Source-to-tap investigation of the occurrence of nontuberculous mycobacteria in a full-scale chloraminated drinking water system.

Nontuberculous mycobacteria (NTM) in drinking water are a significant public health concern. However, an incomplete understanding of the factors that influence the occurrence of NTM in drinking water limits our ability to characterize risk and prevent infection. This study sought to evaluate the influence of season and water treatment, distribution, and stagnation on NTM in drinking water. Samples were collected source-to-tap in a full-scale, chloraminated drinking water system approximately monthly from December 2019 to November 2020. NTM were characterized using culture-dependent (plate culture with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry [MALDI-TOF MS] isolate analysis) and culture-independent methods (quantitative PCR and genome-resolved metagenomics). Sampling locations included source waters, three locations within the treatment plant, and five buildings receiving water from the distribution system. Building plumbing samples consisted of first draw, 5-min flush, and full flush cold-water samples. As the study took place during the COVID-19 pandemic, the influence of reduced water usage in three of the five buildings was also investigated. The highest NTM densities source-to-tap were found in the summer first draw building water samples (107 gene copies/L), which also had the lowest monochloramine concentrations. Flushing was found to be effective for reducing NTM and restoring disinfectant residuals, though flush times necessary to improve water quality varied by building. Clinically relevant NTM species, including Mycobacterium avium, were recovered via plate culture, with increased occurrence observed in buildings with higher water age. Four of five NTM metagenome-assembled genomes were identified to the species level and matched identified isolates.IMPORTANCENTM infections are increasing in prevalence, difficult to treat, and associated with high morbidity and mortality rates. Our lack of understanding of the factors that influence NTM occurrence in drinking water limits our ability to prevent infections, accurately characterize risk, and focus remediation efforts. In this study, we comprehensively evaluated NTM in a full-scale drinking water system, showing that various steps in treatment and distribution influence NTM presence. Stagnant building water contained the highest NTM densities source-to-tap and was associated with low disinfectant residuals. We illustrated the differences in NTM detection and characterization obtained from culture-based and culture-independent methods, highlighting the complementarity between these approaches. We demonstrated that focusing NTM mitigation efforts in building plumbing systems, which have the highest NTM densities source-to-tap, has potential for immediate positive effects. We also identified steps during treatment that increase NTM levels, which provides beneficial information for utilities seeking to reduce NTM in finished water.

Simulating Rumen Conditions Using an Anaerobic Dynamic Membrane Bioreactor to Enhance Hydrolysis of Lignocellulosic Biomass.

An anaerobic dynamic membrane bioreactor (AnDMBR) mimicking rumen conditions was developed to enhance the hydrolysis of lignocellulosic materials and the production of volatile fatty acids (VFAs) when treating food waste. The AnDMBR was inoculated with cow rumen content and operated at a 0.5 day hydraulic retention time, 2-4 day solids retention time, a temperature of 39 °C, and a pH of 6.3, characteristics similar to those of a rumen. Removal rates of neutral detergent fiber and acid detergent fiber of 58.9 ± 8.4 and 69.0 ± 8.6%, respectively, and a VFA yield of 0.55 ± 0.12 g VFA as chemical oxygen demand g volatile solids (VS)fed-1 were observed at an organic loading rate of 18 ± 2 kg VS m-3 day-1. The composition and activity of the microbial community remained consistent after biofilm disruption, bioreactor upset, and reinoculation. Up to 66.7 ± 5.7% of the active microbial populations and 51.0 ± 7.0% of the total microbial populations present in the rumen-mimicking AnDMBR originated from the inoculum. This study offers a strategy to leverage the features of a rumen; the AnDMBR achieved high hydrolysis and fermentation rates even when treating substrates different from those fed to ruminants.

Competitive Reactions during Ethanol Chain Elongation Were Temporarily Suppressed by Increasing Hydrogen Partial Pressure through Methanogenesis Inhibition.

Organic waste streams can be converted into high-value platform chemicals such as medium-chain carboxylic acids (MCCAs) using mixed microbial communities via chain elongation. However, the heterogeneity of waste streams and the use of complex microbial communities can lead to undesirable reactions, thus decreasing process efficiency. We explored suppressing excessive ethanol oxidation to acetate (EEO) by increasing the hydrogen partial pressure (PH2) through hydrogenotrophic methanogenesis inhibition by periodically adding 2-bromoethanesulfonate (2-BES) to an MCCA-producing bioreactor to reach 10 mM of 2-BES upon addition. The bioreactor was fed with pretreated food waste and brewery waste containing high concentrations of short-chain carboxylic acids and ethanol, respectively. While 2-BES addition initially reduced EEO, some methanogens (Methanobrevibacter spp.) persisted and resistant populations were selected over time. Besides changing the methanogenic community structure, adding 2-BES also changed the bacterial community structure due to its impact on PH2. While we demonstrated that PH2 could be manipulated using 2-BES to control EEO, methods that do not require the addition of a chemical inhibitor should be explored to maintain optimum PH2 for long-term suppression of EEO.

Evaluation of Nanaerobic Digestion as a Mechanism to Explain Surplus Methane Production in Animal Rumina and Engineered Digesters.

Nanaerobes are a newly described class of microorganisms that use a unique cytochrome bd oxidase to achieve nanaerobic respiration at <2 µM dissolved oxygen (∼1% of atmospheric oxygen) but are not viable above this value due to the lack of other terminal oxidases. Although sharing an overlapping ecological niche with methanogenic archaea, the role of nanaerobes in methanogenic systems has not been studied so far. To explore their occurrence and significance, we re-analyzed published meta-omic datasets from animal rumina and waste-to-energy digesters, including conventional anaerobic digesters and anaerobic digesters with ultra-low oxygenation. Results show that animal rumina share broad similarities in the microbial community and system performance with oxygenated digesters, rather than with conventional anaerobic digesters, implying that trace levels of oxygen drive the efficient digestion in ruminants. The rumen system serves as an ideal model for the newly named nanaerobic digestion, as it relies on the synergistic co-occurrence of nanaerobes and methanogens for methane yield enhancement. The most abundant ruminal bacterial family Prevotellaceae contains many nanaerobes, which perform not only anaerobic fermentation but also nanaerobic respiration using cytochrome bd oxidase. These nanaerobes generally accompany hydrogenotrophic methanogens to constitute a thermodynamically and physiologically consistent framework for efficient methane generation. Our findings provide new insights into ruminal methane emissions and strategies to enhance methane generation from biomass.

Anaerobic Digestion as a Core Technology in Addressing the Global Sanitation Crisis: Challenges and Opportunities.

Successfully addressing the complex global sanitation problem is a massive undertaking. Anaerobic digestion (AD), coupled with post-treatment, has been identified as a promising technology to contribute to meeting this goal. It offers multiple benefits to the end users, such as the potential inactivation of pathogenic microorganisms in waste and the recovery of resources, including renewable energy and nutrients. This feature article provides an overview of the most frequently applied AD systems for decentralized communities and low- and lower-middle-income countries with an emphasis on sanitation, including technologies for which pathogen inactivation was considered during the design. Challenges to AD use are then identified, such as experience, economics, knowledge/training of personnel and users, and stakeholder analysis. Finally, accelerators for AD implementation are noted, such as the inclusion of field studies in academic journals, analysis of emerging contaminants, the use of sanitation toolboxes and life cycle assessment in design, incorporation of artificial intelligence in monitoring, and expansion of undergraduate and graduate curricula focused on Water, Sanitation, and Hygiene (WASH).

Predictive Modeling of Virus Inactivation by UV.

UV254 disinfection strategies are commonly applied to inactivate pathogenic viruses in water, food, air, and on surfaces. There is a need for methods that rapidly predict the kinetics of virus inactivation by UV254, particularly for emerging and difficult-to-culture viruses. We conducted a systematic literature review of inactivation rate constants for a wide range of viruses. Using these data and virus characteristics, we developed and evaluated linear and nonlinear models for predicting inactivation rate constants. Multiple linear regressions performed best for predicting the inactivation kinetics of (+) ssRNA and dsDNA viruses, with cross-validated root mean squared relative prediction errors similar to those associated with experimental rate constants. We tested the models by predicting and measuring inactivation rate constants of a (+) ssRNA mouse coronavirus and a dsDNA marine bacteriophage; the predicted rate constants were within 7% and 71% of the experimental rate constants, respectively, indicating that the prediction was more accurate for the (+) ssRNA virus than the dsDNA virus. Finally, we applied our models to predict the UV254 rate constants of several viruses for which high-quality UV254 inactivation data are not available. Our models will be valuable for predicting inactivation kinetics of emerging or difficult-to-culture viruses.

UV Disinfection of Human Norovirus: Evaluating Infectivity Using a Genome-Wide PCR-Based Approach.

The removal and inactivation of infectious human norovirus (HuNoV) is a major focus in water purification, but the effectiveness of disinfection processes on norovirus is largely unknown owing to the lack of a readily available infectivity assay. In particular, norovirus behavior through unit processes may be over- or underestimated using current approaches for assessing HuNoV infectivity (e.g., surrogates, molecular methods). Here, we fill a critical knowledge gap by estimating inactivation data for HuNoV after exposure to UV254, a commonly used disinfection process in the water industry. Specifically, we used a PCR-based approach that accurately tracks positive-sense single-stranded RNA virus inactivation without relying on culturing methods. We first confirmed that the approach is valid with a culturable positive-sense single-stranded RNA human virus, coxsackievirus B5, by applying both qPCR- and culture-based methods to measure inactivation kinetics with UV254 treatment. We then applied the qPCR-based method to establish a UV254 inactivation curve for HuNoV (inactivation rate constant = 0.27 cm2 mJ-1). Based on a comparison with previously published data, HuNoV exhibited similar UV254 susceptibility compared with other enteric single-stranded RNA viruses (e.g., Echovirus 12, feline calicivirus) but degraded much faster than MS2 (inactivation rate constant = 0.14 cm2 mJ-1). In addition to establishing a HuNoV inactivation rate constant, we developed an approach using a single qPCR assay that can be applied to estimate HuNoV inactivation in UV254 disinfection systems.

Trends in Antimicrobial Resistance Genes in Manure Blend Pits and Long-Term Storage Across Dairy Farms with Comparisons to Antimicrobial Usage and Residual Concentrations.

The use of antimicrobials by the livestock industry can lead to the release of unmetabolized antimicrobials and antimicrobial resistance genes (ARG) into the environment. However, the relationship between antimicrobial use, residual antimicrobials, and ARG prevalence within manure is not well understood, specifically across temporal and location-based scales. The current study determined ARG abundance in untreated manure blend pits and long-term storage systems from 11 conventional and one antimicrobial-free dairy farms in the Northeastern U.S. at six times over one-year. Thirteen ARGs corresponding to resistance mechanisms for tetracyclines, macrolides-lincosamides, sulfonamides, aminoglycosides, and ß-lactams were quantified using a Custom qPCR Array or targeted qPCR. ARG abundance differed between locations, suggesting farm specific microbial resistomes. ARG abundance also varied temporally. Manure collected during the winter contained lower ARG abundances. Overall, normalized ARG concentrations did not correlate to average antimicrobial usage or tetracycline concentrations across farms and collection dates. Of the 13 ARGs analyzed, only four genes showed a higher abundance in samples from conventional farms and eight ARGs exhibited similar normalized concentrations in the conventional and antimicrobial-free farm samples. No clear trends were observed in ARG abundance between dairy manure obtained from blend pits and long-term storage collected during two drawdown periods (fall and spring), although higher ARG abundances were generally observed in spring compared to fall. This comprehensive study informs future studies needed to determine the contributions of ARGs from dairy manure to the environment.

Biofilms in Full-Scale Drinking Water Ozone Contactors Contribute Viable Bacteria to Ozonated Water.

Concentrations of viable microbial cells were monitored using culture-based and culture-independent methods across multichamber ozone contactors in a full-scale drinking water treatment plant. Membrane-intact and culturable cell concentrations in ozone contactor effluents ranged from 1200 to 3750 cells/mL and from 200 to 3850 colony forming units/mL, respectively. Viable cell concentrations decreased significantly in the first ozone contact chamber, but rose, even as ozone exposure increased, in subsequent chambers. Our results implicate microbial detachment from biofilms on contactor surfaces, and from biomass present within lime softening sediments in a hydraulic dead zone, as a possible reason for increasing cell concentrations in water samples from sequential ozone chambers. Biofilm community structures on baffle walls upstream and downstream from the dead zone were significantly different from each other ( p = 0.017). The biofilms downstream of the dead zone contained a significantly ( p = 0.036) higher relative abundance of bacteria of the genera Mycobacterium and Legionella than the upstream biofilms. These results have important implications as the effluent from ozone contactors is often treated further in biologically active filters and bacteria in ozonated water continuously seed filter microbial communities.

Anaerobic Disposal of Arsenic-Bearing Wastes Results in Low Microbially Mediated Arsenic Volatilization.

The removal of arsenic from drinking water sources produces arsenic-bearing wastes, which are disposed of in a variety of ways. Several disposal options involve anaerobic environments, including mixing arsenic waste with cow dung, landfills, anaerobic digesters, and pond sediments. Though poorly understood, the production of gaseous arsenic species in these environments can be a primary goal (cow dung mixing) or an unintended consequence (anaerobic digesters). Once formed, these gaseous arsenic species are readily diluted in the atmosphere. Arsenic volatilization can be mediated by the enzyme arsenite S-adenosylmethionine methyltransferase (ArsM) or through the enzymes involved in methanogenesis. In this study, methanogenic mesocosms with arsenic-bearing ferric iron waste from an electrocoagulation drinking water treatment system were used to evaluate the role of methanogenesis in arsenic volatilization using methanogen inhibitors. Arsenic volatilization was highest in methanogenic mesocosms, but represented <0.02% of the total arsenic added. 16S rRNA cDNA sequencing, qPCR of mcrA transcripts, and functional gene array-based analysis of arsM expression, revealed that arsenic volatilization correlated with methanogenic activity. Aqueous arsenic concentrations increased in all mesocosms, indicating that unintended contamination may result from disposal in anaerobic environments. This highlights that more research is needed before recommending anaerobic disposal intended to promote arsenic volatilization.

Vinegar-amended anaerobic biosand filter for the removal of arsenic and nitrate from groundwater.

The performance of a vinegar-amended anaerobic biosand filter was evaluated for future application as point-of-use water treatment in rural areas for the removal of arsenic and nitrate from groundwater containing common ions. Due to the importance of sulfate and iron in arsenic removal and their variable concentrations in groundwater, influent sulfate and iron concentrations were varied. Complete removal of influent nitrate (50 mg/L) and over 50% removal of influent arsenic (200 µg/L) occurred. Of all conditions tested, the lowest median effluent arsenic concentration was 88 µg/L. Iron removal occurred completely when 4 mg/L was added, and sulfate concentrations were lowered to a median concentration <2 mg/L from influent concentrations of 22 and 50 mg/L. Despite iron and sulfate removal and the establishment of reducing conditions, arsenic concentrations remained above the World Health Organization's arsenic drinking water standard. Further research is necessary to determine if anaerobic biosand filters can be improved to meet the arsenic drinking water standard and to evaluate practical implementation challenges.

Metatranscriptome of an anaerobic benzene-degrading, nitrate-reducing enrichment culture reveals involvement of carboxylation in benzene ring activation.

The enzymes involved in the initial steps of anaerobic benzene catabolism are not known. To try to elucidate this critical step, a metatranscriptomic analysis was conducted to compare the genes transcribed during the metabolism of benzene and benzoate by an anaerobic benzene-degrading, nitrate-reducing enrichment culture. RNA was extracted from the mixed culture and sequenced without prior mRNA enrichment, allowing simultaneous examination of the active community composition and the differential gene expression between the two treatments. Ribosomal and mRNA sequences attributed to a member of the family Peptococcaceae from the order Clostridiales were essentially only detected in the benzene-amended culture samples, implicating this group in the initial catabolism of benzene. Genes similar to each of two subunits of a proposed benzene-carboxylating enzyme were transcribed when the culture was amended with benzene. Anaerobic benzoate degradation genes from strict anaerobes were transcribed only when the culture was amended with benzene. Genes for other benzoate catabolic enzymes and for nitrate respiration were transcribed in both samples, with those attributed to an Azoarcus species being most abundant. These findings indicate that the mineralization of benzene starts with its activation by a strict anaerobe belonging to the Peptococcaceae, involving a carboxylation step to form benzoate. These data confirm the previously hypothesized syntrophic association between a benzene-degrading Peptococcaceae strain and a benzoate-degrading denitrifying Azoarcus strain for the complete catabolism of benzene with nitrate as the terminal electron acceptor.

Differential resistance of drinking water bacterial populations to monochloramine disinfection.

The impact of monochloramine disinfection on the complex bacterial community structure in drinking water systems was investigated using culture-dependent and culture-independent methods. Changes in viable bacterial diversity were monitored using culture-independent methods that distinguish between live and dead cells based on membrane integrity, providing a highly conservative measure of viability. Samples were collected from lab-scale and full-scale drinking water filters exposed to monochloramine for a range of contact times. Culture-independent detection of live cells was based on propidium monoazide (PMA) treatment to selectively remove DNA from membrane-compromised cells. Quantitative PCR (qPCR) and pyrosequencing of 16S rRNA genes was used to quantify the DNA of live bacteria and characterize the bacterial communities, respectively. The inactivation rate determined by the culture-independent PMA-qPCR method (1.5-log removal at 664 mg·min/L) was lower than the inactivation rate measured by the culture-based methods (4-log removal at 66 mg·min/L). Moreover, drastic changes in the live bacterial community structure were detected during monochloramine disinfection using PMA-pyrosequencing, while the community structure appeared to remain stable when pyrosequencing was performed on samples that were not subject to PMA treatment. Genera that increased in relative abundance during monochloramine treatment include Legionella, Escherichia, and Geobacter in the lab-scale system and Mycobacterium, Sphingomonas, and Coxiella in the full-scale system. These results demonstrate that bacterial populations in drinking water exhibit differential resistance to monochloramine, and that the disinfection process selects for resistant bacterial populations.

Navigating wastewater energy recovery strategies: a life cycle comparison of anaerobic membrane bioreactor and conventional treatment systems with anaerobic digestion.

The objective of this study was to evaluate emerging anaerobic membrane bioreactor (AnMBR) technology in comparison with conventional wastewater energy recovery technologies. Wastewater treatment process modeling and systems analyses were combined to evaluate the conditions under which AnMBR may produce more net energy and have lower life cycle environmental emissions than high rate activated sludge with anaerobic digestion (HRAS+AD), conventional activated sludge with anaerobic digestion (CAS+AD), and an aerobic membrane bioreactor with anaerobic digestion (AeMBR+AD). For medium strength domestic wastewater treatment under baseline assumptions at 15 °C, AnMBR recovered 49% more energy as biogas than HRAS+AD, the most energy positive conventional technology considered, but had significantly higher energy demands and environmental emissions. Global warming impacts associated with AnMBR were largely due to emissions of effluent dissolved methane. For high strength domestic wastewater treatment, AnMBR recovered 15% more net energy than HRAS+AD, and the environmental emissions gap between the two systems was reduced. Future developments of AnMBR technology in low energy fouling control, increased flux, and management of effluent methane emissions would make AnMBR competitive with HRAS+AD. Rapid advancements in AnMBR technology must continue to achieve its full economic and environmental potential as an energy recovery strategy for domestic wastewater.

Toward the autonomous flushing of building plumbing: Characterizing oxidation-reduction potential and temperature sensor dynamics.

Manual flushing of building plumbing is commonly used to address water quality issues that arise from water stagnation. Autonomous flushing informed by sensors has the potential to aid in the management of building plumbing, but a number of knowledge gaps hinder its application. This study evaluates autonomous flushing of building plumbing with online sensor and actuator nodes deployed under kitchen sinks in five residential houses. Online oxidation-reduction potential (ORP) and temperature data were collected for nine weeks during the winter and summer in houses with both free chlorine and chloramine. ORP levels in houses with free chlorine residuals decreased after overnight stagnation. The overnight decrease in ORP was not observed when tap water was automatically flushed for five minutes at 6:00 h every morning. ORP levels in houses with chloramine residuals did not decrease consistently after overnight stagnation, and daily automated flushes did not have an observable effect on the ORP signals. Additional laboratory experiments were carried out to evaluate ORP signals during chlorine decay and after incremental changes in chlorine, as would be expected in building plumbing conditions. Results from the lab and field deployments suggest on-line ORP sensors may be used to detect free chlorine decay due to stagnating water, but are not as effective in detecting chloramine decay. However, field results also suggest ORP may not respond as expected on a timely manner after free chlorine or chloramine have been restored, hindering their applicability in developing control algorithms. In this paper we tested twice-daily five-minute automatic flushing and found that it counteracts water quality degradation associated with overnight stagnation in free chlorine systems. An automatic sensor-based flushing is proposed using online temperature sensor data to determine when flushing has reached water from the main. The results suggest that flushing informed by temperature sensors can reduce the flushing time by 46 % compared to the preset five-minute static flush.

Investigating the suitability of online flow cytometry for monitoring full-scale drinking water ozone system disinfection effectiveness.

While online monitoring of physicochemical parameters has widely been incorporated into drinking water treatment systems, online microbial monitoring has lagged behind, resulting in the use of surrogate parameters (disinfectant residual, applied dose, concentration × time, CT) to assess disinfection system performance. Online flow cytometry (online FCM) allows for automated quantification of total and intact microbial cells. This study sought to investigate the feasibility of online FCM for full-scale drinking water ozone disinfection system performance monitoring. A water treatment plant with high lime solids turbidity in the ozone contactor influent was selected to evaluate the online FCM in challenging conditions. Total and intact cell counts were monitored for 40 days and compared to surrogate parameters (ozone residual, ozone dose, and CT) and grab sample assay results for cellular adenosine triphosphate (cATP), heterotrophic plate counts (HPC), impedance flow cytometry, and 16S rRNA gene sequencing. Online FCM provided insight into the dynamics of the full-scale ozone system, including offering early warning of increased contactor effluent cell concentrations, which was not observed using surrogate measures. Positive correlations were observed between online FCM intact cell counts and cATP levels (Kendall's tau=0.40), HPC (Kendall's tau=0.20), and impedance flow cytometry results (Kendall's tau=0.30). Though a strong correlation between log intact cell removal and CT was not observed, 16S rRNA gene sequencing results showed that passage through the ozone contactor significantly changed the microbial community (p < 0.05). Potential causes of the low overall cell inactivation in the contactor and the significant changes in the microbial community after ozonation include regrowth in the later chambers of the contactor and varied ozone resistance of drinking water microorganisms. This study demonstrates the suitability of direct, online microbial analysis for monitoring full-scale disinfection systems.

Arsenic waste management: a critical review of testing and disposal of arsenic-bearing solid wastes generated during arsenic removal from drinking water.

Water treatment technologies for arsenic removal from groundwater have been extensively studied due to widespread arsenic contamination of drinking water sources. Central to the successful application of arsenic water treatment systems is the consideration of appropriate disposal methods for arsenic-bearing wastes generated during treatment. However, specific recommendations for arsenic waste disposal are often lacking or mentioned as an area for future research and the proper disposal and stabilization of arsenic-bearing waste remains a barrier to the successful implementation of arsenic removal technologies. This review summarizes current disposal options for arsenic-bearing wastes, including landfilling, stabilization, cow dung mixing, passive aeration, pond disposal, and soil disposal. The findings from studies that simulate these disposal conditions are included and compared to results from shorter, regulatory tests. In many instances, short-term leaching tests do not adequately address the range of conditions encountered in disposal environments. Future research directions are highlighted and include establishing regulatory test conditions that align with actual disposal conditions and evaluating nonlandfill disposal options for developing countries.