Biology subjects

van Loosdrecht, M. C. M.

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

At least 19 recordsLinked to original sources

bioRxiv2026

Dark CO2 Fixation via the Ethylmalonyl-CoA Pathway Establishes Metabolic Parity: A Stoichiometric Basis for Compounding Ecosystem Shifts

A central challenge in microbial ecology is understanding how competing guilds coexist and why community structures unexpectedly drift away from theoretical steady states. While polyphosphate accumulation provides energy and redox buffering in many heterotrophs, how competing lineages occupying the same ecological niche without polyphosphate synthesis manage equivalent intracellular redox imbalances remains unposed. Using an enhanced biological phosphorus removal macrocosm, we integrated quantitative stoichiometry with metaproteomics to resolve alternative metabolic strategies that underpin cellular homeostasis in heterotrophs. We show that glycogen-accumulating organisms (GAOs) achieve baseline metabolic parity with polyphosphate-accumulating organisms (PAOs) through a parallel redox-buffering mechanism: heterotrophic CO2/HCO3- re-assimilation via the ethylmalonyl-CoA pathway. This inorganic carbon fixation couples structural carbon conservation with tight redox control, mitigating intracellular electron overflow and eliminating the long-assumed GAO bioenergetic inferiority. The bioenergetic efficiency of GAOs is further fortified by fine-tuned metabolic wiring, featuring energy-efficient high affinity acetate activation, ferredoxin-centered biochemistry, and energy-neutral polyhydroxyalkanoates (PHA) mobilization. Strikingly, minor formate co-feeding disrupted this established PAO/GAO parity. Stoichiometric simulations revealed that this formate supplementation creates an asymmetric bioenergetic niche that grants per-cycle energy gains exclusively to GAOs. Decoupled from hydraulic throughput, solids retention time control retains cells carrying accumulated intracellular inventory, translating subtle per-cycle stoichiometric edges into a multi-generational ratchet and drives a rapid community shift from PAO/GAO co-dominance to GAO dominance. By exposing limitations of traditional single-substrate and single-cycle steady-state models, our findings reveal how inorganic resource management and generational metabolic compounding govern community assembly in biomass-retaining microbial ecosystems.

bioengineering↗

bioRxiv2025

Measuring Temporal Variations of Nucleotide Pools in Microbial Granular Biofilm Performing Enhanced Biological Phosphorous Removal

Microbial communities often face environmental fluctuations that occur on timescales much shorter than their growth rate or proteome turnover. In such cases, cellular responses are likely driven by rapid changes in metabolite pools, particularly energy nucleotides including ATP, ADP, and AMP. However, robust methods to quantify these metabolites in biofilm-forming microbial communities are lacking. Here, we developed and systematically evaluated a metabolomics workflow for a granular biofilm enrichment, which performs Enhanced Biological Phosphorous Removal (EBPR). We combined fast quenching in liquid nitrogen and a boiling water extraction, followed by high resolution mass spectrometry, using porous graphitic carbon chromatography and 13C-labeled internal reference standards. Among tested procedures, a boiling water extraction was most suitable for extraction of nucleotides, as indicated by stable adenylate energy charge (AEC) and isotopic ratios. Applied to an anaerobic-aerobic cycle of a lab scale EBPR system, the method revealed dynamic changes in AEC and uridylate energy charge (UEC) during acetate uptake and polyphosphate degradation. These results demonstrate that energy pool imbalances underlie rapid metabolic switching observed in EBPR systems. Moreover, the established method provides a foundation for performing metabolomic studies of microbial biofilms in general.

systems biology↗

bioRxiv2025

Discovery of microbial glycoside hydrolases via enrichment and metaproteomics

The immense microbial diversity on Earth represents a vast genomic resource, yet discovering novel enzymes from complex environments remains challenging. Here, we combine microbial enrichment with metagenomics and metaproteomics to facilitate the identification of microbial glycoside hydrolases that operate under defined conditions. We enriched microbial communities using the carbohydrate polymer pullulan at elevated temperatures and acidic conditions. Pullulan is a natural polysaccharide composed of maltotriose units linked by -1,6 glycosidic bonds. Along with its hydrolyzing enzymes, it has broad applications across various industries. The enrichment inocula were sampled from thermophilic compost and soil from the bank of a pond. In both cases, Alicyclobacillus emerged as the dominant microorganism. Metaproteomic analysis of the enrichment biomass and secretome identified several pullulan-degrading enzymes from this organism. Notably, these enzymes were absent in the metagenomic analysis of the initial inoculum, underscoring the effectiveness of combining microbial enrichment with multi-omics for uncovering novel enzymes from complex microbial environments.

ecology↗

bioRxiv2025

Wastewater metaproteomics: tracking microbial and human protein biomarkers

Wastewater-based surveillance has become a powerful tool for monitoring the spread of pathogens, antibiotic resistance genes, and measuring population-level exposure to pharmaceuticals and chemicals. While surveillance methods commonly target small molecules, DNA, or RNA, wastewater also contains a vast spectrum of proteins. However, despite recent advances in environmental proteomics, large-scale monitoring of protein biomarkers in wastewater is still far from routine. Analyzing raw wastewater presents a challenge due to its heterogeneous mixture of organic and inorganic substances, microorganisms, cellular debris, and various chemical pollutants. To overcome these obstacles, we developed a wastewater metaproteomics approach including efficient protein extraction and an optimized data-processing pipeline. The pipeline utilizes de novo sequencing to customize large public sequence databases to enable comprehensive metaproteomic coverage. Using this approach, we analyzed wastewater samples collected over three months from two urban locations. This revealed a core microbiome comprising a broad spectrum of microbes, gut bacteria and potential opportunistic pathogens. Additionally, we identified nearly 200 human proteins, including promising population-level health indicators, such as immunoglobulins, uromodulin, and cancer-associated proteins.

ecology↗

bioRxiv2025

Metabolic implications for dual substrate growth in "Candidatus Accumulibacter"

This study explores the metabolic implications of dual substrate uptake in "Candidatus Accumulibacter", focusing on the co-consumption of volatile fatty acids and amino acids under conditions typical of enhanced biological phosphorus removal (EBPR) systems. Combining batch tests from highly enriched "Ca. Accumulibacter" cultures with conditional flux balance analysis (cFBA) predictions, we demonstrated that co-consumption of acetate and aspartate leads to synergistic metabolic interactions, lowering ATP loss compared to individual substrate consumption. The metabolic synergy arises from the complementary roles of acetate and aspartate uptake: acetate uptake provides acetyl-CoA to support aspartate metabolism, while aspartate conversion generates NADH, reducing the need for glycogen degradation during acetate uptake. We termed this type of metabolic interaction as reciprocal synergy. We further expanded our predictions to uncover three types of interactions between catabolic pathways when substrates are co-consumed by "Ca. Accumulibacter": (i) neutral, (ii) one-way synergistic and (iii) reciprocal synergistic interactions. Our results highlight the importance of network topology in determining metabolic interactions and optimizing resource use. These findings provide new insights into the metabolism "Ca. Accumulibacter" and suggest strategies for improving EBPR performance in wastewater treatment plants, where the influent typically contains a mixture of organic carbon compounds. SynopsisThis research demonstrates how dual substrate uptake by "Ca. Accumulibacter" enhances metabolic efficiency in EBPR by reducing global ATP losses through optimization of storage polymer usage.

systems biology↗

bioRxiv2024

Long-term multi-meta-omics resolves the ecophysiological controls of seasonal N2O emissions

The potent greenhouse gas nitrous oxide (N2O) originates primarily from natural and engineered microbiomes. Emission seasonality is widely reported while the underlying metabolic controls remain largely unresolved, hindering effective mitigation. We use biological wastewater treatment as tractable model ecosystem over nearly two years. Long-term metagenomic-resolved metaproteomics is combined with ex situ kinetic and full-scale operational characterization. By leveraging the evidence independently obtained at multiple ecophysiological levels, from individual genetic potential to actual metabolism and emergent community phenotype, the cascade of environmental and operational triggers driving N2O emissions is resolved. We explain the dynamics in nitrite accumulation with the kinetic unbalance between ammonia and nitrite oxidisers, and identify nitrifier denitrification as the prime N2O-producing pathway. The dissolved O2 emerged as the key actionable parameter for emission control. This work exemplifies the yet-to-be-realized potential of multi-meta-omics approaches for the mechanistic understanding and ecological engineering of microbiomes, ultimately advancing sustainable biotechnological developments.

microbiology↗

bioRxiv2024

Selective enrichment of high-affinity clade II N2O-reducers in a mixed culture

Microorganisms encoding for the N2O reductase (NosZ) are the only known biological sink of the potent greenhouse gas N2O, and are central to global N2O mitigation efforts. Yet, the ecological constraints selecting for different N2O-reducers strains and controlling the assembly of N2O-respiring communities remain largely unknown. Of particular biotechnological interest are clade II NosZ populations, which usually feature high N2O affinities and often lack other denitrification genes. Two planktonic N2O-respiring mixed cultures were enriched under limiting and excess dissolved N2O availability to assess the impact of substrate affinity and N2O cytotoxicity, respectively. Genome-resolved metaproteomics was used to infer the metabolism of the enriched populations. We show that clade II N2O-reducers outcompete clade I affiliates for N2O at sufficiently low sludge dilution rates (0.006 h-1), a scenario previously only theorized based on pure-cultures. Under N2O limitation, all enriched N2O-reducers encoded and expressed only clade II NosZ, while also possessing other denitrification genes. Two Azonexus and Thauera genera affiliates dominated the culture. We explain their coexistence with the genome-inferred metabolic exchange of cobalamin intermediates. Conversely, under excess N2O, clade I and II populations coexisted. Notably, the single dominant N2O-reducer (genus Azonexus) expressed most cobalamin biosynthesis marker genes, likely to contrast the continuous cobalamin inactivation by dissolved cytotoxic N2O concentrations (400 {micro}M). Ultimately, we demonstrate that the solids dilution rate controls the selection among NosZ clades, albeit the conditions selecting for genomes possessing the sole nosZ remain elusive. Additionally, we suggest the significance of N2O-cobalamin interactions in shaping the composition of N2O-respiring microbiomes.

systems biology↗

bioRxiv2023

"Candidatus Siderophilus nitratireducens": a psychrophilic, nap-dependent nitrate-reducing iron oxidizer within the new order Siderophiliales

Nitrate leaching from agricultural soils is increasingly found in groundwater, a primary source of drinking water worldwide. This nitrate influx can potentially stimulate the biological oxidation of iron in anoxic groundwater reservoirs. Nitrate-reducing iron-oxidizing (NRFO) bacteria have been extensively studied in laboratory settings, yet their ecophysiology in natural environments remains largely unknown. To this end, we established a pilot-scale filter on nitrate-rich groundwater to elucidate the structure and metabolism of nitrate-reducing iron-oxidizing microbiomes under oligotrophic conditions mimicking natural groundwaters. The enriched community stoichiometrically removed iron and nitrate consistently with NRFO metabolism. Genome-resolved metagenomics revealed the underlying metabolic network between the dominant iron-dependent denitrifying autotrophs and the less abundant organoheterotrophs. The most abundant genome belonged to a new Candidate order, named Siderophiliales. This new species, "Candidatus Siderophilus nitratireducens", carries central genes to iron oxidation (cytochrome c cyc2), carbon fixation (rbc), and for the sole periplasmic nitrate reductase (nap). To our knowledge, this is the first report of nap-based lithoautotrophic growth, and we demonstrate that iron oxidation coupled to dissimilatory reduction of nitrate to nitrite is thermodynamically favourable under realistic Fe3+/Fe2+ and [Formula] concentration ratios. Ultimately, by bridging the gap between laboratory investigations and real-world conditions, this study provides insights into the intricate interplay between nitrate and iron in groundwater ecosystems, and expands our understanding of NRFOs taxonomic diversity and ecological role.

microbiology↗

bioRxiv2023

Aerobic denitrification as N2O source in microbial communities

Nitrous oxide (N2O) is a potent greenhouse gas of primarily microbial origin. Aerobic and anoxic emissions are commonly ascribed to nitrification and denitrification, respectively. Beyond this established dichotomy, we quantitatively prove that heterotrophic denitrification can significantly contribute to aerobic nitrogen turnover and N2O emissions in complex microbiomes exposed to frequent oxic/anoxic transitions. Planktonic, nitrification-inhibited denitrifying enrichments respired over a third of the influent organic substrate with nitrate at high oxygen concentrations. N2O accounted for up to one quarter of the aerobically respired nitrate. The constitutive detection of all denitrification enzymes in both anoxic and oxic periods highlight the selective advantage offered by metabolic preparedness in dynamic environments. We posit that aerobic denitrification and associated N2O formation is currently underestimated in dynamic microbial ecosystems.

microbiology↗

bioRxiv2023

Production of extracellular polymeric substances in granular sludge under selection for Accumulibacter and Competibacter

Granular sludge intensifies the removal of nutrients from wastewater. Granules structured by extracellular polymeric substances (EPS) can be recovered as biomaterial. Links between microbial selection and EPS formation during granulation need to get uncovered. We inoculated anaerobic-aerobic sequencing batch reactors with either flocs or granules to study the relationships between microbial selection, bioaggregation, exopolymer formation, and EPS composition. Selection for slow-growing organisms like the model polyphosphate- accumulating organism "Candidatus Accumulibacter" (max. 83% vs. amplicon sequencing read counts) and glycogen-accumulating organism "Ca. Competibacter" (max. 45%) sustained granulation. Gel-forming exopolymers were produced as high as above 40% of the volatile solids of the biomass by stepwise increase of the organic loading rate (0.3 to 2.0 g CODAc d-1 LR-1). Confocal laser scanning microscopy, FT-IR spectroscopy, and HPAE-PAD chromatography revealed the complex and dynamic chemical compositions of the structural EPS in relation to microbial population shifts along reactor regimes. The analysis of 20 representative genomes of "Ca. Accumulibacter" and "Ca. Competibacter" recovered from public databases revealed their functional potential to produce EPS among other representative wastewater microorganisms. The more than 40 functional gene categories annotated highlight the complexity of EPS metabolic networks from monomers processing to assembly, export, and epimerizations. The combination of ecological engineering principles and systems microbiology will help unravel and direct the production of EPS from wastewater, valorizing residual granular sludge into beneficial biomaterials for the circular economy. HighlightsO_LISelection for slow-growing organisms like PAOs and GAOs fostered a robust granulation. C_LIO_LIStructural EPS were produced above 40% of biomass volatile content under high loading. C_LIO_LIChemical composition of EPS evolved together with the microbial community composition. C_LIO_LIGenomic insights highlighted the genetic potential of PAOs and GAOs for EPS formation. C_LIO_LIMicrobial communities are complex; further are their EPS compositions and metabolisms. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/534144v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@a163e2org.highwire.dtl.DTLVardef@1a4ab94org.highwire.dtl.DTLVardef@1fc93d4org.highwire.dtl.DTLVardef@14d4b7f_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

Catch me if you can: Capturing extracellular DNA transformation in mixed cultures via Hi-C sequencing

Environmental microorganisms evolve constantly under various stressors using different adaptive mechanisms, including horizontal gene transfer. Microorganisms benefit from transferring genetic information that code for antibiotic resistance via mobile genetic elements (plasmids). Due to the complexity of natural microbial ecosystems, quantitative data on the transfer of genetic information in microbial communities remain unclear. Two 1-L chemostats (one control and one test) were inoculated with activated sludge, fed with synthetic wastewater, and operated for 45 days at a hydraulic retention time of 1 day to study the transformation capacity of a rolling-circle plasmid encoding GFP and kanamycin resistance genes, at increasing concentrations of kanamycin (0.01-2.5-50-100 mg L-1) representing environmental, wastewater, lab-selection, and gut or untreated pharmaceutical wastewater discharge environments. The plasmid DNA was spiked daily at 5 {micro}g L-1 in the test chemostat. The evolution of the microbial community composition was analyzed by 16S rRNA gene amplicon sequencing and metagenomics, and the presence of the plasmid by quantitative PCR. We used Hi-C sequencing to identify natural transformant microorganisms under steady-state conditions with low (2.5 mg L-1) and high (50 mg L-1) concentrations of kanamycin. Both chemostats selected for the same 6 predominant families of Spirosomaceae, Comamonadaceae, Rhodocyclaceae, Rhizobiaceae, Microbacteriaceae, and Chitinophagaceae, while biomass formation in the presence of kanamycin was higher with the plasmid. Hence, the antibiotic exerted the main pressure on microbial selection, while the plasmid helped these populations better resist the antibiotic treatment and grow. The kanamycin resistance gene increased in both reactors (log 7 gene copies g VSS-1). When higher antibiotic concentrations were applied, the GFP/16S ratio was increased, highlighting plasmids accumulation in the test reactor over time. The plasmid transformed mainly inside populations of Bosea sp., Runella spp., and Microbacterium sp.. This study made one significant step forward by demonstrating that microorganisms in enrichments from activated sludge biomasses can acquire exogenous synthetic plasmids by transformation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/508255v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1002657org.highwire.dtl.DTLVardef@d535f6org.highwire.dtl.DTLVardef@1c9e045org.highwire.dtl.DTLVardef@a2fcfc_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

Enrichment and application of bacterial sialic acids containing polymers from the extracellular polymeric substances of Candidatus 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 to separate them into different molecular weight fractions. This separation resulted in two high molecular weight (> 5,500 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 suggest that they can serve as source for sepsis treatment drugs, although further purification needs to be evaluated. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/508216v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@1f59ff7org.highwire.dtl.DTLVardef@d933fcorg.highwire.dtl.DTLVardef@1e3ff78org.highwire.dtl.DTLVardef@1994948_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LINulOs in "Ca. Accumulibacter" are likely located in high molecular weight polysaccharides. C_LIO_LISize exclusion chromatography allows to obtain high molecular weight polysaccharide-rich fractions enriched with NulOs. C_LIO_LIEPS and the NulOs-rich fractions can serve as source for sepsis treatment drugs. C_LI

microbiology↗

bioRxiv2022

Turnover of the extracellular polymeric matrix in an EBPR microbial community

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). EPS 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. EPS 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-labelled acetate. Samples were collected at the end of each aerobic phase. EPS 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 secreted 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. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/503576v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@925680org.highwire.dtl.DTLVardef@16d15ccorg.highwire.dtl.DTLVardef@b23a26org.highwire.dtl.DTLVardef@107d2b1_HPS_FORMAT_FIGEXP M_FIG C_FIG 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.

microbiology↗

bioRxiv2022

Comparative metaproteomics demonstrates different views on the complex granular sludge microbiome

The tremendous progress in sequencing technologies has made 16S amplicon and whole metagenome sequencing routine in microbiome studies. Furthermore, advances in mass spectrometric techniques has expanded conventional proteomics into the field of microbial ecology. Commonly referred to as metaproteomics, this approach measures the gene products (i.e., proteins) to subsequently identify the actively-expressed metabolic pathways and the protein-biomass composition of complete microbial communities. However, more systematic studies on metaproteomic and genomic approaches are urgently needed, to determine the orthogonal character of these approaches. Here we describe a deep, comparative metaproteomic study on the complex aerobic granular sludge microbiome obtained from different wastewater treatment plants. Thereby, we demonstrate the different views that can be obtained on the central nutrient-removing organisms depending on the omic approach and reference sequence databases. Furthermore, we demonstrate a homogenized Genome Taxonomy Database (GTDB) that subsequently enables a more accurate interpretation of data from different omics approaches. Ultimately, our systematic study underscores the importance of metaproteomics in the characterization of complex microbiomes; and the necessity of accurate reference sequence databases to improve the comparison between approaches and accuracy in scientific reporting.

systems biology↗

bioRxiv2022

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 safeguard water resources. Nonetheless, many questions remain about their effectiveness to prevent the dissemination of antimicrobial resistance (AMR). 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. In this study, we analyzed the transfer of ARGs and MGEs in a full-scale Nereda(R) 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 hosts of ARGs in the sludge. In the effluent, 478 resistance determinants were detected, of which 89% 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. These can pose a risk for AMR dissemination by transformation into microorganisms of receiving water bodies. Total intracellular ARGs decreased 3-42% as a result of 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. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/482492v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1a5eedcorg.highwire.dtl.DTLVardef@e4da78org.highwire.dtl.DTLVardef@c324bforg.highwire.dtl.DTLVardef@1a18138_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIA DNA database from an AGS reactor was constructed to study the system resistome, mobilome, and microbiome. C_LIO_LIThe genera Pseudomonas and Rhodoferax were the predominant ARG carriers in the system. C_LIO_LIMGEs and ARGs often co-localize on contigs recovered from the exDNA of the effluent. C_LIO_LIAGS plants are efficient at reducing ARB. C_LIO_LIThe exDNA is an underestimated DNA fraction containing ARGs in the effluent. C_LI

bioengineering↗

bioRxiv2020

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 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 PointsO_LI"Ca. Accumulibacter" has the potential to produce a range of nonulosonic acids. C_LIO_LIMass spectrometry and lectin binding can reveal the presence and location of nonulosonic acids. C_LIO_LIRole of nonulosonic acid in non-pathogenic bacteria needs to be studied in detail. C_LI

microbiology↗

bioRxiv2020

Antibiotic resistance genes and mobile genetic elements removal from treated wastewater by sewage-sludge biochar and iron-oxide coated sand

Disinfection of treated wastewater in wastewater treatment plants (WWTPs) is used to minimize emission of coliforms, pathogens, and antibiotic resistant bacteria (ARB) in the environment. However, the fate of free-floating extracellular DNA (eDNA) that do carry antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) is overlooked. Water technologies are central to urban and industrial ecology for sanitation and resource recovery. Biochar produced by pyrolysis of sewage sludge and iron-oxide-coated sands recovered as by-product of drinking water treatment were tested as adsorbents to remove ARGs and MGEs from WWTP effluent. DNA adsorption properties and materials applicability were studied in batch and up-flow column systems at bench scale. Breakthrough curves were measured with ultrapure water and treated wastewater at initial DNA concentrations of 0.1-0.5 mg mL-1 and flow rates of 0.1-0.5 mL min-1. Batch tests with treated wastewater indicated that the adsorption profiles of biochar and iron-oxide coated sand followed a Freundlich isotherm, suggesting a multilayer adsorption of nucleic acids. Sewage-sludge biochar exhibited higher DNA adsorption capacity (1 mg g-1) and longer saturation breakthrough times (4 to 10 times) than iron-oxide coated sand (0.2 mg g-1). The removal of a set of representative ARGs and MGEs was measured by qPCR comparing the inlet and outlet of the plug-flow column fed with treated wastewater. ARGs and MGEs present as free-floating eDNA were adsorbed by sewage-sludge biochar at 85% and iron-oxide coated sand at 54%. From the environmental DNA consisting of the free-floating extracellular DNA plus the intracellular DNA of the cells present in the effluent water, 97% (sewage-sludge biochar) and 66% (iron-oxide coated sand) of the tested genes present were removed. Sewage-sludge biochar displayed interesting properties to minimize the spread of antimicrobial resistances to the aquatic environment while strengthening the role of WWTPs as resource recovery factories. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/302018v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@bdc78borg.highwire.dtl.DTLVardef@177a537org.highwire.dtl.DTLVardef@1ffefdeorg.highwire.dtl.DTLVardef@1dd1457_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LISewage-sludge biochar and iron oxide coated sands were tested to adsorb DNA and cells. C_LIO_LIBiochar removed 97% of genes tested from environmental DNA of unfiltered effluent. C_LIO_LI85% of ARGs and MGEs of free-floating extracellular DNA were retained by biochar. C_LIO_LIBiochar is a WWTP by-product that can be re-used for public health sanitation. C_LI

bioengineering↗

bioRxiv2020

Quantitative profiling of microbial communities by de novo metaproteomics

Metaproteomics has emerged as one of the most promising approaches for determining the composition and metabolic functions of complete microbial communities. Conventional metaproteomics approaches however, rely on the construction of protein sequence databases and efficient peptide-spectrum matching algorithms. Thereby, very large sequence databases impact on computational efforts and sensitivity. More recently, advanced de novo sequencing strategies--which annotate peptide sequences without the requirement for a database--have become (again) increasingly proposed for proteomics applications. Such approaches would vastly expand many metaproteomics applications by enabling rapid community profiling and by capturing unsequenced community members, which otherwise remain inaccessible for further interpretation. Nevertheless, because of the lack of efficient pipelines and validation procedures, those strategies have only rarely been employed for community proteomics. Here we report on a newly established de novo metaproteomics pipeline which was evaluated for its quantitative performance using synthetic and natural communities. Additionally, we introduce a novel validation strategy and investigate the actual content of community members within community proteomics data.

systems biology↗