. dAg was shown to be responsible for the vast majority of the observed Ag-NP toxicity, as determined by abiotic Ag-NP dissolution tests. For the sizes of Ag-NP studied (20 and 80 nm), there appears to be a negligible nanoparticle-specific toxicity. This was further supported by similarities in inhibition mechanisms between Ag(+) and Ag-NP, with both causing decreases in AMO activity and destabilization of the outer-membrane of N. europaea. Finally, equal concentrations of total silver were found to be tightly associated to both Ag(+) and Ag-NP-exposed cells despite Ag-NP concentrations being five times greater, by mass, than Ag(+) concentrations.

Influence of Bovine Serum Albumin and Alginate on Silver Nanoparticle Dissolution and Toxicity to Nitrosomonas europaea

Bovine serum albumin (BSA), a model protein, reduced the toxicity of 20 nm citrate silver nanoparticles (AgNP) toward Nitrosomonas europaea, a model ammonia oxidizing bacteria, through a dual-mode protection mechanism. BSA reduced AgNP toxicity by chelating the silver ions (Ag(+)) released from the AgNPs. BSA further reduced AgNP toxicity by binding to the AgNP surface thus preventing NH3-dependent dissolution from occurring. Due to BSAs affinity toward Ag(+) chemisorbed on the AgNP surface, increased concentrations of BSA lead to increased AgNP dissolution rates. This, however, did not increase AgNP toxicity as the dissolved Ag(+) were adsorbed onto the BSA molecules. Alginate, a model extracellular polysaccharide (EPS), lacks strong Ag(+) ligands and was unable to protect N. europaea from Ag(+) toxicity. However, at high concentrations, alginate reduced AgNP toxicity by binding to the AgNP surface and reducing AgNP dissolution rates. Unlike BSA, alginate only weakly interacted with the AgNP surface and was unable to completely prevent NH3-dependent AgNP dissolution from occurring. Based on these results, AgNP toxicity in high protein environments (e.g., wastewater) is expected to be muted while the EPS layers of wastewater biofilms may provide additional protection from AgNPs, but not from Ag(+) that have already been released.

Expression of merA, amoA and hao in continuously cultured Nitrosomonas europaea cells exposed to zinc chloride additions

The effects of ZnCl2 additions on a mercuric reductase, merA, ammonia monooxygenase, amoA, and hydroxylamine (NH2OH) oxidoreductase, hao, gene expression were examined in continuously cultured Nitrosomonas europaea cells. The reactor was operated for 85 days with a 6.9 d hydraulic retention time and with four successive additions of ZnCl2 achieving maximum concentrations from 3 to 90 µM Zn2+. Continuously cultured N. europaea cells were more resistant to Zn2+ inhibition than previously examined batch cultured cells due to the presence of Mg2+ in the growth media, suggesting that Zn2+ enters the cell through Mg2+ import channels. The maximum merA up‐regulation was 45‐fold and expression increased with increases in Zn2+ concentration and decreased as Zn2+ concentrations decreased. Although Zn2+ irreversibly inactivated ammonia oxidation in N. europaea, the addition of either 600 µM CuSO4 or 2250 µM MgSO4 protected N. europaea from ZnCl2 inhibition, indicating a competition between Zn2+ and Cu2+/Mg2+ for uptake and/or AMO active sites. Since ZnCl2 inhibition is irreversible and amoA was up‐regulated at 30 and 90 µM additions, it is hypothesized that de novo synthesis of the AMO enzyme is needed to overcome inhibition. The up‐regulation of merA during exposure to non‐inhibitory Zn2+ levels indicates that merA is an excellent early warning signal for Zn2+ inhibition. Biotechnol. Bioeng. 2009;102: 546–553.

Influence of ammonia on silver nanoparticle dissolution and toxicity to Nitrosomonas europaea

Nitrosomonas europaea, a model ammonia oxidizing bacterium, was sensitive to both ionic silver (Ag(+)) and 20 nm citrate capped silver nanoparticles (AgNPs). AgNP toxicity has been previously shown to be primarily due to the dissolution of Ag(+). The rate of AgNP dissolution dramatically increased in test medium containing ammonium sulfate ((NH4)2SO4) and HEPES buffer compared to test medium containing either deionized water or HEPES buffer alone. The AgNP dissolution rates accelerated with increases in ammonia (NH3) concentrations either through increases in pH or through higher (NH4)2SO4 concentrations. Ammonia likely participated in the oxidation of the AgNP to form [Formula: see text] in solution leading to the observed increase in AgNP dissolution rates. AgNP toxicity was enhanced as NH3 concentrations increased. However, Ag(+) toxicity was constant at all NH3 concentrations tested. Therefore, it can be concluded that the increased AgNP toxicity was due to increased Ag(+) release and not due to a synergistic effect between NH3 and Ag(+). The results of this study may provide insights in the fate and toxicity of AgNPs in high NH3 environments including wastewater treatment plants, eutrophic waterways and alkaline environments.

Inhibition and Gene Expression of Nitrosomonas europaea Biofilms Exposed to Phenol and Toluene

Pure culture biofilms of the ammonia‐oxidizing bacterium Nitrosomonas europaea were grown in a Drip Flow Biofilm Reactor and exposed to the aromatic hydrocarbons phenol and toluene. Ammonia oxidation rates, as measured by nitrite production in the biofilms, were inhibited 50% when exposed to 56 µM phenol or 100 µM toluene, while 50% inhibition of suspended cells occurred at 8 µM phenol or 20 µM toluene. Biofilm‐grown cells dispersed into liquid medium and immediately exposed to phenol or toluene experienced similar inhibition levels as batch grown cells, indicating that mass transfer may be a factor in N. europaea biofilm resistance. Whole genome microarray analysis of gene expression was used to detect genes up‐regulated in biofilms during toluene and phenol exposure. Two genes, a putative pirin protein (NE1545) and a putative inner membrane protein (NE1546) were up‐regulated during phenol exposure, but no genes were up‐regulated during toluene exposure. Using qRT‐PCR, up‐regulation of NE1545 was detected in biofilms and suspended cells exposed to a range of phenol concentrations and levels of inhibition. In the biofilms, NE1545 expression was up‐regulated an average of 13‐fold over the range of phenol concentrations tested, and was essentially independent of phenol concentration. However, the expression of NE1545 in suspended cells increased from 20‐fold at 7 µM phenol up to 80‐fold at 30 µM phenol. This study demonstrates that biofilms of N. europaea are more resistant than suspended cells to inhibition of ammonia oxidation by phenol and toluene, even though the global transcriptional responses to the inhibitors do not differ in N. europaea between the suspended and attached growth states. Biotechnol. Bioeng. 2011; 108:750–757.

Expression of merA, trxA, amoA, and hao in continuously cultured Nitrosomonas europaea cells exposed to cadmium sulfate additions

The effects of CdSO4 additions on the gene expressions of a mercury reductase, merA, an oxidative stress protein, trxA, the ammonia‐monooxygenase enzyme (AMO), amoA, and the hydroxylamine oxidoreductase enzyme (HAO), hao, were examined in continuously cultured N. europaea cells. The reactor was fed 50 mM NH4+ and was operated for 78 days with a 6.9 days hydraulic retention time. Over this period, six successive batch additions of CdSO4 were made with increasing maximum concentrations ranging from 1 to 60 µM Cd2+. The expression of merA was highly correlated with the level of Cd2+ within the reactor (Rs = 0.90) with significant up‐regulation measured at non‐inhibitory Cd2+ concentrations. Cd2+ appears to target AMO specifically at lower concentrations and caused oxidative stress at higher concentrations, as indicated by the SOURs (specific oxygen uptake rates) and the up‐regulation of trxA. Since Cd2+ inhibition is irreversible and amoA was up‐regulated in response to Cd2+ inhibition, it is hypothesized that de novo synthesis of the AMO enzyme occurred and was responsible for the observed recovery in activity. Continuously cultured N. europaea cells were more resistant to Cd2+ inhibition than previously examined batch cultured cells due to the presence of Mg2+ and Ca2+ in the growth media, suggesting that Cd2+ enters the cell through Mg2+ and Ca2+ import channels. The up‐regulation of merA during exposure to non‐inhibitory Cd2+ levels indicates that merA is an excellent early warning signal for Cd2+ inhibition. Biotechnol. Bioeng. 2009; 104: 1004–1011.

Linking NE1545 gene expression with cell volume changes in Nitrosomonas europaea cells exposed to aromatic hydrocarbons

Nitrosomonas europaea, a model ammonia oxidizing bacterium, was exposed to a wide variety of aromatic hydrocarbons in 3 h batch assays. The expression of NE1545, a phenol sentinel gene involved in fatty acid metabolism, was monitored via quantitative real-time polymerase chain reaction (qRT-PCR) and a Coulter Counter technique was used to monitor changes in cell volume. Decreases in cell volume and NE1545 gene expression correlated strongly with exposure to aromatic hydrocarbons that possessed a single polar group substitution (e.g. phenol and aniline). Aromatic hydrocarbons that contain no polar group substitutions (e.g. toluene) or multiple polar group substitutions (e.g. p-hydroquinone) caused negligible changes in NE1545 expression and cell volume. The oxidation of aromatic hydrocarbons by N. europaea from configurations without a single polar group to one with two polar groups (e.g. p-cresol oxidized to 4-hydroxybenzyl alcohol) and from configurations with no polar groups to one with a single polar group (e.g. ethylbenzene oxidized to 4-ethylphenol) greatly influenced NE1545 gene expression and observed changes in cell volume. Nitrification inhibition in N. europaea by the aromatic hydrocarbons was found to be completely reversible; however, the decreases in cell volume were not reversible suggesting a physical change in cell membrane composition. Ammonia monooxygenase blocking studies showed that the chemical exposure that was responsible for the cell volume decrease and up-regulation in gene expression and not the observed inhibition. N. europaea is the first bacterium shown to experience significant changes in cell volume when exposed to μM concentrations of aromatic hydrocarbons, three orders of magnitude lower than previous studies with other bacteria.

The influence of Corexit 9500A and weathering on Alaska North Slope crude oil toxicity to the ammonia oxidizing bacterium, Nitrosomonas europaea.

The toxicity of the water associated fraction (WAF) of Alaska North Slope Crude oil (ANSC), Corexit 9500A and the dispersant enhanced WAF (DEWAF) of ANSC:Corexit 9500A mixtures were examined on the model ammonia oxidizing bacterium, Nitrosomonas europaea. Corexit 9500A was not toxic at environmentally relevant concentrations. Corexit 9500A greatly increased the toxicity of ANSC by increasing the chemical oxygen demand (COD) of the DEWAF. However, a majority of the DEWAF compounds were not toxic to N. europaea. Weathered WAF and DEWAF were not toxic to N. europaea even though their COD did not change compared to non-weathered controls, suggesting that toxicity was due to a small volatile fraction of the ANSC. The over-expression of the NE1545 gene, a marker for aromatic hydrocarbon exposure, in N. europaea cells exposed to WAF and DEWAF suggests that aromatic hydrocarbons are bioavailable to the cells and may play a role in the observed toxicity.

Investigating Nitrosomonas europaea Stress Biomarkers in Batch, Continuous Culture, and Biofilm Reactors

The understanding of nitrification inhibition in ammonia oxidizing bacteria (AOB) by priority pollutants and emerging contaminants is critical in managing the nitrogen cycle to preserve current water supplies, one of the National Academy of Engineers Grand Challenges in Engineering for the twenty-first century. Nitrosomonas europaea is an excellent model AOB for nitrification inhibition experimentation due to its well-defined NH(3) metabolism and the availability of a wide range of physiological and transcriptional tools that can characterize the mechanism of nitrification inhibition and probe N. europaeas response to the inhibitor. This chapter is a compilation of the physiological and transcriptional methods that have been used to characterize nitrification inhibition of N. europaea under a wide variety of growth conditions including batch, continuously cultured, and in biofilms. The protocols presented here can be applied to other AOB, and may be readily adapted for other autotrophic bacteria (e.g., nitrite oxidizing bacteria).

Transcriptional and Physiological Responses of Nitrosococcus mobilis to Copper Exposure

This research examined the dynamic physiological and transcriptional responses of Nitrosococcus mobilis, an unsquenced ammonia-oxidizing bacterium, to Cu2+ exposure. Cu2+ exposure caused decreases in N. mobilis nitrite production and oxygen uptake rates, and the release of intercellular K+ suggested that Cu2+ damaged the outer-membrane of N. mobilis cells. Shotgun DNA microarrays identified five previously unsequenced genes that were up- or down-regulated in response to Cu2+ exposure. The up-regulated putative efflux protein (HP 1900) and a copper containing NnrS protein (HP 1696) were found to correlate with Cu2+ concentrations. The up-regulation of these genes may be useful as a bioindicator of the presence of Cu2+ in natural and engineered systems. Finally, the expression of amoA and hao genes encoding the sole metabolic enzymes ammonia monooxygenase and hydroxylamine oxidoreductase, respectively, were measured. While hao expression did not change upon exposure to Cu2+, amoA expression increased upon e...