Biology subjects

Dueholm, M. S.

Publications and source records attributed to Dueholm, M. S..

12 recordsLinked to original sources

bioRxiv2024

The predicted secreted proteome of activated sludge microorganisms indicate distinct nutrient niches

In wastewater treatment plants (WWTPs) complex microbial communities process diverse chemical compounds from sewage. Secreted proteins are critical because many are the first to interact with or degrade external (macro)molecules. To better understand microbial functions in WWTPs, we predicted secreted proteomes of WWTP microbiota from more than 1000 high-quality metagenome-assembled genomes (MAGs) from 23 Danish WWTPs with biological nutrient removal. Focus was placed on examining secreted catabolic exoenzymes that target major classes of macromolecules. We demonstrate that Bacteroidota have high potential to digest complex polysaccharides, but also proteins and nucleic acids. Poorly understood activated sludge members of Acidobacteriota and Gemmatimonadota also have high capacities for extracellular polysaccharide digestion. Secreted nucleases are encoded by 61% of MAGs indicating an importance for extracellular DNA and/or RNA digestion in WWTPs. Secreted lipases were the least-common macromolecule-targeting enzymes predicted, encoded mainly by Gammaproteobacteria and Myxococcota. In contrast, diverse taxa encode extracellular peptidases, indicating that proteins are widely used nutrients. Diverse secreted multi-heme cytochromes suggest capabilities for extracellular electron-transfer by various taxa, including some Bacteroidota that encode undescribed cytochromes with >100 heme-binding motifs. Myxococcota have exceptionally large secreted protein complements, probably related to predatory lifestyles and/or complex cell cycles. Many Gammaproteobacteria MAGs (mostly former Betaproteobacteria) encode few or no secreted hydrolases, but many periplasmic substrate-binding proteins and ABC- and TRAP-transporters, suggesting they are mostly sustained by small molecules. Together, this study provides a comprehensive overview of how WWTPs microorganisms interact with the environment, providing new insights into their functioning and niche partitioning. ImportanceWastewater treatment plants are critical biotechnological systems that clean wastewater, allowing the water to reenter the environment and limit eutrophication and pollution. They are also increasingly important for recovery of resources. They function primarily by the activity of microorganisms, which act as a living sponge, taking-up and transforming nutrients, organic material and pollutants. Despite much research, many microorganisms in WWTPs are uncultivated and poorly characterized, limiting our understanding of their functioning. Here, we analyzed a large collection of high-quality metagenome-assembled genomes from WWTPs for encoded secreted enzymes and proteins, with special emphasis on those used to degrade organic material. This analysis showed highly distinct secreted proteome profiles among different major phylogenetic groups of microorganisms, thereby providing new insights into how different groups function and co-exist in activated sludge. This knowledge will contribute to a better understanding of how to efficiently manage and exploit WWTP microbiomes.

microbiology↗

bioRxiv2023

A comprehensive overview of the Chloroflexota community in wastewater treatment plants worldwide

Filamentous Chloroflexota are abundant in activated sludge wastewater treatment plants (WWTPs) worldwide and are occasionally associated with poor solid-liquid separation or foaming, but most of the abundant lineages remain undescribed. Here, we present a comprehensive overview of Chloroflexota abundant in WWTPs worldwide, using high-quality metagenome-assembled genomes (MAGs) and 16S rRNA amplicon data from 740 Danish and global WWTPs. Many novel taxa were described, encompassing 4 families, 13 genera and 29 novel species. These were widely distributed across most continents, influenced by factors such as climate zone and WWTP process design. Visualization by fluorescence in situ hybridization (FISH) confirmed their high abundances in many WWTPs based on the amplicon data and showed a filamentous morphology for nearly all species. Most formed thin and short trichomes integrated into the floc structure, unlikely to form the typical inter-floc bridging that hinders activated sludge floc settling. Metabolic reconstruction of 53 high-quality MAGs, representing most of the novel genera, offered further insights into their versatile metabolisms and suggested a primary role in carbon removal and involvement in nitrogen and sulfur cycling. The presence of glycogen reserves, detected by FISH-Raman microspectroscopy, seemed widespread across the phylum demonstrating that these bacteria likely utilize glycogen as an energy storage to survive periods with limited resources. This study gives a broad overview of the Chloroflexota community in global activated sludge WWTPs and improves our understanding of their roles in these engineered ecosystems. ImportanceChloroflexota are often abundant members of the biomass in wastewater treatment plants (WWTPs) worldwide, typically with a filamentous morphology, forming the backbones of the activated sludge (AS) floc. However, their overgrowth can often cause operational issues connected to poor settling or foaming, impairing effluent quality and increases operational costs. Despite the importance, few Chloroflexota genera have been characterized so far. Here, we present a comprehensive overview of Chloroflexota abundant in WWTPs worldwide and an in-depth characterization of their morphology, phylogeny, and ecophysiology, obtaining a broad understanding of their ecological role in activated sludge.

microbiology↗

bioRxiv2021

MiDAS 4: A global catalogue of full-length 16S rRNA gene sequences and taxonomy for studies of bacterial communities in wastewater treatment plants

Biological wastewater treatment and an increased focus on resource recovery is fundamental for environmental protection, human health, and sustainable development. Microbial communities are responsible for these processes, but our knowledge of their diversity and function is still poor, partly due to the lack of good reference databases and comprehensive global studies. Here, we sequenced more than 5 million high-quality, full-length 16S rRNA gene sequences from 740 wastewater treatment plants (WWTPs) across the world and used the sequences to construct MiDAS 4, a full-length amplicon sequence variant resolved 16S rRNA gene reference database with a comprehensive taxonomy from the domain to species-level for all references. Using a study-independent amplicon dataset from the Global Water Microbiome Consortium project (269 WWTPs), we showed that the MiDAS 4 database provides much better coverage for bacteria in WWTPs worldwide compared to commonly applied universal references databases, and greatly improved the rate of genus and species-level classification. Hence, MiDAS 4 provides a unifying taxonomy for the majority of prokaryotic diversity in WWTPs globally, which can be used for linking microbial identities with their functions across studies. Taking advantage of MiDAS 4, we carried out an amplicon-based, global-scale microbial community profiling of activated sludge plants using two common sets of primers targeting the V1-V3 and V4 region of the 16S rRNA gene. We found that the V1-V3 primers were generally best suited for this ecosystem, and revealed how environmental conditions and biogeography shape the activated sludge microbiota. We also identified process-critical taxa (core and conditionally rare or abundant taxa), encompassing 966 genera and 1530 species. These represented approximately 80% and 50% of the accumulated read abundance, respectively, and represent targets for further investigations. Finally, we showed that for well-studied functional guilds, such as nitrifiers or polyphosphate accumulating organisms, the same genera were prevalent worldwide, with only a few abundant species in each genus.

microbiology↗

bioRxiv2021

Functional amyloids in the microbiomes of a rat Parkinson's disease model and wild-type rats

Cross-seeding between amyloidogenic proteins in the gut is receiving increasing attention as a possible mechanism for initiation or acceleration of amyloid formation by aggregation-prone proteins such as SN, which is central in the development of Parkinsons disease. This is particularly pertinent in view of the growing number of functional (i.e. benign and useful) amyloid proteins discovered in bacteria. Here we identify two functional amyloid proteins, Pr12 and Pr17, in fecal matter from Parkinsons disease transgenic rats and their wild type counterparts, based on their stability against dissolution by formic acid. Both proteins show robust aggregation into ThT-positive aggregates that contain higher-order {beta}-sheets and have a fibrillar morphology, indicative of amyloid proteins. In addition, Pr17 aggregates formed in vitro showed significant resistance against formic acid, suggesting an ability to form highly stable amyloid. Treatment with proteinase K revealed a protected core of approx. 9 kDa. Neither Pr12 nor Pr17, however, affected SN aggregation in vitro. Thus, amyloidogenicity does not per se lead to an ability to cross-seed fibrillation of SN. Our results support the use of proteomics and formic acid to identify amyloid protein in complex mixtures and indicates the existence of numerous functional amyloid proteins in microbiomes. IMPORTANCEThe bacterial microbiome in the gastrointestinal tract is increasingly seen as important for human health and disease. One area of particular interest is that of neurodegenerative diseases such as Parkinsons which involve pathological aggregation into amyloid of human proteins such as - synuclein (SN). Bacteria are known to form benign or functional amyloid, some of which may initiate unwanted aggregation of e.g. SN in the enteric nervous system through cross-seeding via contact with the microbiome. Here we show that the rat microbiome contains several proteins which form this type of amyloid aggregate both in vivo and in vitro. Although the two proteins we investigate in depth do not directly promote SN aggregation, our work shows that the microbiome potentially harbors a significant number of bacterial amyloid which could play a role in human physiology at various levels.

biophysics↗

bioRxiv2020

Overexpression of human alpha-synuclein leads to dysregulated microbiome and metabolites with ageing in a rat model of Parkinson disease

Since Braaks hypothesis stating that sporadic Parkinsons disease follows a specific progression of the pathology from the peripheral to the central nervous system and can be monitored by detecting accumulation of the alpha-Synuclein protein. There is growing interest in understanding how the gut (commensal) microbiome can regulate alpha-Synuclein accumulation which can lead to PD. We studied a transgenic rat model overexpressing the human alpha-Synuclein and found that the protein overexpression resulted in gut alpha-Synuclein expression and aggregation in the gut neurons with advancing age. A progressive gut microbial composition alteration characterized by the reduction of Firmicutes to Bacteroidetes ratio could be detected in the young transgenic rat model and interestingly this ratio was then increased with aging. This observation was accompanied in older animals by intestinal inflammation, increase gut permeability and a robust alteration in metabolites production characterized by the increase of succinate level in the feces and serum. Manipulation of the gut bacteria by short-term antibiotics treatment revealed a complete loss of short-chain fatty acids (SCFAs) and reduction in succinate levels. Although antibiotics treatment did not change alpha-synuclein expression in the enteric nervous system of the colon, it can reduce alpha-synuclein expression in the olfactory bulb of the transgenic rats. In summary, synchronous with ageing, our data emphasize that the gut microbiome dysbiosis leads to a specific alteration of gut metabolites which are reflected in the serum and can be modulated by the environment.

neuroscience↗

bioRxiv2020

Characterizing the growing microorganisms at species level in 46 anaerobic digesters at Danish wastewater treatment plants: A six-year survey on microbiome structure and key drivers

Anaerobic digestion (AD) is a key technology at many wastewater treatment plants (WWTPs) for converting surplus activated sludge to methane-rich biogas. However, the limited number of surveys and the lack of comprehensive data sets have hindered a deeper understanding of the characteristics and associations between key variables and the microbiome composition. Here, we present a six-year survey of 46 anaerobic digesters, located at 22 WWTPs in Denmark, which is the largest known study of the microbial ecology of AD at WWTPs at a regional scale. For three types of AD (mesophilic, mesophilic with thermal hydrolysis pretreatment, and thermophilic), we present the typical value range of 12 key parameters including operational variables and performance parameters. The bacterial and archaeal microbiomes were analyzed at species-level resolution using amplicon sequencing in >1,000 samples and the new ecosystem-specific MiDAS 3 reference database. We detected 42 phyla, 1,600 genera and 3,584 species in the bacterial microbiome, where 70% of the genera and 93% of the species represented uncultivated taxa that were only classified based on MiDAS 3 denovo placeholder taxonomy. More than 40% of the 100 most abundant bacterial species did not grow in the digesters and were only present due to immigration with the feed sludge. Temperature, ammonium concentration, and pH were the main drivers shaping the microbiome clusters of the three types of ADs for both bacteria and for archaea. Within mesophilic digesters, feed sludge composition and other key parameters (organic loading rate, biogas yield, and ammonium concentration) correlated with the growing bacterial microbiome. Furthermore, correlation analysis revealed the main drivers for specific species among growing bacteria and archaea, and revealed the potential ecological function of many novel taxa. Our study highlights the influence of immigration on bacterial AD microbiome. Subsetting the growing microbes improves the understanding of the diversity and main drivers of microbiome assembly, and elucidates functionality of specific species-level microorganisms. This six-year survey provides a comprehensive insight into microbiome structure at species level, engineering and ecological performance, and a foundation for future studies of the ecological significance/characteristics and function of the novel taxa.

microbiology↗

bioRxiv2020

Connecting structure to function with the recovery of over 1000 high-quality activated sludge metagenome-assembled genomes encoding full-length rRNA genes using long-read sequencing

Microorganisms are critical to water recycling, pollution removal and resource recovery processes in the wastewater industry. While the structure of this complex community is increasingly understood based on 16S rRNA gene studies, this structure cannot currently be linked to functional potential due to the absence of high-quality metagenome-assembled genomes (MAGs) with full-length rRNA genes for nearly all species. Here, we sequence 23 Danish full-scale wastewater treatment plant metagenomes, producing >1 Tbp of long-read and >0.9 Tbp of short-read data. We recovered 1083 high-quality MAGs, including 57 closed circular genomes. The MAGs accounted for ~30% of the community, and meet the stringent MIMAG high-quality draft requirements including full-length rRNA genes. We show how novel high-quality MAGs in combination with >13 years of amplicon data, Raman microspectroscopy and fluorescence in situ hybridisation can be used to uncover abundant undescribed lineages belonging to important functional groups.

microbiology↗

bioRxiv2019

Species-level microbiome composition of activated sludge - introducing the MiDAS 3 ecosystem-specific reference database and taxonomy

The function of microbial communities in wastewater treatment systems and anaerobic digesters is dictated by the physiological activity of its members and complex interactions between them. Since functional traits are often conserved at low taxonomic ranks (genus, species, strain), the development of high taxonomic resolution and reliable classification is the first crucial step towards understanding the role of microbes in any ecosystem. Here we present MiDAS 3, a comprehensive 16S rRNA gene reference database based on high-quality full-length sequences derived from activated sludge and anaerobic digester systems. The MiDAS 3 taxonomy proposes unique provisional names for all microorganisms down to species level. MiDAS 3 was applied for the detailed analysis of microbial communities in 20 Danish wastewater treatment plants with nutrient removal, sampled over 12 years, demonstrating community stability and many abundant core taxa. The top 50 most abundant species belonged to genera, of which >50% have no known function in the system, emphasizing the need for more efforts towards elucidating the role of important members of wastewater treatment ecosystems. The MiDAS 3 taxonomic database guided an update of the MiDAS Field Guide - an online resource linking the identity of microorganisms in wastewater treatment systems to available data related to their functional importance. The new field guide contains a complete list of genera (>1,800) and species (>4,200) found in activated sludge and anaerobic digesters. The identity of the microbes is linked to functional information, where available. The website also provides the possibility to BLAST the sequences against MiDAS 3 taxonomy directly online. The MiDAS Field Guide is a collaborative platform acting as an online knowledge repository and facilitating understanding of wastewater treatment ecosystem function.

microbiology↗

bioRxiv2019

Comprehensive ecosystem-specific 16S rRNA gene databases with automated taxonomy assignment (AutoTax) provide species-level resolution in microbial ecology

High-throughput 16S rRNA gene amplicon sequencing is an essential method for studying the diversity and dynamics of microbial communities. However, this method is presently hampered by the lack of high-identity reference sequences for many environmental microbes in the public 16S rRNA gene reference databases, and by the absence of a systematic and comprehensive taxonomy for the uncultured majority. Here we demonstrate how high-throughput synthetic long-read sequencing can be applied to create ecosystem-specific full-length 16S rRNA gene amplicon sequence variant (FL-ASV) reference databases that include high-identity references (>98.7% identity) for nearly all abundant bacteria (>0.01% relative abundance) using Danish wastewater treatment systems and anaerobic digesters as an example. In addition, we introduce a novel sequence identity-based approach for automated taxonomy assignment (AutoTax) that provides a complete seven-rank taxonomy for all reference sequences, using the SILVA taxonomy as a backbone, with stable placeholder names for unclassified taxa. The FL-ASVs are perfectly suited for the evaluation of taxonomic resolution and bias associated with primers commonly used for amplicon sequencing, allowing researchers to choose those that are ideal for their ecosystem. The AutoTax taxonomy greatly improves the classification of short-read 16S rRNA gene amplicon sequence variants (ASVs) at the genus- and species-level, compared to the commonly used universal reference databases. Importantly, the placeholder names provide a way to explore the unclassified environmental taxa at different taxonomic ranks, which in combination with in situ analyses can be used to uncover their ecological roles.

microbiology↗

bioRxiv2019

The Proteome of Tetrasphaera elongata is Prepadapted to Changing Environmental Conditions in Wastewater Treatment Plants

The activated sludge in wastewater treatment plants (WWTP) designed for enhanced biological phosphorus removal (EBPR) experiences periodically changing nutrient and oxygen availability. Tetrasphaera is the most abundant genus in Danish WWTP and represent up to 20-30% of the activated sludge community based on 16S rRNA amplicon sequencing and quantitative fluorescence in situ hybridization analyses, although it is in low abundance in the influent wastewater. Here we investigate how Tetrasphaera can successfully out-compete most other microorganisms in such highly dynamic ecosystems. To achive this, we analyze the physiological adaptations of the WWTP isolate T. elongata str. LP2 during an aerobic to anoxic shift by label-free quantitative proteomics and NMR-metabolomics. Escherichia coli was used as reference organism as it shares several metabolic capabilities and is regularly introduced to wastewater treatment plants, but without succeeding there. When compared to E. coli, only minor changes in the proteome of T. elongata were observed after the switch to anoxic conditions. This indicates that metabolic pathways for anaerobic energy harvest were already expressed during the aerobic growth. This allows continuous growth of Tetrasphaera immediately after the switch to anoxic conditions. Metabolomics furthermore revealed that the substrates provided were exploited far more efficiently by Tetrasphaera than by E. coli. These results suggest that T. elongata prosper in the dynamic WWTP environment due to adaptation to the changing environmental conditions. Significance of the studyMembers of the genus Tetrasphaera are widely distributed and highly abundant in most well-operating WWTPs with EBPR configuration. However, despite their high abundance in situ, little is known about their physiology and ecological role. Although the importance of Tetrasphaera in engineered wastewater treatment systems is slowly being recognized, additional information is needed to understand the full extent of functions the microorganisms have in many of the essential biological processes in the WWTP. Such information may improve available process models and ultimately lead to better wastewater treatment as well as resource recovery. This study supplies proteomic and metabolomic data on the aerobic/anoxic adaptation of Tetrasphaera and provides a hypothesis on how Tetrasphaera might compete in dynamic engineered systems.

microbiology↗

bioRxiv2017

Novel prosthecate bacteria from the candidate phylum Acetothermia revealed by culture-independent genomics and advanced microscopy

Members of the candidate phylum Acetothermia are globally distributed and detected in various habitats. However, little is known about their physiology and ecological importance. In this study, an OTU belonging to Acetothermia was detected at high abundance in two full-scale anaerobic digesters. The first closed genome from this phylum was obtained by differential coverage binning of metagenomes and scaffolding with nanopore data. Genome annotation and metabolic reconstruction suggested an anaerobic chemoheterotrophic lifestyle in which the bacterium obtain energy and carbon via fermentation of peptides, amino acids, and simple sugars to acetate, formate, and hydrogen. The morphology was unusual and composed of a central rod-shaped cell with bipolar prosthecae as revealed by fluorescence in situ hybridization combined with confocal laser scanning microscopy, Raman microspectroscopy and atomic force microscopy. We hypothesize that these prosthecae allow for increased nutrient uptake by greatly expanding the cell surface area, providing a competitive advantage under nutrient-limited conditions.

microbiology↗

bioRxiv2017

Identifying the abundant and active microorganisms common to full scale anaerobic digesters

Anaerobic digestion is widely applied to treat organic waste at wastewater treatment plants. Characterisation of the underlying microbiology represents a source of information to develop strategies for improved operation. To this end, we investigated the microbial community composition of thirty-two full-scale digesters over a six-year period using 16S rRNA gene amplicon sequencing. Sampling of the sludge fed into these systems revealed that several of the most abundant populations were likely inactive and immigrating with the influent. This observation indicates that a failure to consider immigration will interfere with correlation analysis and give an inaccurate picture of the active microbial community. Furthermore, several abundant OTUs could not be classified to genus level with commonly applied taxonomies, making inference of their function unreliable. As such, the existing MiDAS taxonomy was updated to include these abundant phylotypes. The communities of individual plants surveyed were remarkably similar - with only 300 OTUs representing 80% of the total reads across all plants, and 15% of these identified as likely inactive immigrating microbes. By identifying the abundant and active taxa in anaerobic digestion, this study paves the way for targeted characterisation of the process important organisms towards an in-depth understanding of the microbial ecology of these biotechnologically important systems.

microbiology↗