Rasmus Hansen Kirkegaard

Complete nitrification by Nitrospira bacteria

Nitrification, the oxidation of ammonia via nitrite to nitrate, has always been considered to be a two-step process catalysed by chemolithoautotrophic microorganisms oxidizing either ammonia or nitrite. No known nitrifier carries out both steps, although complete nitrification should be energetically advantageous. This functional separation has puzzled microbiologists for a century. Here we report on the discovery and cultivation of a completely nitrifying bacterium from the genus Nitrospira, a globally distributed group of nitrite oxidizers. The genome of this chemolithoautotrophic organism encodes the pathways both for ammonia and nitrite oxidation, which are concomitantly activated during growth by ammonia oxidation to nitrate. Genes affiliated with the phylogenetically distinct ammonia monooxygenase and hydroxylamine dehydrogenase genes of Nitrospira are present in many environments and were retrieved on Nitrospira-contigs in new metagenomes from engineered systems. These findings fundamentally change our picture of nitrification and point to completely nitrifying Nitrospira as key components of nitrogen-cycling microbial communities.

Back to Basics – The Influence of DNA Extraction and Primer Choice on Phylogenetic Analysis of Activated Sludge Communities

DNA extraction and primer choice have a large effect on the observed community structure in all microbial amplicon sequencing analyses. Although the biases are well known, no comprehensive analysis has been conducted in activated sludge communities. In this study we systematically explored the impact of a number of parameters on the observed microbial community: bead beating intensity, primer choice, extracellular DNA removal, and various PCR settings. In total, 176 samples were subjected to 16S rRNA amplicon sequencing, and selected samples were investigated through metagenomics and metatranscriptomics. Quantitative fluorescence in situ hybridization was used as a DNA extraction-independent method for qualitative comparison. In general, an effect on the observed community was found on all parameters tested, although bead beating and primer choice had the largest effect. The effect of bead beating intensity correlated with cell-wall strength as seen by a large increase in DNA from Gram-positive bacteria (up to 400%). However, significant differences were present at lower phylogenetic levels within the same phylum, suggesting that additional factors are at play. The best primer set based on in silico analysis was found to underestimate a number of important bacterial groups. For 16S rRNA gene analysis in activated sludge we recommend using the FastDNA SPIN Kit for Soil with four times the normal bead beating and V1-3 primers.

Genomic insights into members of the candidate phylum Hyd24-12 common in mesophilic anaerobic digesters.

Members of the candidate phylum Hyd24-12 are globally distributed, but no genomic information or knowledge about their morphology, physiology or ecology is available. In this study, members of the Hyd24-12 lineage were shown to be present and abundant in full-scale mesophilic anaerobic digesters at Danish wastewater treatment facilities. In some samples, a member of the Hyd24-12 lineage was one of the most abundant genus-level bacterial taxa, accounting for up to 8% of the bacterial biomass. Three closely related and near-complete genomes were retrieved using metagenome sequencing of full-scale anaerobic digesters. Genome annotation and metabolic reconstruction showed that they are Gram-negative bacteria likely involved in acidogenesis, producing acetate and hydrogen from fermentation of sugars, and may play a role in the cycling of sulphur in the digesters. Fluorescence in situ hybridization revealed single rod-shaped cells dispersed within the flocs. The genomic information forms a foundation for a more detailed understanding of their role in anaerobic digestion and provides the first insight into a hitherto undescribed branch in the tree of life.

Retrieval of a million high-quality, full-length microbial 16S and 18S rRNA gene sequences without primer bias

Small subunit ribosomal RNA (SSU rRNA) genes, 16S in bacteria and 18S in eukaryotes, have been the standard phylogenetic markers used to characterize microbial diversity and evolution for decades. However, the reference databases of full-length SSU rRNA gene sequences are skewed to well-studied ecosystems and subject to primer bias and chimerism, which results in an incomplete view of the diversity present in a sample. We combine poly(A)-tailing and reverse transcription of SSU rRNA molecules with synthetic long-read sequencing to generate high-quality, full-length SSU rRNA sequences, without primer bias, at high throughput. We apply our approach to samples from seven different ecosystems and obtain more than a million SSU rRNA sequences from all domains of life, with an estimated raw error rate of 0.17%. We observe a large proportion of novel diversity, including several deeply branching phylum-level lineages putatively related to the Asgard Archaea. Our approach will enable expansion of the SSU rRNA reference databases by orders of magnitude, and contribute to a comprehensive census of the tree of life.

mmgenome: a toolbox for reproducible genome extraction from metagenomes

Summary Recovery of population genomes is becoming a standard analysis in metagenomics and a multitude of different approaches exists. However, the workflows are complex, requiring data generation, binning, validation and finishing to generate high quality population genome bins. In addition, several different approaches are often used on the same dataset as the optimal strategy to extract a specific population genome varies. Here we introduce mmgenome: a toolbox for reproducible genome extraction from metagenomes. At the core of mmgenome is an R package that facilitates effortless integration of different binning strategies by collecting information on scaffolds. Genome binning is facilitated through integrated tools that support effortless visualizations, validation and calculation of key statistics. Full reproducibility and transparency is obtained through Rmarkdown, whereby every step can be recreated. Availability and implementation The binning framework of mmge-nome is implemented in R. Wrapper scripts for data generation and finishing is written in Perl. The mmgenome toolbox and associated step-by-step guides are available at http://madsal-bertsen.github.io/mmgenome/. Contact ma@bio.aau.dk Supplementary information Supplementary data are available at Bioinformatics online.

The impact of immigration on microbial community composition in 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. Hence, we investigated microbial communities of thirty-two full-scale anaerobic 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 growing microbial community. Furthermore, several abundant OTUs could not be classified to genus level with commonly applied taxonomies, making inference of their function unreliable and comparison to other studies problematic. As such, the existing MiDAS taxonomy was updated to include these abundant phylotypes. The communities of individual digesters surveyed were remarkably similar – with only 300 OTUs representing 80% of the total reads across all plants, and 15% of these identified as non-growing and possibly inactive immigrating microbes. By identifying abundant and growing taxa in anaerobic digestion, this study paves the way for targeted characterisation of the process-important organisms towards an in-depth understanding of the microbiology.

MiDAS 2.0: an ecosystem-specific taxonomy and online database for the organisms of wastewater treatment systems expanded for anaerobic digester groups

Abstract Wastewater is increasingly viewed as a resource, with anaerobic digester technology being routinely implemented for biogas production. Characterising the microbial communities involved in wastewater treatment facilities and their anaerobic digesters is considered key to their optimal design and operation. Amplicon sequencing of the 16S rRNA gene allows high-throughput monitoring of these systems. The MiDAS field guide is a public resource providing amplicon sequencing protocols and an ecosystem-specific taxonomic database optimized for use with wastewater treatment facility samples. The curated taxonomy endeavours to provide a genus-level-classification for abundant phylotypes and the online field guide links this identity to published information regarding their ecology, function and distribution. This article describes the expansion of the database resources to cover the organisms of the anaerobic digester systems fed primary sludge and surplus activated sludge. The updated database includes descriptions of the abundant genus-level-taxa in influent wastewater, activated sludge and anaerobic digesters. Abundance information is also included to allow assessment of the role of emigration in the ecology of each phylotype. MiDAS is intended as a collaborative resource for the progression of research into the ecology of wastewater treatment, by providing a public repository for knowledge that is accessible to all interested in these biotechnologically important systems. Database URL: http://www.midasfieldguide.org

Integrative microbial community analysis reveals full-scale enhanced biological phosphorus removal under tropical conditions

Management of phosphorus discharge from human waste is essential for the control of eutrophication in surface waters. Enhanced biological phosphorus removal (EBPR) is a sustainable, efficient way of removing phosphorus from waste water without employing chemical precipitation, but is assumed unachievable in tropical temperatures due to conditions that favour glycogen accumulating organisms (GAOs) over polyphosphate accumulating organisms (PAOs). Here, we show these assumptions are unfounded by studying comparative community dynamics in a full-scale plant following systematic perturbation of operational conditions, which modified community abundance, function and physicochemical state. A statistically significant increase in the relative abundance of the PAO Accumulibacter was associated with improved EBPR activity. GAO relative abundance also increased, challenging the assumption of competition. An Accumulibacter bin-genome was identified from a whole community metagenomic survey, and comparative analysis against extant Accumulibacter genomes suggests a close relationship to Type II. Analysis of the associated metatranscriptome data revealed that genes encoding proteins involved in the tricarboxylic acid cycle and glycolysis pathways were highly expressed, consistent with metabolic modelling results. Our findings show that tropical EBPR is indeed possible, highlight the translational potential of studying competition dynamics in full-scale waste water communities and carry implications for plant design in tropical regions.

Thousands of primer-free, high-quality, full-length SSU rRNA sequences from all domains of life

Ribosomal RNA (rRNA) genes are the consensus marker for determination of microbial diversity on the planet, invaluable in studies of evolution and, for the past decade, high-throughput sequencing of variable regions of ribosomal RNA genes has become the backbone of most microbial ecology studies. However, the underlying reference databases of full-length rRNA gene sequences are underpopulated, ecosystem skewed1, and subject to primer bias2, which hamper our ability to study the true diversity of ecosystems. Here we present an approach that combines reverse transcription of full-length small subunit (SSU) rRNA genes and synthetic long read sequencing by molecular tagging, to generate primer-free, full-length SSU rRNA gene sequences from all domains of life, with a median raw error rate of 0.17%. We generated thousands of full-length SSU rRNA sequences from five well-studied ecosystems (soil, human gut, fresh water, anaerobic digestion, and activated sludge) and obtained sequences covering all domains of life and the majority of all described phyla. Interestingly, 30% of all bacterial operational taxonomic units were novel, compared to the SILVA database (less than 97% similarity). For the Eukaryotes, the novelty was even larger with 63% of all OTUs representing novel taxa. In addition, 15% of the 18S rRNA OTUs were highly novel sequences with less than 80% similarity to the databases. The generation of primer-free full-length SSU rRNA sequences enabled eco-system specific estimation of primer-bias and, especially for eukaryotes, showed a dramatic discrepancy between the in-silico evaluation and primer-free data generated in this study. The large amount of novel sequences obtained here reaffirms that there is still vast, untapped microbial diversity lacking representatives in the SSU rRNA databases and that there might be more than millions after all1, 3. With our new approach, it is possible to readily expand the rRNA databases by orders of magnitude within a short timeframe. This will, for the first time, enable a broad census of the tree of life.

Genomic and in situ investigations of the novel uncultured Chloroflexi associated with 0092 morphotype filamentous bulking in activated sludge

Overgrowth of filamentous bacteria in activated sludge wastewater treatment plants (WWTPs) leads to impaired sludge settleability, a condition known as bulking, which is a common operational problem worldwide. Filaments with the Eikelboom 0092 morphotype are commonly associated with such bulking episodes. Members of the uncultured B45 phylotype, which is embraced within the phylum Chloroflexi, were recently shown to exhibit this morphology. Although these organisms are among the most abundant populations recorded in activated sludge processes, nothing is known about their metabolic characteristics. In this study, a genome sequence, representing the B45 phylotype, was retrieved from a metagenome generated from an activated sludge WWTP. The genome consisted of two chromosomes and one plasmid, which were 4.0, 1.0 and 0.04 Mbps in size, respectively. A metabolic model was constructed for this organism, based on annotation of its genome, showing its ability to generate energy by respiration, utilizing oxygen, nitrite or nitrous oxide as electron acceptors, or by fermentation of sugars. The ability of B45 members to ferment sugars under anaerobic conditions was validated in situ with microautoradiography—fluorescence in situ hybridization. The provisional name of ‘Candidatus Promineofilum breve’ is proposed for this species. This study represents the first detailed information on an uncultured genus of filamentous organisms from activated sludge.