Introducing ARTiMiS: A Low-Cost Flow Imaging Microscope for Microalgal Monitoring.

Manual microscopy is the gold standard for phytoplankton monitoring in diverse engineered and natural environments. However, it is both labor-intensive and requires specialized training for accuracy and consistency, and therefore difficult to implement on a routine basis without significant time investment. Automation can reduce this burden by simplifying the measurement to a single indicator (e.g., chlorophyll fluorescence) measurable by a probe, or by processing samples on an automated cytometer for more granular information. The cost of commercially available flow imaging cytometers, however, poses a steep financial barrier to adoption. To overcome these labor and cost barriers, we developed ARTiMiS: the Autonomous Real-Time Microbial 'Scope. The ARTiMiS is a low-cost flow imaging microscopy-based platform with onboard software capable of providing taxonomically resolved quantitation of phytoplankton communities in real-time. ARTiMiS leverages novel multimodal imaging and onboard machine learning-based data processing that is currently optimized for a curated and expandable database of industrially relevant microalgae. We demonstrate its operational limits, performance in identification of laboratory-cultivated microalgae, and potential for continuous monitoring of complex microalgal communities in full-scale industrial cultivation systems.

Metatranscriptomic Analysis Reveals Synergistic Activities of Comammox and Anammox Bacteria in Full-Scale Attached Growth Nitrogen Removal System.

Leveraging comammox Nitrospira and anammox bacteria for shortcut nitrogen removal can drastically lower the carbon footprint of wastewater treatment facilities by decreasing aeration energy, carbon, alkalinity, and tank volume requirements while also potentially reducing nitrous oxide emissions. However, their co-occurrence as dominant nitrifying bacteria is rarely reported in full-scale wastewater treatment. As a result, there is a poor understanding of how operational parameters, in particular, dissolved oxygen, impact their activity and synergistic behavior. Here, we report the impact of dissolved oxygen concentration (DO = 2, 4, 6 mg/L) on the microbial community's transcriptomic expression in a full-scale integrated fixed film activated sludge (IFAS) municipal wastewater treatment facility where nitrogen removal is predominantly performed by comammox Nitrospira and anammox bacterial populations. 16S rRNA transcript compositions revealed anammox bacteria and Nitrospira were significantly more active in IFAS biofilms compared to suspended sludge biomass. In IFAS biofilms, anammox bacteria significantly increased hzo expression at lower dissolved oxygen concentrations and this increase was highly correlated with the amoA expression levels of comammox bacteria. Interestingly, the genes involved in nitrite oxidation by comammox bacteria were significantly more upregulated, relative to the genes involved in ammonia oxidation with decreasing dissolved oxygen concentrations. Ultimately, our findings suggest that comammox Nitrospira supplies anammox bacteria with nitrite via ammonia oxidation and that this synergistic behavior is dependent on dissolved oxygen concentrations.

Intensive Microalgal Cultivation and Tertiary Phosphorus Recovery from Wastewaters via the EcoRecover Process.

Mixed community microalgal wastewater treatment technologies have the potential to advance the limits of technology for biological nutrient recovery while producing a renewable carbon feedstock, but a deeper understanding of their performance is required for system optimization and control. In this study, we characterized the performance of a 568 m3·day-1 Clearas EcoRecover system for tertiary phosphorus removal (and recovery as biomass) at an operating water resource recovery facility (WRRF). The process consists of a (dark) mix tank, photobioreactors (PBRs), and a membrane tank with ultrafiltration membranes for the separation of hydraulic and solids residence times. Through continuous online monitoring, long-term on-site monitoring, and on-site batch experiments, we demonstrate (i) the importance of carbohydrate storage in PBRs to support phosphorus uptake under dark conditions in the mix tank and (ii) the potential for polyphosphate accumulation in the mixed algal communities. Over a 3-month winter period with limited outside influences (e.g., no major upstream process changes), the effluent total phosphorus (TP) concentration was 0.03 ± 0.03 mg-P·L-1 (0.01 ± 0.02 mg-P·L-1 orthophosphate). Core microbial community taxa included Chlorella spp., Scenedesmus spp., and Monoraphidium spp., and key indicators of stable performance included near-neutral pH, sufficient alkalinity, and a diel rhythm in dissolved oxygen.

Co-Occurrence and Cooperation between Comammox and Anammox Bacteria in a Full-Scale Attached Growth Municipal Wastewater Treatment Process.

Cooperation between comammox and anammox bacteria for nitrogen removal has been recently reported in laboratory-scale systems, including synthetic community constructs; however, there are no reports of full-scale municipal wastewater treatment systems with such cooperation. Here, we report intrinsic and extant kinetics as well as genome-resolved community characterization of a full-scale integrated fixed film activated sludge (IFAS) system where comammox and anammox bacteria co-occur and appear to drive nitrogen loss. Intrinsic batch kinetic assays indicated that majority of the aerobic ammonia oxidation was driven by comammox bacteria (1.75 ± 0.08 mg-N/g TS-h) in the attached growth phase, with minimal contribution by ammonia-oxidizing bacteria. Interestingly, a portion of total inorganic nitrogen (∼8%) was consistently lost during these aerobic assays. Aerobic nitrite oxidation assays eliminated the possibility of denitrification as a cause of nitrogen loss, while anaerobic ammonia oxidation assays resulted in rates consistent with anammox stoichiometry. Full-scale experiments at different dissolved oxygen (DO = 2 - 6 mg/L) setpoints indicated persistent nitrogen loss that was partly sensitive to DO concentrations. Genome-resolved metagenomics confirmed the high abundance (relative abundance 6.53 ± 0.34%) of two Brocadia-like anammox populations, while comammox bacteria within the Ca. Nitrospira nitrosa cluster were lower in abundance (0.37 ± 0.03%) and Nitrosomonas-like ammonia oxidizers were even lower (0.12 ± 0.02%). Collectively, our study reports for the first time the co-occurrence and cooperation of comammox and anammox bacteria in a full-scale municipal wastewater treatment system.

Identifying Eukaryotes and Factors Influencing Their Biogeography in Drinking Water Metagenomes.

The biogeography of eukaryotes in drinking water systems is poorly understood relative to that of prokaryotes or viruses, limiting the understanding of their role and management. A challenge with studying complex eukaryotic communities is that metagenomic analysis workflows are currently not as mature as those that focus on prokaryotes or viruses. In this study, we benchmarked different strategies to recover eukaryotic sequences and genomes from metagenomic data and applied the best-performing workflow to explore the factors affecting the relative abundance and diversity of eukaryotic communities in drinking water distribution systems (DWDSs). We developed an ensemble approach exploiting k-mer- and reference-based strategies to improve eukaryotic sequence identification and identified MetaBAT2 as the best-performing binning approach for their clustering. Applying this workflow to the DWDS metagenomes showed that eukaryotic sequences typically constituted small proportions (i.e., <1%) of the overall metagenomic data with higher relative abundances in surface water-fed or chlorinated systems with high residuals. The α and ß diversities of eukaryotes were correlated with those of prokaryotic and viral communities, highlighting the common role of environmental/management factors. Finally, a co-occurrence analysis highlighted clusters of eukaryotes whose members' presence and abundance in DWDSs were affected by disinfection strategies, climate conditions, and source water types.

Gradual Recovery of Building Plumbing-Associated Microbial Communities after Extended Periods of Altered Water Demand during the COVID-19 Pandemic.

COVID-19 pandemic-related building restrictions heightened drinking water microbiological safety concerns post-reopening due to the unprecedented nature of commercial building closures. Starting with phased reopening (i.e., June 2020), we sampled drinking water for 6 months from three commercial buildings with reduced water usage and four occupied residential households. Samples were analyzed using flow cytometry and full-length 16S rRNA gene sequencing along with comprehensive water chemistry characterization. Prolonged building closures resulted in 10-fold higher microbial cell counts in the commercial buildings [(2.95 ± 3.67) × 105 cells mL-1] than in residential households [(1.11 ± 0.58) × 104 cells mL-1] with majority intact cells. While flushing reduced cell counts and increased disinfection residuals, microbial communities in commercial buildings remained distinct from those in residential households on the basis of flow cytometric fingerprinting [Bray-Curtis dissimilarity (dBC) = 0.33 ± 0.07] and 16S rRNA gene sequencing (dBC = 0.72 ± 0.20). An increase in water demand post-reopening resulted in gradual convergence in microbial communities in water samples collected from commercial buildings and residential households. Overall, we find that the gradual recovery of water demand played a key role in the recovery of building plumbing-associated microbial communities as compared to short-term flushing after extended periods of reduced water demand.

Drift dynamics in microbial communities and the effective community size.

The structure and diversity of all open microbial communities are shaped by individual births, deaths, speciation and immigration events; the precise timings of these events are unknowable and unpredictable. This randomness is manifest as ecological drift in the population dynamics, the importance of which has been a source of debate for decades. There are theoretical reasons to suppose that drift would be imperceptible in large microbial communities, but this is at odds with circumstantial evidence that effects can be seen even in huge, complex communities. To resolve this dichotomy we need to observe dynamics in simple systems where key parameters, like migration, birth and death rates can be directly measured. We monitored the dynamics in the abundance of two genetically modified strains of Escherichia coli, with tuneable growth characteristics, that were mixed and continually fed into 10 identical chemostats. We demonstrated that the effects of demographic (non-environmental) stochasticity are very apparent in the dynamics. However, they do not conform to the most parsimonious and commonly applied mathematical models, where each stochastic event is independent. For these simple models to reproduce the observed dynamics we need to invoke an 'effective community size', which is smaller than the census community size.

Impact of Hurricane Maria on Drinking Water Quality in Puerto Rico.

This study performed a comprehensive assessment of the impact of Hurricane Maria (HM) on drinking water quality in Puerto Rico (PR) by integrating targeted chemical analysis of both inorganic (18 trace elements) and organic trace pollutants (200 micropollutants) with high-throughput quantitative toxicogenomics and in vitro biomarkers-based toxicity assays. Average concentrations of 14 detected trace elements and 20 organic micropollutants showed elevation after HM. Arsenic, sucralose, perfluorooctanoic acid (PFOA), atrazine-2-hydroxy, benzotriazole, acesulfame, and prometon were at significantly (p < 0.05) higher levels in the post-HM than in the pre-HM samples. Thirteen micropollutants, including four pesticides, were only detected in posthurricane samples. Spatial comparison showed higher pollutant and toxicity levels in the samples from northern PR (where eight Superfund sites are located) than in those from southern PR. Distinctive pathway-specific molecular toxicity fingerprints for water extracts before and after HM and at different locations revealed changes in toxicity nature that likely resulted from the impact of HM on drinking water composition. Correlation analysis and Maximum Cumulative Ratio assessment suggested that metals (i.e., arsenic) and PFOA were the top ranked pollutants that have the potential to cause increased risk after HM, providing a possible direction for future water resource management and epidemiological studies.

Comparable Nutrient Uptake across Diel Cycles by Three Distinct Phototrophic Communities.

The capacity of microalgae to advance the limit of technology of nutrient recovery and accumulate storage carbon make them promising candidates for wastewater treatment. However, the extent to which these capabilities are influenced by microbial community composition remains poorly understood. To address this knowledge gap, 3 mixed phototrophic communities sourced from distinct latitudes within the continental United States (28° N, Tampa, FL; 36° N, Durham, NC; and 40° N, Urbana, IL) were operated in sequencing batch reactors (8 day solids residence time, SRT) subjected to identical diel light cycles with media addition at the start of the nighttime period. Despite persistent differences in community structure as determined via 18S rRNA (V4 and V8-V9 hypervariable regions) and 16S rRNA (V1-V3) gene amplicon sequencing, reactors achieved similar and stable nutrient recovery after 2 months (8 SRTs) of operation. Intrinsic carbohydrate and lipid storage capacity and maximum specific carbon storage rates differed significantly across communities despite consistent levels of observed carbon storage across reactors. This work supports the assertion that distinct algal communities cultivated under a common selective environment can achieve consistent performance while maintaining independent community structures and intrinsic carbon storage capabilities, providing further motivation for the development of engineered phototrophic processes for wastewater management.

Design and Evaluation of Illumina MiSeq-Compatible, 18S rRNA Gene-Specific Primers for Improved Characterization of Mixed Phototrophic Communities.

UNLABELLED: The use of high-throughput sequencing technologies with the 16S rRNA gene for characterization of bacterial and archaeal communities has become routine. However, the adoption of sequencing methods for eukaryotes has been slow, despite their significance to natural and engineered systems. There are large variations among the target genes used for amplicon sequencing, and for the 18S rRNA gene, there is no consensus on which hypervariable region provides the most suitable representation of diversity. Additionally, it is unclear how much PCR/sequencing bias affects the depiction of community structure using current primers. The present study amplified the V4 and V8-V9 regions from seven microalgal mock communities as well as eukaryotic communities from freshwater, coastal, and wastewater samples to examine the effect of PCR/sequencing bias on community structure and membership. We found that degeneracies on the 3' end of the current V4-specific primers impact read length and mean relative abundance. Furthermore, the PCR/sequencing error is markedly higher for GC-rich members than for communities with balanced GC content. Importantly, the V4 region failed to reliably capture 2 of the 12 mock community members, and the V8-V9 hypervariable region more accurately represents mean relative abundance and alpha and beta diversity. Overall, the V4 and V8-V9 regions show similar community representations over freshwater, coastal, and wastewater environments, but specific samples show markedly different communities. These results indicate that multiple primer sets may be advantageous for gaining a more complete understanding of community structure and highlight the importance of including mock communities composed of species of interest. IMPORTANCE: The quantification of error associated with community representation by amplicon sequencing is a critical challenge that is often ignored. When target genes are amplified using currently available primers, differential amplification efficiencies result in inaccurate estimates of community structure. The extent to which amplification bias affects community representation and the accuracy with which different gene targets represent community structure are not known. As a result, there is no consensus on which region provides the most suitable representation of diversity for eukaryotes. This study determined the accuracy with which commonly used 18S rRNA gene primer sets represent community structure and identified particular biases related to PCR amplification and Illumina MiSeq sequencing in order to more accurately study eukaryotic microbial communities.

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.

Efficacy and Mechanism of Antibiotic Resistance Gene Degradation and Cell Membrane Damage during Ultraviolet Advanced Oxidation Processes.

Combinations of UV with oxidants can initiate advanced oxidation processes (AOPs) and enhance bacterial inactivation. However, the effectiveness and mechanisms of UV-AOPs in damaging nucleic acids (e.g., antibiotic resistance genes (ARGs)) and cell integrity represent a knowledge gap. This study comprehensively compared ARG degradation and cell membrane damage under three different UV-AOPs. The extracellular ARG (eARG) removal efficiency followed the order of UV/chlorine > UV/H2O2 > UV/peracetic acid (PAA). Hydroxyl radical (•OH) and reactive chlorine species (RCS) largely contributed to eARG removal, while organic radicals made a minor contribution. For intracellular ARGs (iARGs), UV/H2O2 did not remove better than UV alone due to the scavenging of •OH by cell components, whereas UV/PAA provided a modest synergism, likely due to diffusion of PAA into cells and intracellular •OH generation. Comparatively, UV/chlorine achieved significant synergistic iARG removal, suggesting the critical role of the RCS in resisting cellular scavenging and inactivating ARGs. Additionally, flow cytometry analysis demonstrated that membrane damage was mainly attributed to chlorine oxidation, while the impacts of radicals, H2O2, and PAA were negligible. These results provide mechanistic insights into bacterial inactivation and fate of ARGs during UV-AOPs, and shed light on the suitability of quantitative polymerase chain reaction (qPCR) and flow cytometry in assessing disinfection performance.

Nitrogen source influences the interactions of comammox bacteria with aerobic nitrifiers.

While the co-existence of comammox Nitrospira with canonical nitrifiers is well documented in diverse ecosystems, there is still a dearth of knowledge about the mechanisms underpinning their interactions. Understanding these interaction mechanisms is important as they may play a critical role in governing nitrogen biotransformation in natural and engineered ecosystems. In this study, we tested the ability of two environmentally relevant factors (nitrogen source and availability) to shape interactions between strict ammonia and nitrite-oxidizing bacteria and comammox Nitrospira in continuous flow column reactors. The composition of inorganic nitrogen species in reactors fed either ammonia or urea was similar during the lowest input nitrogen concentration (1 mg-N/L), but higher concentrations (2 and 4 mg-N/L) promoted significant differences in nitrogen species composition and nitrifier abundances. The abundance and diversity of comammox Nitrospira were dependent on both nitrogen source and input concentrations as multiple comammox Nitrospira populations were preferentially enriched in the urea-fed system. In contrast, their abundance was reduced in response to higher nitrogen concentrations in the ammonia-fed system. The preferential enrichment of comammox Nitrospira in the urea-fed system could be associated with their ureolytic activity calibrated to their ammonia oxidation rates, thus minimizing ammonia accumulation, which may be partially inhibitory. However, an increased abundance of comammox Nitrospira was not associated with a reduced abundance of nitrite oxidizers in the urea-fed system while a negative correlation was found between them in the ammonia-fed system, the latter dynamic likely emerging from reduced availability of nitrite to strict nitrite oxidizers at low ammonia concentrations. IMPORTANCE: Nitrification is an essential biological process in drinking water and wastewater treatment systems for treating nitrogen pollution. The discovery of comammox Nitrospira and their detection alongside canonical nitrifiers in these engineered ecosystems have made it necessary to understand the environmental conditions that regulate their abundance and activity relative to other better-studied nitrifiers. This study aimed to evaluate two important factors that could potentially influence the behavior of nitrifying bacteria and, therefore, impact nitrification processes. Column reactors fed with either ammonia or urea were systematically monitored to capture changes in nitrogen biotransformation and the nitrifying community as a function of influent nitrogen concentration, nitrogen source, and reactor depth. Our findings show that with increased ammonia availability, comammox Nitrospira decreased in abundance while nitrite oxidizers abundance increased. Yet, in systems with increasing urea availability, comammox Nitrospira abundance and diversity increased without an associated reduction in the abundance of canonical nitrifiers.

Metagenomic evaluation of the performance of passive Moore swabs for sewage monitoring relative to composite sampling over time resolved deployments.

Moore swabs have re-emerged as a versatile tool in the field of wastewater-based epidemiology during the COVID-19 pandemic and offer unique advantages for monitoring pathogens in sewer systems, especially at the neighborhood-level. However, whether Moore swabs provide comparable results to more commonly used composite samples remains to be rigorously tested including the optimal duration of Moore swab deployment. This study provides new insights into these issues by comparing the results from Moore swab samples to those of paired composite samples collected from the same sewer lines continuously over six to seventy-two hours post-deployment, during low COVID-19 prevalence periods. Our results show that Moore swabs accumulated approximately 10-fold higher PMMoV concentrations (on a basis of mL of Moore swab squeezed filtrate to mL of composite sewage) and showed comparable trends in terms of bacterial species abundance when compared to composite samples. Moore swabs also generally captured higher SARS-CoV-2 N1/N2 RNA concentrations than composite samples. Moore swabs showed comparable trends in terms of abundance dynamics of the sewage microbiome to composite samples and variable signs of saturation over time that were site and/or microbial population-specific. Based on our dual ddRT-PCR and shotgun metagenomic approach, we find that Moore swabs at our sites were optimally deployed for 6 h at a time at two sites.

Community structure and function during periods of high performance and system upset in a full-scale mixed microalgal wastewater resource recovery facility.

Microalgae have the potential to exceed current nutrient recovery limits from wastewater, enabling water resource recovery facilities (WRRFs) to achieve increasingly stringent effluent permits. The use of photobioreactors (PBRs) and the separation of hydraulic retention and solids residence time (HRT/SRT) further enables increased biomass in a reduced physical footprint while allowing operational parameters (e.g., SRT) to select for desired functional communities. However, as algal technology transitions to full-scale, there is a need to understand the effect of operational and environmental parameters on complex microbial dynamics among mixotrophic microalgae, bacterial groups, and pests (i.e., grazers and pathogens) and to implement robust process controls for stable long-term performance. Here, we examine a full-scale, intensive WRRF utilizing mixed microalgae for tertiary treatment in the US (EcoRecover, Clearas Water Recovery Inc.) during a nine-month monitoring campaign. We investigated the temporal variations in microbial community structure (18S and 16S rRNA genes), which revealed that stable system performance of the EcoRecover system was marked by a low-diversity microalgal community (DINVSIMPSON = 2.01) dominated by Scenedesmus sp. (MRA = 55 %-80 %) that achieved strict nutrient removal (effluent TP < 0.04 mg·L-1) and steady biomass concentration (TSSmonthly avg. = 400-700 mg·L-1). Operational variables including pH, alkalinity, and influent ammonium (NH4+), correlated positively (p < 0.05, method = Spearman) with algal community during stable performance. Further, the use of these parameters as operational controls along with N/P loading and SRT allowed for system recovery following upset events. Importantly, the presence or absence of bacterial nitrification did not directly impact algal system performance and overall nutrient recovery, but partial nitrification (potentially resulting from NO2- accumulation) inhibited algal growth and should be considered during long-term operation. The microalgal communities were also adversely affected by zooplankton grazers (ciliates, rotifers) and fungal parasites (Aphelidium), particularly during periods of upset when algal cultures were experiencing culture turnover or stress conditions (e.g., nitrogen limitation, elevated temperature). Overall, the active management of system operation in order to maintain healthy algal cultures and high biomass productivity can result in significant periods (>4 months) of stable system performance that achieve robust nutrient recovery, even in winter months in northern latitudes (WI, USA).

Long-term impact of Hurricane Maria on point-of-use drinking water quality in Puerto Rico and associated potential adverse health effects.

Drinking water security in Puerto Rico (PR) is increasingly challenged by both regulated and emerging anthropogenic contaminants, which was exacerbated by the Hurricane Maria (HM) due to impaired regional water cycle and damaged water infrastructure. Leveraging the NIEHS PROTECT (Puerto Rico Testsite for Exploring Contamination Threats) cohort, this study assessed the long-term tap water (TW) quality changes from March 2018 to November 2018 after HM in PR, by innovatively integrating two different effect-based quantitative toxicity assays with a targeted analysis of 200 organic and 22 inorganic pollutants. Post-hurricane PR TW quality showed recovery after >6-month period as indicated by the decreased number of contaminants showing elevated average concentrations relative to pre-hurricane samples, with significant difference of both chemical and toxicity levels between northern and southern PR. Molecular toxicity profiling and correlation revealed that the HM-accelerated releases of certain pesticides and PPCPs could exert increased cellular oxidative and/or AhR (aryl hydrocarbon receptor)-mediated activities that may persist for more than six months after HM. Maximum cumulative ratio and adverse outcome pathway (AOP) assessment identified the top ranked detected TW contaminants (Cu, Sr, V, perfluorooctanoic acid) that potentially associated with different adverse health effects such as inflammation, impaired reproductive systems, cancers/tumors, and/or organ toxicity. These insights can be incorporated into the regulatory framework for post-disaster risk assessment, guiding water quality control and management for public health protection.

Effects of error, chimera, bias, and GC content on the accuracy of amplicon sequencing.

IMPORTANCE: Amplicon sequencing of targeted genes is the predominant approach to estimate the membership and structure of microbial communities. However, accurate reconstruction of community composition is difficult due to sequencing errors, and other methodological biases and effective approaches to overcome these challenges are essential. Using a mock community of 33 phylogenetically diverse strains, this study evaluated the effect of GC content on sequencing results and tested different approaches to improve overall sequencing accuracy while characterizing the pros and cons of popular amplicon sequence data processing approaches. The sequencing results from this study can serve as a benchmarking data set for future algorithmic improvements. Furthermore, the new insights on sequencing error, chimera formation, and GC bias from this study will help enhance the quality of amplicon sequencing studies and support the development of new data analysis approaches.

Low diversity and microdiversity of comammox bacteria in wastewater systems suggest specific adaptations within the Ca. Nitrospira nitrosa cluster.

Studies have found Ca. Nitrospira nitrosa-like bacteria to be the principal or sole comammox bacteria in nitrogen removal systems for wastewater treatment. In contrast, multiple populations of strict ammonia and nitrite oxidizers co-exist in similar systems. This apparent lack of diversity is surprising and could impact the feasibility of leveraging comammox bacteria for nitrogen removal. We used full-length 16S rRNA gene sequencing and genome-resolved metagenomics to compare the species-level diversity of comammox bacteria with that of strict nitrifiers in full-scale wastewater treatment systems and assess whether this comparison is consistent or diverged at the strain-level. Full-length 16S rRNA gene sequencing indicated that Nitrosomonas-like bacteria exhibited higher species-level diversity in comparison with other nitrifying bacteria, while the strain-level diversity (also called microdiversity) of most Nitrospira-like bacteria were higher than Nitrosomonas-like bacteria with few exceptions (one Nitrospira lineage II population). Comammox bacterial metagenome assembled genomes (MAGs) were associated with Ca. Nitrospira nitrosa. The average amino acid identity between principal comammox bacterial MAGs (93% ± 3) across systems was significantly higher than that of the Nitrosomonas-like ammonia oxidizers (73% ± 8), the Nitrospira_A-like nitrite oxidizer (85% ± 4), and the Nitrospira_D-like nitrite oxidizer (83% ± 1). This demonstrated the low species-level diversity of comammox bacteria compared with strict nitrifiers and further suggests that the same comammox population was detected in all systems. Comammox bacteria (Nitrospira lineage II), Nitrosomonas and, Nitrospira_D (Nitrospira lineage II) MAGs were significantly less microdiverse than the Nitrospira_A (lineage I) MAGs. Interestingly, strain-resolved analysis also indicates that different nitrogen removal systems harbor different comammox bacterial strains within the Ca. Nitrospira nitrosa cluster. These results suggest that comammox bacteria associated with Ca. Nitrospira nitrosa have low species- and strain-level diversity in nitrogen removal systems and may thus harbor specific adaptations to the wastewater ecosystem.

Parallel deployment of passive and composite samplers for surveillance and variant profiling of SARS-CoV-2 in sewage.

Wastewater-based epidemiology during the COVID-19 pandemic has proven useful for public health decision-making but is often hampered by sampling methodology constraints, particularly at the building- or neighborhood-level. Time-weighted composite samples are commonly used; however, autosamplers are expensive and can be affected by intermittent flows in sub-sewershed contexts. In this study, we compared time-weighted composite, grab, and passive sampling via Moore swabs, at four locations across a college campus to understand the utility of passive sampling. After optimizing the methods for sample handling and processing for viral RNA extraction, we quantified SARS-CoV-2 N1 and N2, as well as a fecal strength indicator, PMMoV, by ddRT-PCR and applied tiled amplicon sequencing of the SARS-CoV-2 genome. Passive samples compared favorably with composite samples in our study area: for samples collected concurrently, 42 % of the samples agreed between Moore swab and composite samples and 58 % of the samples were positive for SARS-CoV-2 using Moore swabs while composite samples were below the limit of detection. Variant profiles from Moore swabs showed a shift from variant BA.1 to BA.2, consistent with in-person saliva samples. These data have implications for the broader implementation of sewage surveillance without advanced sampling technologies and for the utilization of passive sampling approaches for other emerging pathogens.

Bioreactor function under perturbation scenarios is affected by interactions between bacteria and protozoa.

This study investigated the impact of transient cadmium perturbations on the structure and function of the microbial community in an activated sludge system. The impact of cadmium perturbation on the bioreactor performance, bacterial activity, bacterial community structure, and bacteria-protozoa interactions was examined. The bacterial community exhibited a short-term inhibition following a pulse perturbation of cadmium. Process recovery was associated with an increase in bacterial abundance above the unperturbed control reactor, followed by high biomass activity after the washout of cadmium. This trend was seen for multiple experiments at both laboratory- and pilot-scale. The increase in biomass activity could not be explained by changes in bacterial community structure. Independent experiments showed that the increase in bacterial abundance, and by association biomass activity, was caused by the decrease in the protozoal grazing due to the higher inhibition of ciliated protozoa as compared to bacteria when exposed to cadmium. This paper highlights the importance of expanding the investigative boundaries of the microbial ecology of bioengineered systems to include protozoal grazing, especially under perturbation scenarios.