Turnover of the extracellular polymeric matrix of granules performing biological phosphate removal.

Polyphosphate accumulating organisms (PAOs) are responsible for enhanced biological phosphate removal (EBPR) from wastewater, where they grow embedded in a matrix of extracellular polymeric substances (EPS). EPSs comprise a mixture of biopolymers like polysaccharides or (glyco)proteins. Despite previous studies, little is known about the dynamics of EPS in mixed cultures, and their production by PAOs and potential consumption by flanking microbes. EPSs are biodegradable and have been suggested to be a substrate for other organisms in the community. Studying EPS turnover can help elucidate their biosynthesis and biodegradation cycles. We analyzed the turnover of proteins and polysaccharides in the EPS of an enrichment culture of PAOs relative to the turnover of internal proteins. An anaerobic-aerobic sequencing batch reactor (SBR) simulating EBPR conditions was operated to enrich for PAOs. After achieving a stable culture, carbon source was switched to uniformly 13C-labeled acetate. Samples were collected at the end of each aerobic phase. EPSs were extracted by alkaline treatment. 13C enrichment in proteins and sugars (after hydrolysis of polysaccharides) in the extracted EPS were measured by mass spectrometry. The average turnover rate of sugars and proteins (0.167 and 0.192 d-1 respectively) was higher than the expected value based on the solid removal rate (0.132 d-1), and no significant difference was observed between intracellular and extracellular proteins. This indicates that EPS from the PAO enriched community is not selectively degraded by flanking populations under stable EBPR process conditions. Instead, we observed general decay of biomass, which corresponds to a value of 0.048 d-1. KEY POINTS: • Proteins showed a higher turnover rate than carbohydrates. • Turnover of EPS was similar to the turnover of intracellular proteins. • EPS is not preferentially consumed by flanking populations.

Enrichment and application of extracellular nonulosonic acids containing polymers of Accumulibacter.

Pseudaminic and legionaminic acids are a subgroup of nonulosonic acids (NulOs) unique to bacterial species. There is a lack of advances in the study of these NulOs due to their complex synthesis and production. Recently, it was seen that "Candidatus Accumulibacter" can produce Pse or Leg analogues as part of its extracellular polymeric substances (EPS). In order to employ a "Ca. Accumulibacter" enrichment as production platform for bacterial sialic acids, it is necessary to determine which fractions of the EPS of "Ca. Accumulibacter" contain NulOs and how to enrich and/or isolate them. We extracted the EPS from granules enriched with "Ca. Accumulibcater" and used size-exclusion chromatography (SEC) to separate them into different molecular weight (MW) fractions. This separation resulted in two high molecular weight (> 5500 kDa) fractions dominated by polysaccharides, with a NulO content up to 4 times higher than the extracted EPS. This suggests that NulOs in "Ca. Accumulibacter" are likely located in high molecular weight polysaccharides. Additionally, it was seen that the extracted EPS and the NulO-rich fractions can bind and neutralize histones. This opens the possibility of EPS and NulO-rich fractions as potential source for sepsis treatment drugs. KEY POINTS: • NulOs in "Ca. Accumulibacter" are likely located in high MW polysaccharides • SEC allows to obtain high MW polysaccharide-rich fractions enriched with NulOs • EPS and the NulOs-rich fractions are a potential source for sepsis treatment drugs.

Catch me if you can: capturing microbial community transformation by extracellular DNA using Hi-C sequencing.

The transformation of environmental microorganisms by extracellular DNA is an overlooked mechanism of horizontal gene transfer and evolution. It initiates the acquisition of exogenous genes and propagates antimicrobial resistance alongside vertical and conjugative transfers. We combined mixed-culture biotechnology and Hi-C sequencing to elucidate the transformation of wastewater microorganisms with a synthetic plasmid encoding GFP and kanamycin resistance genes, in the mixed culture of chemostats exposed to kanamycin at concentrations representing wastewater, gut and polluted environments (0.01-2.5-50-100 mg L-1). We found that the phylogenetically distant Gram-negative Runella (102 Hi-C links), Bosea (35), Gemmobacter (33) and Zoogloea (24) spp., and Gram-positive Microbacterium sp. (90) were transformed by the foreign plasmid, under high antibiotic exposure (50 mg L-1). In addition, the antibiotic pressure shifted the origin of aminoglycoside resistance genes from genomic DNA to mobile genetic elements on plasmids accumulating in microorganisms. These results reveal the power of Hi-C sequencing to catch and surveil the transfer of xenogenetic elements inside microbiomes.

Operational Strategies to Selectively Produce Purple Bacteria for Microbial Protein in Raceway Reactors.

Purple non-sulfur bacteria (PNSB) show potential for microbial protein production on wastewater as animal feed. They offer good selectivity (i.e., low microbial diversity and high abundance of one species) when grown anaerobically in the light. However, the cost of closed anaerobic photobioreactors is prohibitive for protein production. Although open raceway reactors are cheaper, their feasibility to selectively grow PNSB is thus far unexplored. This study developed operational strategies to boost PNSB abundance in the biomass of a raceway reactor fed with volatile fatty acids. For a flask reactor run at a 2 day sludge retention time (SRT), matching the chemical oxygen demand (COD) loading rate to the removal rate in the light period prevented substrate availability during the dark period and increased the PNSB abundance from 50-67 to 88-94%. A raceway reactor run at a 2 day SRT showed an increased PNSB abundance from 14 to 56% when oxygen supply was reduced (no stirring at night). The best performance was achieved at the highest surface-to-volume ratio (10 m2 m-3 increased light availability) showing productivities up to 0.2 g protein L-1 day-1 and a PNSB abundance of 78%. This study pioneered in PNSB-based microbial protein production in raceway reactors, yielding high selectivity while avoiding the combined availability of oxygen, COD, and darkness.

Production of nonulosonic acids in the extracellular polymeric substances of "Candidatus Accumulibacter phosphatis".

Nonulosonic acids (NulOs) are a family of acidic carbohydrates with a nine-carbon backbone, which include different related structures, such as sialic acids. They have mainly been studied for their relevance in animal cells and pathogenic bacteria. Recently, sialic acids have been discovered as an important compound in the extracellular matrix of virtually all microbial life and in "Candidatus Accumulibacter phosphatis", a well-studied polyphosphate-accumulating organism, in particular. Here, bioaggregates highly enriched with these bacteria (approx. 95% based on proteomic data) were used to study the production of NulOs in an enrichment of this microorganism. Fluorescence lectin-binding analysis, enzymatic quantification, and mass spectrometry were used to analyze the different NulOs present, showing a wide distribution and variety of these carbohydrates, such as sialic acids and bacterial NulOs, in the bioaggregates. Phylogenetic analysis confirmed the potential of "Ca. Accumulibacter" to produce different types of NulOs. Proteomic analysis showed the ability of "Ca. Accumulibacter" to reutilize and reincorporate these carbohydrates. This investigation points out the importance of diverse NulOs in non-pathogenic bacteria, which are normally overlooked. Sialic acids and other NulOs should be further investigated for their role in the ecology of "Ca. Accumulibacter" in particular, and biofilms in general. KEY POINTS: •"Ca. Accumulibacter" has the potential to produce a range of nonulosonic acids. •Mass spectrometry and lectin binding can reveal the presence and location of nonulosonic acids. •The role of nonulosonic acid in non-pathogenic bacteria needs to be studied in detail.

Modeling hydraulic transport and anaerobic uptake by PAOs and GAOs during wastewater feeding in EBPR granular sludge reactors.

New-generation bioprocesses using granular sludge aim for a high-rate removal of nutrients from wastewater with low footprint. Achieving enhanced biological phosphorus removal (EBPR) relies on the design of sludge beds and wastewater feeding conditions to optimally load the biomass and to select for polyphosphate- (PAOs) over glycogen-accumulating organisms (GAOs) and over other heterotrophs. A hydraulic-metabolic mathematical model was developed to elucidate the impact of hydraulic transport patterns and environmental conditions on the PAO/GAO competition during up-flow feeding through an EBPR granular sludge bed. Tracer experiments highlighted plug-flow regimes with dispersion under both rapid (9 m h-1 , Rebed = 1.6, Pez = 7.2, Pet = 4.6) and slow (0.9 m h-1 , Rebed = 0.2, Pez = 21.3, Pet = 3.4) feeding. Non-turbulent regimes (Rebed << 103 ) promote a safe implementation of simultaneous fill/draw. Feeding time, pH, and temperature significantly impacted bacterial competition for carbon uptake under anaerobic slow feeding. Feeding duration should be designed to avoid full depletion of intracellular storage polymers within static granules. PAOs bear twice longer feeding than GAOs by using both polyphosphate and glycogen hydrolysis to sustain anaerobic C-uptake. Alkaline conditions (pH 7.25-8.0) by, e.g., dosing lime in the feed select for PAOs independently of temperature (10-30°C). A twice higher bed is required for full anaerobic conversions at 10 rather than 20°C. Biosystem responses for anaerobic C-uptake can be anticipated using the model toward designing robust anaerobic selectors to manage the microbial resource in EBPR granular sludge. Biotechnol. Bioeng. 2017;114: 1688-1702. © 2017 Wiley Periodicals, Inc.

Growth of Nitrosococcus-Related Ammonia Oxidizing Bacteria Coincides with Extremely Low pH Values in Wastewater with High Ammonia Content.

Ammonia oxidation decreases the pH in wastewaters where alkalinity is limited relative to total ammonia. The activity of ammonia oxidizing bacteria (AOB), however, typically decreases with pH and often ceases completely in slightly acidic wastewaters. Nevertheless, nitrification at low pH has been reported in reactors treating human urine, but it has been unclear which organisms are involved. In this study, we followed the population dynamics of ammonia oxidizing organisms and reactor performance in synthetic fully hydrolyzed urine as the pH decreased over time in response to a decrease in the loading rate. Populations of the ß-proteobacterial Nitrosomonas europaea lineage were abundant at the initial pH close to 6, but the growth of a possibly novel Nitrosococcus-related AOB genus decreased the pH to the new level of 2.2, challenging the perception that nitrification is inhibited entirely at low pH values, or governed exclusively by ß-proteobacterial AOB or archaea. With the pH shift, nitrite oxidizing bacteria were not further detected, but nitrous acid (HNO2) was still removed through chemical decomposition to nitric oxide (NO) and nitrate. The growth of acid-tolerant γ-proteobacterial AOB should be prevented, by keeping the pH above 5.4, which is a typical pH limit for the N. europaea lineage. Otherwise, the microbial community responsible for high-rate nitrification can be lost, and strong emissions of hazardous volatile nitrogen compounds such as NO are likely.

Hospital-Use Pharmaceuticals in Swiss Waters Modeled at High Spatial Resolution.

A model to predict the mass flows and concentrations of pharmaceuticals predominantly used in hospitals across a large number of sewage treatment plant (STP) effluents and river waters was developed at high spatial resolution. It comprised 427 geo-referenced hospitals and 742 STPs serving 98% of the general population in Switzerland. In the modeled base scenario, domestic, pharmaceutical use was geographically distributed according to the population size served by the respective STPs. Distinct hospital scenarios were set up to evaluate how the predicted results were modified when pharmaceutical use in hospitals was allocated differently; for example, in proportion to number of beds or number of treatments in hospitals. The hospital scenarios predicted the mass flows and concentrations up to 3.9 times greater than in the domestic scenario for iodinated X-ray contrast media (ICM) used in computed tomography (CT), and up to 6.7 times greater for gadolinium, a contrast medium used in magnetic resonance imaging (MRI). Field measurements showed that ICM and gadolinium were predicted best by the scenarios using number of beds or treatments in hospitals with the specific facilities (i.e., CT and/or MRI). Pharmaceuticals used both in hospitals and by the general population (e.g., cyclophosphamide, sulfamethoxazole, carbamazepine, diclofenac) were predicted best by the scenario using the number of beds in all hospitals, but the deviation from the domestic scenario values was only small. Our study demonstrated that the bed number-based hospital scenarios were effective in predicting the geographical distribution of a diverse range of pharmaceuticals in STP effluents and rivers, while the domestic scenario was similarly effective on the scale of large river-catchments.

Influence of Different Sewer Biofilms on Transformation Rates of Drugs.

To estimate drug consumption more reliably, wastewater-based epidemiology would benefit from a better understanding of drug residue stability during in-sewer transport. We conducted batch experiments with real, fresh wastewater and sewer biofilms. Experimental conditions mimic small to medium-sized gravity sewers with a relevant ratio of biofilm surface area to wastewater volume (33 m2 m-3). The influences of biological, chemical, and physical processes on the transformation of 30 illicit drug and pharmaceutical residues were quantified. Rates varied among locations and over time. Three substances were not stable-that is, >20% transformation, mainly due to biological processes-at least for one type of tested biofilm for a residence time ≤2 h: amphetamine, 6-acetylcodeine, and 6-monoacetylmorphine. Cocaine, ecgonine methyl ester, norcocaine, cocaethylene, and mephedrone were mainly transformed by chemical hydrolysis and, hence, also unstable in sewers. In contrast, ketamine, norketamine, O-desmethyltramadol, diclofenac, carbamazepine, and methoxetamine were not substantially affected by in-sewer processes under all tested conditions and residence times up to 12 h. Our transformation rates include careful quantification of uncertainty and can be used to identify situations in which specific compounds are not stable. This will improve accuracy and uncertainty estimates of drug consumption when applied to the back-calculation.

Multilevel correlations in the biological phosphorus removal process: From bacterial enrichment to conductivity-based metabolic batch tests and polyphosphatase assays.

Enhanced biological phosphorus removal (EBPR) from wastewater relies on the preferential selection of active polyphosphate-accumulating organisms (PAO) in the underlying bacterial community continuum. Efficient management of the bacterial resource requires understanding of population dynamics as well as availability of bioanalytical methods for rapid and regular assessment of relative abundances of active PAOs and their glycogen-accumulating competitors (GAO). A systems approach was adopted here toward the investigation of multilevel correlations from the EBPR bioprocess to the bacterial community, metabolic, and enzymatic levels. Two anaerobic-aerobic sequencing-batch reactors were operated to enrich activated sludge in PAOs and GAOs affiliating with "Candidati Accumulibacter and Competibacter phosphates", respectively. Bacterial selection was optimized by dynamic control of the organic loading rate and the anaerobic contact time. The distinct core bacteriomes mainly comprised populations related to the classes Betaproteobacteria, Cytophagia, and Chloroflexi in the PAO enrichment and of Gammaproteobacteria, Alphaproteobacteria, Acidobacteria, and Sphingobacteria in the GAO enrichment. An anaerobic metabolic batch test based on electrical conductivity evolution and a polyphosphatase enzymatic assay were developed for rapid and low-cost assessment of the active PAO fraction and dephosphatation potential of activated sludge. Linear correlations were obtained between the PAO fraction, biomass specific rate of conductivity increase under anaerobic conditions, and polyphosphate-hydrolyzing activity of PAO/GAO mixtures. The correlations between PAO/GAO ratios, metabolic activities, and conductivity profiles were confirmed by simulations with a mathematical model developed in the aqueous geochemistry software PHREEQC.

Selecting for a high lipid accumulating microalgae culture by dual growth limitation in a continuous bioreactor.

A dual-growth-limited continuous operated bioreactor (chemostat) was used to enhance lipid accumulation in an enrichment culture of microalgae. The light intensity and nitrogen concentration where both limiting factors resulting in high lipid accumulation in the mixed culture. Both conditions of light and nitrogen excess and deficiency were tested. Strategies to selectively enrich for a phototrophic lipid-storing community, based on the use of different nitrogen sources (ammonium vs. nitrate) and vitamin B supplementation in the growth medium, were evaluated. The dual limitation of both nitrogen and light enhanced the accumulation of storage compounds. Ammoniacal nitrogen was the preferred nitrogen source. Vitamin B supplementation led to a doubling of the lipid productivity. The availability of vitamins played a key role in selecting an efficient lipid-storing community, primarily consisting of Trebouxiophyceae (with an 82 % relative abundance among eukaryotic microorganisms). The obtained lipid volumetric productivity (387 mg L-1 d-1) was among the highest reported in literature for microalgae bioreactors. Lipid production by the microalgae enrichment surpassed the efficiencies reported for continuous microalgae pure cultures, highlighting the benefits of mixed-culture photo-biotechnologies for fuels and food ingredients in the circular economy.

Microbiome, resistome and mobilome of chlorine-free drinking water treatment systems.

Drinking water treatment plants (DWTPs) are designed to remove physical, chemical, and biological contaminants. However, until recently, the role of DWTPs in minimizing the cycling of antibiotic resistance determinants has got limited attention. In particular, the risk of selecting antibiotic-resistant bacteria (ARB) is largely overlooked in chlorine-free DWTPs where biological processes are applied. Here, we combined high-throughput quantitative PCR and metagenomics to analyze the abundance and dynamics of microbial communities, antibiotic resistance genes (ARGs), and mobile genetic elements (MGEs) across the treatment trains of two chlorine-free DWTPs involving dune-based and reservoir-based systems. The microbial diversity of the water increased after all biological unit operations, namely rapid and slow sand filtration (SSF), and granular activated carbon filtration. Both DWTPs reduced the concentration of ARGs and MGEs in the water by circa 2.5 log gene copies mL-1, despite their relative increase in the disinfection sub-units (SSF in dune-based and UV treatment in reservoir-based DWTPs). The total microbial concentration was also reduced (2.5 log units), and none of the DWTPs enriched for bacteria containing genes linked to antibiotic resistance. Our findings highlight the effectiveness of chlorine-free DWTPs in supplying safe drinking water while reducing the concentration of antibiotic resistance determinants. To the best of our knowledge, this is the first study that monitors the presence and dynamics of antibiotic resistance determinants in chlorine-free DWTPs.

A novel mechanistic modelling approach for microbial selection dynamics: Towards improved design and control of raceway reactors for purple bacteria.

Purple phototrophic bacteria (PPB) show an underexplored potential for resource recovery from wastewater. Raceway reactors offer a more affordable full-scale solution on wastewater and enable useful additional aerobic processes. Current mathematical models of PPB systems provide useful mechanistic insights, but do not represent the full metabolic versatility of PPB and thus require further advancement to simulate the process for technology development and control. In this study, a new modelling approach for PPB that integrates the photoheterotrophic, and both anaerobic and aerobic chemoheterotrophic metabolic pathways through an empirical parallel metabolic growth constant was proposed. It aimed the modelling of microbial selection dynamics in competition with aerobic and anaerobic microbial community under different operational scenarios. A sensitivity analysis was carried out to identify the most influential parameters within the model and calibrate them based on experimental data. Process perturbation scenarios were simulated, which showed a good performance of the model.

PyroTRF-ID: a novel bioinformatics methodology for the affiliation of terminal-restriction fragments using 16S rRNA gene pyrosequencing data.

BACKGROUND: In molecular microbial ecology, massive sequencing is gradually replacing classical fingerprinting techniques such as terminal-restriction fragment length polymorphism (T-RFLP) combined with cloning-sequencing for the characterization of microbiomes. Here, a bioinformatics methodology for pyrosequencing-based T-RF identification (PyroTRF-ID) was developed to combine pyrosequencing and T-RFLP approaches for the description of microbial communities. The strength of this methodology relies on the identification of T-RFs by comparison of experimental and digital T-RFLP profiles obtained from the same samples. DNA extracts were subjected to amplification of the 16S rRNA gene pool, T-RFLP with the HaeIII restriction enzyme, 454 tag encoded FLX amplicon pyrosequencing, and PyroTRF-ID analysis. Digital T-RFLP profiles were generated from the denoised full pyrosequencing datasets, and the sequences contributing to each digital T-RF were classified to taxonomic bins using the Greengenes reference database. The method was tested both on bacterial communities found in chloroethene-contaminated groundwater samples and in aerobic granular sludge biofilms originating from wastewater treatment systems. RESULTS: PyroTRF-ID was efficient for high-throughput mapping and digital T-RFLP profiling of pyrosequencing datasets. After denoising, a dataset comprising ca. 10'000 reads of 300 to 500 bp was typically processed within ca. 20 minutes on a high-performance computing cluster, running on a Linux-related CentOS 5.5 operating system, enabling parallel processing of multiple samples. Both digital and experimental T-RFLP profiles were aligned with maximum cross-correlation coefficients of 0.71 and 0.92 for high- and low-complexity environments, respectively. On average, 63±18% of all experimental T-RFs (30 to 93 peaks per sample) were affiliated to phylotypes. CONCLUSIONS: PyroTRF-ID profits from complementary advantages of pyrosequencing and T-RFLP and is particularly adapted for optimizing laboratory and computational efforts to describe microbial communities and their dynamics in any biological system. The high resolution of the microbial community composition is provided by pyrosequencing, which can be performed on a restricted set of selected samples, whereas T-RFLP enables simultaneous fingerprinting of numerous samples at relatively low cost and is especially adapted for routine analysis and follow-up of microbial communities on the long run.

Light intensity defines growth and photopigment content of a mixed culture of purple phototrophic bacteria.

Purple bacteria (PPB), anoxygenic photoorganoheterotrophic organisms with a hyper-versatile metabolism and high biomass yields over substrate, are promising candidates for the recovery of nutrient resources from wastewater. Infrared light is a pivotal parameter to control and design PPB-based resource recovery. However, the effects of light intensities on the physiology and selection of PPB in mixed cultures have not been studied to date. Here, we examined the effect of infrared irradiance on PPB physiology, enrichment, and growth over a large range of irradiance (0 to 350 W m-2) in an anaerobic mixed-culture sequencing batch photobioreactor. We developed an empirical mathematical model that suggests higher PPB growth rates as response to higher irradiance. Moreover, PPB adapted to light intensity by modulating the abundances of their phototrophic complexes. The obtained results provide an in-depth phylogenetic and metabolic insight the impact of irradiance on PPB. Our findings deliver the fundamental information for guiding the design of light-driven, anaerobic mixed-culture PPB processes for wastewater treatment and bioproduct valorization.

Monitoring SARS-CoV-2 in sewage: Toward sentinels with analytical accuracy.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemia has been one of the most difficult challenges humankind has recently faced. Wastewater-based epidemiology has emerged as a tool for surveillance and mitigation of potential viral outbreaks, circumventing biases introduced by clinical patient testing. Due to the situation urgency, protocols followed for isolating viral RNA from sewage were not adapted for such sample matrices. In parallel to their implementation for fast collection of data to sustain surveillance and mitigation decisions, molecular protocols need to be harmonized to deliver accurate, reproducible, and comparable analytical outputs. Here we studied analytical variabilities linked to viral RNA isolation methods from sewage. Three different influent wastewater volumes were used to assess the effects of filtered volumes (50, 100 or 500 mL) for capturing viral particles. Three different concentration strategies were tested: electronegative membranes, polyethersulfone membranes, and anion-exchange diethylaminoethyl cellulose columns. To compare the number of viral particles, different RNA isolation methods (column-based vs. magnetic beads) were compared. The effect of extra RNA purification steps and different RT-qPCR strategies (one step vs. two-step) were also evaluated. Results showed that the combination of 500 mL filtration volume through electronegative membranes and without multiple RNA purification steps (using column-based RNA purification) using two-step RT-qPCR avoided false negatives when basal viral load in sewage are present and yielded more consistent results during the surveillance done during the second-wave in Delft (The Hague area, The Netherlands). By paving the way for standardization of methods for the sampling, concentration and molecular detection of SARS-CoV-2 viruses from sewage, these findings can help water and health surveillance authorities to use and trust results coming from wastewater based epidemiology studies in order to anticipate SARS-CoV-2 outbreaks.

Metagenomic profiling and transfer dynamics of antibiotic resistance determinants in a full-scale granular sludge wastewater treatment plant.

In the One Health context, wastewater treatment plants (WWTPs) are central to safeguarding water resources. Nonetheless, many questions remain about their effectiveness in preventing antimicrobial resistance (AMR) dissemination. Most surveillance studies monitor the levels and removal of selected antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in intracellular DNA (iDNA) extracted from WWTP influents and effluents. The role of extracellular free DNA (exDNA) in wastewater is mostly overlooked. This study analyzed the transfer of ARGs and MGEs in a full-scale Nereda® reactor removing nutrients with aerobic granular sludge. We tracked the composition and fate of the iDNA and exDNA pools of influent, sludge, and effluent samples. Metagenomics was used to profile the microbiome, resistome, and mobilome signatures of iDNA and exDNA extracts. Selected ARGs and MGEs were analyzed by qPCR. From 2,840 ARGs identified, the genes arr-3 (2%), tetC (1.6%), sul1 (1.5%), oqxB (1.2%), and aph(3")-Ib (1.2%) were the most abundant among all sampling points and bioaggregates. Pseudomonas, Acinetobacter, Aeromonas, Acidovorax, Rhodoferax, and Streptomyces populations were the main potential hosts of ARGs in the sludge. In the effluent, 478 resistance determinants were detected, of which 89% were from exDNA potentially released by cell lysis during aeration in the reactor. MGEs and multiple ARGs were co-localized on the same extracellular genetic contigs. Total intracellular ARGs decreased 3-42% due to wastewater treatment. However, the ermB and sul1 genes increased by 2 and 1 log gene copies mL-1, respectively, in exDNA from influent to effluent. The exDNA fractions need to be considered in AMR surveillance, risk assessment, and mitigation strategies.

Catabolism of sialic acids in an environmental microbial community.

Sialic acids are a family of nine-carbon negatively charged carbohydrates. In animals, they are abundant on mucosa surfaces as terminal carbohydrates of mucin glycoproteins. Some commensal and pathogenic bacteria are able to release, take up and catabolize sialic acids. Recently, sialic acids have been discovered to be widespread among most microorganisms. Although the catabolism of sialic acids has been intensively investigated in the field of host-microbe interactions, very limited information is available on microbial degradation of sialic acids produced by environmental microorganisms. In this study, the catabolic pathways of sialic acids within a microbial community dominated by 'Candidatus Accumulibacter' were evaluated. Protein alignment tools were used to detect the presence of the different proteins involved in the utilization of sialic acids in the flanking populations detected by 16S rRNA gene amplicon sequencing. The results showed the ability of Clostridium to release sialic acids from the glycan chains by the action of a sialidase. Clostridium and Chryseobacterium can take up free sialic acids and utilize them as nutrient. Interestingly, these results display similarities with the catabolism of sialic acids by the gut microbiota. This study points at the importance of sialic acids in environmental communities in the absence of eukaryotic hosts.

Effects of micro-aeration on microbial niches and antimicrobial resistances in blackwater anaerobic digesters.

Anaerobic digestion (AD) of source-diverted blackwater (toilet flush) at ambient room temperature presents challenges for fast hydrolysis of particulate matters. This study investigated the effect of different micro-aeration dosages for blackwater AD. Sequencing batch reactors were operated at ambient room temperature (22 ± 1°C) with micro-aeration (0, 5, 10, 50, and 150 mg O2 g-1 CODfeed per cycle) and gradually reduced hydraulic retention times from 5 d to 2 d. The methanogenesis efficiencies were greater at low oxygen dosages (i.e., 0, 5, 10) while the volatile fatty acids (VFAs) accumulated more at high oxygen dosages (i.e., 50, 150). Microbial communities were significantly different under different oxygen dosages (p<0.05), with segregation of microbial ecological niches in low and high oxygen dosage communities. The low-oxygen-dosage niche (0, 5, and 10 mg g-1 CODfeed) was inhabited by fermenting and syntrophic bacteria (e.g., Cytophaga, Syntrophomonas) and methanogens (e.g., Methanobacterium, Methanolinea, Methanosaeta). The high-oxygen-dosage niche (50 and 150 mg g-1 CODfeed) had significantly (p<0.05) more facultative anaerobic bacteria (Ignavibacteriales and Cloacamonales), and aerobic bacteria (Rhodocyclales). Moreover, blackwater can be a source of antimicrobial resistance genes (ARGs), which are affected by different oxygen dosages. The ARG variation correlated with the microbial community composition (p<0.05). Low-oxygen-dosage communities contained a higher prevalence of mobile gene elements (intI1 and korB) and tetM, ermB, sul1, sul2, and blaCTX-M than the high-oxygen-dosage communities, indicating that oxygen dosage influenced the prevalence of populations carrying ARGs. These findings suggest that application of micro-aeration to AD can be used to control ARG profiles.

Upgrading residues from wastewater and drinking water treatment plants as low-cost adsorbents to remove extracellular DNA and microorganisms carrying antibiotic resistance genes from treated effluents.

Wastewater treatment is challenged by the continuous emergence of chemical and biological contaminants. Disinfection, advanced oxidation, and activated carbon technologies are accessible in high-income countries to suppress them. Low-cost, easily implementable, and scalable solutions are needed for sanitation across regions. We studied the properties of low-cost absorbents recycled from drinking water and wastewater treatment plant residues to remove environmental DNA and xenogenetic elements from used water. Materials characteristics and DNA adsorption properties of used iron-oxide-coated sands and of sewage-sludge biochar obtained by pyrolysis of surplus activated sludge were examined in bench-scale batch and up-flow column systems. Adsorption profiles followed Freundlich isotherms, suggesting a multilayer adsorption of nucleic acids on these materials. Sewage-sludge biochar exhibited high DNA adsorption capacity (1 mg g-1) and long saturation breakthrough times compared to iron-oxide-coated sand (0.2 mg g-1). Selected antibiotic resistance genes and mobile genetic elements present on the free-floating extracellular DNA fraction and on the total environmental DNA (i.e., both extra/intracellular) were removed at 85% and 97% by sewage-sludge biochar and at 54% and 66% by iron-oxide-coated sand, respectively. Sewage-sludge biochar is attractive as low-cost adsorbent to minimize the spread of antimicrobial resistances to the aquatic environment while strengthening the role of sewage treatment plants as resource recovery factories.