Biology subjects

Dueholm, M. K. D.

Publications and source records attributed to Dueholm, M. K. D..

6 recordsLinked to original sources

bioRxiv2023

Global abundance patterns, diversity, and ecology of Patescibacteria in wastewater treatment plants

BackgroundMicroorganisms are responsible for nutrient removal and resource recovery in wastewater treatment plants (WWTPs), and their diversity is often studied by 16S rRNA gene amplicon sequencing. However, this approach underestimates the abundance and diversity of Patescibacteria due to the low coverage of commonly used PCR primers for this highly divergent bacterial phylum. Therefore, our current understanding of the global diversity, distribution, and ecological role of Patescibacteria in WWTPs is very incomplete. This is particularly relevant as Patescibacteria are considered to be associated with microbial host cells and can therefore influence the abundance and temporal variability of other microbial groups that are important for WWTP functioning. ResultsHere, we evaluated the in silico coverage of widely used 16S rRNA gene-targeted primer pairs and redesigned a primer pair targeting the V4 region of bacterial and archaeal 16S rRNA genes to expand its coverage for Patescibacteria. We then experimentally evaluated and compared the performance of the original and modified V4-targeted primers on 565 WWTP samples from the MiDAS global sample collection. Using the modified primer pair, the percentage of ASVs classified as Patescibacteria increased from 5.9% to 23.8%, and the number of detected patescibacterial genera increased from 560 to 1,576, while the detected diversity of the remaining microbial community remained similar. Due to this significantly improved coverage of Patescibacteria, we identified 23 core genera of Patescibacteria in WWTPs and described the global distribution pattern of these unusual microbes in these systems. Finally, correlation network analysis revealed potential host organisms that might be associated with Patescibacteria in WWTPs. Interestingly, strong indications were found for an association between Patescibacteria of the Saccharimonadia and globally abundant polyphosphate-accumulating organisms of the genus Ca. Phosporibacter. ConclusionsOur study (i) provides an improved 16S rRNA gene V4 region-targeted amplicon primer pair inclusive of Patescibacteria with little impact on the detection of other taxa, (ii) reveals the diversity and distribution patterns of Patescibacteria in WWTPs on a global scale, and (iii) provides new insights into the ecological role and potential hosts of Patescibacteria in WWTPs.

ecology↗

bioRxiv2023

Benchmarking long-read sequencing strategies for obtaining ASV-resolved rRNA operons from environmental microeukaryotes

The use of short-read metabarcoding for classifying microeukaryotes is challenged by the lack of comprehensive 18S rRNA reference databases. While recent advances in high-throughput long-read sequencing provide the potential to greatly increase the phylogenetic coverage of these databases, the performance of different sequencing technologies and subsequent bioinformatics processing remain to be evaluated, primarily because of the absence of well-defined eukaryotic mock communities. To address this challenge, we created a eukaryotic rRNA operons clone-library and turned it into a precisely defined synthetic eukaryotic mock community. This mock community was then used to evaluate the performance of three long-read sequencing techniques (PacBio HiFi, and Nanopore UMI with/without clonal pre-amplification) and three tools for resolving amplicons sequence variants (ASVs) (Uchime3, Unoise3, and DADA2). We investigated the sensitivity of the sequencing techniques based on the number of detected mock taxa, and the accuracy of the different ASV-calling tools with a specific focus on the presence of chimera among the final rRNA operon ASVs. Based on our findings, we provide recommendations and best practice protocols for how to cost-effectively obtain essential error-free rRNA operons in high-throughput. An agricultural soil sample was used to demonstrate that the sequencing and bioinformatic results from the mock community also translates to highly diverse natural samples.

microbiology↗

bioRxiv2023

MiDAS 5: Global diversity of bacteria and archaea in anaerobic digesters

Anaerobic digestion represents a key biotechnology for the transformation of organic waste into renewable energy (biogas) and relies on complex microbial communities that work in concert to degrade the complex substrates into methane and carbon dioxide. Here, we sequenced more than half a million high-quality, full-length 16S rRNA gene sequences from 285 full-scale anaerobic digesters (ADs) across the world to expand our knowledge about diversity and function of the bacteria and archaea in ADs. The sequences were processed into full-length 16S rRNA amplicon sequence variants (FL-ASVs), which were added to the MiDAS 4 database for bacteria and archaea in wastewater treatment systems to create MiDAS 5. The expansion of the MiDAS database significantly increased the coverage for bacteria and archaea in ADs worldwide, leading to an improved rate of genus and species-level classification. Using MiDAS 5, we carried out an amplicon-based, global-scale microbial community profiling of the sampled ADs using three common sets of primers targeting different regions of the 16S rRNA gene in bacteria and/or archaea. We revealed how environmental conditions and biogeography shape the AD microbiota. We also identify core and conditionally rare or abundant taxa, encompassing 692 genera and 1013 species. These represent 84-99% and 18-61% of the accumulated read abundance respectively, across samples depending on the amplicon primers used. Finally, we examined the global diversity of functional groups with known importance for the anaerobic digestion process. Our online global MiDAS Field Guide presents the data generated in this study and summarizes present knowledge about all taxa.

microbiology↗

bioRxiv2023

Amplicon-guided isolation and cultivation of previously uncultured microbial species from activated sludge

Microbes are fundamental for biological wastewater treatment. However, most microbial species found in activated sludge (AS) from wastewater treatment plants (WWTPs) have never been isolated and grown as pure cultures, thus limiting our understanding of the underlying biological processes. To change this, we here introduce an experimental setup where the plating of dispersed AS bacteria are combined with 16S rRNA gene amplicon sequencing of total plate biomass for rapid identification of growth conditions that allow for the isolation of key microbial species in AS. We show that agarose plates composed of AS fluid supplemented with various carbon sources support the growth of many previously uncultivated AS bacteria. To confirm that the approach can also be used to isolate previously uncultured species, we picked 200 colonies from the plates for growth in liquid medium. This resulted in 185 growing cultures representing 102 strains based on unique 16S rRNA gene V1-V3 amplicon sequence variants (ASVs). Classification of the ASVs with the MiDAS 4 database revealed 48 distinct genera, including the previously uncultured AAP99, Ca. Propionivibrio, Ellin6067, midas_g_12, and Ca. Brachybacter. Among the ASVs that obtained species-level classification, we observed 43 unique species of which 29 were only classified based on the MiDAS placeholder taxonomy highlighting the potential for culturing many novel taxa. Preparation of glycerol stocks and subsequent validation by restreaking on plates resulted in 10 pure cultures of which six represent core or conditional rare or abundant (CRAT) species observed within the MiDAS global survey of WWTPs. ImportanceBiological wastewater treatment relies on complex microbial communities that assimilate nutrients and break down pollutants in the wastewater. Knowledge about the physiology and metabolism of bacteria in wastewater treatment plants (WWTPs) may therefore be used to improve the efficacy and economy of wastewater treatment. Our current knowledge is largely based on 16S rRNA gene amplicon profiling, fluorescence in situ hybridization studies, and predictions based on metagenome-assembled genomes. Bacterial isolates are often required to validate genome-based predictions as they allow researchers to analyze a specific species without interference from other bacteria and with simple bulk measurements. Unfortunately, there are currently very few pure cultures of microbes commonly found in WWTPs. To address this, we introduce an isolation strategy that takes advantage of state-of-the-art microbial profiling techniques to uncover suitable growth conditions for key WWTP microbes. We furthermore demonstrate that this information can be used to isolate key organisms representing global WWTPs.

microbiology↗

bioRxiv2023

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

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

bioengineering↗

bioRxiv2021

Re-evaluation of the phylogenetic diversity and global distribution of the genus Candidatus Accumulibacter

Candidatus Accumulibacter was the first microorganism identified as a polyphosphate-accumulating organism (PAO), important for phosphorus removal from wastewater. This genus is diverse, and the current phylogeny and taxonomic framework appears complicated, with the majority of publicly available genomes classified as "Candidatus Accumulibacter phosphatis", despite notable phylogenetic divergence. The ppk1 marker gene allows for a finer scale differentiation into different "types" and "clades", nevertheless taxonomic assignments remain confusing and inconsistent across studies. Therefore, a comprehensive re-evaluation is needed to establish a common understanding of this genus, both in terms of naming and basic conserved physiological traits. Here, we provide this re-assessment using a comparison of genome, ppk1, and 16S rRNA gene-based approaches from comprehensive datasets. We identified 15 novel species, along with the well-known Ca. A. phosphatis, Ca. A. deltensis and Ca. A. aalborgensis. To compare the species in situ, we designed new species-specific FISH probes and revealed their morphology and arrangement in activated sludge. Based on the MiDAS global survey, Ca. Accumulibacter species were widespread in WWTPs with phosphorus removal, indicating the process design as a major driver for their abundance. Genome mining for PAO related pathways and FISH-Raman microspectroscopy confirmed the potential for the PAO metabolism in all Ca. Accumulibacter species, with detection in situ of the typical PAO storage polymers. Genome annotation further revealed fine-scale differences in the nitrate/nitrite reduction pathways. This provides insights into the niche differentiation of these lineages, potentially explaining their coexistence in the same ecosystem while contributing to overall phosphorus and nitrogen removal. ImportanceCandidatus Accumulibacter is the most studied PAO organism, with a primary role in biological nutrient removal. However, the species-level taxonomy of this lineage is convoluted due to the use of different phylogenetic markers or genome sequencing. Here, we redefined the phylogeny of these organisms, proposing a comprehensive approach which could be used to address the classification of other diverse and uncultivated lineages. Using genome-resolved phylogeny, compared to 16S rRNA gene- and other phylogenetic markers phylogeny, we obtained a higher resolution taxonomy and established a common understanding of this genus. Furthermore, genome mining of gene and pathways of interest, validated in situ by application of a new set of FISH probes and Raman micromicrospectroscopy, provided additional high-resolution metabolic insights into these organisms.

microbiology↗