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Martin Pabst
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Journal article
(2026)
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Yujia Zhai, Xun Wang, Xuhan Deng, Xiaoming Li, Bin Hu, Walter van der Meer, Mark C.M. van Loosdrecht, Gang Liu, Martin Pabst
Riverbank filtration is a nature-based water treatment strategy known for its effective removal of organic micropollutants. Yet, the mechanisms governing their biodegradation, especially the role of redox transitions in mediating biotransformation, remain insufficiently understood. Here, we integrate metagenomic profiling with chemical analytics in a 10 m simulated riverbank filtration system to demonstrate how sequential oxidizing–reducing degradation enhances organic micropollutant transformation. Oxygen stratification structured distinct microbial and enzymatic pathways: oxidizing zones (>+200 mV redox potential) facilitated cytochrome P450-mediated oxidation (oxidizing condition, OXD), while subsequent redox shifts to reducing conditions (←400 mV, sequential oxidizing–reducing (SOR) conditions) activated reductive transformations (e.g., via nitronate monooxygenase and aldehyde dehydrogenase) and conjugation pathways. These SOR conditions significantly enhanced the removal of recalcitrant compounds, including irbesartan (+25.3%), benzotriazole (13.4%), and gabapentin (+9.7%). Metagenomic analysis revealed redox-driven microbial specialization, with Pseudomonadota and Nitrospirota dominating in oxidizing zones and reducing microzones enriched in pathways associated with nitrotoluene and ethylbenzene degradation, providing genomic evidence for sequential organic micropollutant breakdown. These findings establish a mechanistic framework for harnessing oxidizing–reducing microbial partnerships to amplify organic micropollutant removal in nature-based water treatment systems, which can be used for riverbank filtration site selection and well field construction and optimization.
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Riverbank filtration is a nature-based water treatment strategy known for its effective removal of organic micropollutants. Yet, the mechanisms governing their biodegradation, especially the role of redox transitions in mediating biotransformation, remain insufficiently understood. Here, we integrate metagenomic profiling with chemical analytics in a 10 m simulated riverbank filtration system to demonstrate how sequential oxidizing–reducing degradation enhances organic micropollutant transformation. Oxygen stratification structured distinct microbial and enzymatic pathways: oxidizing zones (>+200 mV redox potential) facilitated cytochrome P450-mediated oxidation (oxidizing condition, OXD), while subsequent redox shifts to reducing conditions (←400 mV, sequential oxidizing–reducing (SOR) conditions) activated reductive transformations (e.g., via nitronate monooxygenase and aldehyde dehydrogenase) and conjugation pathways. These SOR conditions significantly enhanced the removal of recalcitrant compounds, including irbesartan (+25.3%), benzotriazole (13.4%), and gabapentin (+9.7%). Metagenomic analysis revealed redox-driven microbial specialization, with Pseudomonadota and Nitrospirota dominating in oxidizing zones and reducing microzones enriched in pathways associated with nitrotoluene and ethylbenzene degradation, providing genomic evidence for sequential organic micropollutant breakdown. These findings establish a mechanistic framework for harnessing oxidizing–reducing microbial partnerships to amplify organic micropollutant removal in nature-based water treatment systems, which can be used for riverbank filtration site selection and well field construction and optimization.
Journal article
(2026)
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Le Min Chen, Sunanda Keisham, Hiroaki Tateno, Gijs Y. Kleine, Martin Pabst, Mario Pronk, Mark C.M. van Loosdrecht, Yuemei Lin
The long-term effects of environmental conditions, such as seawater salinity, on the extracellular investigated EPS changes during a stepwise increase in salinity (0–4%), renewing over 90% of biomass at each condition. Stable granulation, complete anaerobic acetate uptake, and phosphate removal were maintained throughout. FT-IR of granules showed significant changes in glycans (1025 cm⁻¹) and sialic acid (1730 cm⁻¹), which were reflected in the EPS. Lectin microarray revealed that increasing salinity reduced glycan diversity in EPS glycoproteins, while increasing negatively charged groups, including sialic acids and sulfated groups. At 4% salinity, EPS negative charge increased by 19.8% compared to 0%. Microbial community composition shifted from a diverse mix (Dechloromonas; 23%, “Candidatus Competibacter”; 13%, “Candidatus Accumulibacter”; 28%) at 0% to a dominant (69% – 75%) unclassified Accumulibacter clade I species at 1 - 4% salinity. Metaproteomic analysis showed strong upregulation of genes of “Ca. Accumulibacter” involved in monosaccharide, lipopolysaccharide, and peptidoglycan biosynthesis from 3% - 4% salinity, indicating its adaptation to salinity stress. Dechloromonas and “Ca. Competibacter” represented a minor or a non-significant fraction of those proteins related to glycan synthesis across the salinities. Despite that no glycoprotein biosynthesis pathways were identified in the metaproteomic data, three putative glycoproteins produced by “Ca. Accumulibacter” were detected across all conditions. They were downregulated as the salinity increased. These findings highlight how “Ca.Accumulibacter” dynamically adapts its EPS, particularly glycoprotein glycans, in response to increasing salinity, offering new insights into EPS adaptation under environmental stress.
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The long-term effects of environmental conditions, such as seawater salinity, on the extracellular investigated EPS changes during a stepwise increase in salinity (0–4%), renewing over 90% of biomass at each condition. Stable granulation, complete anaerobic acetate uptake, and phosphate removal were maintained throughout. FT-IR of granules showed significant changes in glycans (1025 cm⁻¹) and sialic acid (1730 cm⁻¹), which were reflected in the EPS. Lectin microarray revealed that increasing salinity reduced glycan diversity in EPS glycoproteins, while increasing negatively charged groups, including sialic acids and sulfated groups. At 4% salinity, EPS negative charge increased by 19.8% compared to 0%. Microbial community composition shifted from a diverse mix (Dechloromonas; 23%, “Candidatus Competibacter”; 13%, “Candidatus Accumulibacter”; 28%) at 0% to a dominant (69% – 75%) unclassified Accumulibacter clade I species at 1 - 4% salinity. Metaproteomic analysis showed strong upregulation of genes of “Ca. Accumulibacter” involved in monosaccharide, lipopolysaccharide, and peptidoglycan biosynthesis from 3% - 4% salinity, indicating its adaptation to salinity stress. Dechloromonas and “Ca. Competibacter” represented a minor or a non-significant fraction of those proteins related to glycan synthesis across the salinities. Despite that no glycoprotein biosynthesis pathways were identified in the metaproteomic data, three putative glycoproteins produced by “Ca. Accumulibacter” were detected across all conditions. They were downregulated as the salinity increased. These findings highlight how “Ca.Accumulibacter” dynamically adapts its EPS, particularly glycoprotein glycans, in response to increasing salinity, offering new insights into EPS adaptation under environmental stress.
Aerobic Granular Sludge (AGS) is an innovative and efficient biotechnology for wastewater treatment that has been successfully applied on full-scale worldwide. Full-scale municipal AGS systems typically contain both granular sludge (granules) and flocculent sludge (flocs). Studies on the different roles of granules and flocs remain limited. In this study, a laboratory-scale AGS reactor fed with complex synthetic wastewater was operated to simulate full-scale AGS systems and to study the different functional roles of granules and flocs. The laboratory reactor achieved a coexistence of granules and flocs with a floc mass fraction of 17 %. The activities of different size fractions were evaluated using batch experiments and compared for carbon, nitrogen, and phosphorus removal: flocs (FL; <0.2 mm), small granules (SG; 0.2∼1.0 mm), medium granules (MG; 1.0∼2.0 mm), and large granules (LG; >2.0 mm). During feeding, large granules and medium granules exhibited more substrate uptake than small granules and flocs due to preferential substrate access. For aerobic conversion, flocs and small granules showed higher biomass-specific nitrification rates, while medium granules and large granules showed higher phosphorus uptake and denitrification capacity. Furthermore, large granules and medium granules showed stronger mass transfer limitation of oxygen, which limits their nitrification capability. Microbial community analysis using metagenomics and metaproteomics was performed across size fractions, and distinct communities in granules and flocs were shown. Granules showed a high abundance of Candidatus Accumulibacter (polyphosphate-accumulating organisms, PAOs) and Candidatus Competibacter (glycogen-accumulating organisms, GAOs). Flocs showed a high abundance of Nitrosomonas (ammonium-oxidizing bacteria, AOB) and Tetrasphaera (fermentative PAOs) and a low abundance of Ca. Accumulibacter. The distribution of microbial activities and microbial community over sludge size fractions in the laboratory reactor is similar to full-scale AGS systems, indicating that this laboratory setup can simulate full-scale systems and can be used for future research. Overall, this study highlights the importance of maintaining a good balance between different granule sizes and flocs to optimize nutrient removal.
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Aerobic Granular Sludge (AGS) is an innovative and efficient biotechnology for wastewater treatment that has been successfully applied on full-scale worldwide. Full-scale municipal AGS systems typically contain both granular sludge (granules) and flocculent sludge (flocs). Studies on the different roles of granules and flocs remain limited. In this study, a laboratory-scale AGS reactor fed with complex synthetic wastewater was operated to simulate full-scale AGS systems and to study the different functional roles of granules and flocs. The laboratory reactor achieved a coexistence of granules and flocs with a floc mass fraction of 17 %. The activities of different size fractions were evaluated using batch experiments and compared for carbon, nitrogen, and phosphorus removal: flocs (FL; <0.2 mm), small granules (SG; 0.2∼1.0 mm), medium granules (MG; 1.0∼2.0 mm), and large granules (LG; >2.0 mm). During feeding, large granules and medium granules exhibited more substrate uptake than small granules and flocs due to preferential substrate access. For aerobic conversion, flocs and small granules showed higher biomass-specific nitrification rates, while medium granules and large granules showed higher phosphorus uptake and denitrification capacity. Furthermore, large granules and medium granules showed stronger mass transfer limitation of oxygen, which limits their nitrification capability. Microbial community analysis using metagenomics and metaproteomics was performed across size fractions, and distinct communities in granules and flocs were shown. Granules showed a high abundance of Candidatus Accumulibacter (polyphosphate-accumulating organisms, PAOs) and Candidatus Competibacter (glycogen-accumulating organisms, GAOs). Flocs showed a high abundance of Nitrosomonas (ammonium-oxidizing bacteria, AOB) and Tetrasphaera (fermentative PAOs) and a low abundance of Ca. Accumulibacter. The distribution of microbial activities and microbial community over sludge size fractions in the laboratory reactor is similar to full-scale AGS systems, indicating that this laboratory setup can simulate full-scale systems and can be used for future research. Overall, this study highlights the importance of maintaining a good balance between different granule sizes and flocs to optimize nutrient removal.
Journal article
(2026)
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Carlota Vijande, Sabela Balboa, Massimo Lazzari, Juan Manuel Lema, Martin Pabst
Plastic pollution is a growing concern, especially poly(ethylene terephthalate) (PET), one of the most produced plastic polymers. Although several microorganisms capable of degrading PET have been identified, little is known about those present in wastewater treatment plants (WWTPs). This study explores their ability to degrade PET and the enzymes involved. Activated sludge from two facilities-one urban WWTP and one industrial WWTP-was cultivated with PET of different crystallinities. The inoculum source primarily determined differences in microbial community composition. Metagenomics revealed more than 300 genes homologous to PET-degrading enzymes in all biofilms; however, metaproteomics confirmed expression of only a few of these enzymes in industrial WWTP-derived biofilms. This inoculum demonstrated the ability to degrade PET breakdown products within 24 h. In addition, FTIR analysis revealed initial signs of surface alteration. In conclusion, this study reveals the presence of microorganisms in industrial wastewater treatment sludge that possess the genetic potential to degrade PET.
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Plastic pollution is a growing concern, especially poly(ethylene terephthalate) (PET), one of the most produced plastic polymers. Although several microorganisms capable of degrading PET have been identified, little is known about those present in wastewater treatment plants (WWTPs). This study explores their ability to degrade PET and the enzymes involved. Activated sludge from two facilities-one urban WWTP and one industrial WWTP-was cultivated with PET of different crystallinities. The inoculum source primarily determined differences in microbial community composition. Metagenomics revealed more than 300 genes homologous to PET-degrading enzymes in all biofilms; however, metaproteomics confirmed expression of only a few of these enzymes in industrial WWTP-derived biofilms. This inoculum demonstrated the ability to degrade PET breakdown products within 24 h. In addition, FTIR analysis revealed initial signs of surface alteration. In conclusion, this study reveals the presence of microorganisms in industrial wastewater treatment sludge that possess the genetic potential to degrade PET.
Journal article
(2026)
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Mengqing Fan, Qiang Xu, Xiaoxuan Wang, Zhiwei Fang, Mark C.M. van Loosdrecht, Martin Pabst, Yu Tao, Joan B. Rose, Walter van der Meer, Gang Liu
Fire hydrants are widely installed in drinking water distribution systems, where stagnant water forms multiple ‘high-risk zones’. The stagnant water quality at hydrant terminals has been poorly studied. Here we show that stagnant water exhibited an 18-fold increase in manganese, a 40-fold increase in total cell counts, a 13-fold increase in adenosine triphosphate and enrichment of opportunistic pathogens compared with flowing water. Notable changes were also observed in microbial communities and dissolved organic matter composition, including shifts in dominant bacterial taxa, transformation of saturated oxidized compounds and generation of unsaturated reduced compounds. This study also explored the ecological mechanisms underlying the covariation of microorganisms and dissolved organic matter after water stagnation. This finding provides an additional possibility for drinking water quality deterioration in drinking water distribution systems, highlighting the potential threat posed by stagnant water in non-consumer terminals (fire hydrants) to water safety.
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Fire hydrants are widely installed in drinking water distribution systems, where stagnant water forms multiple ‘high-risk zones’. The stagnant water quality at hydrant terminals has been poorly studied. Here we show that stagnant water exhibited an 18-fold increase in manganese, a 40-fold increase in total cell counts, a 13-fold increase in adenosine triphosphate and enrichment of opportunistic pathogens compared with flowing water. Notable changes were also observed in microbial communities and dissolved organic matter composition, including shifts in dominant bacterial taxa, transformation of saturated oxidized compounds and generation of unsaturated reduced compounds. This study also explored the ecological mechanisms underlying the covariation of microorganisms and dissolved organic matter after water stagnation. This finding provides an additional possibility for drinking water quality deterioration in drinking water distribution systems, highlighting the potential threat posed by stagnant water in non-consumer terminals (fire hydrants) to water safety.
Decoding Sugars
Mass Spectrometric Advances in the Analysis of the Sugar Alphabet
Monosaccharides play a central role in metabolic networks and in the biosynthesis of glycomolecules, which perform essential functions across all domains of life. Thus, identifying and quantifying these building blocks is crucial in both research and industry. Routine methods have been established to facilitate the analysis of common monosaccharides. However, despite the presence of common metabolites, most organisms utilize distinct sets of monosaccharides and derivatives. These molecules therefore display a large diversity, potentially numbering in the hundreds or thousands, with many still unknown. This complexity presents significant challenges in the study of glycomolecules, particularly in microbes, including pathogens and those with the potential to serve as novel model organisms. This review discusses mass spectrometric techniques for the isomer-sensitive analysis of monosaccharides, their derivatives, and activated forms. Although mass spectrometry allows for untargeted analysis and sensitive detection in complex matrices, the presence of stereoisomers and extensive modifications necessitates the integration of advanced chromatographic, electrophoretic, ion mobility, or ion spectroscopic methods. Furthermore, stable-isotope incorporation studies are critical in elucidating biosynthetic routes in novel organisms.
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Monosaccharides play a central role in metabolic networks and in the biosynthesis of glycomolecules, which perform essential functions across all domains of life. Thus, identifying and quantifying these building blocks is crucial in both research and industry. Routine methods have been established to facilitate the analysis of common monosaccharides. However, despite the presence of common metabolites, most organisms utilize distinct sets of monosaccharides and derivatives. These molecules therefore display a large diversity, potentially numbering in the hundreds or thousands, with many still unknown. This complexity presents significant challenges in the study of glycomolecules, particularly in microbes, including pathogens and those with the potential to serve as novel model organisms. This review discusses mass spectrometric techniques for the isomer-sensitive analysis of monosaccharides, their derivatives, and activated forms. Although mass spectrometry allows for untargeted analysis and sensitive detection in complex matrices, the presence of stereoisomers and extensive modifications necessitates the integration of advanced chromatographic, electrophoretic, ion mobility, or ion spectroscopic methods. Furthermore, stable-isotope incorporation studies are critical in elucidating biosynthetic routes in novel organisms.
Wastewater metaproteomics
Tracking microbial and human protein biomarkers
Journal article
(2026)
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Claudia G. Tugui, Filine Cordesius, Willem van Holthe, Mark C.M. Van Loosdrecht, Martin Pabst
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 approximately 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.
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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 approximately 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.
Journal article
(2026)
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Zi Xuan Hu, Jia Yu Zhang, Jitske van Ede, Yao Yao Zhang, Yu Quan Li, Mattia Ghirardello, M. Carmen Galan, Martin Pabst, Li Liu, Josef Voglmeir
Sialic acids are a diverse family of acidic sugars typically found at the terminal positions of glycan chains, mediating key physiological and pathological processes across animals - particularly vertebrates - including cell signaling and host-pathogen interactions. The distribution of sialic acids in lower animals such as mollusks, however, remains largely unresolved. Here, we report the discovery of unconjugated 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN), a deaminated analogue of N-acetylneuraminic acid, in the muscle tissue of Pacific oysters (Magallana gigas). Using UPLC-ESI-MS/MS fingerprinting, we identified naturally occurring free KDN at a concentration of 1.2 ± 0.1 nmol/100 mg of oyster muscle tissue. To investigate the biosynthetic pathway, four candidate genes were identified in the M. gigas genome, and the corresponding recombinant proteins were expressed and characterized. Enzymatic assays revealed that one putative sialic acid aldolase (MgNPL) specifically catalyzes the cleavage of KDN into mannose and pyruvate. To our knowledge, this represents the first molecular evidence of KDN metabolism in mollusks and highlights both the unexpected conservation of substrate-specific aldolase activity and distinct sialic acid utilization mechanisms compared to vertebrates.
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Sialic acids are a diverse family of acidic sugars typically found at the terminal positions of glycan chains, mediating key physiological and pathological processes across animals - particularly vertebrates - including cell signaling and host-pathogen interactions. The distribution of sialic acids in lower animals such as mollusks, however, remains largely unresolved. Here, we report the discovery of unconjugated 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN), a deaminated analogue of N-acetylneuraminic acid, in the muscle tissue of Pacific oysters (Magallana gigas). Using UPLC-ESI-MS/MS fingerprinting, we identified naturally occurring free KDN at a concentration of 1.2 ± 0.1 nmol/100 mg of oyster muscle tissue. To investigate the biosynthetic pathway, four candidate genes were identified in the M. gigas genome, and the corresponding recombinant proteins were expressed and characterized. Enzymatic assays revealed that one putative sialic acid aldolase (MgNPL) specifically catalyzes the cleavage of KDN into mannose and pyruvate. To our knowledge, this represents the first molecular evidence of KDN metabolism in mollusks and highlights both the unexpected conservation of substrate-specific aldolase activity and distinct sialic acid utilization mechanisms compared to vertebrates.
Journal article
(2026)
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Le Min Chen, Sunanda Keisham, Hiroaki Tateno, Gijs Y. Kleine, Martin Pabst, Mario Pronk, Mark C.M. van Loosdrecht, Yuemei Lin
The authors would like to inform readers that a sentence was inadvertently omitted from the ‘Abstract’ section during the production process. The correct ‘Abstract’ section should read as follows: The long-term effects of environmental conditions, such as seawater salinity, on the extracellular polymeric substances (EPS) of aerobic granular sludge (AGS) remain poorly understood. This study investigated EPS changes during a stepwise increase in salinity (0–4%), renewing over 90% of biomass at each condition. Stable granulation, complete anaerobic acetate uptake, and phosphate removal were maintained throughout. FT-IR of granules showed significant changes in glycans (1025 cm⁻¹) and sialic acid (1730 cm⁻¹), which were reflected in the EPS. Lectin microarray revealed that increasing salinity reduced glycan diversity in EPS glycoproteins, while increasing negatively charged groups, including sialic acids and sulfated groups. At 4% salinity, EPS negative charge increased by 19.8% compared to 0%. Microbial community composition shifted from a diverse mix (Dechloromonas; 23%, “Candidatus Competibacter”; 13%, “Candidatus Accumulibacter”; 28%) at 0% to a dominant (69% – 75%) unclassified Accumulibacter clade I species at 1 - 4% salinity. Metaproteomic analysis showed strong upregulation of genes of “Ca. Accumulibacter” involved in monosaccharide, lipopolysaccharide, and peptidoglycan biosynthesis from 3% - 4% salinity, indicating its adaptation to salinity stress. Dechloromonas and “Ca. Competibacter” represented a minor or a non-significant fraction of those proteins related to glycan synthesis across the salinities. Despite that no glycoprotein biosynthesis pathways were identified in the metaproteomic data, three putative glycoproteins produced by “Ca. Accumulibacter” were detected across all conditions. They were downregulated as the salinity increased. These findings highlight how “Ca. Accumulibacter” dynamically adapts its EPS, particularly glycoprotein glycans, in response to increasing salinity, offering new insights into EPS adaptation under environmental stress.
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The authors would like to inform readers that a sentence was inadvertently omitted from the ‘Abstract’ section during the production process. The correct ‘Abstract’ section should read as follows: The long-term effects of environmental conditions, such as seawater salinity, on the extracellular polymeric substances (EPS) of aerobic granular sludge (AGS) remain poorly understood. This study investigated EPS changes during a stepwise increase in salinity (0–4%), renewing over 90% of biomass at each condition. Stable granulation, complete anaerobic acetate uptake, and phosphate removal were maintained throughout. FT-IR of granules showed significant changes in glycans (1025 cm⁻¹) and sialic acid (1730 cm⁻¹), which were reflected in the EPS. Lectin microarray revealed that increasing salinity reduced glycan diversity in EPS glycoproteins, while increasing negatively charged groups, including sialic acids and sulfated groups. At 4% salinity, EPS negative charge increased by 19.8% compared to 0%. Microbial community composition shifted from a diverse mix (Dechloromonas; 23%, “Candidatus Competibacter”; 13%, “Candidatus Accumulibacter”; 28%) at 0% to a dominant (69% – 75%) unclassified Accumulibacter clade I species at 1 - 4% salinity. Metaproteomic analysis showed strong upregulation of genes of “Ca. Accumulibacter” involved in monosaccharide, lipopolysaccharide, and peptidoglycan biosynthesis from 3% - 4% salinity, indicating its adaptation to salinity stress. Dechloromonas and “Ca. Competibacter” represented a minor or a non-significant fraction of those proteins related to glycan synthesis across the salinities. Despite that no glycoprotein biosynthesis pathways were identified in the metaproteomic data, three putative glycoproteins produced by “Ca. Accumulibacter” were detected across all conditions. They were downregulated as the salinity increased. These findings highlight how “Ca. Accumulibacter” dynamically adapts its EPS, particularly glycoprotein glycans, in response to increasing salinity, offering new insights into EPS adaptation under environmental stress.
Journal article
(2026)
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Aswin Muralidharan, Ana Rita Costa, Desi Fierlier, Daan Frits van den Berg, Halewijn van den Bossche, Adja Damba Zoumaro-Djayoon, Martin Pabst, Martin Pacesa, Stan J. J. Brouns, More authors...
Jumbo phages protect their genomes from DNA-sensing bacterial defense systems by enclosing them within vesicles and nucleus-like compartments. Very little is known about defense systems specialized to counter these phages. Here, we show that AVAST type 5 (Avs5) systems, part of the signal transduction ATPases of numerous domains (STAND) superfamily, confer conserved immunity against jumbo phages. Using fluorescence microscopy and biotin proximity labeling, we demonstrate that Avs5 localizes to early infection vesicles, where it senses an essential, early-expressed phage protein named JADA (Jumbo phage Avs5 Defense Activator). Recognition of phage infection triggers the Sir2-like effector domain of Avs5 across three Avs5 clades, resulting in rapid NAD + hydrolysis, disruption of phage nucleus formation, and arrest of infection. These findings reveal a spatially coordinated bacterial immune strategy that targets an early vulnerability in jumbo phage infection.
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Jumbo phages protect their genomes from DNA-sensing bacterial defense systems by enclosing them within vesicles and nucleus-like compartments. Very little is known about defense systems specialized to counter these phages. Here, we show that AVAST type 5 (Avs5) systems, part of the signal transduction ATPases of numerous domains (STAND) superfamily, confer conserved immunity against jumbo phages. Using fluorescence microscopy and biotin proximity labeling, we demonstrate that Avs5 localizes to early infection vesicles, where it senses an essential, early-expressed phage protein named JADA (Jumbo phage Avs5 Defense Activator). Recognition of phage infection triggers the Sir2-like effector domain of Avs5 across three Avs5 clades, resulting in rapid NAD + hydrolysis, disruption of phage nucleus formation, and arrest of infection. These findings reveal a spatially coordinated bacterial immune strategy that targets an early vulnerability in jumbo phage infection.
A Special Issue of Mass Spectrometry Reviews to Honor Professor Renato Zenobi
A Lifetime of Mentorship and Innovation in Mass Spectrometry
Journal article
(2025)
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Daphne Keulen, Myrto Apostolidi, Geoffroy Geldhof, Olivier Le Bussy, Martin Pabst, Marcel Ottens
The challenging task of designing biopharmaceutical downstream processes is initially to select the type of unit operations, followed by optimizing their operating conditions. For complex flowsheet optimizations, the strategy becomes crucial in terms of duration and outcome. In this study, we compared three optimization strategies, namely, simultaneous, top-to-bottom, and superstructure decomposition. Moreover, all strategies were evaluated by either using chromatographic Mechanistic Models (MMs) or Artificial Neural Networks (ANNs). An overall evaluation of 39 flowsheets was performed, including a buffer-exchange step between the chromatography operations. All strategies identified orthogonal structures to be optimal, and the weighted overall performance values were generally consistent between the MMs and ANNs. In terms of time-efficiency, the decomposition method with MMs stands out when utilizing multiple cores on a multiprocessing system for simulations. This study analyses the influence of different optimization strategies on flowsheet optimization and advices on suitable strategies and modeling techniques for specific scenarios.
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The challenging task of designing biopharmaceutical downstream processes is initially to select the type of unit operations, followed by optimizing their operating conditions. For complex flowsheet optimizations, the strategy becomes crucial in terms of duration and outcome. In this study, we compared three optimization strategies, namely, simultaneous, top-to-bottom, and superstructure decomposition. Moreover, all strategies were evaluated by either using chromatographic Mechanistic Models (MMs) or Artificial Neural Networks (ANNs). An overall evaluation of 39 flowsheets was performed, including a buffer-exchange step between the chromatography operations. All strategies identified orthogonal structures to be optimal, and the weighted overall performance values were generally consistent between the MMs and ANNs. In terms of time-efficiency, the decomposition method with MMs stands out when utilizing multiple cores on a multiprocessing system for simulations. This study analyses the influence of different optimization strategies on flowsheet optimization and advices on suitable strategies and modeling techniques for specific scenarios.
Journal article
(2025)
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Priya Chaudhary, Annapurna Bhattacharjee, Y. S. Shivay, Martin Pabst, Shilpi Sharma
Purpose: Organic farming practices enhance soil health by the addition of bio-amendments, which improve microbial diversity and abundance. Improved soil health, due to enhanced dynamic interactions between abiotic and biotic components of the environment, impedes the progression of diseases caused by soil-borne phytopathogens. The present study aimed to characterize the critical microbial and edaphic factors in correlation with phytopathogen suppression in soil from mustard fields managed under different farming practices. Methods and results: Organic soil exhibited better suppression of phytopathogens, availability of macronutrients, and high biocontrol enzymatic activities than soil from conventionally managed field. In terms of maximal phytopathogen suppression, Fusarium solani was suppressed to 85% by fungal fraction of organic soil, whereas F. fujikori was suppressed to 77% by bacterial fraction of organic soil. However, available micronutrients were higher in conventionally managed soils. Positive correlations between enhanced biocontrol enzyme activities and organic farming practice were deciphered, highlighting improved disease suppressive potential of organic soil. Metagenomic sequencing of the rhizosphere soil from mustard plants grown using “Jeevan Jyoti” bio-amendment and conventional farming regimes revealed that microbial communities could play key roles in modulating general disease suppressiveness of soil. Functional annotation enabled the identification of dominant biological processes, impacting plant and soil health positively. Specifically, the open reading frames coding for bacterial proteins involved in transport and key cellular processes were enriched in datasets of organic farm soil. Conclusion: The evidences from this study delineate the correlation between microbial components present in rhizosphere soil and edaphic factors, in the context of general disease suppressiveness.
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Purpose: Organic farming practices enhance soil health by the addition of bio-amendments, which improve microbial diversity and abundance. Improved soil health, due to enhanced dynamic interactions between abiotic and biotic components of the environment, impedes the progression of diseases caused by soil-borne phytopathogens. The present study aimed to characterize the critical microbial and edaphic factors in correlation with phytopathogen suppression in soil from mustard fields managed under different farming practices. Methods and results: Organic soil exhibited better suppression of phytopathogens, availability of macronutrients, and high biocontrol enzymatic activities than soil from conventionally managed field. In terms of maximal phytopathogen suppression, Fusarium solani was suppressed to 85% by fungal fraction of organic soil, whereas F. fujikori was suppressed to 77% by bacterial fraction of organic soil. However, available micronutrients were higher in conventionally managed soils. Positive correlations between enhanced biocontrol enzyme activities and organic farming practice were deciphered, highlighting improved disease suppressive potential of organic soil. Metagenomic sequencing of the rhizosphere soil from mustard plants grown using “Jeevan Jyoti” bio-amendment and conventional farming regimes revealed that microbial communities could play key roles in modulating general disease suppressiveness of soil. Functional annotation enabled the identification of dominant biological processes, impacting plant and soil health positively. Specifically, the open reading frames coding for bacterial proteins involved in transport and key cellular processes were enriched in datasets of organic farm soil. Conclusion: The evidences from this study delineate the correlation between microbial components present in rhizosphere soil and edaphic factors, in the context of general disease suppressiveness.
Journal article
(2025)
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Nina Roothans, Martin Pabst, Menno van Diemen, Claudia Herrera Mexicano, Marcel Zandvoort, Thomas Abeel, Mark C.M. van Loosdrecht, Michele Laureni
Nitrous oxide (N2O) is the third most important greenhouse gas and originates primarily from natural and engineered microbiomes. Effective emission mitigations are currently hindered by the largely unresolved ecophysiological controls of coexisting N2O-converting metabolisms in complex communities. To address this, we used biological wastewater treatment as a model ecosystem and combined long-term metagenome-resolved metaproteomics with ex situ kinetic and full-scale operational characterization over nearly 2 years. 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 seasonal N2O emissions has ultimately been resolved. We identified nitrifier denitrification as the dominant N2O-producing pathway and dissolved O2 as the prime operational parameter, paving the way to the design and fostering of robust emission control strategies. This work exemplifies the untapped potential of multi-meta-omics in the mechanistic understanding and ecological engineering of microbiomes towards reducing anthropogenic impacts and advancing sustainable biotechnological developments.
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Nitrous oxide (N2O) is the third most important greenhouse gas and originates primarily from natural and engineered microbiomes. Effective emission mitigations are currently hindered by the largely unresolved ecophysiological controls of coexisting N2O-converting metabolisms in complex communities. To address this, we used biological wastewater treatment as a model ecosystem and combined long-term metagenome-resolved metaproteomics with ex situ kinetic and full-scale operational characterization over nearly 2 years. 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 seasonal N2O emissions has ultimately been resolved. We identified nitrifier denitrification as the dominant N2O-producing pathway and dissolved O2 as the prime operational parameter, paving the way to the design and fostering of robust emission control strategies. This work exemplifies the untapped potential of multi-meta-omics in the mechanistic understanding and ecological engineering of microbiomes towards reducing anthropogenic impacts and advancing sustainable biotechnological developments.
Journal article
(2025)
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Michele Laureni, Francesc Corbera-Rubio, Mark C.M. van Loosdrecht, DaeHyun Daniel Kim, Savanna Browne, Nina Roothans, David G Weissbrodt, Karel Olavarria, Nadieh de Jonge, Sukhwan Yoon, Martin Pabst
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. Clade II NosZ populations are of particular biotechnological interest as they usually feature high N2O affinities and often lack other denitrification genes. We focus on the yet-unresolved ecological constraints selecting for different N2O-reducers strains and controlling the assembly of N2O-respiring communities. Two planktonic N2O-respiring mixed cultures were enriched at low dilution rates 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. Under N2O limitation, clade II N2O-reducers fully outcompeted clade I affiliates, a scenario previously only theorized based on pure-cultures. All enriched N2O-reducers encoded and expressed the sole clade II NosZ, while also possessing other denitrification genes. Two Azonexus and Thauera genera affiliates dominated the culture, and we hypothesize their coexistence to be explained by the genome-inferred metabolic exchange of cobalamin intermediates. Under excess N2O, clade I and II populations coexisted; yet, proteomic evidence suggests that clade II affiliates respired most of the N2O, de facto outcompeting clade I affiliates. The single dominant N2O-reducer (genus Azonexus) notably expressed most cobalamin biosynthesis marker genes, likely to contrast the continuous cobalamin inactivation by dissolved cytotoxic N2O concentrations (400 μM). Ultimately, our results strongly suggest the solids dilution rate to play a pivotal role in controlling the selection among NosZ clades, albeit the conditions selecting for genomes possessing the sole nosZ remain elusive. We furthermore highlight the potential significance of N2O-cobalamin interactions in shaping the composition of N2O-respiring microbiomes.
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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. Clade II NosZ populations are of particular biotechnological interest as they usually feature high N2O affinities and often lack other denitrification genes. We focus on the yet-unresolved ecological constraints selecting for different N2O-reducers strains and controlling the assembly of N2O-respiring communities. Two planktonic N2O-respiring mixed cultures were enriched at low dilution rates 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. Under N2O limitation, clade II N2O-reducers fully outcompeted clade I affiliates, a scenario previously only theorized based on pure-cultures. All enriched N2O-reducers encoded and expressed the sole clade II NosZ, while also possessing other denitrification genes. Two Azonexus and Thauera genera affiliates dominated the culture, and we hypothesize their coexistence to be explained by the genome-inferred metabolic exchange of cobalamin intermediates. Under excess N2O, clade I and II populations coexisted; yet, proteomic evidence suggests that clade II affiliates respired most of the N2O, de facto outcompeting clade I affiliates. The single dominant N2O-reducer (genus Azonexus) notably expressed most cobalamin biosynthesis marker genes, likely to contrast the continuous cobalamin inactivation by dissolved cytotoxic N2O concentrations (400 μM). Ultimately, our results strongly suggest the solids dilution rate to play a pivotal role in controlling the selection among NosZ clades, albeit the conditions selecting for genomes possessing the sole nosZ remain elusive. We furthermore highlight the potential significance of N2O-cobalamin interactions in shaping the composition of N2O-respiring microbiomes.
Journal article
(2025)
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Le Min Chen, Tessa Hofstra, Jelle Langedijk, Sebastian Andrei, Martin Pabst, Mario Pronk, Mark C.M. van Loosdrecht, Yuemei Lin
Identifying structural proteins within the extracellular polymeric substances (EPS) will provide a better understanding of the stability of aerobic granular sludge (AGS) and biofilms in general. In this work, an abundant surface protein was identified and localized in the extracellular matrix of seawater-adapted AGS. Granules with good phosphate removal were cultivated in a sequencing batch bubble column reactor with acetate as a carbon source dissolved in seawater. “Candidatus Accumulibacter” was observed as the most dominant community member through fluorescent in-situ hybridization. A surface protein of 74.5 kDa was identified in the EPS extract of the seawater-adapted AGS by SDS-PAGE and mass spectrometry. The surface protein was produced by an Accumulibacter species and showed homology to S-layer proteins. A type 1 secretion system was found adjacent to the gene encoding for the surface protein, suggesting a possible export system. Antibodies generated from a unique peptide of the surface protein confirmed the extracellular location of the surface protein. Microscopy observations with antibody staining showed the surface protein forms dense structures within the Accumulibacter microcolonies and larger fiber structures around the microcolonies. These observations highlight the importance of the protein for the structural properties of the granule. To detect more structural proteins in the EPS, optimization of the EPS extraction and in situ imaging validation methods are essential.
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Identifying structural proteins within the extracellular polymeric substances (EPS) will provide a better understanding of the stability of aerobic granular sludge (AGS) and biofilms in general. In this work, an abundant surface protein was identified and localized in the extracellular matrix of seawater-adapted AGS. Granules with good phosphate removal were cultivated in a sequencing batch bubble column reactor with acetate as a carbon source dissolved in seawater. “Candidatus Accumulibacter” was observed as the most dominant community member through fluorescent in-situ hybridization. A surface protein of 74.5 kDa was identified in the EPS extract of the seawater-adapted AGS by SDS-PAGE and mass spectrometry. The surface protein was produced by an Accumulibacter species and showed homology to S-layer proteins. A type 1 secretion system was found adjacent to the gene encoding for the surface protein, suggesting a possible export system. Antibodies generated from a unique peptide of the surface protein confirmed the extracellular location of the surface protein. Microscopy observations with antibody staining showed the surface protein forms dense structures within the Accumulibacter microcolonies and larger fiber structures around the microcolonies. These observations highlight the importance of the protein for the structural properties of the granule. To detect more structural proteins in the EPS, optimization of the EPS extraction and in situ imaging validation methods are essential.
Journal article
(2025)
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Robert Heyer, Maximilian Wolf, Dirk Benndorf, Sergio Uzzau, Jana Seifert, Lucia Grenga, Martin Pabst, Heike Schmitt, Bart Mesuere, More authors...
One Health seeks to integrate and balance the health of humans, animals, and environmental systems, which are intricately linked through microbiomes. These microbial communities exchange microbes and genes, influencing not only human and animal health but also key environmental, agricultural, and biotechnological processes. Preventing the emergence of pathogens as well as monitoring and controlling the composition of microbiomes through microbial effectors including virulence factors, toxins, antibiotics, non-ribosomal peptides, and viruses holds transformative potential. However, the mechanisms by which these microbial effectors shape microbiomes and their broader functional consequences for host and ecosystem health remain poorly understood. Metaproteomics offers a novel methodological framework as it provides insights into microbial dynamics by quantifying microbial biomass composition, metabolic functions, and detecting effectors like viruses, antimicrobial resistance proteins, and non-ribosomal peptides. Here, we highlight the potential of metaproteomics in elucidating microbial effectors and their impact on microbiomes and discuss their potential for modulating microbiomes to foster desired functions.
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One Health seeks to integrate and balance the health of humans, animals, and environmental systems, which are intricately linked through microbiomes. These microbial communities exchange microbes and genes, influencing not only human and animal health but also key environmental, agricultural, and biotechnological processes. Preventing the emergence of pathogens as well as monitoring and controlling the composition of microbiomes through microbial effectors including virulence factors, toxins, antibiotics, non-ribosomal peptides, and viruses holds transformative potential. However, the mechanisms by which these microbial effectors shape microbiomes and their broader functional consequences for host and ecosystem health remain poorly understood. Metaproteomics offers a novel methodological framework as it provides insights into microbial dynamics by quantifying microbial biomass composition, metabolic functions, and detecting effectors like viruses, antimicrobial resistance proteins, and non-ribosomal peptides. Here, we highlight the potential of metaproteomics in elucidating microbial effectors and their impact on microbiomes and discuss their potential for modulating microbiomes to foster desired functions.
Journal article
(2025)
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Francesc Corbera-Rubio, Alje S. Boersma, Weren de Vet, Martin Pabst, Paul W.J.J. van der Wielen, Maartje A.H.J. van Kessel, Mark C.M. van Loosdrecht, Doris van Halem, Sebastian Lücker, Michele Laureni
Methane removal is an essential step in drinking water production from methane-rich groundwaters. Conventional aeration-based stripping results in significant direct methane emissions, contributing up to one-third of a treatment plant's total carbon footprint. To address this, a full-scale trickling filter was operated for biological methane oxidation upstream of a submerged sand filter, and its performance was compared to a conventional aeration–submerged sand filtration set-up. Full-scale data were combined with ex-situ batch assays and metagenome-resolved metaproteomics to quantify the individual contribution of the main (a)biotic processes and characterize the enriched microbial communities. Both treatment setups fully removed methane, iron, ammonium, and manganese, yet the underlying mechanisms differed significantly. Methane was completely removed from the effluent after trickling filtration, with stripping and biological oxidation each accounting for half of the removal, thereby halving overall methane emissions. Methane-oxidizing bacteria not only outcompeted nitrifiers in the trickling filter, but also likely contributed directly to ammonia oxidation. In contrast to the submerged filter preceded by methane stripping, signatures of biological iron oxidation were almost completely absent in the trickling filter, suggesting that the presence of methane directly or indirectly promotes chemical iron oxidation. All systems had similar ex-situ manganese oxidation capacities, yet removal occurred only in the submerged filters but not the trickling filter. Ultimately, our results demonstrate that trickling filtration is effective in promoting biological methane oxidation at comparable produced drinking water quality, highlighting its potential for advancing sustainable drinking water production.
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Methane removal is an essential step in drinking water production from methane-rich groundwaters. Conventional aeration-based stripping results in significant direct methane emissions, contributing up to one-third of a treatment plant's total carbon footprint. To address this, a full-scale trickling filter was operated for biological methane oxidation upstream of a submerged sand filter, and its performance was compared to a conventional aeration–submerged sand filtration set-up. Full-scale data were combined with ex-situ batch assays and metagenome-resolved metaproteomics to quantify the individual contribution of the main (a)biotic processes and characterize the enriched microbial communities. Both treatment setups fully removed methane, iron, ammonium, and manganese, yet the underlying mechanisms differed significantly. Methane was completely removed from the effluent after trickling filtration, with stripping and biological oxidation each accounting for half of the removal, thereby halving overall methane emissions. Methane-oxidizing bacteria not only outcompeted nitrifiers in the trickling filter, but also likely contributed directly to ammonia oxidation. In contrast to the submerged filter preceded by methane stripping, signatures of biological iron oxidation were almost completely absent in the trickling filter, suggesting that the presence of methane directly or indirectly promotes chemical iron oxidation. All systems had similar ex-situ manganese oxidation capacities, yet removal occurred only in the submerged filters but not the trickling filter. Ultimately, our results demonstrate that trickling filtration is effective in promoting biological methane oxidation at comparable produced drinking water quality, highlighting its potential for advancing sustainable drinking water production.
Journal article
(2025)
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Berdien van Olst, Simon A. Eerden, Nella A. Eštok, Samarpita Roy, Ben Abbas, Yuemei Lin, Mark C.M. van Loosdrecht, Martin Pabst
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 those used in biological wastewater treatment. Limited proteolysis of whole granules and metaproteome isolation from the culture's supernatant successfully classified over 50% of the identified protein biomass to be secreted. Moreover, structural and sequence-based classification identified 387 proteins, corresponding to over 50% of the secreted protein biomass, with characteristics that could aid the formation of aggregates, including filamentous, beta-barrel containing, and cell surface proteins. While various of these aggregate-forming proteins originated from Ca. Accumulibacter, some proteins associated with other taxa. 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.
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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 those used in biological wastewater treatment. Limited proteolysis of whole granules and metaproteome isolation from the culture's supernatant successfully classified over 50% of the identified protein biomass to be secreted. Moreover, structural and sequence-based classification identified 387 proteins, corresponding to over 50% of the secreted protein biomass, with characteristics that could aid the formation of aggregates, including filamentous, beta-barrel containing, and cell surface proteins. While various of these aggregate-forming proteins originated from Ca. Accumulibacter, some proteins associated with other taxa. 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.
Journal article
(2025)
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Jitske M. van Ede, Suzanne van der Steen, Geert M. van der Kraan, Mark C.M. van Loosdrecht, Martin Pabst
The immense microbial diversity on Earth represents a vast genomic resource, yet discovering novel enzymes from complex environments remains challenging. Here, we combine a microbial enrichment with metagenomics and metaproteomics to facilitate the identification of microbial glycoside hydrolases that operate under defined conditions. We enriched microbial communities on the carbohydrate polymer pullulan at elevated temperatures under acidic conditions. Pullulan is a natural polysaccharide composed of maltotriose units linked by α-1,6-glycosidic bonds. Pullulan, along with its hydrolyzing enzymes, has broad applications across various industries. The enrichment inocula were sampled from thermophilic compost and from soil from the bank of a pond. In both cases, Alicyclobacillus was identified as the dominant microorganism. Metaproteomic analysis of the enriched biomass and secretome enabled the identification of several pullulan-degrading enzyme candidates from this organism. These enzymes were absent in the metagenomic analysis of the initial inoculum, which is highly complex with a wide diversity of species. This underscores the effectiveness of combining microbial enrichment with multi-omics for uncovering novel enzymes and sequence variants that operate under defined conditions from complex microbial environments.
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The immense microbial diversity on Earth represents a vast genomic resource, yet discovering novel enzymes from complex environments remains challenging. Here, we combine a microbial enrichment with metagenomics and metaproteomics to facilitate the identification of microbial glycoside hydrolases that operate under defined conditions. We enriched microbial communities on the carbohydrate polymer pullulan at elevated temperatures under acidic conditions. Pullulan is a natural polysaccharide composed of maltotriose units linked by α-1,6-glycosidic bonds. Pullulan, along with its hydrolyzing enzymes, has broad applications across various industries. The enrichment inocula were sampled from thermophilic compost and from soil from the bank of a pond. In both cases, Alicyclobacillus was identified as the dominant microorganism. Metaproteomic analysis of the enriched biomass and secretome enabled the identification of several pullulan-degrading enzyme candidates from this organism. These enzymes were absent in the metagenomic analysis of the initial inoculum, which is highly complex with a wide diversity of species. This underscores the effectiveness of combining microbial enrichment with multi-omics for uncovering novel enzymes and sequence variants that operate under defined conditions from complex microbial environments.