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.

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.

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.

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.

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.

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.

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.

Microbial Nitrogen Metabolism in Chloraminated Drinking Water Reservoirs.

Ammonia availability due to chloramination can promote the growth of nitrifying organisms, which can deplete chloramine residuals and result in operational problems for drinking water utilities. In this study, we used a metagenomic approach to determine the identity and functional potential of microorganisms involved in nitrogen biotransformation within chloraminated drinking water reservoirs. Spatial changes in the nitrogen species included an increase in nitrate concentrations accompanied by a decrease in ammonium concentrations with increasing distance from the site of chloramination. This nitrifying activity was likely driven by canonical ammonia-oxidizing bacteria (i.e., Nitrosomonas) and nitrite-oxidizing bacteria (i.e., Nitrospira) as well as by complete-ammonia-oxidizing (i.e., comammox) Nitrospira-like bacteria. Functional annotation was used to evaluate genes associated with nitrogen metabolism, and the community gene catalogue contained mostly genes involved in nitrification, nitrate and nitrite reduction, and nitric oxide reduction. Furthermore, we assembled 47 high-quality metagenome-assembled genomes (MAGs) representing a highly diverse assemblage of bacteria. Of these, five MAGs showed high coverage across all samples, which included two Nitrosomonas, Nitrospira, Sphingomonas, and Rhizobiales-like MAGs. Systematic genome-level analyses of these MAGs in relation to nitrogen metabolism suggest that under ammonia-limited conditions, nitrate may be also reduced back to ammonia for assimilation. Alternatively, nitrate may be reduced to nitric oxide and may potentially play a role in regulating biofilm formation. Overall, this study provides insight into the microbial communities and their nitrogen metabolism and, together with the water chemistry data, improves our understanding of nitrogen biotransformation in chloraminated drinking water distribution systems.IMPORTANCE Chloramines are often used as a secondary disinfectant when free chlorine residuals are difficult to maintain. However, chloramination is often associated with the undesirable effect of nitrification, which results in operational problems for many drinking water utilities. The introduction of ammonia during chloramination provides a potential source of nitrogen either through the addition of excess ammonia or through chloramine decay. This promotes the growth of nitrifying microorganisms and provides a nitrogen source (i.e., nitrate) for the growth for other organisms. While the roles of canonical ammonia-oxidizing and nitrite-oxidizing bacteria in chloraminated drinking water systems have been extensively investigated, those studies have largely adopted a targeted gene-centered approach. Further, little is known about the potential long-term cooccurrence of complete-ammonia-oxidizing (i.e., comammox) bacteria and the potential metabolic synergies of nitrifying organisms with their heterotrophic counterparts that are capable of denitrification and nitrogen assimilation. This study leveraged data obtained for genome-resolved metagenomics over a time series to show that while nitrifying bacteria are dominant and likely to play a major role in nitrification, their cooccurrence with heterotrophic organisms suggests that nitric oxide production and nitrate reduction to ammonia may also occur in chloraminated drinking water systems.

Disinfection exhibits systematic impacts on the drinking water microbiome.

Limiting microbial growth during drinking water distribution is achieved either by maintaining a disinfectant residual or through nutrient limitation without using a disinfectant. The impact of these contrasting approaches on the drinking water microbiome is not systematically understood. We use genome-resolved metagenomics to compare the structure, metabolic traits, and population genomes of drinking water microbiome samples from bulk drinking water across multiple full-scale disinfected and non-disinfected drinking water systems. Microbial communities cluster at the structural- and functional potential-level based on the presence/absence of a disinfectant residual. Disinfectant residual alone explained 17 and 6.5% of the variance in structure and functional potential of the drinking water microbiome, respectively, despite including multiple drinking water systems with variable source waters and source water communities and treatment strategies. The drinking water microbiome is structurally and functionally less diverse and variable across disinfected compared to non-disinfected systems. While bacteria were the most abundant domain, archaea and eukaryota were more abundant in non-disinfected and disinfected systems, respectively. Community-level differences in functional potential were driven by enrichment of genes associated with carbon and nitrogen fixation in non-disinfected systems and γ-aminobutyrate metabolism in disinfected systems likely associated with the recycling of amino acids. Genome-level analyses for a subset of phylogenetically-related microorganisms suggests that disinfection selects for microorganisms capable of using fatty acids, presumably from microbial decay products, via the glyoxylate cycle. Overall, we find that disinfection exhibits systematic selective pressures on the drinking water microbiome and may select for microorganisms able to utilize microbial decay products originating from disinfection-inactivated microorganisms. Video abstract.

Long solids retention times and attached growth phase favor prevalence of comammox bacteria in nitrogen removal systems.

The discovery of the complete ammonia oxidizing (comammox) bacteria overturns the traditional two-organism nitrification paradigm which largely underpins the design and operation of nitrogen removal during wastewater treatment. Quantifying the abundance, diversity, and activity of comammox bacteria in wastewater treatment systems is important for ensuring a clear understanding of the nitrogen biotransformations responsible for ammonia removal. To this end, we conducted a yearlong survey of 14 full-scale nitrogen removal systems including mainstream conventional and simultaneous nitrification-denitrification and side-stream partial nitrification-anammox systems with varying process configurations. Metagenomics and genome-resolved metagenomics identified comammox bacteria in mainstream conventional and simultaneous nitrification-denitrification systems, with no evidence for their presence in side-stream partial nitrification-anammox systems. Further, comammox bacterial diversity was restricted to clade A and these clade A comammox bacteria were detected in systems with long solids retention times (>10 days) and/or in the attached growth phase. Using a newly designed qPCR assay targeting the amoB gene of clade A comammox bacteria in combination with quantitation of other canonical nitrifiers, we show that long solids retention time is the key process parameter associated with the prevalence and abundance of comammox bacteria. The increase in comammox bacterial abundance was not associated with concomitant decrease in the abundance of canonical nitrifiers; however, systems with comammox bacteria showed significantly better and temporally stable ammonia removal compared to systems where they were not detected. Finally, in contrast to recent studies, we do not find any significant association of comammox bacterial prevalence and abundance with dissolved oxygen concentrations in this study.

Applying biotechnology for drinking water biofiltration: advancing science and practice.

Drinking water biofiltration processes have evolved over time, moving from unintentional to deliberate, with careful filter media selection, nutrient and trace metal supplementation, oxidant amendment, and bioaugmentation of key microorganisms, to achieve improvements in water quality. Biofiltration is on the precipice of a revolution that aims to customize the microbial community for targeted functional outcomes. These outcomes might be to enhance or introduce target functional activity for contaminant removal, to avoid hydraulic challenges, or to shape beneficially the downstream microbial community. Moving from the foundational molecular techniques that are commonly applied to biofiltration processes, such as amplicon sequencing and quantitative, real-time polymerase chain reaction, the biofiltration revolution will be facilitated by modern biotechnological tools, including metagenomics, metatranscriptomics, and metaproteomics. The application of such tools will provide a rich knowledge base of microbial community structure/function data under various water quality and operational conditions, where this information will be utilized to select biofilter conditions that promote the enrichment and maintenance of microorganisms with the desired functions.

Drinking Water Microbiome Project: Is it Time?

Now is an opportune time to foster collaborations across sectors and geographical boundaries to enable development of best practices for drinking water (DW) microbiome research, focusing on accuracy and reproducibility of meta-omic techniques (while learning from past microbiome projects). A large-scale coordinated effort that builds on this foundation will enable the urgently needed comprehensive spatiotemporal understanding and control of DW microbiomes by engineering interventions to protect public health. This opinion paper highlights the need to initiate and conduct a large-scale coordinated DW microbiome project by addressing key knowledge gaps and recommends a roadmap for this effort.

Impact of solids residence time on community structure and nutrient dynamics of mixed phototrophic wastewater treatment systems.

Suspended growth, mixed community phototrophic wastewater treatment systems (including high-rate algal ponds and photobioreactors) have the potential to achieve biological nitrogen and phosphorus recovery with effluent nutrient concentrations below the current limit-of-technology. In order to achieve reliable and predictive performance, it is necessary to establish a thorough understanding of how design and operational decisions influence the complex community structure governing nutrient recovery in these systems. Solids residence time (SRT), a critical operational parameter governing growth rate, was leveraged as a selective pressure to shape microbial community structure in laboratory-scale photobioreactors fed secondary effluent from a local wastewater treatment plant. In order to decouple the effects of SRT and hydraulic retention time (HRT), nutrient loading was fixed across all experimental conditions and the effect of changing SRT on microbial community structure, diversity, and stability, as well as its impact on nutrient recovery, was characterized. Reactors were operated at distinct SRTs (5, 10, and 15 days) with diurnal lighting over long-term operation (>6 SRTs), and in-depth examination of the eukaryotic and bacterial community structure was performed using amplicon-based sequencing of the 18S and 16S rRNA genes, respectively. In order to better represent the microalgal community structure, this study leveraged improved 18S rRNA gene primers that have been shown to provide a more accurate representation of the wastewater process-relevant algal community members. Long-term operation resulted in distinct eukaryotic communities across SRTs, independent of the relative abundance of Operational Taxonomic Units (OTUs) in the inoculum. The longest SRT (15 days, SRT 15) resulted in a more stable algal community along with stable bacterial nitrification, while the shortest SRT (5 days, SRT 5) resulted in a less stable, more dynamic community. Although SRT was not strongly associated with overall bacterial diversity, the eukaryotic community of SRT 15 was significantly less diverse and less even than SRT 5, with a few dominant OTUs making up a majority of the eukaryotic community structure in the former. Overall, although longer SRTs promote stable bacterial nitrification, short SRTs promote higher eukaryotic diversity, increased functional stability, and better total N removal via biomass assimilation. These results indicate that SRT may be a key factor in not only controlling microalgal community membership, but community diversity and functional stability as well. Ultimately, the efficacy and reliability of NH4+ removal may be in tension with TN removal in mixed phototrophic systems given that lower SRTs may achieve better total N removal (via biomass assimilation) through increased eukaryotic diversity, biomass productivity, and functional stability.

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.

NanoAmpli-Seq: a workflow for amplicon sequencing for mixed microbial communities on the nanopore sequencing platform.

Background: Amplicon sequencing on Illumina sequencing platforms leverages their deep sequencing and multiplexing capacity but is limited in genetic resolution due to short read lengths. While Oxford Nanopore or Pacific Biosciences sequencing platforms overcome this limitation, their application has been limited due to higher error rates or lower data output. Results: In this study, we introduce an amplicon sequencing workflow, i.e., NanoAmpli-Seq, that builds on the intramolecular-ligated nanopore consensus sequencing (INC-Seq) approach and demonstrate its application for full-length 16S rRNA gene sequencing. NanoAmpli-Seq includes vital improvements to the INC-Seq protocol that reduces sample processing time while significantly improving sequence accuracy. The developed protocol includes chopSeq software for fragmentation and read orientation correction of INC-Seq consensus reads while nanoClust algorithm was designed for read partitioning-based de novo clustering and within cluster consensus calling to obtain accurate full-length 16S rRNA gene sequences. Conclusions: NanoAmpli-Seq accurately estimates the diversity of tested mock communities with average consensus sequence accuracy of 99.5% for 2D and 1D2 sequencing on the nanopore sequencing platform. Nearly all residual errors in NanoAmpli-Seq sequences originate from deletions in homopolymer regions, indicating that homopolymer aware base calling or error correction may allow for sequencing accuracy comparable to short-read sequencing platforms.

The active microbial community more accurately reflects the anaerobic digestion process: 16S rRNA (gene) sequencing as a predictive tool.

BACKGROUND: Amplicon sequencing methods targeting the 16S rRNA gene have been used extensively to investigate microbial community composition and dynamics in anaerobic digestion. These methods successfully characterize amplicons but do not distinguish micro-organisms that are actually responsible for the process. In this research, the archaeal and bacterial community of 48 full-scale anaerobic digestion plants were evaluated on DNA (total community) and RNA (active community) level via 16S rRNA (gene) amplicon sequencing. RESULTS: A significantly higher diversity on DNA compared with the RNA level was observed for archaea, but not for bacteria. Beta diversity analysis showed a significant difference in community composition between the DNA and RNA of both bacteria and archaea. This related with 25.5 and 42.3% of total OTUs for bacteria and archaea, respectively, that showed a significant difference in their DNA and RNA profiles. Similar operational parameters affected the bacterial and archaeal community, yet the differentiating effect between DNA and RNA was much stronger for archaea. Co-occurrence networks and functional prediction profiling confirmed the clear differentiation between DNA and RNA profiles. CONCLUSIONS: In conclusion, a clear difference in active (RNA) and total (DNA) community profiles was observed, implying the need for a combined approach to estimate community stability in anaerobic digestion.

The control of disinfection byproducts and their precursors in biologically active filtration processes.

While disinfection provides hygienically safe drinking water, the disinfectants react with inorganic or organic precursors, leading to the formation of harmful disinfection byproducts (DBPs). Biological filtration is a process in which an otherwise conventional granular filter is designed to remove not only fine particulates but also dissolved organic matters (e.g., DBP precursors) through microbially mediated degradation. Recently, applications of biofiltration in drinking water treatment have increased significantly. This review summarizes the effectiveness of biofiltration in removing DBPs and their precursors and identifies potential factors in biofilters that may control the removal or contribute to formation of DBP and their precursors during drinking water treatment. Biofiltration can remove a fraction of the precursors of halogenated DBPs (trihalomethanes, haloacetic acids, haloketones, haloaldehydes, haloacetonitriles, haloacetamides, and halonitromethanes), while also demonstrating capability in removing bromate and halogenated DBPs, except for trihalomethanes. However, the effectiveness of biofiltration mediated removal of nitrosamine and its precursors appears to be variable. An increase in nitrosamine precursors after biofiltration was ascribed to the biomass sloughing off from media or direct nitrosamine formation in the biofilter under certain denitrifying conditions. Operating parameters, such as pre-ozonation, media type, empty bed contact time, backwashing, temperature, and nutrient addition may be optimized to control the regulated DBPs in the biofilter effluent while minimizing the formation of unregulated emerging DBPs. While summarizing the state of knowledge of biofiltration mediated control of DBPs, this review also identifies several knowledge gaps to highlight future research topics of interest.

Electrochemical sensors for identifying pyocyanin production in clinical Pseudomonas aeruginosa isolates.

In clinical practice, delays in obtaining culture results impact patient care and the ability to tailor antibiotic therapy. Despite the advancement of rapid molecular diagnostics, the use of plate cultures inoculated from swab samples continues to be the standard practice in clinical care. Because the inoculation culture process can take between 24 and 48h before a positive identification test can be run, there is an unmet need to develop rapid throughput methods for bacterial identification. Previous work has shown that pyocyanin can be used as a rapid, redox-active biomarker for identifying Pseudomonas aeruginosa in clinical infections. However, further validation is needed to confirm pyocyanin production occurs in all clinical strains of P. aeruginosa. Here, we validate this electrochemical detection strategy using clinical isolates obtained from patients with hospital-acquired infections or with cystic fibrosis. Square-wave voltammetric scans of 94 different clinical P. aeruginosa isolates were taken to measure the concentration of pyocyanin. The results showed that all isolates produced measureable concentrations of pyocyanin with production rates correlated with patient symptoms and comorbidity. Further bioinformatics analysis confirmed that 1649 genetically sequenced strains (99.9%) of P. aeruginosa possess the two genes (PhzM and PhzS) necessary to produce pyocyanin, supporting the specificity of this biomarker. Confirming the production of pyocyanin by all clinically-relevant strains of P. aeruginosa is a significant step towards validating this strategy for rapid, point-of-care diagnostics.