Activated sludge microbial community assembly: the role of influent microbial community immigration.

Wastewater treatment plants (WWTPs) are host to diverse microbial communities and receive a constant influx of microbes from influent wastewater. However, the impact of immigrants on the structure and activities of the activated sludge (AS) microbial community remains unclear. To gain insight on this phenomenon known as perpetual community coalescence, the current study utilized controlled manipulative experiments that decoupled the influent wastewater composition from the microbial populations to reveal the fundamental mechanisms involved in immigration between sewers and AS-WWTP. The immigration dynamics of heterotrophs were analyzed by harvesting wastewater biomass solids from three different sewer systems and adding to synthetic wastewater. Immigrating influent populations were observed to contribute up to 14% of the sequencing reads in the AS. By modeling the net growth rate of taxa, it was revealed that immigrants primarily exhibited low or negative net growth rates. By developing a protocol to reproducibly grow AS-WWTP communities in the lab, we have laid down the foundational principles for the testing of operational factors creating community variations with low noise and appropriate replication. Understanding the processes that drive microbial community diversity and assembly is a key question in microbial ecology. In the future, this knowledge can be used to manipulate the structure of microbial communities and improve system performance in WWTPs.IMPORTANCEIn biological wastewater treatment processes, the microbial community composition is essential in the performance and stability of the system. This study developed a reproducible protocol to investigate the impact of influent immigration (or perpetual coalescence of the sewer and activated sludge communities) with appropriate reproducibility and controls, allowing intrinsic definitions of core and immigrant populations to be established. The method developed herein will allow sequential manipulative experiments to be performed to test specific hypothesis and optimize wastewater treatment processes to meet new treatment goals.

Short-chain polyphosphates: Extraction effects on migration and size estimation of Saccharomyces cerevisiae extracts with polyacrylamide gel electrophoresis.

Polyacrylamide gel electrophoresis is commonly used to characterize the chain length of polyphosphates (polyP), more generally called condensed phosphates. After separation, nonradioactive, optical polyP staining is limited to chain lengths greater than 15 PO 3 - ${\rm{PO}}_3^ - $ monomers with toluidine blue or 4',6-diamidino-2-phenylindole. PolyP chain lengths longer than 62 PO 3 - $\;{\rm{PO}}_3^ - $ monomers were correlated to the shortest DNA ladders. In this study, synthetic linear polyPs (Sigma-Aldrich "Type 45", estimated mean length of 45 PO 3 - ${\rm{PO}}_3^ - $ monomers), trimetaphosphate (trimetaP: 3 PO 3 - ${\rm{PO}}_3^ - $ ring), tripolyphosphate (tripolyP), pyrophosphate (PPi ), and inorganic orthophosphate (o-Pi ) were visualized after separation by an in situ hydrolytic degradation process to o-Pi that was subsequently stained with methyl green. Statistically insignificant migration reduction of synthetic short-chain polyP after perchloric acid or phenol-chloroform extraction was confirmed with the Friedman test. 31 P diffusion-ordered NMR spectroscopy confirmed that extraction also reduced PPi diffusivity by <10%. Linear regression between the Rf peak migration value and the logarithm of synthetic polyP molecular weights enabled estimation of extracted polyP chain lengths from 2 to 45 PO 3 - ${\rm{PO}}_3^ - $ monomers. Linear polyP extracts from Saccharomyces cerevisiae grown in aerobic conditions were generally shorter than extracts cultured in anaerobic conditions. Extractions from both aerobic and anaerobic S. cerevisiae included tripolyP and o-Pi , but no PPi .

Toward a Universal Unit for Quantification of Antibiotic Resistance Genes in Environmental Samples.

Surveillance of antibiotic resistance genes (ARGs) has been increasingly conducted in environmental sectors to complement the surveys in human and animal sectors under the "One-Health" framework. However, there are substantial challenges in comparing and synthesizing the results of multiple studies that employ different test methods and approaches in bioinformatic analysis. In this article, we consider the commonly used quantification units (ARG copy per cell, ARG copy per genome, ARG density, ARG copy per 16S rRNA gene, RPKM, coverage, PPM, etc.) for profiling ARGs and suggest a universal unit (ARG copy per cell) for reporting such biological measurements of samples and improving the comparability of different surveillance efforts.

Extraction processes reduce polyphosphate ion migration, dispersion and diffusion as detected with gel electrophoresis and 31 P DOSY NMR.

Inorganic polyphosphates (polyPs) have been identified in eukaryotic and prokaryotic cells alike. Various extraction methods have been optimized as a necessary step before identification and measurement of these polymers. Three commercially available sodium polyP glasses were either dissolved or dissolved and extracted by two commonly used polyP extraction techniques - perchloric acid or buffered phenol-chloroform. The products were separated by polyacrylamide gel electrophoresis (PAGE), stained with toluidine blue O, and the migration results quantitatively compared. Both extraction processes reduced the relative migration distances of the peak and leading edges, and the stained band lengths, suggesting reduced polyP migration and dispersion. 31 P diffusion-ordered spectroscopy nuclear magnetic resonance confirmed that polyP extraction by perchloric acid or phenol-chloroform processes reduced polyP diffusion coefficients and suggested hydrolytic degradation with stronger end-chain signals. Reduced polyP diffusivity after extraction makes possible an overestimation of synthetic polyP chain length assignment when compared to unextracted polyP ladders with PAGE. The mechanism(s) for reduced synthetic polyP diffusion after extraction and intracellular chemical environment effects on migration are not known.

Impact of Joule Heating and pH on Biosolids Electro-Dewatering.

Electro-dewatering (ED) is a novel technology to reduce the overall costs of residual biosolids processing, transport, and disposal. In this study, we investigated Joule heating and pH as parameters controlling the dewaterability limit, dewatering rate, and energy efficiency. Temperature-controlled electrodes revealed that Joule heating enhances water removal by increasing evaporation and electro-osmotic flow. High temperatures increased the dewatering rate, but had little impact on the dewaterability limit and energy efficiency. Analysis of horizontal layers after 15-min ED suggests electro-osmotic flow reversal, as evidenced by a shifting of the point of minimum moisture content from the anode toward the cathode. This flow reversal was also confirmed by the pH at the anode being below the isoelectric point, as ascertained by pH titration. The important role of pH on ED was further studied by adding acid/base solutions to biosolids prior to ED. An acidic pH reduced the biosolids charge while simultaneously increasing the dewatering efficiency. Thus, process optimization depends on trade-offs between speed and efficiency, according to physicochemical properties of the biosolids microstructure.

Impact of UV and peracetic acid disinfection on the prevalence of virulence and antimicrobial resistance genes in uropathogenic Escherichia coli in wastewater effluents.

Wastewater discharges may increase the populations of pathogens, including Escherichia coli, and of antimicrobial-resistant strains in receiving waters. This study investigated the impact of UV and peracetic acid (PAA) disinfection on the prevalence of virulence and antimicrobial resistance genes in uropathogenic Escherichia coli (UPEC), the most abundant E. coli pathotype in municipal wastewaters. Laboratory disinfection experiments were conducted on wastewater treated by physicochemical, activated sludge, or biofiltration processes; 1,766 E. coli isolates were obtained for the evaluation. The target disinfection level was 200 CFU/100 ml, resulting in UV and PAA doses of 7 to 30 mJ/cm(2) and 0.9 to 2.0 mg/liter, respectively. The proportions of UPECs were reduced in all samples after disinfection, with an average reduction by UV of 55% (range, 22% to 80%) and by PAA of 52% (range, 11% to 100%). Analysis of urovirulence genes revealed that the decline in the UPEC populations was not associated with any particular virulence factor. A positive association was found between the occurrence of urovirulence and antimicrobial resistance genes (ARGs). However, the changes in the prevalence of ARGs in potential UPECs were different following disinfection, i.e., UV appears to have had no effect, while PAA significantly reduced the ARG levels. Thus, this study showed that both UV and PAA disinfections reduced the proportion of UPECs and that PAA disinfection also reduced the proportion of antimicrobial resistance gene-carrying UPEC pathotypes in municipal wastewaters.

Should activated sludge models consider influent seeding of nitrifiers? Field characterization of nitrifying bacteria.

This study revealed the presence of nitrifying bacteria in influent municipal wastewaters reaching full-scale biological wastewater treatment plants. Respirometric assays showed that the influent nitrifiers were active following a 5- to 8-hour period of metabolic induction. Diversity analyses by pyrosequencing of functional gene PCR (polymerase chain reaction) amplicon suggested that the nitrifiers in the influent stream likely seeded activated sludge bioreactors since the most abundant operational taxonomic units in the influent and mixed liquor were the same. Based on the estimated seeding intensity of 0.3 g of nitrifiers per day per gram of nitrifiers already present, the absolute minimum solids retention time (SRT) was reduced by approximately 56% at 5 °C as compared to non-seeding conditions. This can have important repercussions on the design and sizing of bioreactors operating in cold climates and calls for a need to fine-tune process modelling by considering the contribution of autotrophic nitrifying biomass from municipal influent streams.

Biological and physicochemical wastewater treatment processes reduce the prevalence of virulent Escherichia coli.

Effluents discharged from wastewater treatment plants are possible sources of pathogenic bacteria, including Escherichia coli, in the freshwater environment, and determining the possible selection of pathogens is important. This study evaluated the impact of activated sludge and physicochemical wastewater treatment processes on the prevalence of potentially virulent E. coli. A total of 719 E. coli isolates collected from four municipal plants in Québec before and after treatment were characterized by using a customized DNA microarray to determine the impact of treatment processes on the frequency of specific pathotypes and virulence genes. The percentages of potentially pathogenic E. coli isolates in the plant influents varied between 26 and 51%, and in the effluents, the percentages were 14 to 31%, for a reduction observed at all plants ranging between 14 and 45%. Pathotypes associated with extraintestinal pathogenic E. coli (ExPEC) were the most abundant at three of the four plants and represented 24% of all isolates, while intestinal pathogenic E. coli pathotypes (IPEC) represented 10% of the isolates. At the plant where ExPEC isolates were not the most abundant, a large number of isolates were classified as both ExPEC and IPEC; overall, 6% of the isolates were classified in both groups, with the majority being from the same plant. The reduction of the proportion of pathogenic E. coli could not be explained by the preferential loss of one virulence gene or one type of virulence factor; however, the quinolone resistance gene (qnrS) appears to enhance the loss of virulence genes, suggesting a mechanism involving the loss of pathogenicity islands.

Genome-scale metabolic modeling to provide insight into the production of storage compounds during feast-famine cycles of activated sludge.

Studying storage metabolism during feast-famine cycles of activated sludge treatment systems provides profound insight in terms of both operational issues (e.g., foaming and bulking) and process optimization for the production of value added by-products (e.g., bioplastics). We examined the storage metabolism (including poly-ß-hydroxybutyrate [PHB], glycogen, and triacylglycerols [TAGs]) during feast-famine cycles using two genome-scale metabolic models: Rhodococcus jostii RHA1 (iMT1174) and Escherichia coli K-12 (iAF1260) for growth on glucose, acetate, and succinate. The goal was to develop the proper objective function (OF) for the prediction of the main storage compound produced in activated sludge for given feast-famine cycle conditions. For the flux balance analysis, combinations of three OFs were tested. For all of them, the main OF was to maximize growth rates. Two additional sub-OFs were used: (1) minimization of biochemical fluxes, and (2) minimization of metabolic adjustments (MoMA) between the feast and famine periods. All (sub-)OFs predicted identical substrate-storage associations for the feast-famine growth of the above-mentioned metabolic models on a given substrate when glucose and acetate were set as sole carbon sources (i.e., glucose-glycogen and acetate-PHB), in agreement with experimental observations. However, in the case of succinate as substrate, the predictions depended on the network structure of the metabolic models such that the E. coli model predicted glycogen accumulation and the R. jostii model predicted PHB accumulation. While the accumulation of both PHB and glycogen was observed experimentally, PHB showed higher dynamics during an activated sludge feast-famine growth cycle with succinate as substrate. These results suggest that new modeling insights between metabolic predictions and population ecology will be necessary to properly predict metabolisms likely to emerge within the niches of activated sludge communities. Nonetheless, we believe that the development of this approach will help guide the optimization of the production of storage compounds as valuable by-products of wastewater treatment.

Cellular RNA levels define heterotrophic substrate-uptake rate sub-guilds in activated sludge microbial communities.

A heterotrophic-specialist model was proposed previously to divide wastewater treatment plant (WWTP) heterotrophs into sub-guilds of consumers of readily or slowly degradable substrates (RDS or SDS, respectively). The substrate degradation rate model coupled to metabolic considerations predicted that RNA and polyhydroxyalkanoate (PHA) levels would be positively correlated in the activated sludge communities with high RNA and PHA occurring in RDS-consumers, and low RNA with no PHA accumulation occurring in SDS-consumers because their external substrates are always present. This prediction was verified in previous studies and in the current one. Thus, RNA and PHA levels were used as biomarkers of the RDS- and SDS-consumer sub-guilds for cell sorting using flow cytometry of samples from three WWTPs. Subsequently, 16S rRNA gene amplicon sequencing revealed that the sorted groups were highly similar over time and among WWTPs, and demonstrated a clear segregation by RNA levels. Predicted ecophysiological traits based on 16S rRNA phylogeny suggested that the high-RNA population showed RDS-consumer traits such as higher rrn copy numbers per genome. Using a mass-flow immigration model, it appeared that the high-RNA populations exhibited high immigration rates more frequently than low-RNA populations, but the differences in frequencies were less with increasing solids residence times.

Antibiotic resistance gene sequencing is necessary to reveal the complex dynamics of immigration from sewers to activated sludge.

Microbial community composition has increasingly emerged as a key determinant of antibiotic resistance gene (ARG) content. However, in activated sludge wastewater treatment plants (AS-WWTPs), a comprehensive understanding of the microbial community assembly process and its impact on the persistence of antimicrobial resistance (AMR) remains elusive. An important part of this process is the immigration dynamics (or community coalescence) between the influent and activated sludge. While the influent wastewater contains a plethora of ARGs, the persistence of a given ARG depends initially on the immigration success of the carrying population, and the possible horizontal transfer to indigenously resident populations of the WWTP. The current study utilized controlled manipulative experiments that decoupled the influent wastewater composition from the influent microbial populations to reveal the fundamental mechanisms involved in ARG immigration between sewers and AS-WWTP. A novel multiplexed amplicon sequencing approach was used to track different ARG sequence variants across the immigration interface, and droplet digital PCR was used to quantify the impact of immigration on the abundance of the targeted ARGs. Immigration caused an increase in the abundance of over 70 % of the quantified ARGs. However, monitoring of ARG amplicon sequence variants (ARG-ASVs) at the immigration interface revealed various immigration patterns such as (i) suppression of the indigenous mixed liquor ARG-ASV by the immigrant, or conversely (ii) complete immigration failure of the influent ARG-ASV. These immigration profiles are reported for the first time here and highlight the crucial information that can be gained using our novel multiplex amplicon sequencing techniques. Future studies aiming to reduce AMR in WWTPs should consider the impact of influent immigration in process optimisation and design.

Wastewater-based epidemiology: the crucial role of viral shedding dynamics in small communities.

Background: Wastewater surveillance (WWS) of pathogens is a rapidly evolving field owing to the 2019 coronavirus disease pandemic, which brought about a paradigm shift in public health authorities for the management of pathogen outbreaks. However, the interpretation of WWS in terms of clinical cases remains a challenge, particularly in small communities where large variations in pathogen concentrations are routinely observed without a clear relation to clinical incident cases. Methods: Results are presented for WWS from six municipalities in the eastern part of Canada during the spring of 2021. We developed a numerical model based on viral kinetics reduction functions to consider both prevalent and incident cases to interpret the WWS data in light of the reported clinical cases in the six surveyed communities. Results: The use of the proposed numerical model with a viral kinetics reduction function drastically increased the interpretability of the WWS data in terms of the clinical cases reported for the surveyed community. In line with our working hypothesis, the effects of viral kinetics reduction modeling were more important in small communities than in larger communities. In all but one of the community cases (where it had no effect), the use of the proposed numerical model led to a change from a +1.5% (for the larger urban center, Quebec City) to a +48.8% increase in the case of a smaller community (Drummondville). Conclusion: Consideration of prevalent and incident cases through the proposed numerical model increases the correlation between clinical cases and WWS data. This is particularly the case in small communities. Because the proposed model is based on a biological mechanism, we believe it is an inherent part of any wastewater system and, hence, that it should be used in any WWS analysis where the aim is to relate WWS measurement to clinical cases.

Impact of pH and removed filtrate on E. coli regrowth and microbial community during storage of electro-dewatered biosolids.

Residual biosolids can be land applied if they meet microbiological requirements at the time of application. Electro-dewatering technology is shown to reduce biosolids bacterial counts to detection limits with little potential for bacterial regrowth during incubations. Here, we investigated the impacts on Escherichia coli regrowth and microbial communities of biosolids pH, removed nutrients via the filtrate, and inhibitory compounds produced in electro-dewatered biosolids. Findings suggest pH as the primary mechanism impacting E. coli regrowth in electro-dewatered biosolids. Propidium monoazide treatments were effective at removing DNA from dead cells, based on the removal of obligate anaerobes observed after anaerobic incubation. Analyses of high throughput sequenced data showed lower alpha-diversities associated with electro-dewatering treatment and incubation time. Moreover, biosolids pH and incubation period were the main factors contributing to the variations in microbial community compositions after incubation. Results highlight the role of electro-dewatered biosolids' low pH on inhibiting the regrowth of culturable bacteria as well as reducing the microbial community variance.

Evaluation of a new model for the reduction of excess sludge production by ozonation of return activated sludge: what solids COD fraction is affected?

This paper aims at clarifying the effect of ozone on the RAS solids to model activated sludge systems equipped with RAS-ozonation processes for the reduction of sludge production. A common hypothesis is that ozone only affects active biomass by promoting cryptic growth. Data from a pilot-scale study were used to test this and two other model extensions to IWA-ASM3. All model extensions were able to simulate the observed linear reduction in sludge production with increasing ozone dose when the MLVSS are kept constant. However, model simulations showed the inconsistency of the cryptic growth hypothesis with the extent of sludge reduction. The second tested model extensions assumes that ozone affects all the solids fractions (active biomass, endogenous residue, and influent inert particulate COD) equally. This extension could properly simulate the observed sludge reduction, but it failed to predict the trends in effluent BOD5, ATP/VSS, and nitrification rates. A third tested model extension, which performed better, assumes that biomass is inactivated at a specific rate higher than the specific rate of transformation by ozone of the other solids fractions. Finally, the predictions from this model extension were most accurate if either (i) the nitrifiers were inactivated at a lower rate then heterotrophs, (ii) the nitrifiers model parameters (e.g., maximum growth rate) were changed under ozone (i.e., metabolic adaptation, (iii) or both.

Dynamics of integron structures across a wastewater network - Implications to resistance gene transfer.

Class 1 and other integrons are common in wastewater networks, often being associated with antibiotic resistance genes (ARGs). However, the importance of different integron structures in ARG transfer within wastewater systems has only been implied, especially between community and hospital sources, among wastewater treatment plant compartments, and in receiving waters. This uncertainty is partly because current clinical class 1 integron qPCR assays (i.e., that target human-impacted structures, i.e., clintI1) poorly delineate clintI1 from non-impacted class 1 integron structures. They also say nothing about their ARG content. To fill these technical gaps, new real-time qPCR assays were developed for "impacted" class 1 structures (called aint1; i.e., anthropogenic class 1 integrons) and empty aint1 structures (i.e., carry no ARGs; called eaint1). The new assays and other integron assays then were used to examine integron dynamics across a wastewater network. 16S metagenomic sequencing also was performed to characterise associated microbiomes. aint1 abundances per bacterial cell were about 10 times greater in hospital wastewaters compared with other compartments, suggesting aint1 enrichment with ARGs in hospital sources. Conversely, the relative abundance of eaint1 structures were over double in recycled activated sludge compared with other compartments, except receiving waters (RAS; ∼30% of RAS class 1 structures did not carry ARGs). Microbiome analysis showed that human-associated bacterial taxa with mobile integrons also differed in RAS and river sediments. Further, class 1 integrons in RAS bacteria appear to have released ARGs, whereas hospital bacteria have accumulated ARGs. Results show that quantifying integron dynamics can help explain where ARG transfer occurs in wastewater networks, and should be considered in future studies on antibiotic resistance in the environment.

Microbial immigration in wastewater treatment systems: analytical considerations and process implications.

Microbial immigration from sewers to wastewater treatment systems is attracting increasing attention for understanding community assembly mechanisms, and improving process modeling and operation. While there is no consensus on approaches to analyze immigration, we suggest to classify them as relevant to either rare (non-observable) diffusive immigration or to time-continuous high-rate mass flow immigration (i.e. mass effects). When analyzed by a mass flow approach, heterotrophs appear to be strongly influenced by deterministic selection, suggesting that the heterotrophs should be subdivided into several functional guilds when assessing their assembly mechanisms. Conversely, nitrifiers appear to transfer neutrally from sewer to activated sludge, and this immigration can restore full nitrification in otherwise non-nitrifying reactors. With further refinement, these findings could be included in predictive process models with various objectives.

Wastewater microbial community structure and functional traits change over short timescales.

Wastewater contains microorganisms coming from various sources, e.g. feces discharges, soil infiltrations and sewer biofilms and sediments. The primary objective of this work was to determine if end-of-pipe wastewater microbial community structures exhibits short-timescale variation, and assess possible microbial origins. To this end, we measured hourly physicochemical characteristics of wastewater influent for 2 days and analyzed the microbial community at 4-h intervals using 16S rRNA gene amplicon sequencing. Results showed large variations in the microbial community composition at phylum and genus levels, i.e. Proteobacteria ranged from 44 to 63% of the total relative abundance and Arcobacter ranged from 11 to 22%. Diurnal patterns were observed in the alpha-diversity, beta-diversity and the prevalence of several taxa. Wastewater physicochemical characteristics explained 61% of the total microbial community variance by Canonical Correspondence Analysis (CCA), with flow rate being the main explanatory variable exhibiting a clear diurnal profile. Comparison with public databases using closed reference OTUs revealed that only 7.3% of the sequences were shared with human gut microbiota and 21.7% with soil microbiota, the majority being from the sewer biofilms and sediments. The functional trait, weighted average ribosomal RNA operon (rrn) copy number per genome, was found to be relatively high in the wastewater microbiota (average 3.6, soil 2.1, and human gut 2.6) and significantly correlated with flow, inferring active microbial enrichments in the sewer. The prevalence of Methylophilaceae, methanol oxidation genes and denitrification genes were related to high influent methanol and NO3- concentration in the influent wastewater. These functional organisms and genes indicate important carbon and nutrient removal related functions in the sewer. Together, the observed temporal patterns of the microbial community and functional traits suggest that high wastewater flow causes greater transport of active sewer microorganisms which are functionally important.

Predicting the accumulation of storage compounds by Rhodococcus jostii RHA1 in the feast-famine growth cycles using genome-scale flux balance analysis.

Feast-famine cycles in biological wastewater resource recovery systems select for bacterial species that accumulate intracellular storage compounds such as poly-ß-hydroxybutyrate (PHB), glycogen, and triacylglycerols (TAG). These species survive better the famine phase and resume rapid substrate uptake at the beginning of the feast phase faster than microorganisms unable to accumulate storage. However, ecophysiological conditions favouring the accumulation of either storage compounds remain to be clarified, and predictive capabilities need to be developed to eventually rationally design reactors producing these compounds. Using a genome-scale metabolic modelling approach, the storage metabolism of Rhodococcus jostii RHA1 was investigated for steady-state feast-famine cycles on glucose and acetate as the sole carbon sources. R. jostii RHA1 is capable of accumulating the three storage compounds (PHB, TAG, and glycogen) simultaneously. According to the experimental observations, when glucose was the substrate, feast phase chemical oxygen demand (COD) accumulation was similar for the three storage compounds; when acetate was the substrate, however, PHB accumulation was 3 times higher than TAG accumulation and essentially no glycogen was accumulated. These results were simulated using the genome-scale metabolic model of R. jostii RHA1 (iMT1174) by means of flux balance analysis (FBA) to determine the objective functions capable of predicting these behaviours. Maximization of the growth rate was set as the main objective function, while minimization of total reaction fluxes and minimization of metabolic adjustment (environmental MOMA) were considered as the sub-objective functions. The environmental MOMA sub-objective performed better than the minimization of total reaction fluxes sub-objective function at predicting the mixture of storage compounds accumulated. Additional experiments with 13C-labelled bicarbonate (HCO3-) found that the fluxes through the central metabolism reactions during the feast phases were similar to the ones during the famine phases on acetate due to similarity in the carbon sources in the feast and famine phases (i.e., acetate and poly-ß-hydroxybutyrate, respectively); this suggests that the environmental MOMA sub-objective function could be used to analyze successive environmental conditions such as the feast and famine cycles while the metabolically similar carbon sources are taken up by microorganisms.

Composition of heterotrophic specialized sub-guilds defined by a positive RNA and polyhydroxyalkanoate correlation in activated sludge.

Microbial heterotrophic guilds in activated sludge wastewater treatment systems have complex population structures and functions. A previously proposed heterotrophic-specialist model states that heterotrophs consist of sub-guilds specialized in consuming specific classes of compounds, either readily degradable substrate (RDS) or slowly degradable substrate (SDS) according to current mathematical modeling practices for wastewater treatment processes. It follows from metabolic considerations that the levels of RNA and polyhydroxyalkanoate (PHA) are correlated for strains of the same species growing in different environments; a conjecture previously tested. The proposed classification of heterotrophs into RDS or SDS consumers predicts that the same correlation would also be found across heterotrophic species in conventional activated sludge systems; this prediction was tested in the current study. The positive correlation between the RNA and PHA levels was observed in 9 conventional activated sludge plants in two independent sampling times and it was also found stable over a 6-month regular sampling period at one of these plants. Together, these results imply that the levels of RNA and PHA can be used to define heterotrophic-specialist sub-guilds. In order to gain insight in the species composition of the defined sub-guilds, flow cytometry cell sorting was used to further analyze one of the activated sludge samples. Four sorted sub-samples were obtained (high-RNA/high-PHA, low-RNA/high-PHA, high-RNA/low-PHA, and low-RNA/low-PHA), and the phylogenetic composition of each was determined using 16S rRNA gene amplicon pyrosequencing. Heterotrophic genera were identified across 12 phyla, and their representation in each sorted sub-sample showed that the high-RNA/high-PHA and low-RNA/low-PHA groups were most dissimilar. The enriched genera in these sorted sub-samples are suggested to represent the composition of heterotrophic-specialized sub-guilds defined by the kinetics of substrate consumption.

Water and sanitation: an essential battlefront in the war on antimicrobial resistance.

Water and sanitation represent a key battlefront in combatting the spread of antimicrobial resistance (AMR). Basic water sanitation infrastructure is an essential first step towards protecting public health, thereby limiting the spread of pathogens and the need for antibiotics. AMR presents unique human health risks, meriting new risk assessment frameworks specifically adapted to water and sanitation-borne AMR. There are numerous exposure routes to AMR originating from human waste, each of which must be quantified for its relative risk to human health. Wastewater treatment plants play a vital role in centralized collection and treatment of human sewage, but there are numerous unresolved issues in terms of the microbial ecological processes occurring within them and the extent to which they attenuate or amplify AMR. Research is needed to advance understanding of the fate of resistant bacteria and antibiotic resistance genes in various waste management systems, depending on the local constraints and intended reuse applications. World Health Organization and national AMR action plans would benefit from a more holistic 'One Water' understanding. In this article we provide a framework for research, policy, practice and public engagement aimed at limiting the spread of AMR from water and sanitation in low-, medium- and high-income countries.