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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.

Understanding the microbial ecology of a system requires that the observed population dynamics can be linked to their metabolic functions. However, functional characterization is laborious and the choice of organisms should be prioritized to those that are frequently abundant (core) or transiently abundant, which are therefore putatively make the greatest contribution to carbon turnover in the system. We analyzed the microbial communities in 13 Danish wastewater treatment plants with nutrient removal in consecutive years and a single plant periodically over 6 years, using Illumina sequencing of 16S ribosomal RNA amplicons of the V4 region. The plants contained a core community of 63 abundant genus-level operational taxonomic units (OTUs) that made up 68% of the total reads. A core community consisting of abundant OTUs was also observed within the incoming wastewater to three plants. The net growth rate for individual OTUs was quantified using mass balance, and it was found that 10% of the total reads in the activated sludge were from slow or non-growing OTUs, and that their measured abundance was primarily because of immigration with the wastewater. Transiently abundant organisms were also identified. Among them the genus Nitrotoga (class Betaproteobacteria) was the most abundant putative nitrite oxidizer in a number of activated sludge plants, which challenges previous assumptions that Nitrospira (phylum Nitrospirae) are the primary nitrite-oxidizers in activated sludge systems with nutrient removal.

Microorganisms play crucial roles in water recycling, pollution removal and resource recovery in the wastewater industry. The structure of these microbial communities is increasingly understood based on 16S rRNA amplicon sequencing data. However, such data cannot be linked to functional potential in the absence of high-quality metagenome-assembled genomes (MAGs) for nearly all species. Here, we use long-read and short-read sequencing to recover 1083 high-quality MAGs, including 57 closed circular genomes, from 23 Danish full-scale wastewater treatment plants. The MAGs account for ~30% of the community based on relative abundance, and meet the stringent MIMAG high-quality draft requirements including full-length rRNA genes. We use the information provided by these MAGs in combination with >13 years of 16S rRNA amplicon sequencing data, as well as Raman microspectroscopy and fluorescence in situ hybridisation, to uncover abundant undescribed lineages belonging to important functional groups. Microbes play key roles in wastewater treatment. Here, Singleton et al. use long-read and short-read sequencing to recover 1083 high-quality metagenome-assembled genomes from 23 wastewater treatment plants, and combine this information with amplicon data, Raman microspectroscopy and FISH to reveal functionally important lineages.

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. Until now, the oxidation steps necessary for complete nitrification have always been observed to occur in two separate microorganisms in a cross-feeding interaction; here, together with the study by van Kessel et al ., Daims et al . report the enrichment and characterization of Nitrospira species that encode all of the enzymes necessary to catalyse complete nitrification, a phenotype referred to as “comammox” (for complete ammonia oxidation). Time to rethink nitrification Two groups this week report the enrichment and characterization of Nitrospira species that encode all of the enzymes necessary to catalyse complete nitrification, a phenotype referred to as 'comammox' (for complete ammonia oxidation). Until now, this two-step reaction was thought to involve two organisms in a cross-feeding interaction. Phylogenetic analyses suggest that comammox Nitrospira are present in a number of diverse environments, so these findings have the potential to fundamentally change our view of the nitrogen cycle and open a new frontier in nitrification research.

Until now, the oxidation steps necessary for complete nitrification had always been observed to occur in two separate microorganisms in a cross-feeding interaction; here, together with the study by Daims et al ., van Kessel et al . report the enrichment and characterization of Nitrospira species that encode all of the enzymes necessary to catalyse complete nitrification, a phenotype referred to as ‘comammox’ (for complete ammonia oxidation). Time to rethink nitrification Two groups this week report the enrichment and characterization of Nitrospira species that encode all of the enzymes necessary to catalyse complete nitrification, a phenotype referred to as 'comammox' (for complete ammonia oxidation). Until now, this two-step reaction was thought to involve two organisms in a cross-feeding interaction. Phylogenetic analyses suggest that comammox Nitrospira are present in a number of diverse environments, so these findings have the potential to fundamentally change our view of the nitrogen cycle and open a new frontier in nitrification research. Nitrification is a two-step process where ammonia is first oxidized to nitrite by ammonia-oxidizing bacteria and/or archaea, and subsequently to nitrate by nitrite-oxidizing bacteria. Already described by Winogradsky in 1890 1 , this division of labour between the two functional groups is a generally accepted characteristic of the biogeochemical nitrogen cycle 2 . Complete oxidation of ammonia to nitrate in one organism (complete ammonia oxidation; comammox) is energetically feasible, and it was postulated that this process could occur under conditions selecting for species with lower growth rates but higher growth yields than canonical ammonia-oxidizing microorganisms 3 . Still, organisms catalysing this process have not yet been discovered. Here we report the enrichment and initial characterization of two Nitrospira species that encode all the enzymes necessary for ammonia oxidation via nitrite to nitrate in their genomes, and indeed completely oxidize ammonium to nitrate to conserve energy. Their ammonia monooxygenase (AMO) enzymes are phylogenetically distinct from currently identified AMOs, rendering recent acquisition by horizontal gene transfer from known ammonia-oxidizing microorganisms unlikely. We also found highly similar amoA sequences (encoding the AMO subunit A) in public sequence databases, which were apparently misclassified as methane monooxygenases. This recognition of a novel amoA sequence group will lead to an improved understanding of the environmental abundance and distribution of ammonia-oxidizing microorganisms. Furthermore, the discovery of the long-sought-after comammox process will change our perception of the nitrogen cycle.

More of the diversity present in any microbiome is revealed by a method to sequence 16S rRNA that avoids 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.

The glycogen-accumulating organism (GAO) ‘ Candidatus Competibacter’ (Competibacter) uses aerobically stored glycogen to enable anaerobic carbon uptake, which is subsequently stored as polyhydroxyalkanoates (PHAs). This biphasic metabolism is key for the Competibacter to survive under the cyclic anaerobic-‘feast’: aerobic-‘famine’ regime of enhanced biological phosphorus removal (EBPR) wastewater treatment systems. As they do not contribute to phosphorus (P) removal, but compete for resources with the polyphosphate-accumulating organisms (PAO), thought responsible for P removal, their proliferation theoretically reduces the EBPR capacity. In this study, two complete genomes from Competibacter were obtained from laboratory-scale enrichment reactors through metagenomics. Phylogenetic analysis identified the two genomes, ‘ Candidatus Competibacter denitrificans’ and ‘ Candidatus Contendobacter odensis’, as being affiliated with Competibacter-lineage subgroups 1 and 5, respectively. Both have genes for glycogen and PHA cycling and for the metabolism of volatile fatty acids. Marked differences were found in their potential for the Embden–Meyerhof–Parnas and Entner–Doudoroff glycolytic pathways, as well as for denitrification, nitrogen fixation, fermentation, trehalose synthesis and utilisation of glucose and lactate. Genetic comparison of P metabolism pathways with sequenced PAOs revealed the absence of the Pit phosphate transporter in the Competibacter-lineage genomes—identifying a key metabolic difference with the PAO physiology. These genomes are the first from any GAO organism and provide new insights into the complex interaction and niche competition between PAOs and GAOs in EBPR systems.

Thaumarchaeota have been detected in several industrial and municipal wastewater treatment plants (WWTPs), despite the fact that ammonia-oxidizing archaea (AOA) are thought to be adapted to low ammonia environments. However, the activity, physiology and metabolism of WWTP-associated AOA remain poorly understood. We report the cultivation and complete genome sequence of Candidatus Nitrosocosmicus exaquare, a novel AOA representative from a municipal WWTP in Guelph, Ontario (Canada). In enrichment culture, Ca. N. exaquare oxidizes ammonia to nitrite stoichiometrically, is mesophilic, and tolerates at least 15 m m of ammonium chloride or sodium nitrite. Microautoradiography (MAR) for enrichment cultures demonstrates that Ca . N. exaquare assimilates bicarbonate in association with ammonia oxidation. However, despite using inorganic carbon, the ammonia-oxidizing activity of Ca. N. exaquare is greatly stimulated in enrichment culture by the addition of organic compounds, especially malate and succinate. Ca. N. exaquare cells are coccoid with a diameter of ~1–2 μm. Phylogenetically, Ca. N. exaquare belongs to the Nitrososphaera sister cluster within the Group I.1b Thaumarchaeota , a lineage which includes most other reported AOA sequences from municipal and industrial WWTPs. The 2.99 Mbp genome of Ca. N. exaquare encodes pathways for ammonia oxidation, bicarbonate fixation, and urea transport and breakdown. In addition, this genome encodes several key genes for dealing with oxidative stress, including peroxidase and catalase. Incubations of WWTP biofilm demonstrate partial inhibition of ammonia-oxidizing activity by 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), suggesting that Ca. N. exaquare-like AOA may contribute to nitrification in situ . However, CARD-FISH-MAR showed no incorporation of bicarbonate by detected Thaumarchaeaota , suggesting that detected AOA may incorporate non-bicarbonate carbon sources or rely on an alternative and yet unknown metabolism.

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 from  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.

Serpentinization, a geochemical process found on modern and ancient Earth, provides an ultra-reducing environment that can support microbial methanogenesis and acetogenesis. Several groups of archaea, such as the order Methanocellales , are characterized by their ability to produce methane. Here, we generate metagenomic sequences from serpentinized springs in The Cedars, California, and construct a circularized metagenome-assembled genome of a Methanocellales archaeon, termed Met12, that lacks essential methanogenesis genes. The genome includes genes for an acetyl-CoA pathway, but lacks genes encoding methanogenesis enzymes such as methyl-coenzyme M reductase, heterodisulfide reductases and hydrogenases. In situ transcriptomic analyses reveal high expression of a multi-heme c- type cytochrome, and heterologous expression of this protein in a model bacterium demonstrates that it is capable of accepting electrons. Our results suggest that Met12, within the order Methanocellales , is not a methanogen but a CO 2 -reducing, electron-fueled acetogen without electron bifurcation. Several groups of archaea, such as the order Methanocellales , are characterised by their ability to produce methane. Here, Suzuki et al. identify a Methanocellales archaeon that lacks essential methanogenesis genes and seems to be instead a CO 2 -reducing, electron-fueled acetogen.