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.

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.

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.

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.

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.

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.

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.

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.

A snapshot of the global drinking water virome: Diversity and metabolic potential vary with residual disinfectant use.

Viruses are important drivers of microbial community ecology and evolution, influencing microbial mortality, metabolism, and horizontal gene transfer. However, the effects of viruses remain largely unknown in many environments, including in drinking water systems. Drinking water metagenomic studies have offered a whole community perspective of bacterial impacts on water quality, but have not yet considered the influences of viruses. In this study, we address this gap by mining viral DNA sequences from publicly available drinking water metagenomes from distribution systems in six countries around the world. These datasets provide a snapshot of the taxonomic diversity and metabolic potential of the global drinking water virome; and provide an opportunity to investigate the effects of geography, climate, and drinking water treatment practices on viral diversity. Both environmental conditions and differences in sample processing were found to influence the viral composition. Using free chlorine as the residual disinfectant was associated with clear differences in viral taxonomic diversity and metabolic potential, with significantly fewer viral populations and less even viral community structures than observed in distribution systems without residual disinfectant. Additionally, drinking water viruses carry antibiotic resistance genes (ARGs), as well as genes to survive oxidative stress and nitrogen limitation. Through this study, we have demonstrated that viral communities are diverse across drinking water systems and vary with the use of residual disinfectant. Our findings offer directions for future research to develop a more robust understanding of how virus-bacteria interactions in drinking water distribution systems affect water quality.

Comammox Nitrospira bacteria outnumber canonical nitrifiers irrespective of electron donor mode and availability in biofiltration systems.

Complete ammonia oxidizing bacteria coexist with canonical ammonia and nitrite oxidizing bacteria in a wide range of environments. Whether this is due to competitive or cooperative interactions, or a result of niche separation is not yet clear. Understanding the factors driving coexistence of nitrifiers is critical to manage nitrification processes occurring in engineered and natural ecosystems. In this study, microcosm-based experiments were used to investigate the impact of nitrogen source and loading on the population dynamics of nitrifiers in drinking water biofilter media. Shotgun sequencing of DNA followed by co-assembly and reconstruction of metagenome assembled genomes revealed clade A2 comammox bacteria were likely the primary nitrifiers within microcosms and increased in abundance over Nitrosomonas-like ammonia and Nitrospira-like nitrite oxidizing bacteria irrespective of nitrogen source type or loading. Changes in comammox bacterial abundance did not correlate with either ammonia or nitrite oxidizing bacterial abundance in urea-amended systems, where metabolic reconstruction indicated potential for cross-feeding between strict ammonia and nitrite oxidizers. In contrast, comammox bacterial abundance demonstrated a negative correlation with nitrite oxidizers in ammonia-amended systems. This suggests potentially weaker synergistic relationships between strict ammonia and nitrite oxidizers might enable comammox bacteria to displace strict nitrite oxidizers from complex nitrifying communities.

Simple Affinity-Based Method for Concentrating Viruses from Wastewater Using Engineered Curli Fibers.

Wastewater surveillance is a proven method for tracking community spread and prevalence of some infectious viral diseases. A primary concentration step is often used to enrich viral particles from wastewater prior to subsequent viral quantification and/or sequencing. Here, we present a simple procedure for concentrating viruses from wastewater using bacterial biofilm protein nanofibers known as curli fibers. Through simple genetic engineering, we produced curli fibers functionalized with single-domain antibodies (also known as nanobodies) specific for the coat protein of the model virus bacteriophage MS2. Using these modified fibers in a simple spin-down protocol, we demonstrated efficient concentration of MS2 in both phosphate-buffered saline (PBS) and in the wastewater matrix. Additionally, we produced nanobody-functionalized curli fibers capable of binding the spike protein of SARS-CoV-2, showing the versatility of the system. Our concentration protocol is simple to implement, can be performed quickly under ambient conditions, and requires only components produced through bacterial culture. We believe this technology represents an attractive alternative to existing concentration methods and warrants further research and optimization for field-relevant applications.

Evaluating de Novo Assembly and Binning Strategies for Time Series Drinking Water Metagenomes.

Reconstructing microbial genomes from metagenomic short-read data can be challenging due to the unknown and uneven complexity of microbial communities. This complexity encompasses highly diverse populations, which often includes strain variants. Reconstructing high-quality genomes is a crucial part of the metagenomic workflow, as subsequent ecological and metabolic inferences depend on their accuracy, quality, and completeness. In contrast to microbial communities in other ecosystems, there has been no systematic assessment of genome-centric metagenomic workflows for drinking water microbiomes. In this study, we assessed the performance of a combination of assembly and binning strategies for time series drinking water metagenomes that were collected over 6 months. The goal of this study was to identify the combination of assembly and binning approaches that result in high-quality and -quantity metagenome-assembled genomes (MAGs), representing most of the sequenced metagenome. Our findings suggest that the metaSPAdes coassembly strategies had the best performance, as they resulted in larger and less fragmented assemblies, with at least 85% of the sequence data mapping to contigs greater than 1 kbp. Furthermore, a combination of metaSPAdes coassembly strategies and MetaBAT2 produced the highest number of medium-quality MAGs while capturing at least 70% of the metagenomes based on read recruitment. Utilizing different assembly/binning approaches also assists in the reconstruction of unique MAGs from closely related species that would have otherwise collapsed into a single MAG using a single workflow. Overall, our study suggests that leveraging multiple binning approaches with different metaSPAdes coassembly strategies may be required to maximize the recovery of good-quality MAGs. IMPORTANCE Drinking water contains phylogenetic diverse groups of bacteria, archaea, and eukarya that affect the esthetic quality of water, water infrastructure, and public health. Taxonomic, metabolic, and ecological inferences of the drinking water microbiome depend on the accuracy, quality, and completeness of genomes that are reconstructed through the application of genome-resolved metagenomics. Using time series metagenomic data, we present reproducible genome-centric metagenomic workflows that result in high-quality and -quantity genomes, which more accurately signifies the sequenced drinking water microbiome. These genome-centric metagenomic workflows will allow for improved taxonomic and functional potential analysis that offers enhanced insights into the stability and dynamics of drinking water microbial communities.

Oligotyping and metagenomics reveal distinct Candidatus Accumulibacter communities in side-stream versus conventional full-scale enhanced biological phosphorus removal (EBPR) systems.

Candidatus Accumulibacter phosphatis (CAP) and its clade-level micro-diversity has been associated with and implicated in functional differences in phosphorus removal performance in enhanced biological phosphorus removal (EBPR) systems. Side-stream EBPR (S2EBPR) is an emerging process that has been shown to present a suite of advantages over the conventional EBPR design, however, large knowledge gaps remain in terms of its underlying ecological mechanisms. Here, we compared and revealed the higher-resolution differences in microbial ecology of CAP between a full-scale side-stream EBPR configuration and a conventional A2O EBPR process that were operated in parallel and with the same influent feed. Even though the relative abundance of CAP, revealed by 16S rRNA gene amplicon sequencing, was similar in both treatment trains, a clade-level analysis, using combined 16S rRNA-gene based amplicon sequencing and oligotyping analysis and metagenomics analysis, revealed the distinct CAP microdiversity between the S2EBPR and A2O configurations that likely attributed to the improved performance in S2EBPR in comparison to conventional EBPR. Furthermore, genome-resolved metagenomics enabled extraction of three metagenome-assembled genomes (MAGs) belonging to CAP clades IIB (RCAB4-2), IIC (RC14) and II (RC18), from full-scale EBPR sludge for the first time, including a distinct Ca. Accumulibacter clade that is dominant and associated only with the S2EBPR configuration. The results also revealed the temporally increasing predominance of RC14, which belonged to Clade IIC, during the implementation of the S2EBPR configuration. Finally, we also show the existence of previously uncharacterized diversity of clades of CAP, namely the clades IIB and as yet unidentified clade of type II, in full-scale EBPR communities, highlighting the unknown diversity of CAP communities in full-scale EBPR systems.

Spatial-temporal targeted and non-targeted surveys to assess microbiological composition of drinking water in Puerto Rico following Hurricane Maria.

Loss of basic utilities, such as drinking water and electricity distribution, were sustained for months in the aftermath of Hurricane Maria's (HM) landfall in Puerto Rico (PR) in September 2017. The goal of this study was to assess if there was deterioration in biological quality of drinking water due to these disruptions. This study characterized the microbial composition of drinking water following HM across nine drinking water systems (DWSs) in PR and utilized an extended temporal sampling campaign to determine if changes in the drinking water microbiome were indicative of HM associated disturbance followed by recovery. In addition to monitoring water chemistry, the samples were subjected to culture independent targeted and non-targeted microbial analysis including quantitative PCR (qPCR) and genome-resolved metagenomics. The qPCR results showed that residual disinfectant was the major driver of bacterial concentrations in tap water with marked decrease in concentrations from early to late sampling timepoints. While Mycobacterium avium and Pseudomonas aeruginosa were not detected in any sampling locations and timepoints, genetic material from Leptospira and Legionella pneumophila were transiently detected in a few sampling locations. The majority of metagenome assembled genomes (MAGs) recovered from these samples were not associated with pathogens and were consistent with bacterial community members routinely detected in DWSs. Further, whole metagenome-level comparisons between drinking water samples collected in this study with samples from other full-scale DWS indicated no significant deviation from expected community membership of the drinking water microbiome. Overall, our results suggest that disruptions due to HM did not result in significant and sustained deterioration of biological quality of drinking water at our study sites.

Prospects for multi-omics in the microbial ecology of water engineering.

Advances in high-throughput sequencing technologies and bioinformatics approaches over almost the last three decades have substantially increased our ability to explore microorganisms and their functions - including those that have yet to be cultivated in pure isolation. Genome-resolved metagenomic approaches have enabled linking powerful functional predictions to specific taxonomical groups with increasing fidelity. Additionally, related developments in both whole community gene expression surveys and metabolite profiling have permitted for direct surveys of community-scale functions in specific environmental settings. These advances have allowed for a shift in microbiome science away from descriptive studies and towards mechanistic and predictive frameworks for designing and harnessing microbial communities for desired beneficial outcomes. Water engineers, microbiologists, and microbial ecologists studying activated sludge, anaerobic digestion, and drinking water distribution systems have applied various (meta)omics techniques for connecting microbial community dynamics and physiologies to overall process parameters and system performance. However, the rapid pace at which new omics-based approaches are developed can appear daunting to those looking to apply these state-of-the-art practices for the first time. Here, we review how modern genome-resolved metagenomic approaches have been applied to a variety of water engineering applications from lab-scale bioreactors to full-scale systems. We describe integrated omics analysis across engineered water systems and the foundations for pairing these insights with modeling approaches. Lastly, we summarize emerging omics-based technologies that we believe will be powerful tools for water engineering applications. Overall, we provide a framework for microbial ecologists specializing in water engineering to apply cutting-edge omics approaches to their research questions to achieve novel functional insights. Successful adoption of predictive frameworks in engineered water systems could enable more economically and environmentally sustainable bioprocesses as demand for water and energy resources increases.

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.

Episodic Decrease in Temperature Increases mcy Gene Transcription and Cellular Microcystin in Continuous Cultures of Microcystis aeruginosa PCC 7806.

Microcystins produced during harmful cyanobacterial blooms are a public health concern. Although patterns are emerging, the environmental cues that stimulate production of microcystin remain confusing, hindering our ability to predict fluctuations in bloom toxicity. In earlier work, growth at cool temperatures relative to optimum (18°C vs. 26°C) was confirmed to increase microcystin quota in batch cultures of Microcystis aeruginosa NIES-843. Here, we tested this response in M. aeruginosa PCC 7806 using continuous cultures to examine temporal dynamics and using RNA-sequencing to investigate the physiological nature of the response. A temperature reduction from 26 to 19°C increased microcystin quota ∼2-fold, from an average of ∼464 ag µm-3 cell volume to ∼891 ag µm-3 over a 7-9 d period. Reverting the temperature to 26°C returned the cellular microcystin quota to ∼489 ag µm-3. Long periods (31-42 d) at 19°C did not increase or decrease microcystin quota beyond that observed at 7-9 d. Nitrogen concentration had little effect on the overall response. RNA sequencing indicated that the decrease in temperature to 19°C induced a classic cold-stress response in M. aeruginosa PCC 7806, but this operated on a different timescale than the increased microcystin production. Microcystin quota showed a strong 48- to 72-h time-lag correlation to mcy gene expression, but no correlation to concurrent mcy expression. This work confirms an effect of temperature on microcystin quota and extends our understanding of the physiological nature of the response.

Differential prevalence and host-association of antimicrobial resistance traits in disinfected and non-disinfected drinking water systems.

Antimicrobial resistance (AMR) in drinking water has received less attention than its counterparts in the urban water cycle. While culture-based techniques or gene-centric PCR have been used to probe the impact of treatment approaches (e.g., disinfection) on AMR in drinking water, to our knowledge there is no systematic comparison of AMR trait distribution and prevalence between disinfected and disinfectant residual-free drinking water systems. We used metagenomics to assess the associations between disinfectant residuals and AMR prevalence and its host association in full-scale drinking water distribution systems (DWDSs) with and without disinfectant residuals. While the differences in AMR profiles between DWDSs were associated with the presence or absence of disinfectant, they were also associated with overall water chemistry and more importantly with microbial community structure. AMR genes and mechanisms differentially abundant in disinfected systems were primarily associated with nontuberculous mycobacteria (NTM). Finally, evaluation of metagenome assembled genomes (MAGs) also suggests that NTM possessing AMR genes conferring intrinsic resistance to key antibiotics were prevalent in disinfected systems, whereas such NTM genomes were not detected in disinfectant residual free DWDSs. Altogether, our findings provide insights into the drinking water resistome and its association with potential opportunistic pathogens, particularly in systems with disinfectant residual.