Evaluation of molecular-based methods for the detection and quantification of Cryptosporidium spp. in wastewater.

Cryptosporidium poses significant public health risks as a cause of waterborne disease worldwide. Clinical surveillance of cryptosporidiosis is largely underreported due to the asymptomatic and mildly symptomatic infections, clinical misdiagnoses, and barriers to access testing. Wastewater surveillance overcomes these limitations and could serve as an effective tool for identifying cryptosporidiosis at the population level. Despite its potential, the lack of standardized wastewater surveillance methods for Cryptosporidium spp. challenges implementation design and the comparability between studies. Thus, this study compared and contrasted Cryptosporidium wastewater surveillance methods for concentrating wastewater oocysts, extracting oocyst DNA, and detecting Cryptosporidium genetic markers. The evaluated concentration methods included electronegative membrane filtration, Envirocheck HV capsule filtration, centrifugation, and Nanotrap Microbiome Particles, with and without additional immunomagnetic separation purification (except for the Nanotrap Microbiome Particles). Oocyst DNA extraction by either the DNeasy Powersoil Pro kit and the QIAamp DNA Mini kit were evaluated and the impact of bead beating and freeze-thaw pretreatments on DNA recoveries was assessed. Genetic detection via qPCR assays targeting either the Cryptosporidium 18S rRNA gene or the Cryptosporidium oocyst wall protein gene were tested. Oocyst recovery percentages were highest for centrifugation (39-77 %), followed by the Nanotrap Microbiome Particles (24 %), electronegative filtration with a PBST elution (22 %), and Envirocheck HV capsule filtration (13 %). Immunomagnetic separation purification was found to be unsuitable due to interference from the wastewater matrix. Bead-beating pretreatment enhanced DNA recoveries from both the DNeasy Powersoil Pro kit (314 gc/µL DNA) and the QIAamp DNA Mini kit (238 gc/µL DNA). In contrast, freeze-thaw pretreatment reduced DNA recoveries to under 92 gc/µL DNA, likely through DNA degradation. Finally, while both qPCR assays were specific to Cryptosporidium spp., the 18S rRNA assay had a 5-fold lower detection limit and could detect a wider range of Cryptosporidium spp. than the Cryptosporidium oocyst wall protein assay.

Representative Public Health Surveys Pose Several Challenges: Lessons Learned Across 9 Communities During the COVID-19 Pandemic.

Community surveillance surveys offer an opportunity to obtain important and timely public health information that may help local municipalities guide their response to public health threats. The objective of this paper is to present approaches, challenges, and solutions from SARS-CoV-2 surveillance surveys conducted in different settings by 2 research teams. For rapid assessment of a representative sample, a 2-stage cluster sampling design was developed by an interdisciplinary team of researchers at Oregon State University between April 2020 and June 2021 across 6 Oregon communities. In 2022, these methods were adapted for New York communities by a team of veterinary, medical, and public health practitioners. Partnerships were established with local medical facilities, health departments, COVID-19 testing sites, and health and public safety staff. Field staff were trained using online modules, field manuals describing survey methods and safety protocols, and in-person meetings with hands-on practice. Private and secure data integration systems and public awareness campaigns were implemented. Pilot surveys and field previews revealed challenges in survey processes that could be addressed before surveys proceeded. Strong leadership, robust trainings, and university-community partnerships proved critical to successful outcomes. Cultivating mutual trust and cooperation among stakeholders is essential to prepare for the next pandemic.

Wastewater surveillance for bacterial targets: current challenges and future goals.

Wastewater-based epidemiology (WBE) expanded rapidly in response to the COVID-19 pandemic. As the public health emergency has ended, researchers and practitioners are looking to shift the focus of existing wastewater surveillance programs to other targets, including bacteria. Bacterial targets may pose some unique challenges for WBE applications. To explore the current state of the field, the National Science Foundation-funded Research Coordination Network (RCN) on Wastewater Based Epidemiology for SARS-CoV-2 and Emerging Public Health Threats held a workshop in April 2023 to discuss the challenges and needs for wastewater bacterial surveillance. The targets and methods used in existing programs were diverse, with twelve different targets and nine different methods listed. Discussions during the workshop highlighted the challenges in adapting existing programs and identified research gaps in four key areas: choosing new targets, relating bacterial wastewater data to human disease incidence and prevalence, developing methods, and normalizing results. To help with these challenges and research gaps, the authors identified steps the larger community can take to improve bacteria wastewater surveillance. This includes developing data reporting standards and method optimization and validation for bacterial programs. Additionally, more work is needed to understand shedding patterns for potential bacterial targets to better relate wastewater data to human infections. Wastewater surveillance for bacteria can help provide insight into the underlying prevalence in communities, but much work is needed to establish these methods.IMPORTANCEWastewater surveillance was a useful tool to elucidate the burden and spread of SARS-CoV-2 during the pandemic. Public health officials and researchers are interested in expanding these surveillance programs to include bacterial targets, but many questions remain. The NSF-funded Research Coordination Network for Wastewater Surveillance of SARS-CoV-2 and Emerging Public Health Threats held a workshop to identify barriers and research gaps to implementing bacterial wastewater surveillance programs.

Effectiveness of the tropical plants Rhynchospora corymbosa and Coix lacryma-jobi in vertical flow constructed wetlands for municipal primary sewage effluent treatment.

The performance of two tropical plants, Rhynchospora corymbosa L. (RC) and Coix lacryma-jobi, L (CL) in treatment of primary sewage effluent in lab-scale vertical-flow constructed wetlands (VFCW) along with no plant control wetland was investigated. A batch-flow VFCWs were operated under batch fill and drain hydraulic loading system with hydraulic retention times (HRT) of 0.5, 1, and 2 days and fill rate of 8 L/day. Removal of solids, organics, nutrients, and pathogens were monitored. The volumetric contaminant removal rates were best described by 1st order kinetics except for ammonia and phosphate, which was best described by Stover-Kincannon kinetics. Influent TSS, PO43-, COD, BOD5, and total coliform concentration were low but high in NH4+ concentration. CL was better in nutrient removal as HRT increases compared to RC. RC was more efficient at TSS, turbidity, and organics removal. Pathogen removal was independent of plant type but HRT. Solids and organic removal were lower in CL planted CWs due to preferential flow paths created by their bulky root. CL planted CWs removed more nutrients followed by RC planted CWs and then no-plant control CWs. The results of these tests demonstrate that both CL and RC are suitable for the treatment of municipal wastewater in VFCW system.

Correlation between Clinical and Wastewater SARS-CoV-2 Genomic Surveillance, Oregon, USA.

SARS-CoV-2 variant proportions in a population can be estimated through genomic sequencing of clinical specimens or wastewater samples. We demonstrate strong pairwise correlation between statewide variant estimates in Oregon, USA, derived from both methods (correlation coefficient 0.97). Our results provide crucial evidence of the effectiveness of community-level genomic surveillance.

Anaerobic digester microbiome dynamics in response to moderate and failure-inducing shock loads of fats, oils and greases.

Accidental organic overloading (shock loading) is common during the anaerobic co-digestion of fats, oils and greases (FOG) and may lead to decreased performance or reactor failure due to the effects on the microbiome. Here, adapted and non-adapted lab-scale anaerobic digesters were exposed to FOG shocks of varying organic strengths. The microbiome was sequenced during the recovery periods employed between each shock event. Non-failure-inducing shocks resulted in enrichment of fermentative bacteria, and acetoclastic and methylotrophic methanogens. However, sub-dominant bacterial populations were largely responsible for increased biogas production observed after adaptation. Following failure events, early recovery communities were dominated by Pseudomonas and Methanosaeta while late recovery communities shifted toward sub-dominant bacterial taxa and Methanosarcina. Generally, the recovered microbiome structure diverged from that of both the initial and optimized microbiomes. Thus, while non-failure-inducing FOG shocks can be beneficial, the adaptations gained are lost after a failure event and adaptation must begin again.

Evaluation of a Wastewater-Based Epidemiological Approach to Estimate the Prevalence of SARS-CoV-2 Infections and the Detection of Viral Variants in Disparate Oregon Communities at City and Neighborhood Scales.

BACKGROUND: Positive correlations have been reported between wastewater SARS-CoV-2 concentrations and a community's burden of infection, disease or both. However, previous studies mostly compared wastewater to clinical case counts or nonrepresentative convenience samples, limiting their quantitative potential. OBJECTIVES: This study examined whether wastewater SARS-CoV-2 concentrations could provide better estimations for SARS-CoV-2 community prevalence than reported cases of COVID-19. In addition, this study tested whether wastewater-based epidemiology methods could identify neighborhood-level COVID-19 hotspots and SARS-CoV-2 variants. METHODS: Community SARS-CoV-2 prevalence was estimated from eight randomized door-to-door nasal swab sampling events in six Oregon communities of disparate size, location, and demography over a 10-month period. Simultaneously, wastewater SARS-CoV-2 concentrations were quantified at each community's wastewater treatment plant and from 22 Newport, Oregon, neighborhoods. SARS-CoV-2 RNA was sequenced from all positive wastewater and nasal swab samples. Clinically reported case counts were obtained from the Oregon Health Authority. RESULTS: Estimated community SARS-CoV-2 prevalence ranged from 8 to 1,687/10,000 persons. Community wastewater SARS-CoV-2 concentrations ranged from 2.9 to 5.1 log10 gene copies per liter. Wastewater SARS-CoV-2 concentrations were more highly correlated (Pearson's r=0.96; R2=0.91) with community prevalence than were clinically reported cases of COVID-19 (Pearson's r=0.85; R2=0.73). Monte Carlo simulations indicated that wastewater SARS-CoV-2 concentrations were significantly better than clinically reported cases at estimating prevalence (p<0.05). In addition, wastewater analyses determined neighborhood-level COVID-19 hot spots and identified SARS-CoV-2 variants (B.1 and B.1.399) at the neighborhood and city scales. DISCUSSION: The greater reliability of wastewater SARS-CoV-2 concentrations over clinically reported case counts was likely due to systematic biases that affect reported case counts, including variations in access to testing and underreporting of asymptomatic cases. With these advantages, combined with scalability and low costs, wastewater-based epidemiology can be a key component in public health surveillance of COVID-19 and other communicable infections. https://doi.org/10.1289/EHP10289.

Resistome characterization of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolated from wastewater treatment utilities in Oregon.

Infections resistant to broad spectrum antibiotics due to the emergence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae is of global concern. This study characterizes the resistome (i.e., entire ecology of resistance determinants) of 11 ESBL-producing Escherichia coli isolates collected from eight wastewater treatment utilities across Oregon. Whole genome sequencing was performed to identify the most abundant antibiotic resistance genes including ESBL-associated genes, virulence factors, as well as their sequence types. Moreover, the phenotypes of antibiotic resistance were characterized. ESBL-associated genes (i.e., blaCMY, blaCTX, blaSHV, blaTEM) were found in all but one of the isolates with five isolates carrying two of these genes (four with blaCTX and blaTEM; one with blaCMY and blaTEM). The ampC gene and virulence factors were present in all the E. coli isolates. Across all the isolates, 31 different antibiotic resistance genes were identified. Additionally, all E. coli isolates harbored phenotypic resistance to beta-lactams (penicillins and cephalosporins), while 8 of the 11 isolates carried multidrug resistance phenotypes (resistance to three or more classes of antibiotics). Findings highlight the risks associated with the presence of ESBL-producing E. coli isolates in wastewater systems that have the potential to enter the environment and may pose direct or indirect risks to human health.

Detection of SARS-CoV-2 B.1.351 (Beta) Variant through Wastewater Surveillance before Case Detection in a Community, Oregon, USA.

Genomic surveillance has emerged as a critical monitoring tool during the SARS-CoV-2 pandemic. Wastewater surveillance has the potential to identify and track SARS-CoV-2 variants in the community, including emerging variants. We demonstrate the novel use of multilocus sequence typing to identify SARS-CoV-2 variants in wastewater. Using this technique, we observed the emergence of the B.1.351 (Beta) variant in Linn County, Oregon, USA, in wastewater 12 days before this variant was identified in individual clinical specimens. During the study period, we identified 42 B.1.351 clinical specimens that clustered into 3 phylogenetic clades. Eighteen of the 19 clinical specimens and all wastewater B.1.351 specimens from Linn County clustered into clade 1. Our results provide further evidence of the reliability of wastewater surveillance to report localized SARS-CoV-2 sequence information.

Impact of Sampling Type, Frequency, and Scale of the Collection System on SARS-CoV-2 Quantification Fidelity.

With the rapid onset of the COVID-19 pandemic, wastewater-based epidemiology sampling methodologies for SARS-CoV-2 were often implemented quickly and may not have considered the unique drainage catchment characteristics. This study assessed the impact of grab versus composite sampling on the detection and quantification of SARS-CoV-2 in four different catchment scales with flow rates ranging from high flow (wastewater treatment plant influent) to medium flow (neighborhood scale) to low-flow (city block scale) to ultralow flow (building scale). At the high-flow site, grab samples were comparable to 24 h composite samples with SARS-CoV-2 detected in all samples and differed in concentration from the composite by <1 log 10 unit. However, as the size of the catchment decreased, the percentage of negative grab samples increased despite all respective composites being positive, and the SARS-CoV-2 concentrations of grab samples varied from those of the composites by up to almost 2 log 10 units. At the ultra-low-flow site, increased sampling frequencies generated composite samples with higher fidelity to the 5 min composite, which is the closest estimate of the true SARS-CoV-2 composite concentration that could be measured. Thus, composite sampling is more likely to compensate for temporal signal variability while grab samples do not, especially as the catchment basin size decreases.

Standardizing data reporting in the research community to enhance the utility of open data for SARS-CoV-2 wastewater surveillance.

SARS-CoV-2 RNA detection in wastewater is being rapidly developed and adopted as a public health monitoring tool worldwide. With wastewater surveillance programs being implemented across many different scales and by many different stakeholders, it is critical that data collected and shared are accompanied by an appropriate minimal amount of metainformation to enable meaningful interpretation and use of this new information source and intercomparison across datasets. While some databases are being developed for specific surveillance programs locally, regionally, nationally, and internationally, common globally-adopted data standards have not yet been established within the research community. Establishing such standards will require national and international consensus on what metainformation should accompany SARS-CoV-2 wastewater measurements. To establish a recommendation on minimum information to accompany reporting of SARS-CoV-2 occurrence in wastewater for the research community, the United States National Science Foundation (NSF) Research Coordination Network on Wastewater Surveillance for SARS-CoV-2 hosted a workshop in February 2021 with participants from academia, government agencies, private companies, wastewater utilities, public health laboratories, and research institutes. This report presents the primary two outcomes of the workshop: (i) a recommendation on the set of minimum meta-information that is needed to confidently interpret wastewater SARS-CoV-2 data, and (ii) insights from workshop discussions on how to improve standardization of data reporting.

Impact of biosolids amendment and wastewater effluent irrigation on enteric antibiotic-resistant bacteria - a greenhouse study.

Reuse of wastewater effluent and biosolids in agriculture is essential to sustainable water and nutrient resource management practices. Wastewater and biosolids, however, are reportedly the recipients, reservoirs, and sources of antibiotic-resistant enteric pathogens. While decay rates of fecal bacterial indicators in soil are frequently studied, very few studies have reported on the persistence of the antibiotic-resistant sub-populations. Little is known about how multi-drug resistance phenotypes of enteric bacteria in agricultural soil change over time. In this study, germinated carrot seeds were planted in soil that received biosolids amendment and/or wastewater effluent irrigation in a greenhouse setting. We quantified total and antibiotic-resistant fecal bacterial indicators (Escherichia coli and enterococci) weekly in soil and total E. coli at harvest (day 77) on carrots. Antibiotic susceptibility of 121 E. coli and 110 enterococci collected isolates were determined. E. coli or enterococci were not recovered from the soil without biosolids amendment regardless of the irrigation water source. After biosolids amendment, soil E. coli and enterococci concentrations increased more than 3 log10 CFU/g-TS within the first week, declined slowly over time, but stayed above the detection limit (0.39 CFU/g-TS) over the entirety of the study. No statistical difference was found between effluent wastewater or water irrigation in soil total and antibiotic-resistant E. coli and enterococci concentrations or carrots E. coli levels. Soil antibiotic-resistant E. coli and enterococci decayed significantly faster than total E. coli and enterococci. Moreover, the prevalence of multi-drug resistant (resistance to three or more antibiotics) E. coli declined significantly over time, while almost all collected enterococci isolates showed multi-drug resistance phenotypes. At harvest, E. coli were present on carrots; the majority of which were resistant to ampicillin. The survival of antibiotic-resistant enteric bacteria in soil and on harvested carrots indicates there are transmission risks associated with biosolids amendment use in root crops.

Wastewater Surveillance for SARS-CoV-2 on College Campuses: Initial Efforts, Lessons Learned, and Research Needs.

Wastewater surveillance for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging approach to help identify the risk of a coronavirus disease (COVID-19) outbreak. This tool can contribute to public health surveillance at both community (wastewater treatment system) and institutional (e.g., colleges, prisons, and nursing homes) scales. This paper explores the successes, challenges, and lessons learned from initial wastewater surveillance efforts at colleges and university systems to inform future research, development and implementation. We present the experiences of 25 college and university systems in the United States that monitored campus wastewater for SARS-CoV-2 during the fall 2020 academic period. We describe the broad range of approaches, findings, resources, and impacts from these initial efforts. These institutions range in size, social and political geographies, and include both public and private institutions. Our analysis suggests that wastewater monitoring at colleges requires consideration of local information needs, sewage infrastructure, resources for sampling and analysis, college and community dynamics, approaches to interpretation and communication of results, and follow-up actions. Most colleges reported that a learning process of experimentation, evaluation, and adaptation was key to progress. This process requires ongoing collaboration among diverse stakeholders including decision-makers, researchers, faculty, facilities staff, students, and community members.

Wastewater surveillance for SARS-CoV-2 on college campuses: Initial efforts, lessons learned and research needs.

Background: Wastewater surveillance for SARS-CoV-2 is an emerging approach to help identify the risk of a COVID-19 outbreak. This tool can contribute to public health surveillance at both community (wastewater treatment system) and institutional (e.g., colleges, prisons, nursing homes) scales. Objectives: This research aims to understand the successes, challenges, and lessons learned from initial wastewater surveillance efforts at colleges and university systems to inform future research, development and implementation. Methods: This paper presents the experiences of 25 college and university systems in the United States that monitored campus wastewater for SARS-CoV-2 during the fall 2020 academic period. We describe the broad range of approaches, findings, resource needs, and lessons learned from these initial efforts. These institutions range in size, social and political geographies, and include both public and private institutions. Discussion: Our analysis suggests that wastewater monitoring at colleges requires consideration of information needs, local sewage infrastructure, resources for sampling and analysis, college and community dynamics, approaches to interpretation and communication of results, and follow-up actions. Most colleges reported that a learning process of experimentation, evaluation, and adaptation was key to progress. This process requires ongoing collaboration among diverse stakeholders including decision-makers, researchers, faculty, facilities staff, students, and community members.

Influence of Water Hardness on Silver Ion and Silver Nanoparticle Fate and Toxicity Toward Nitrosomonas europaea.

This study investigated the influence of water hardness (Mg2+ and Ca2+) on the fate and toxicity of 20 nm citrate silver nanoparticles (AgNPs) and Ag+ toward Nitrosomonas europaea, a model ammonia-oxidizing bacterium. Nitrification inhibition of N. europaea by 1 ppm AgNPs and 0.5 ppm Ag+ was reduced from 80% and 83%, respectively, in the absence of Mg2+ to 2% and 33%, respectively, in the presence of 730 µM Mg2+. Introduction of Mg2+ resulted in the rapid aggregation of the AgNP suspensions and reduced the 3 h Ag+ dissolution rates from 30%, in the absence of Mg2+, to 9%, in the presence of 730 µM Mg2+. Reduced AgNP dissolution rates resulted in decreased concentrations of silver that were found adsorbed to N. europaea cells. Increasing AgNP concentrations in the presence of Mg2+ increased the observed inhibition of nitrification, but was always less than what was observed in the absence of Mg2+. The presence of Mg2+ also reduced the adsorption of Ag+ to cells, possibly due to multiple mechanisms, including a reduction in the negative surface charge of the N. europaea membrane and a competition between Mg2+ and Ag+ for membrane binding sites and transport into the cells. Ca2+ demonstrated similar protection mechanisms, as Ag+ toxicity was reduced and AgNP suspensions aggregated and decreased their dissolution rates. These results indicate that the toxicity of Ag+ and AgNPs to nitrifying bacteria in wastewater treatment would be less pronounced in systems with hard water.

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 BSA's 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.

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.

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.

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.

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. europaea's 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).