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.

The fate of sulfamethoxazole and trimethoprim in a micro-aerated anaerobic membrane bioreactor and the occurrence of antibiotic resistance in the permeate.

This study investigates the effects, conversions, and resistance induction, following the addition of 150 µg·L-1 of two antibiotics, sulfamethoxazole (SMX) and trimethoprim (TMP), in a laboratory-scale micro-aerated anaerobic membrane bioreactor (MA-AnMBR). TMP and SMX were removed at 97 and 86%, indicating that micro-aeration did not hamper their removal. These antibiotics only affected the pH and biogas composition of the process, with a significant change in pH from 7.8 to 7.5, and a decrease in biogas methane content from 84 to 78%. TMP was rapidly adsorbed onto the sludge and subsequently degraded during the long solids retention time of 27 days. SMX adsorption was minimal, but the applied hydraulic retention time of 2.6 days was sufficiently long to biodegrade SMX. The levels of three antibiotic-resistant genes (ARGs) (sul1, sul2, and dfrA1) and one mobile genetic element biomarker (intI1) were analyzed by qPCR. Additions of the antibiotics increased the relative abundances of all ARGs and intI1 in the MA-AnMBR sludge, with the sul2 gene folding 15 times after 310 days of operation. The MA-AnMBR was able to reduce the concentration of antibiotic-resistant bacteria (ARB) in the permeate by 3 log.

Candidate biomarkers of antibiotic resistance for the monitoring of wastewater and the downstream environment.

Urban wastewater treatment plants (UWTPs) are essential for reducing the pollutants load and protecting water bodies. However, wastewater catchment areas and UWTPs emit continuously antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs), with recognized impacts on the downstream environments. Recently, the European Commission recommended to monitor antibiotic resistance in UWTPs serving more than 100 000 population equivalents. Antibiotic resistance monitoring in environmental samples can be challenging. The expected complexity of these systems can jeopardize the interpretation capacity regarding, for instance, wastewater treatment efficiency, impacts of environmental contamination, or risks due to human exposure. Simplified monitoring frameworks will be essential for the successful implementation of analytical procedures, data analysis, and data sharing. This study aimed to test a set of biomarkers representative of ARG contamination, selected based on their frequent human association and, simultaneously, rare presence in pristine environments. In addition to the 16S rRNA gene, ten potential biomarkers (intI1, sul1, ermB, ermF, aph(3'')-Ib, qacEΔ1, uidA, mefC, tetX, and crAssphage) were monitored in DNA extracts (n = 116) from raw wastewater, activated sludge, treated wastewater, and surface water (upstream and downstream of UWTPs) samples collected in the Czech Republic, Denmark, Israel, the Netherlands, and Portugal. Each biomarker was sensitive enough to measure decreases (on average by up to 2.5 log-units gene copy/mL) from raw wastewater to surface water, with variations in the same order of magnitude as for the 16S rRNA gene. The use of the 10 biomarkers allowed the typing of water samples whose origin or quality could be predicted in a blind test. The results show that, based on appropriate biomarkers, qPCR can be used for a cost-effective and technically accessible approach to monitoring wastewater and the downstream environment.

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.

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.

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.

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.

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.

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.

Effect of temperature on the compositions of ladderane lipids in globally surveyed anammox populations.

The adaptation of bacteria involved in anaerobic ammonium oxidation (anammox) to low temperatures will enable more efficient removal of nitrogen from sewage across seasons. At lower temperatures, bacteria typically tune the synthesis of their membrane lipids to promote membrane fluidity. However, such adaptation of anammox bacteria lipids, including unique ladderane phospholipids and especially shorter ladderanes with absent phosphatidyl headgroup, is yet to be described in detail. We investigated the membrane lipids composition (UPLC-HRMS/MS) and dominant anammox populations (16S rRNA gene amplicon sequencing, Fluorescence in situ hybridization) in 14 anammox enrichments cultivated at 10-37 °C. "Candidatus Brocadia" appeared to be the dominant organism in all but two laboratory enrichments of "Ca. Scalindua" and "Ca. Kuenenia". At lower temperatures, the membranes of all anammox populations were composed of shorter [5]-ladderane ester (reduced chain length demonstrated by decreased fraction of C20/(C18 + C20)). This confirmed the previous preliminary evidence on the prominent role of this ladderane fatty acid in low-temperature adaptation. "Ca. Scalindua" and "Ca. Kuenenia" had distinct profile of ladderane lipids compared to "Ca. Brocadia" biomasses with potential implications for adaptability to low temperatures. "Ca. Brocadia" membranes contained a much lower amount of C18 [5]-ladderane esters than reported in the literature for "Ca. Scalindua" at similar temperature and measured here, suggesting that this could be one of the reasons for the dominance of "Ca. Scalindua" in cold marine environments. Furthermore, we propose additional and yet unreported mechanisms for low-temperature adaptation of anammox bacteria, one of which involves ladderanes with absent phosphatidyl headgroup. In sum, we deepen the understanding of cold anammox physiology by providing for the first time a consistent comparison of anammox-based communities across multiple environments.

Monitoring antibiotic resistance genes in wastewater environments: The challenges of filling a gap in the One-Health cycle.

Antibiotic resistance (AR) is a global problem requiring international cooperation and coordinated action. Global monitoring must rely on methods available and comparable across nations to quantify AR occurrence and identify sources and reservoirs, as well as paths of AR dissemination. Numerous analytical tools that are gaining relevance in microbiology, have the potential to be applied to AR research. This review summarizes the state of the art of AR monitoring methods, considering distinct needs, objectives and available resources. Based on the overview of distinct approaches that are used or can be adapted to monitor AR, it is discussed the potential to establish reliable and useful monitoring schemes that can be implemented in distinct contexts. This discussion places the environmental monitoring within the One-Health approach, where two types of risk, dissemination across distinct environmental compartments, and transmission to humans, must be considered. The plethora of methodological approaches to monitor AR and the variable features of the monitored sites challenge the capacity of the scientific community and policy makers to reach a common understanding. However, the dialogue between different methods and the production of action-oriented data is a priority. The review aims to warm up this discussion.

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.

Temperature and Nutrient Limitations Decrease Transfer of Conjugative IncP-1 Plasmid pKJK5 to Wild Escherichia coli Strains.

Plasmid-mediated dissemination of antibiotic resistance among fecal Enterobacteriaceae in natural ecosystems may contribute to the persistence of antibiotic resistance genes in anthropogenically impacted environments. Plasmid transfer frequencies measured under laboratory conditions might lead to overestimation of plasmid transfer potential in natural ecosystems. This study assessed differences in the conjugative transfer of an IncP-1 (pKJK5) plasmid to three natural Escherichia coli strains carrying extended-spectrum beta-lactamases, by filter mating. Matings were performed under optimal laboratory conditions (rich LB medium and 37°C) and environmentally relevant temperatures (25, 15 and 9°C) or nutrient regimes mimicking environmental conditions and limitations (synthetic wastewater and soil extract). Under optimal nutrient conditions and temperature, two recipients yielded high transfer frequencies (5 × 10-1) while the conjugation frequency of the third strain was 1000-fold lower. Decreasing mating temperatures to psychrophilic ranges led to lower transfer frequencies, albeit all three strains conjugated under all the tested temperatures. Low nutritive media caused significant decreases in transconjugants (-3 logs for synthetic wastewater; -6 logs for soil extract), where only one of the strains was able to produce detectable transconjugants. Collectively, this study highlights that despite less-than-optimal conditions, fecal organisms may transfer plasmids in the environment, but the transfer of pKJK5 between microorganisms is limited mainly by low nutrient conditions.

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.

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.

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.

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.

Annual dynamics of antimicrobials and resistance determinants in flocculent and aerobic granular sludge treatment systems.

The occurrence and removal patterns of 24 antimicrobial agents and antimicrobial resistant determinants namely 6 antibiotic resistance genes (ARGs) and 2 mobile genetic elements (MGEs), and the fecal indicator E. coli were investigated in three full-scale wastewater treatment plants. Their waterlines and biosolids lines (including secondary treatment based on both granular and activated sludge) were sampled monthly throughout one year. Samples were analyzed by means of LC-MS/MS, qPCR and cell enumeration, respectively. The influence of rainfall, temperature, and turbidity on the occurrence and removal of the aforementioned agents was assessed through statistical linear mixed models. Ten of the antimicrobial agents (macrolides, fluoroquinolones, tetracyclines, and sulfonamides) were commonly found in influent in concentrations of 0.1-2 µg L-1, and the predominant ARGs were ermB and sul1 (6.4 and 5.9 log10 mL-1 respectively). Warmer temperatures slightly reduced gene concentrations in influent whilst increasing that of E. coli and produced an uneven effect on the antimicrobial concentrations across plants. Rainfall diluted both E. coli (-0.25 logs, p < 0.001) and antimicrobials but not genes. The wastewater treatment reduced the absolute abundance of both genes (1.86 logs on average) and E. coli (2.31 logs on average). The antimicrobials agents were also partly removed, but 8 of them were still detectable after treatment, and 6 accumulated in the biosolids. ARGs were also found in biosolids with patterns resembling those of influent. No significant differences in the removal of antimicrobials, genes and E. coli were observed when comparing conventional activated sludge with aerobic granular sludge. Irrespective of the type of sludge treatment, the removal of genes was significantly reduced with increasing hydraulic loads caused by rainfall (-0.35 logs per ∆ average daily flow p < 0.01), and slightly decreased with increasing turbidity (-0.02 logs per ∆1 nephelometric turbidy unit p < 0.05) .