Biology subjects

van Loosdrecht, M.

Publications and source records attributed to van Loosdrecht, M..

14 recordsLinked to original sources

bioRxiv2026

SugarBase: mapping glycomolecule precursors in microbes

Glycan biosynthesis relies on nucleotide-activated sugars, essential metabolites across all domains of life, yet their usage in microbes is poorly understood. Here we present SugarBase, a mass spectrometry and bioinformatic pipeline for untargeted exploration of microbial nucleotide sugar networks. SugarBase resolves the chemical complexity of microbial metabolism by combining narrow-window DIA fragmentation with a chemistry-informed parent ion identification algorithm. Applying SugarBase across a broad phylogenetic range of microbes revealed extensive, species-specific nucleotide sugar profiles, including many candidates with no existing annotation, generating the most comprehensive inventory of nucleotide sugars to date. SugarBase guided identification of gene clusters and allowed discrimination between pseudaminic- and legionaminic acid-producing strains, where genomic and proteomic data provided only ambiguous information. We resolved distinct nonulosonic acid profiles in several Campylobacter jejuni strains, sugars which may alter susceptibility towards distinct flagellotropic phages. We further identify previously undescribed CMP-activated higher-carbon ulosonic acids in Magnetospirillum, expanding the known chemical space in glycan biosynthesis. In summary, SugarBase supports scalable discovery of microbial nucleotide sugar pathways and enzymes, expanding access to chemically complex glycans and providing new targets for antimicrobial development.

systems biology↗

bioRxiv2024

Metaproteomic profiling of the secretome of a granule-forming Ca. Accumulibacter enrichment

Extracellular proteins are supposed to play crucial roles in the formation and structure of biofilms and aggregates. However, often little is known about these proteins, in particular for microbial communities. Here, we use two advanced metaproteomic approaches to study the extracellular proteome in a granular Candidatus Accumulibacter enrichment as a proxy for microbial communities that form solid microbial granules, such as used in biological wastewater treatment. Limited proteolysis of whole granules and metaproteome isolation from the cultures supernatant successfully identified over 50% of the protein biomass to be secreted. Moreover, structural and sequence-based classification identified 387 proteins, corresponding to over 50% of the secreted biomass, with characteristics that could aid the formation of aggregates, including filamentous, beta-barrel containing, and cell surface proteins. However, while most filamentous proteins originated from Ca. Accumulibacter, among others cell surface proteins did not. This suggests that not only a range of different proteins, but also multiple organisms contribute to granular biofilm formation. Therefore, the obtained extracellular metaproteome data from the granular Ca. Accumulibacter enrichment provides a resource for exploring proteins that potentially support the formation and stability of granular biofilms, whereas the demonstrated approaches can be applied to explore biofilms of microbial communities in general. SIGNIFICANCEBiofilm-forming microbial communities are widespread and pose both challenges and opportunities in various settings in life. Structure-providing, extracellular proteins likely play a crucial role in the formation of the biofilm matrix, but these proteins are challenging to characterise due to the dynamic and complex nature of these communities. We used two advanced metaproteomic approaches to enrich for the extracellular proteins in a granule-forming Candidatus Accumulibacter enrichment culture as a proxy for granule-forming communities present in wastewater treatment plants. The extracellular proteins were additionally classified using structure and sequence-based annotation tools, which identified multiple different protein categories that potentially aid in granule formation, but also may provide structure to the biofilm matrix. Interestingly, although the granules were highly enriched for Ca. Accumulibacter, several structure-providing protein categories originated from other organisms. The obtained metaproteomic data contribute to the understanding of proteins and processes that are potentially involved in granule formation. This could further help to optimise processes involving granular biofilms and identify candidates for the recovery of novel biopolymers for biotechnological applications.

systems biology↗

bioRxiv2024

FT-IR micro-spectroscopy for imaging the extracellular matrix composition in biofilms

Microorganisms form granules by embedding themselves in an extracellular matrix through the secretion of extracellular polymeric substances (EPS). The extracellular matrix is a complex structure comprising of e.g. proteins, polysaccharides, lipids, and extracellular DNA. Understanding the function of individual EPS components within the matrix not only requires knowledge on the composition of the extracellular matrix, but also on the spatial distribution of said components. Molecular imaging like e.g. fluorescence microscopy have been used for the visualization of the extracellular matrix, but these target specific molecules. Untargeted approaches like FT-IR micro-spectroscopy would allow for a broader exploration. In this study FT-IR micro-spectroscopy analysis was implemented on sliced anaerobic granular sludge to explore the EPS distribution. Visualization of single wavenumber absorbance showed a higher polysaccharide content in the EPS at the granule perimeter, shifting to a higher protein concentration toward the centre. The boundary of this shift was approximately 150 {micro}m from the surface, which was in accordance with the layer of fermentative bacteria described in literature. The complexity in the polymer composition meant that many functional groups were overlapping, making FT-IR annotation challenging. To address this, principal component analysis and two-dimensional correlation spectroscopy analysis were included in the analysis. These methods enabled the identification of overlapping functional groups and correlations between functional groups. Positive correlations between protein and polysaccharide functional groups suggested the presence of glycoproteins, which has been regularly described in chemical EPS analysis studies. Additionally, correlations between sulfated compounds and protein/polysaccharide functional groups indicated potential co-localization in the extracellular matrix. Differences in positive correlations of sialic acids with polysaccharides suggest variations in polysaccharide compositions, possibly caused by differences in the microbial community.

microbiology↗

bioRxiv2024

NovoLign: metaproteomics by sequence alignment

Tremendous advances in mass spectrometric and bioinformatic approaches have expanded proteomics into the field of microbial ecology. The commonly used spectral annotation method for metaproteomics data relies on database searching, which requires sample-specific databases obtained from whole metagenome sequencing experiments. However, creating these databases is complex, time-consuming, and prone to errors, potentially biasing experimental outcomes and conclusions. This asks for alternative approaches that can provide rapid and orthogonal insights into metaproteomics data. Here we present NovoLign, a de novo metaproteomics pipeline that performs sequence alignment of de novo sequences from complete metaproteomics experiments. The pipeline enables rapid taxonomic profiling of complex communities and evaluates the taxonomic coverage of metaproteomics outcomes obtained from database searches. Furthermore, the NovoLign pipeline supports the creation of reference sequence databases for database searching to ensure comprehensive coverage. The NovoLign pipeline is publicly available via: https://github.com/hbckleikamp/NovoLign.

systems biology↗

bioRxiv2024

A difficult coexistence: resolving the iron-induced nitrification delay in groundwater filters

Rapid sand filters (RSF) are an established and widely applied technology for the removal of dissolved iron (Fe2+) and ammonium (NH +) in groundwater treatment. Most often, biological NH + oxidation is delayed and starts only upon complete Fe2+ depletion. However, the mechanism(s) responsible for the inhibition of NH + oxidation by Fe2+ or its oxidation (by)products remains elusive, hindering further process control and optimization. We used batch assays, lab-scale columns, and full-scale filter characterizations to resolve the individual impact of the main Fe2+ oxidizing mechanisms and the resulting products on biological NH + oxidation. Modelling of the obtained datasets allowed to quantitatively assess the hydraulic implications of Fe2+ oxidation. Dissolved Fe2+ and the reactive oxygen species formed as byproducts during Fe2+ oxidation had no direct effect on nitrification. The Fe3+ oxides on the sand grain coating, commonly assumed to be the main cause for inhibited nitrification, seemed instead to enhance nitrification by providing additional surface area for biofilm growth. Modelling allowed to exclude mass transfer limitations induced by accumulation of iron flocs and consequent filter clogging as the cause for delayed nitrification. We unequivocally identify the inhibition of NH +oxidizing organisms by the Fe3+ flocs generated during Fe2+ oxidation as the main cause for the commonly observed nitrification delay. The addition of Fe3+ flocs inhibited NH + oxidation both in batch and column tests, and the removal of Fe3+ flocs by backwashing completely re-established the NH + removal capacity, suggesting that the inhibition is reversible. In conclusion, our findings not only identify the iron form that causes the inhibition, albeit the biological mechanism remains to be identified, but also highlight the ecological importance of iron cycling in nitrifying environments. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/581000v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@65ca28org.highwire.dtl.DTLVardef@bb3da3org.highwire.dtl.DTLVardef@cbd32dorg.highwire.dtl.DTLVardef@1956207_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightO_LIDissolved Fe2+ and reactive oxygen species do not affect NH + oxidation C_LIO_LIFe oxide coating aids sand grain colonization by NH +-oxidizing bacteria C_LIO_LIFe3+ flocs inhibit NH + oxidation by reducing the nitrifying capacity of AOB C_LIO_LIChanges in transport patterns due to clogging do not play a major role in NH + oxidation C_LIO_LIThe inhibition of NH4+ oxidation is reversible and reduced by backwashing C_LI

microbiology↗

bioRxiv2024

Shifting to biology promotes highly efficient iron removal in groundwater filters

Rapid sand filters are established and widely applied technologies for groundwater treatment. Conventionally, intensive aeration is employed to provide oxygen for the oxidation and removal of the main groundwater contaminants. While effective, intensive aeration promotes flocculent iron removal, which results in iron flocs that rapidly clog the filter. In this study, we operated two parallel full-scale sand filters at different aeration intensities to resolve the relative contribution of homogeneous, heterogeneous and biological iron removal pathways, and identify their operational controls. Our results show that mild aeration in the LOW filter (5 mg/L O2pH 6.9) promoted biological iron removal and enabled iron oxidation at twice the rate compared to the intensively aerated HIGH filter (>10mg/L O2,pH 7.4). ESEM images showed distinctive twisted stalk-like Fe solids, biosignatures of Gallionella ferruginea, both in the LOW filter sand coatings as well as in its backwash solids. In accordance, 10 times higher DNA copy numbers of G. ferruginea were found in the LOW filter effluent. Clogging by biogenic FeOx was slower than by chemical FeOx flocs, resulting in lower backwash frequencies and yielding four times more water per run. Ultimately, our results reveal that biological Fe2+ oxidation can be actively controlled and favoured over competing physico-chemical routes. The resulting operational benefits are only starting to be appreciated, with the counterintuitive higher oxidation rates and water yields at lower aeration regimes, and the production of more compact and practically valuable FeOx solids being of outmost interest.

microbiology↗

bioRxiv2024

Exploring the metabolic potential of Aeromonas to utilise the carbohydrate polymer chitin

Members of the Aeromonas genus are commonly found in natural aquatic ecosystems. However, they are also frequently present in non-chlorinated drinking water distribution systems. High densities of these bacteria indicate favorable conditions for microbial regrowth, which is considered undesirable. Studies have indicated that the presence of Aeromonas is associated with loose deposits and the presence of invertebrates, specifically Asellus aquaticus. Therefore, a potential source of nutrients in these nutrient poor environments is chitin, the structural shell component in these invertebrates. In this study, we demonstrate the ability of two Aeromonas strains, commonly encountered in drinking water distribution systems, to effectively degrade and utilize chitin as a sole carbon and nitrogen source. We conducted a quantitative proteomics study on the cell biomass and secretome of both strains, revealing a dedicated and diverse spectrum of hydrolytic enzymes and pathways for the uptake and metabolism of chitin. Furthermore, when the primary nutrient source was switched from glucose to chitin, more than half of the Aeromonas proteome showed significant changes. Additionally, a genomic analysis of Aeromonas species found in drinking water distribution systems suggests a general potential ability of this genus to degrade and utilize a variety of carbohydrate biopolymers. This study indicates the relation between the utilization of chitin by Aeromonas and their association with invertebrates such as A. aquaticus in loose deposits in drinking water distribution systems. This knowledge provides the foundation for the development of more effective water sanitation strategies.

microbiology↗

bioRxiv2022

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 being treated 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 about 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 were enriched for antibiotic resistant bacteria. 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.

microbiology↗

bioRxiv2022

Meta-omics profiling of full-scale groundwater rapid sand filters explains stratification of iron, ammonium and manganese removals

Rapid sand filters (RSF) are an established and widely applied technology for groundwater treatment. Yet, the underlying interwoven biological and physical-chemical reactions controlling the sequential removal of iron, ammonia and manganese remain poorly understood. To resolve the contribution and interactions between the individual reactions, we studied two full-scale drinking water treatment plant configurations, namely (i) one dual-media (anthracite and quartz sand) filter and (ii) two single-media (quartz sand) filters in series. In situ and ex situ activity tests were combined with mineral coating characterization and metagenome-guided metaproteomics along the depth of each filter. Both plants exhibited comparable performances and process compartmentalization, with most of ammonium and manganese removal occurring only after complete iron depletion. Within each compartment, the homogeneity of the media coating and genome-based microbial composition highlighted the effect of backwashing on filter media mixing. In stark contrast, intra-compartment contaminant removal was highly stratified following decreasing substrate availability along the filter height. This apparent and long-standing conflict was resolved by quantifying the expressed proteome at different filter heights, revealing a consistent stratification of proteins catalysing ammonia oxidation and protein-based relative abundances of nitrifying genera. This implies that microorganisms adapt their protein pool to the available nutrient load at a faster rate than the backwash mixing frequency. Ultimately, these results show the unique and complementary potential of metaproteomics to understand metabolic adaptations and interactions in highly dynamic ecosystems.

microbiology↗

bioRxiv2022

Antibiotic resistance response to sulfamethoxazole from the intracellular and extracellular DNA fractions of activated sludge

In activated sludge, the antibiotic resistance genes (ARGs) can be present either in the intracellular (iDNA) or extracellular DNA fraction (exDNA). Recent advances in the exDNA extraction methodology allow a better profiling of the pool of ARGs. However, little is known about how stress conditions modify the distribution of ARGs between both DNA fractions. Here, we performed two batch tests for analyzing the effects of two different stress conditions, namely nutrient starvation and high concentrations of sulfamethoxazole (1, 10 and 150 mg L-1) in activated sludge. We tracked by qPCR the resulting relative abundances of four target genes, namely the universal 16S rRNA gene, the class 1 integron-integrase gene intI1, and the sulfonamide resistance genes sul1 and sul2 in both the iDNA and exDNA fractions. In the exDNA pool, unlike starvation, which provoked a decrease of 1-2 log10 [copies] ng DNA-1 in the concentration of sul1 and intI1, the presence of sulfamethoxazole did not influence the abundances of sul1 and sul2. However, high concentrations of sulfamethoxazole (150 mg L- 1) selected for microorganisms harboring sul1 and, more remarkably, sul2 genes in their iDNA during their exponential growth phase. The abundances of intI1 and sul1 were positively correlated in the exDNA fraction (r>0.7), whereas no significant correlation (p<0.05) between the abundance of these two genes was found in the iDNA fraction of the sludge. High SMX concentrations influenced the abundance of ARGs in the iDNA; their abundance in the exDNA was influenced by nutrient limitations. Further studies should consider the profiling of exDNA fractions because of the relationship between ARGs and mobile genetic elements. Besides, the surveillance of antimicrobial resistance is encouraged in wastewater treatment plants facing high antibiotic concentrations. HighlightsO_LIStarvation caused a decrease in the exDNA concentration of the activated sludge. C_LIO_LIA positive correlation in the abundances of intI1 and sul1 was found in the exDNA fraction. C_LIO_LIHigh concentrations of SMX selected for antibiotic resistant microorganisms. C_LI

bioengineering↗

bioRxiv2021

Adaptation of anammox bacteria to low temperature via gradual acclimation and cold shocks: distinctions in protein expression, membrane composition and activities

Anammox bacteria enable an efficient removal of nitrogen from sewage in processes involving partial nitritation and anammox (PN/A) or nitrification, partial denitrification, and anammox (N-PdN/A). In mild climates, anammox bacteria must be adapted to [≤]15 {degrees}C, typically by gradual temperature decrease; however, this takes months or years. To reduce the time necessary for the adaptation, an unconventional method of cold shocks is promising, involving hours-long exposure of anammox biomass to extremely low temperatures. We compared the efficacies of gradual temperature decrease and cold shocks to increase the metabolic activity of anammox (fed batch reactor, planktonic "Ca. Kuenenia"). We assessed the cold shock mechanism on the level of protein expression (quantitative shot-gun proteomics, LC-HRMS/MS) and structure of membrane lipids (UPLC-HRMS/MS). The shocked culture was more active (0.66{+/-}0.06 vs 0.48{+/-}0.06 kg-N/kg-VSS/d) and maintained the relative content of N-respiration proteins at levels consistent levels with the initial state, whereas the content of these proteins decreased in gradually acclimated culture. Cold shocks also induced a more efficient up-regulation of cold shock proteins (e.g. CspB, TypA, ppiD). Ladderane lipids characteristic for anammox evolved to a similar end-point in both cultures which confirms their role in anammox bacteria adaptation to cold and indicates a three-pronged adaptation mechanism involving ladderane lipids (ladderane alkyl length, introduction of shorter non-ladderane alkyls, polar headgroup). Overall, we show the outstanding potential of cold shocks for low-temperature adaptation of anammox bacteria and provide yet unreported detailed mechanisms of anammox adaptation to low temperatures. HighlightsO_LIAnammox bacteria were adapted to low T by gradual acclimation and cold shocks C_LIO_LIThe shocked culture was more active (0.66{+/-}0.06 vs 0.48{+/-}0.06 kg-N/kg-VSS/d) C_LIO_LIN-respiration proteins content decreased in gradually acclimated bacteria C_LIO_LISeveral cold shock proteins were upregulated more efficiently by cold shocks C_LIO_LIAt {downarrow}T, anammox adjusted ladderane membrane lipid composition in three aspects C_LI Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

bioengineering↗

bioRxiv2020

A general approach to explore prokaryotic protein glycosylation reveals the unique surface layer modulation of an anammox bacterium.

The enormous chemical diversity and strain variability of prokaryotic protein glycosylation makes a large-scale exploration exceptionally challenging. Therefore, despite the universal relevance of protein glycosylation across all domains of life, the understanding of their biological significance and the evolutionary forces shaping oligosaccharide structures remains highly limited. Here, we report on a newly established mass binning glycoproteomics approach that establishes the chemical identity of the carbohydrate components and performs untargeted exploration of prokaryotic oligosaccharides from large-scale proteomics data directly. We demonstrate our approach by exploring an enrichment culture of the globally relevant anaerobic ammonium-oxidizing bacterium Ca. Kuenenia stuttgartiensis. By doing so we resolved a remarkable array of oligosaccharides, produced by two entirely unrelated glycosylation machineries targeting the same surface-layer protein (SLP) simultaneously. More intriguingly, the investigated strain also accomplished modulation of highly specialized sugars, supposedly in response to its energy metabolism--the anaerobic oxidation of ammonium --which depends on the acquisition of substrates of opposite charge. Ultimately, we provide a systematic approach for the compositional exploration of prokaryotic protein glycosylation, and reveal for the first time a remarkable balance between maximising cellular protection through a complex array of oligosaccharides and adhering to the requirements of the metabolic lifestyle.

systems biology↗

bioRxiv2020

Effects of light/dark diel cycles on the photoorganoheterotrophic metabolism of Rhodopseudomonas palustris for differential electron allocation to PHAs and H2

Light/dark cycles can impact the electron distribution in Rhodopseudomonas palustris, a hyperversatile photoorganoheterotrophic purple non-sulfur bacterium (PNSB). Dynamic conditions during diel cycles are important for the physiology of PNSB, but the coupling between illumination patterns and redox balancing has not been extensively studied. For survival and growth, Rhodopseudomonas has developed different mechanisms to allocate electrons under dynamic growth conditions. Products such as hydrogen and poly-{beta}-hydroxyalkanoates (PHAs) can form alternative electron sinks. A continuous culture, fed with a balanced nutrients medium, was exposed to three different conditions: 24 h continuous infrared illumination, 16h light/8h dark, and 8h light/16h dark. Light and dark phase durations in a cycle determined the energy availability level (light) and the attainment of a stationary state. Under long dark phases, the acetate substrate accumulated to levels that could not be depleted by growth in the light. Under short dark phases, acetate was rapidly consumed in the light with most of the phototrophic growth occurring under acetate-limiting conditions. Under diel cycles, substrate uptake and growth were unbalanced and Rhodopseudomonas shunted the excess of carbon and electron flow first toward PHAs production. Only secondarily, when PHA storage got saturated, the electron excess was redirected toward H2. A numerical model described well the dynamics of biomass and nutrients during the different light/dark cycle regimes. The model simulations allowed determination of stoichiometric and kinetic parameters for conversion by Rhodopseudomonas. Understanding the inherent process dynamics of diel light cycles in purple sulfur bacteria cultures would enable optimization procedures for targeted bioproduct formation. ImportancePurple non-sulfur bacteria (PNSB) are important anoxygenic phototrophic microorganisms that take part in numerous environmental processes, based on their metabolic versatility. Rhodopseudomonas palustris is a model photosynthetic bacterium of the PNSB guild. Light cycles influence deeply its physiology. Poly-{beta}-hydroxyalkanoates (PHAs) and biohydrogen are two of the most studied metabolic products of Rhodopseudomonas, because of their biotechnology potential besides involvement in carbon and electron allocations in its metabolism. Their production mechanisms have often been described as competitive, but the rationale behind the production of one or the other compound has not been elucidated. Here, we found that under light / dark cycles an excess of organic substrate was first directed toward PHAs production, and only when this pathway was saturated H2 was produced. Understanding the dynamics of carbon and electron allocation under intermittent light cycles enhances our knowledge on PNSB metabolisms and paves ways to manage the formation of targeted bioproducts.

microbiology↗

bioRxiv2020

A novel method to isolate free-floating extracellular DNA from wastewater for quantitation and metagenomic profiling of mobile genetic elements and antibiotic resistance genes

Antibiotic resistant genes (ARGs) and mobile genetic elements (MGEs) can be found in the free-floating extracellular DNA (eDNA) fraction of microbial systems. These xenogenic components can generate bacterial cells resistant to one or more antibiotics by natural transformation. Because of low concentration in wastewater, the obtaining of a high quality and a high yield of eDNA extract is challenging. We developed a method using chromatography to isolate eDNA without causing cell lysis (often unchecked) from complex wastewater matrices. The chromatographic step involved a diethylaminoethyl-cellulose-monolithic column to capture the eDNA found in cell-free filtered wastewater samples (e.g. influent wastewater, activated sludge and treated effluent wastewaster). Free-floating eDNA yields from 1 L of influent, activated sludge and treated effluent water reached 12.5 {+/-} 1.9 g, 12.3 {+/-} 1 g and 5.6 {+/-} 2.9 g of raw eDNA and 9.0 {+/-} 0.7 g, 5.6 {+/-} 0.46 g and 2.6 {+/-} 1.3 g of purified eDNA, respectively. In order to check the suitability of free-floating eDNA extracts for molecular analysis, qPCR and metagenomics were performed. eDNA extracts from treated effluent water were analyzed by qPCR to quantify a selected panel of ARGs and MGEs. Microbiome, resistome, and mobilome profiles from activated sludge free-floating eDNA were measured by metagenomic sequencing. Between iDNA and eDNA fractions, qPCR showed differences of 0.94, 1.11, 1.92 and 1.32 log10 gene copies mL-1 for sulfonamides resistant genes (sul1 and sul2), {beta}-lactamase resistance gene blaCTXM, and the class 1 integron-integrase (intI1) MGE, respectively. These differences highlighted the crucial need for an isolation method to discern both iDNA and eDNA to understand ARGs persistence and quantity in complex cultures. The eDNA yields obtained from 1 L of activated sludge (3.6 g of total suspended solids L-1) samples were substantially higher than the amount of DNA template needed for high-throughput sequencing (>1 g) in service facilities. Subsystems classification showed that the eDNA metagenome was mainly composed by MGEs (65.1%). The 35.9% rest related to traditional functional genetic signatures. It was the first time the resistome from the eDNA fraction was analyzed showing lower number of primary aligned reads when compared to the iDNA and a predominance of aminoglycosides and {beta}-lactamams. Metagenome results showed that eDNA can not be discarded as a pool of ARGs and MGEs for horizontal gene transfer. This novel isolation method was powerful to elucidate the molecular compositions of free-floating eDNA fractions in complex environmental samples such as wastewater environments at different microbial densities. Data obtained using this extraction method will foster xenogenic and microbial risk assessments across urban and natural water systems. This will support water authorities in the delineation of measures to adopt at wastewater treatment plants to remove them and safeguard environmental and public health. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/072397v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1905305org.highwire.dtl.DTLVardef@3a774borg.highwire.dtl.DTLVardef@6e1262org.highwire.dtl.DTLVardef@790c42_HPS_FORMAT_FIGEXP M_FIG Picture created with BioRender C_FIG

molecular biology↗