Biology subjects

Nielsen, P. H.

Publications and source records attributed to Nielsen, P. H..

At least 37 records · Page 2Linked to original sources

bioRxiv2022

Signatures of Microbial Diversity at Multiple Scales of Resolution within Engineered Enrichment Communities

Microbial community dynamics are dictated by both abiotic environmental conditions and biotic interactions. These communities consist of individual microorganisms across the continuum of phylogenetic diversity, ranging from coexisting members of different domains of life and phyla to multiple strains with only a handful of single nucleotide variants. Ecological forces act on a shifting template of population-level diversity that is shaped by evolutionary processes. However, understanding the ecological and evolutionary forces contributing to microbial community interactions and overall ecosystem function is difficult to interrogate for complex, naturally occurring microbial communities. Here, we use two time series of lab-scale engineered enrichment microbial communities simulating phosphorus removal to explore signatures of microbial diversity at multiple phylogenetic scales. We characterized microbial community dynamics and diversity over the course of reactor start-up and long-term dynamics including periods of eubiosis and dysbiosis as informed by the intended ecosystem function of phosphorus removal. We then compared these signatures to lineages from full-scale WWTPs performing phosphorus removal. We found that enriched lineages in lab-scale bioreactors harbor less intra-population diversity than lineages from the full-scale WWTP overall. Our work establishes a foundation for using engineered enrichment microbial communities as a semi-complex model system for addressing the fundamental ecological and evolutionary processes necessary for developing stable microbiome based biotechnologies.

microbiology↗

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↗

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

Global warming readiness: Feasibility of enhanced biological phosphorus removal from wastewater at 35oC

Recent research has shown enhanced biological phosphorus removal (EBPR) from municipal wastewater at warmer temperatures around 30{degrees}C to be stable in both laboratory-scale reactors and full-scale treatment plants. In the context of a changing climate, the feasibility of EBPR at even higher temperatures is of interest. We operated two lab-scale EBPR sequencing batch reactors with alternating anaerobic and aerobic phases for over 300 days at 30{degrees}C and 35{degrees}C, respectively, and followed the dynamics of the communities of phosphorus accumulating organisms (PAOs) and competing glycogen accumulating organisms (GAOs) using a combination of 16S rRNA gene metabarcoding, quantitative PCR and fluorescent in-situ hybridization analyses. Stable and nearly complete P removal was achieved at 30{degrees}C; similarly, long term P removal was stable at 35{degrees}C with effluent PO43--P concentrations < 0.5 mg/L on half of all monitored days. Diverse and abundant Ca. Accumulibacter amplicon sequence variants were closely related to those found in temperate environments, suggesting that EBPR at this temperature does not require a highly specialized PAO community. The slow-feeding strategy used effectively limited the carbon uptake rates of GAOs, allowing PAOs to outcompete GAOs at both temperatures. Candidatus Competibacter was the main GAO, along with cluster III Defluviicoccus members. These organisms withstood the slow-feeding regime, suggesting that their bioenergetic characteristics of carbon uptake differ from those of their tetrad-forming relatives. This specific lineage of GAOs warrants further study to establish how complete P removal can be maintained. Comparative cycle studies at two temperatures for each reactor revealed higher activity of Ca. Accumulibacter when the temperature was increased from 30{degrees}C to 35{degrees}C, suggesting that the stress was a result of the higher carbon (and/or P) metabolic rates of PAOs and GAOs, the resultant carbon deficiency, and additional community competition. An increase in the TOC to PO43--P ratio (from 25:1 to 40:1) effectively eased the carbon deficiency and benefited the proliferation of PAOs. In general, the slow-feeding strategy and sufficiently high carbon input benefited a high and stable EBPR at elevated temperature and represent basic conditions for full-scale applications.

microbiology↗

bioRxiv2021

Quantification of biologically and chemically bound phosphorus in activated sludge from full-scale plants with biological P-removal

Large amounts of phosphorus (P) are present in activated sludge from municipal wastewater treatment plants, where it exists in the form of metal salt precipitates or biologically bound into the biomass as nucleic acids, cell membrane components, and the extracellular polymeric substances or, in special polyphosphate-accumulating organisms (PAOs), as intracellular polyphosphate. Only recently, methods that reliably allow an absolute quantification of the different P-fractions, such as sequential extraction, Raman microspectroscopy, solid-state 31P magic angle spinning (MAS) NMR, and solution state 31P NMR have been developed. This study combines these techniques to obtain a comprehensive P mass-balance of activated sludge from four wastewater treatment plants with enhanced biological phosphate removal (EBPR). The total content of P and various cations was measured by chemical analysis (ICP-OES), and different P fractions were extracted for chemical characterization. Chemically bound P constituted 38-69% of total P, most likely in the form of Fe, Mg, or Al minerals, while organically bound P constituted 7-9%. By using Raman microspectroscopy and solution state 31P NMR and 31P MAS NMR spectroscopy before and after anaerobic P-release experiments, poly-P was quantified and constituted 22-54% of total P in the activated sludges and was found in approx. 25% of all bacterial cells. Moreover, Raman microspectroscopy in combination with fluorescence in situ hybridization (FISH) was used to quantify the species-specific intracellular poly-P of known PAO genera (Tetrasphaera, Ca. Accumulibacter, Dechloromonas) and other microorganisms known to possess high level of poly-P, such as the filamentous Ca. Microthrix. They were all abundant, as measured by quantitative-FISH and amplicon sequencing, and accumulated large amount of poly-P, depending on their cell-size, contributing substantially to the P-removal. Interestingly, in all four EBPR plants investigated, only 1-13% of total poly-P was stored by unidentified PAO, highlighting that most PAOs in the full-scale EBPR plants investigated are now known. HighlightsO_LIExhaustive P mass-balance of main organic and inorganic P-species in four EBPR plants C_LIO_LIQuantification of poly-P of FISH-defined PAO and other species with high P content C_LIO_LITotal P content was 36-50 mgP/gSS of which 31-62% was in biomass and as poly-P C_LIO_LIA high fraction of all cells (25-30%) contained a high content of poly-P C_LIO_LIKnown PAOs contained almost all poly-P in the EBPR plants investigated C_LI

microbiology↗

bioRxiv2020

Candidatus Dechloromonas phosphatis and Candidatus Dechloromonas phosphovora, two novel polyphosphate accumulating organisms abundant in wastewater treatment systems

Members of the genus Dechloromonas are often abundant in enhanced biological phosphorus removal (EBPR) systems and are recognized putative polyphosphate accumulating organisms (PAOs), but their role in phosphate (P) removal is still unclear. Here, we used 16S rRNA gene sequencing and fluorescence in situ hybridization (FISH) to investigate the abundance and distribution of Dechloromonas spp. in Danish wastewater treatment plants. Two species were abundant, novel, and uncultured, and could be targeted by existing FISH probes. Raman microspectroscopy of probe-defined organisms (FISH-Raman) revealed the levels and dynamics of important intracellular storage polymers in abundant Dechloromonas spp. in the activated sludge from four full-scale EBPR plants and from a lab-scale sequencing batch reactor fed with different carbon sources (acetate, glucose, glycine, and glutamate). Moreover, 7 distinct Dechloromonas species were determined from a set of 10 high-quality metagenome-assembled genomes (MAGs) from Danish EBPR plants, each encoding the potential for poly-P, glycogen, and polyhydroxyalkanoates (PHA) accumulation. The two most abundant species exhibited an in situ phenotype in complete accordance with the metabolic information retrieved by the MAGs, with dynamic levels of poly-P, glycogen, and PHA during feast-famine anaerobic-aerobic cycling, legitimately placing these microorganisms among the important PAOs. As no isolates are available for the two species, we propose the names Candidatus Dechloromonas phosphatis and Candidatus Dechloromonas phosphovora.

microbiology↗

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

A refined set of rRNA-targeted oligonucleotide probes for in situ detection and quantification of ammonia-oxidizing bacteria

Ammonia-oxidizing bacteria (AOB) of the betaproteobacterial genera Nitrosomonas and Nitrosospira are key nitrifying microorganisms in many natural and engineered ecosystems. Since many AOB remain uncultured, fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes has been one of the most widely used approaches to study the community composition, abundance, and other features of AOB directly in environmental samples. However, the established and widely used AOB-specific 16S rRNA-targeted FISH probes were designed up to two decades ago, based on much smaller rRNA gene sequence datasets than available today. Several of these probes cover their target AOB lineages incompletely and suffer from a weak target specificity, which causes cross-hybridization of probes that should detect different AOB lineages. Here, a set of new highly specific 16S rRNA-targeted oligonucleotide probes was developed and experimentally evaluated that complements the existing probes and enables the specific detection and differentiation of the known, major phylogenetic clusters of betaproteobacterial AOB. The new probes were successfully applied to visualize and quantify AOB in activated sludge and biofilm samples from seven pilot- and full-scale wastewater treatment systems. Based on its improved target group coverage and specificity, the refined probe set will facilitate future in situ analyses of AOB.

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↗

bioRxiv2020

Exploring the upper pH limits of nitrite oxidation: diversity, ecophysiology, and adaptive traits of haloalkalitolerant Nitrospira

Nitrite-oxidizing bacteria of the genus Nitrospira are key players of the biogeochemical nitrogen cycle. However, little is known about their occurrence and survival strategies in extreme pH environments. Here, we report on the discovery of physiologically versatile, haloalkalitolerant Nitrospira that drive nitrite oxidation at exceptionally high pH. Nitrospira distribution, diversity, and ecophysiology were studied in hypo- and subsaline (1.3-12.8 g salt/l), highly alkaline (pH 8.9-10.3) lakes by amplicon sequencing, metagenomics, and cultivation-based approaches. Surprisingly, not only were Nitrospira populations detected, but they were also considerably diverse with presence of members of Nitrospira lineages I, II and IV. Furthermore, the ability of Nitrospira enrichment cultures to oxidize nitrite at neutral to highly alkaline pH of 10.5 was demonstrated. Metagenomic analysis of a newly enriched Nitrospira lineage IV species, "Candidatus Nitrospira alkalitolerans", revealed numerous adaptive features of this organism to its extreme environment. Among them were a sodium-dependent N-type ATPase and NADH:quinone oxidoreductase next to the proton-driven forms usually found in Nitrospira. Other functions aid in pH and cation homeostasis and osmotic stress defense. "Ca. Nitrospira alkalitolerans" also possesses group 2a and 3b [NiFe] hydrogenases, suggesting it can use hydrogen as alternative energy source. These results reveal how Nitrospira cope with strongly fluctuating pH and salinity conditions and expand our knowledge of nitrogen cycling in extreme habitats.

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

Characterization of a thaumarchaeal symbiont that drives incomplete nitrification in the tropical sponge Ianthella basta

Marine sponges represent one of the few eukaryotic groups that frequently harbor symbiotic members of the Thaumarchaeota, which are important chemoautotrophic ammonia-oxidizers in many environments. However, in most studies, direct demonstration of ammonia-oxidation by these archaea within sponges is lacking, and little is known about sponge-specific adaptations of ammonia-oxidizing archaea (AOA). Here, we characterized the thaumarchaeal symbiont of the marine sponge Ianthella basta using metaproteogenomics, fluorescence in situ hybridization, qPCR and isotope-based functional assays. "Candidatus Nitrosospongia bastadiensis" is only distantly related to cultured AOA. It is an abundant symbiont that is solely responsible for nitrite formation from ammonia in I. basta that surprisingly does not harbor nitrite-oxidizing microbes. Furthermore, this AOA is equipped with an expanded set of extracellular subtilisin-like proteases, a metalloprotease unique among archaea, as well as a putative branched-chain amino acid ABC transporter. This repertoire is strongly indicative of a mixotrophic lifestyle and is (with slight variations) also found in other sponge-associated, but not in free-living AOA. We predict that this feature as well as an expanded and unique set of secreted serpins (protease inhibitors), a unique array of eukaryotic-like proteins, and a DNA-phosporothioation system, represent important adaptations of AOA to life within these ancient filter-feeding animals. Originality-Significance StatementMany marine sponges harbor symbiotic members of the Thaumarchaeota, but there is generally only indirect evidence available about their functional role within these filter-feeding animals. Furthermore, the specific adaptations of thaumarchaeal symbionts to their sponge hosts are incompletely understood. In this study, we thoroughly characterized a thaumarchaeal symbiont residing in the reef sponge Ianthella basta and demonstrate by using a combination of molecular tools and isotope techniques, that it is the only ammonia-oxidizer in its host. In contrast to other sponges, I. basta does not contain nitrite-oxidizing microbes and thus excretes considerable amounts of nitrite. Furthermore, using metagenomics and metaproteomics we reveal important adaptations of this symbiont, that represents a new genus within the Thaumarchaeota, and conclude that it most likely lives as a mixotroph in its sponge host.

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↗

bioRxiv2018

Resolving the individual contribution of key microbial populations to enhanced biological phosphorus removal with Raman-FISH

Enhanced biological phosphorus removal (EBPR) is a globally important biotechnological process and relies on the massive accumulation of phosphate within special microorganisms. Candidatus Accumulibacter conform to classical physiology model for polyphosphate accumulating organisms and are widely believed to be the most important player for the process in full-scale EBPR systems. However, it was impossible till now to quantify the contribution of specific microbial clades to EBPR. In this study, we have developed a new tool to directly link the identity of microbial cells to the absolute quantification of intracellular poly-P and other polymers under in situ conditions, and applied it to eight full-scale EBPR plants. Besides Ca. Accumulibacter, members of the genus Tetrasphaera were found to be important microbes for P accumulation, and in six plants they were the most important. As these Tetrasphaera cells did not exhibit the classical phenotype of poly-P accumulating microbes, our entire understanding of the microbiology of the EBPR process has to be revised. Furthermore, our new single-cell approach can now also be applied to quantify storage polymer dynamics in individual populations in situ in other ecosystems and might become a valuable tool for many environmental microbiologists.

ecology↗

bioRxiv2018

The ecology of the Chloroflexi in full-scale activated sludge wastewater treatment plants

Filamentous bacteria belonging to the phylum Chloroflexi have received considerable attention in wastewater treatment systems for their suggested role in operational problem of impaired sludge settleability known as bulking. Their consistently high abundance in full-scale systems, even in the absence of bulking, indicates that they make a substantial contribution to the nutrient transformations during wastewater treatment. In this study, extensive 16S rRNA amplicon surveys of full-scale Danish WWTPs were screened to identify the most numerically important Chloroflexi genera. Fluorescence in situ hybridization probes were designed for their in situ characterization. All abundant phylotypes of the phylum were identified as facultative anaerobic chemoorganotrophs involved in fermentation of sugars. These groups were all filamentous but differed in their morphology and spatial arrangement. Candidatus Villigracilis was predominantly located within the activated sludge flocs, where they possibly have structural importance, and their abundance was relatively stable. Conversely, the abundance of Candidatus Amarolinea was highly dynamic, relative to other genera, sometimes reaching abundances in excess of 30% of the biovolume, suggesting their likely role in bulking episodes. This study gives an important insight into the role of Chloroflexi in WWTPs, thus contributing to the broader goal of understanding the ecology of these biotechnologically important 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

Peatland Acidobacteria with a dissimilatory sulfur metabolism

Sulfur-cycling microorganisms impact organic matter decomposition in wetlands and consequently greenhouse gas emissions from these globally relevant environments. However,their identities and physiological properties are largely unknown. By applying a functional metagenomics approach to an acidic peatland, we recovered draft genomes of seven novel Acidobacteria species with the potential for dissimilatory sulfite (dsrAB, dsrC, dsrD, dsrN, dsrT, dsrMKJOP) or sulfate respiration (sat, aprBA, qmoABC plus dsr genes). Surprisingly, the genomes also encoded dsrL, a unique gene of the sulfur oxidation pathway. Metatranscriptome analysis demonstrated expression of acidobacterial sulfur-metabolism genes in native peat soil and their upregulation in diverse anoxic microcosms. This indicated an active sulfate respiration pathway, which, however, could also operate in reverse for sulfur oxidation as recently shown for other microorganisms. Acidobacteria that only harbored genes for sulfite reduction additionally encoded enzymes that liberate sulfite from organosulfonates, which suggested organic sulfur compounds as complementary energy sources. Further metabolic potentials included polysaccharide hydrolysis and sugar utilization, aerobic respiration, several fermentative capabilities, and hydrogen oxidation. Our findings extend both, the known physiological and genetic properties of Acidobacteria and the known taxonomic diversity of microorganisms with a DsrAB-based sulfur metabolism, and highlight new fundamental niches for facultative anaerobic Acidobacteria in wetlands based on exploitation of inorganic and organic sulfur molecules for energy conservation.

microbiology↗