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Mechanistic models mostly focus on the target protein and some selected process- or product-related impurities. For a better process understanding, however, it is advantageous to describe also reoccurring host cell protein impurities. Within the purification of biopharmaceuticals, the binding of host cell proteins to a chromatographic resin is far from being described comprehensively. For a broader coverage of the binding characteristics, large-scale proteomic data and systems level knowledge on protein interactions are key. However, a method for determining binding parameters of the entire host cell proteome to selected chromatography resins is still lacking. In this work, we have developed a method to determine binding parameters of all detected individual host cell proteins in an Escherichia coli harvest sample from large-scale proteomics experiments. The developed method was demonstrated to model abundant and problematic proteins, which are crucial impurities to be removed. For these 15 proteins covering varying concentration ranges, the model predicts the independently measured retention time during the validation gradient well. Finally, we optimized the anion exchange chromatography capture step in silico using the determined isotherm parameters of the persistent host cell protein contaminants. From these results, strategies can be developed to separate abundant and problematic impurities from the target antigen.
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Climate change-driven sea level rise threatens freshwater ecosystems and elicits salinity stress in microbiomes. Methane emissions in these systems are largely mitigated by methane-oxidizing microorganisms. Here, we characterized the physiological and metabolic response of freshwater methanotrophic archaea to salt stress. In our microcosm experiments, inhibition of methanotrophic archaea started at 1%. However, during gradual increase of salt up to 3% in a reactor over 12 weeks, the culture continued to oxidize methane. Using gene expression profiles and metabolomics, we identified a pathway for salt-stress response that produces the osmolyte of anaerobic methanotrophic archaea: N(ε)-acetyl-ß-L-lysine. An extensive phylogenomic analysis on N(ε)-acetyl-ß-L-lysine-producing enzymes revealed that they are widespread across both bacteria and archaea, indicating a potential horizontal gene transfer and a link to BORG extrachromosomal elements. Physicochemical analysis of bioreactor biomass further indicated the presence of sialic acids and the consumption of intracellular polyhydroxyalkanoates in anaerobic methanotrophs during salt stress.
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Archaea , Agua Dulce , Metano , Osmorregulación , Filogenia , Estrés Salino , Metano/metabolismo , Agua Dulce/microbiología , Anaerobiosis , Archaea/metabolismo , Archaea/genética , Archaea/clasificación , Oxidación-ReducciónRESUMEN
Nitrous oxide (N2O) is a potent greenhouse gas of primarily microbial origin. Oxic and anoxic emissions are commonly ascribed to autotrophic nitrification and heterotrophic denitrification, respectively. Beyond this established dichotomy, we quantitatively show that heterotrophic denitrification can significantly contribute to aerobic nitrogen turnover and N2O emissions in complex microbiomes exposed to frequent oxic/anoxic transitions. Two planktonic, nitrification-inhibited enrichment cultures were established under continuous organic carbon and nitrate feeding, and cyclic oxygen availability. Over a third of the influent organic substrate was respired with nitrate as electron acceptor at high oxygen concentrations (>6.5 mg/L). N2O accounted for up to one-quarter of the nitrate reduced under oxic conditions. The enriched microorganisms maintained a constitutive abundance of denitrifying enzymes due to the oxic/anoxic frequencies exceeding their protein turnover-a common scenario in natural and engineered ecosystems. The aerobic denitrification rates are ascribed primarily to the residual activity of anaerobically synthesised enzymes. From an ecological perspective, the selection of organisms capable of sustaining significant denitrifying activity during aeration shows their competitive advantage over other heterotrophs under varying oxygen availabilities. Ultimately, we propose that the contribution of heterotrophic denitrification to aerobic nitrogen turnover and N2O emissions is currently underestimated in dynamic environments.
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Bacterias , Desnitrificación , Microbiota , Óxido Nitroso , Óxido Nitroso/metabolismo , Aerobiosis , Bacterias/metabolismo , Bacterias/genética , Bacterias/clasificación , Procesos Heterotróficos , Nitratos/metabolismo , Oxígeno/metabolismo , Nitrógeno/metabolismo , NitrificaciónRESUMEN
Metal cofactors are essential for catalysis and enable countless conversions in nature. Interestingly, the metal cofactor is not always static but mobile with movements of more than 4 Å. These movements of the metal can have different functions. In the case of the xylose isomerase and medium-chain dehydrogenases, it clearly serves a catalytic purpose. The metal cofactor moves during substrate activation and even during the catalytic turnover. On the other hand, in class II aldolases, the enzymes display resting states and active states depending on the movement of the catalytic metal cofactor. This movement is caused by substrate docking, causing the metal cofactor to take the position essential for catalysis. As these metal movements are found in structurally and mechanistically unrelated enzymes, it has to be expected that this metal movement is more common than currently perceived. KEY POINTS: ⢠Metal ions are essential cofactors that can move during catalysis. ⢠In class II aldolases, the metal cofactors can reside in a resting state and an active state. ⢠In MDR, the movement of the metal cofactor is essential for substrate docking.
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Coenzimas , Metales , Metales/metabolismo , Coenzimas/metabolismo , Isomerasas Aldosa-Cetosa/metabolismo , Isomerasas Aldosa-Cetosa/química , Isomerasas Aldosa-Cetosa/genética , Catálisis , Oxidorreductasas/metabolismo , Oxidorreductasas/químicaRESUMEN
Industrial wastewater often has high levels of salt, either due to seawater or e.g. sodium chloride (NaCl) usage in the processing. Previous work indicated that aerobic granular sludge (AGS) is differently affected by seawater or saline water at similar osmotic strength. Here we investigate in more detail the impact of NaCl concentrations and seawater on the granulation and conversion processes for AGS wastewater treatment. Glycerol was used as the carbon source since it is regularly present in industrial wastewaters, and to allow the evaluation of microbial interactions that better reflect real conditions. Long-term experiments were performed to evaluate and compare the effect of salinity on granulation, anaerobic conversions, phosphate removal, and the microbial community. Smooth and stable granules as well as enhanced biological phosphorus removal (EBPR) were achieved up to 20 g/L NaCl or when using seawater. However, at NaCl levels comparable to seawater strength (30 g/L) incomplete anaerobic glycerol uptake and aerobic phosphate uptake were observed, the effluent turbidity increased, and filamentous granules began to appear. The latter is likely due to the direct aerobic growth on the leftover substrate after the anaerobic feeding period. In all reactor conditions, except the reactor with 30 g/L NaCl, Ca. Accumulibacter was the dominant microorganism. In the reactor with 30 g/L NaCl, the relative abundance of Ca. Accumulibacter decreased to ≤1 % and an increase in the genus Zoogloea was observed. Throughout all reactor conditions, Tessaracoccus and Micropruina, both actinobacteria, were present which were likely responsible for the anaerobic conversion of glycerol into volatile fatty acids. None of the glycerol metabolizing proteins were detected in Ca. Accumulibacter which supports previous findings that glycerol can not be directly utilized by Ca. Accumulibacter. The proteome profile of the dominant taxa was analysed and the results are further discussed. The exposure of salt-adapted biomass to hypo-osmotic conditions led to significant trehalose and PO43--P release which can be related to the osmoregulation of the cells. Overall, this study provides insights into the effect of salt on the operation and stability of the EBPR and AGS processes. The findings suggest that maintaining a balanced cation ratio is likely to be more important for the operational stability of EBPR and AGS systems than absolute salt concentrations.
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Glicerol , Fósforo , Salinidad , Aguas del Alcantarillado , Aguas del Alcantarillado/microbiología , Fósforo/metabolismo , Glicerol/metabolismo , Aerobiosis , Reactores Biológicos , Eliminación de Residuos LíquidosRESUMEN
The Craspase CRISPR-Cas effector consists of the RNA-guided ribonuclease gRAMP and the protease TPR-CHAT, coupling target RNA recognition to protease activation. The natural substrate of Craspase is Csx30, a protein cleaved in two fragments that subsequently activates downstream antiviral pathways. Here, we determined the protease substrate specificity of Craspase from Candidatus "Jettenia caeni" (Jc-Craspase). We find that Jc-Craspase cleaves Jc-Csx30 in a target RNA-dependent fashion in A|S, which is different from the sites found in two other studied Craspases (L|D and M|K for Candidatus "Scalindua brodae" and Desulfonema ishimotonii, respectively). The fact that Craspase cleaves a nonconserved site across orthologs indicates the evolution of specific protein interactions between Craspase and its respective Csx30 target protein. The Craspase family thus represents a panel of proteases with different substrate specificities, which we exploited for the development of a readout for multiplexed RNA detection.
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Sistemas CRISPR-Cas , Especificidad por Sustrato , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Asociadas a CRISPR/metabolismo , Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/genéticaRESUMEN
In the beginning was the word. But there were no words for N-glycans, at least, no simple words. Next to chemical formulas, the IUPAC code can be regarded as the best, most reliable and yet immediately comprehensible annotation of oligosaccharide structures of any type from any source. When it comes to N-glycans, the venerable IUPAC code has, however, been widely supplanted by highly simplified terms for N-glycans that count the number of antennae or certain components such as galactoses, sialic acids and fucoses and give only limited room for exact structure description. The highly illustrative - and fortunately now standardized - cartoon depictions gained much ground during the last years. By their very nature, cartoons can neither be written nor spoken. The underlying machine codes (e.g., GlycoCT, WURCS) are definitely not intended for direct use in human communication. So, one might feel the need for a simple, yet intelligible and precise system for alphanumeric descriptions of the hundreds and thousands of N-glycan structures. Here, we present a system that describes N-glycans by defining their terminal elements. To minimize redundancy and length of terms, the common elements of N-glycans are taken as granted. The preset reading order facilitates definition of positional isomers. The combination with elements of the condensed IUPAC code allows to describe even rather complex structural elements. Thus, this "proglycan" coding could be the missing link between drawn structures and software-oriented representations of N-glycan structures. On top, it may greatly facilitate keyboard-based mining for glycan substructures in glycan repositories.
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Proteins are the primary functional actors of the cell. While proteoform diversity is known to be highly biologically relevant, current protein analysis methods are of limited use for distinguishing proteoforms. Mass spectrometric methods, in particular, often provide only ambiguous information on post-translational modification sites, and sequences of co-existing modifications may not be resolved. Here we demonstrate fluorescence resonance energy transfer (FRET)-based single-molecule protein fingerprinting to map the location of individual amino acids and post-translational modifications within single full-length protein molecules. Our data show that both intrinsically disordered proteins and folded globular proteins can be fingerprinted with a subnanometer resolution, achieved by probing the amino acids one by one using single-molecule FRET via DNA exchange. This capability was demonstrated through the analysis of alpha-synuclein, an intrinsically disordered protein, by accurately quantifying isoforms in mixtures using a machine learning classifier, and by determining the locations of two O-GlcNAc moieties. Furthermore, we demonstrate fingerprinting of the globular proteins Bcl-2-like protein 1, procalcitonin and S100A9. We anticipate that our ability to perform proteoform identification with the ultimate sensitivity may unlock exciting new venues in proteomics research and biomarker-based diagnosis.
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Transferencia Resonante de Energía de Fluorescencia , Transferencia Resonante de Energía de Fluorescencia/métodos , Humanos , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Intrínsecamente Desordenadas/química , Imagen Individual de Molécula/métodos , Aprendizaje Automático , Mapeo Peptídico/métodosRESUMEN
Understanding the biological role of protein-linked glycans requires the reliable identification of glycans. Isomer separation and characterization often entail mass spectrometric detection preceded by high-performance chromatography on porous graphitic carbon. To this end, stable isotope-labeled glycans have emerged as powerful tools for retention time normalization. Hitherto, such standards were obtained by chemoenzymatic or purely enzymatic methods, which introduce, e.g., 13C-containing N-acetyl groups or galactose into native glycans. Glycan release with anhydrous hydrazine opens another route for heavy isotope introduction via concomitant de-N-acetylation. Here, we describe that de-N-acetylation can also be achieved with hydrazine hydrate, which is a more affordable and less hazardous reagent. Despite the slower reaction rate, complete conversion is achievable in 72 h at 100 °C for glycans with biantennary glycans with or without sialic acids. Shorter incubation times allow for the isolation of intermediate products with a defined degree of free amino groups, facilitating introduction of different numbers of heavy isotopes. Mass encoded glycans obtained by this versatile approach can serve a broad range of applications, e.g., as internal standards for isomer-specific studies of N-glycans, O-glycans, and human milk oligosaccharide by LC-MS on either porous graphitic carbon orâfollowing permethylationâon reversed phase.
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Grafito , Polisacáridos , Humanos , Polisacáridos/química , Espectrometría de Masas , Oligosacáridos/análisis , Carbono/química , Grafito/química , IsótoposRESUMEN
Enhanced biological phosphate removal and aerobic sludge granulation are commonly studied with fatty acids as substrate. Fermentative substrates such as glucose have received limited attention. In this work, glucose conversion by aerobic granular sludge and its impact on phosphate removal was studied. Long-term stable phosphate removal and successful granulation were achieved. Glucose was rapidly taken up (273 mg/gVSS/h) at the start of the anaerobic phase, while phosphate was released during the full anaerobic phase. Some lactate was produced during glucose consumption, which was anaerobically consumed once glucose was depleted. The phosphate release appeared to be directly proportional to the uptake of lactate. The ratio of phosphorus released to glucose carbon taken up over the full anaerobic phase was 0.25 Pmol/Cmol. Along with glucose and lactate uptake in the anaerobic phase, polyhydroxy-alkanoates and glycogen storage were observed. There was a linear correlation between glucose consumption and lactate formation. While lactate accounted for approximately 89 % of the observed products in the bulk liquid, minor quantities of formate (5 %), propionate (4 %), and acetate (3 %) were also detected (mass fraction). Formate was not consumed anaerobically. Quantitative fluorescence in-situ hybridization (qFISH) revealed that polyphosphate accumulating organisms (PAO) accounted for 61 ± 15 % of the total biovolume. Metagenome evaluation of the biomass indicated a high abundance of Micropruina and Ca. Accumulibacter in the system, which was in accordance with the microscopic observations and the protein mass fraction from metaproteome analysis. Anaerobic conversions were evaluated based on theoretical ATP balances to provide the substrate distribution amongst the dominant genera. This research shows that aerobic granular sludge technology can be applied to glucose-containing effluents and that glucose is a suitable substrate for achieving phosphate removal. The results also show that for fermentable substrates a microbial community consisting of fermentative organisms and PAO develop.
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Glucosa , Aguas del Alcantarillado , Reactores Biológicos , Polifosfatos/metabolismo , Fósforo/metabolismo , LactatosRESUMEN
The tremendous progress in sequencing technologies has made DNA sequencing routine for microbiome studies. Additionally, advances in mass spectrometric techniques have extended conventional proteomics into the field of microbial ecology. However, systematic studies that provide a better understanding of the complementary nature of these 'omics' approaches, particularly for complex environments such as wastewater treatment sludge, are urgently needed. Here, we describe a comparative metaomics study on aerobic granular sludge from three different wastewater treatment plants. For this, we employed metaproteomics, whole metagenome, and 16S rRNA amplicon sequencing to study the same granule material with uniform size. We furthermore compare the taxonomic profiles using the Genome Taxonomy Database (GTDB) to enhance the comparability between the different approaches. Though the major taxonomies were consistently identified in the different aerobic granular sludge samples, the taxonomic composition obtained by the different omics techniques varied significantly at the lower taxonomic levels, which impacts the interpretation of the nutrient removal processes. Nevertheless, as demonstrated by metaproteomics, the genera that were consistently identified in all techniques cover the majority of the protein biomass. The established metaomics data and the contig classification pipeline are publicly available, which provides a valuable resource for further studies on metabolic processes in aerobic granular sludge.
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Microbiota , Aguas del Alcantarillado , Aguas del Alcantarillado/química , ARN Ribosómico 16S/genética , Reactores Biológicos , Metagenoma , Metagenómica/métodosRESUMEN
Hexanoate is a valuable chemical that can be produced by microorganisms that convert short-chain- to medium-chain carboxylic acids through a process called chain elongation. These microorganisms usually produce mixtures of butyrate and hexanoate from ethanol and acetate, but direct conversion of ethanol to hexanoate is theoretically possible. Steering microbial communities to ethanol-only elongation to hexanoate circumvents the need for acetate addition and simplifies product separation. The biological feasibility of ethanol elongation to hexanoate was validated in batch bioreactor experiments with a Clostridium kluyveri-dominated enrichment culture incubated with ethanol, acetate and butyrate in different ratios. Frequent liquid sampling combined with high-resolution off-gas measurements allowed to monitor metabolic behavior. In experiments with an initial ethanol-to-acetate ratio of 6:1, acetate depletion occurred after ± 35 h of fermentation, which triggered a metabolic shift to direct conversion of ethanol to hexanoate despite the availability of butyrate (± 40 mCmol L-1). When only ethanol and no external electron acceptor was supplied, stable ethanol to hexanoate conversion could be maintained until 60-90 mCmol L-1 of hexanoate was produced. After this, transient production of either acetate and butyrate or butyrate and hexanoate was observed, requiring a putative reversal of the Rnf complex. This was not observed before acetate depletion or in presence of low concentrations (40-60 mCmol L-1) of butyrate, suggesting a stabilizing or regulatory role of butyrate or butyrate-related catabolic intermediates. This study sheds light on previously unknown versatility of chain elongating microbes and provides new avenues for optimizing (waste) bioconversion for hexanoate production.
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Caproatos , Etanol , Caproatos/metabolismo , Etanol/metabolismo , Ácidos Carboxílicos , Butiratos/metabolismo , Acetatos/metabolismo , FermentaciónRESUMEN
Proteomics has greatly advanced the understanding of the cellular biochemistry of microorganisms. The thermoalkaliphile Caldalkalibacillus thermarum TA2.A1 is an organism of interest for studies into how alkaliphiles adapt to their extreme lifestyles, as it can grow from pH 7.5 to pH 11. Within most classes of microbes, the membrane-bound electron transport chain (ETC) enables a great degree of adaptability and is a key part of metabolic adaptation. Knowing what membrane proteins are generally expressed is crucial as a benchmark for further studies. Unfortunately, membrane proteins are the category of proteins hardest to detect using conventional cellular proteomics protocols. In part, this is due to the hydrophobicity of membrane proteins as well as their general lower absolute abundance, which hinders detection. Here, we performed a combination of whole cell lysate proteomics and proteomics of membrane extracts solubilised with either SDS or FOS-choline-12 at various temperatures. The combined methods led to the detection of 158 membrane proteins containing at least a single transmembrane helix (TMH). Within this data set we revealed a full oxidative phosphorylation pathway as well as an alternative NADH dehydrogenase type II (Ndh-2) and a microaerophilic cytochrome oxidase ba3. We also observed C. thermarum TA2.A1 expressing transporters for ectoine and glycine betaine, compounds that are known osmolytes that may assist in maintaining a near neutral internal pH when the external pH is highly alkaline.
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Dissimilatory nitrate reduction to ammonia (DNRA) is a common biochemical process in the nitrogen cycle in natural and man-made habitats, but its significance in wastewater treatment plants is not well understood. Several ammonifying Trichlorobacter strains (former Geobacter) were previously enriched from activated sludge in nitrate-limited chemostats with acetate as electron (e) donor, demonstrating their presence in these systems. Here, we isolated and characterized the new species Trichlorobacter ammonificans strain G1 using a combination of low redox potential and copper-depleted conditions. This allowed purification of this DNRA organism from competing denitrifiers. T. ammonificans is an extremely specialized ammonifier, actively growing only with acetate as e-donor and carbon source and nitrate as e-acceptor, but H2 can be used as an additional e-donor. The genome of G1 does not encode the classical ammonifying modules NrfAH/NrfABCD. Instead, we identified a locus encoding a periplasmic nitrate reductase immediately followed by an octaheme cytochrome c that is conserved in many Geobacteraceae species. We purified this octaheme cytochrome c protein (TaNiR), which is a highly active dissimilatory ammonifying nitrite reductase loosely associated with the cytoplasmic membrane. It presumably interacts with two ferredoxin subunits (NapGH) that donate electrons from the menaquinol pool to the periplasmic nitrate reductase (NapAB) and TaNiR. Thus, the Nap-TaNiR complex represents a novel type of highly functional DNRA module. Our results indicate that DNRA catalyzed by octaheme nitrite reductases is a metabolic feature of many Geobacteraceae, representing important community members in various anaerobic systems, such as rice paddy soil and wastewater treatment facilities.
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Amoníaco , Nitratos , Humanos , Nitratos/metabolismo , Oxidación-Reducción , Citocromos c/metabolismo , Nitrato Reductasas/química , Nitrato Reductasas/genética , Nitrato Reductasas/metabolismo , DesnitrificaciónRESUMEN
Mass-spectrometry-based proteomics is increasingly employed to monitor purification processes or to detect critical host cell proteins in the final drug substance. This approach is inherently unbiased and can be used to identify individual host cell proteins without prior knowledge. In process development for the purification of new biopharmaceuticals, such as protein subunit vaccines, a broader knowledge of the host cell proteome could promote a more rational process design. Proteomics can establish qualitative and quantitative information on the complete host cell proteome before purification (i.e., protein abundances and physicochemical properties). Such information allows for a more rational design of the purification strategy and accelerates purification process development. In this study, we present an extensive proteomic characterisation of two E. coli host cell strains widely employed in academia and industry to produce therapeutic proteins, BLR and HMS174. The established database contains the observed abundance of each identified protein, information relating to their hydrophobicity, the isoelectric point, molecular weight, and toxicity. These physicochemical properties were plotted on proteome property maps to showcase the selection of suitable purification strategies. Furthermore, sequence alignment allowed integration of subunit information and occurrences of post-translational modifications from the well-studied E. coli K12 strain.
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Escherichia coli , Proteoma , Escherichia coli/metabolismo , Proteoma/metabolismo , Proteómica , Espectrometría de MasasRESUMEN
An optimal purification process for biopharmaceutical products is important to meet strict safety regulations, and for economic benefits. To find the global optimum, it is desirable to screen the overall design space. Advanced model-based approaches enable to screen a broad range of the design-space, in contrast to traditional statistical or heuristic-based approaches. Though, chromatographic mechanistic modeling (MM), one of the advanced model-based approaches, can be speed-limiting for flowsheet optimization, which evaluates every purification possibility (e.g., type and order of purification techniques, and their operating conditions). Therefore, we propose to use artificial neural networks (ANNs) during global optimization to select the most optimal flowsheets. So, the number of flowsheets for final local optimization is reduced and consequently the overall optimization time. Employing ANNs during global optimization proved to reduce the number of flowsheets from 15 to only 3. From these three, one flowsheet was optimized locally and similar final results were found when using the global outcome of either the ANN or MM as starting condition. Moreover, the overall flowsheet optimization time was reduced by 50% when using ANNs during global optimization. This approach accelerates the early purification process design; moreover, it is generic, flexible, and regardless of sample material's type.
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The yeast Saccharomyces cerevisiae is a widely-used eukaryotic model organism and a promising cell factory for industry. However, despite decades of research, the regulation of its metabolism is not yet fully understood, and its complexity represents a major challenge for engineering and optimizing biosynthetic routes. Recent studies have demonstrated the potential of resource and proteomic allocation data in enhancing models for metabolic processes. However, comprehensive and accurate proteome dynamics data that can be used for such approaches are still very limited. Therefore, we performed a quantitative proteome dynamics study to comprehensively cover the transition from exponential to stationary phase for both aerobically and anaerobically grown yeast cells. The combination of highly controlled reactor experiments, biological replicates, and standardized sample preparation procedures ensured reproducibility and accuracy. In addition, we selected the CEN.PK lineage for our experiments because of its relevance for both fundamental and applied research. Together with the prototrophic standard haploid strain CEN.PK113-7D, we also investigated an engineered strain with genetic minimization of the glycolytic pathway, resulting in the quantitative assessment of 54 proteomes. The anaerobic cultures showed remarkably less proteome-level changes compared with the aerobic cultures, during transition from the exponential to the stationary phase as a consequence of the lack of the diauxic shift in the absence of oxygen. These results support the notion that anaerobically growing cells lack resources to adequately adapt to starvation. This proteome dynamics study constitutes an important step toward better understanding of the impact of glucose exhaustion and oxygen on the complex proteome allocation process in yeast. Finally, the established proteome dynamics data provide a valuable resource for the development of resource allocation models as well as for metabolic engineering efforts.
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Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Anaerobiosis , Proteómica/métodos , Reproducibilidad de los Resultados , Glucosa/metabolismoRESUMEN
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. The homogeneity of the media coating and genome-based microbial composition within each compartment highlighted the effect of backwashing, namely the complete vertical mixing of the filter media. In stark contrast to this homogeneity, the removal of the contaminants was strongly stratified within each compartment, and decreased along the filter height. This apparent and longstanding 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 (up to 2 orders of magnitude difference between top and bottom samples). 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.