Feeding strategy and feed protein level affect the gut microbiota of common carp (Cyprinus carpio).

Common carp (Cyprinus carpio) were fed food with different protein concentrations following different feeding regimes, which were previously shown to affect growth, nitrogen excretion and amino acid catabolism. 16S rRNA gene amplicon sequencing was performed to investigate the gut microbiota of these fish. Lower dietary protein content increased microbial richness, while the combination of demand feeding and dietary protein content affected the composition of the gut microbiota. Hepatic glutamate dehydrogenase (GDH) activity was correlated to the composition of the gut microbiota in all dietary treatments. We found that demand-fed carp fed a diet containing 39% protein had a significantly higher abundance of Beijerinckiaceae compared to other dietary groups. Network analysis identified this family and two Rhizobiales families as hubs in the microbial association network. In demand-fed carp, the microbial association network had significantly fewer connections than in batch-fed carp. In contrast to the large effects of the feeding regime and protein content of the food on growth and nitrogen metabolism, it had only limited effects on gut microbiota composition. However, correlations between gut microbiota composition and liver GDH activity showed that host physiology and gut microbiota are connected, which warrants functional studies into the role of the gut microbiota in fish physiology.

Synthesis and Performance of Bio-Based Amphoteric Surfactants.

As the global surfactant market continues to expand, there is an increasing need to develop bio-based alternatives in the shift towards a circular economy. This study focuses on the synthesis of polar, amphoteric, amine-oxide surfactants starting from biomass-derived monosaccharides and demonstrating their potential in various applications. The synthesis involved a reductive amination of the sugars with an alkylamine and formaldehyde followed by oxidation to produce N-oxide surfactants. These bio-based surfactants exhibited promising properties, including high solubility, foamability, surface tension reduction, and critical micelle concentration. In particular, N-GalA1.10 and N-GalA1.12 showed comparable performance to commercial surfactants. Furthermore, these bio-based surfactants demonstrated significantly lower skin irritation potential when compared to petrochemical-derived counterparts like sodium laureth sulfate (SLES), making them potentially suitable for personal care products. The biodegradability assessment revealed that N-GalA1.12 exhibited good biodegradation, indicating its potential environmental compatibility. In conclusion, this study highlights the potential of bio-based N-oxide surfactants derived from monosaccharides as sustainable and skin-friendly alternatives to traditional amphoteric surfactants, like cocamidopropyl betaine (CAPB).

Unveiling unique microbial nitrogen cycling and nitrification driver in coastal Antarctica.

Largely removed from anthropogenic delivery of nitrogen (N), Antarctica has notably low levels of nitrogen. Though our understanding of biological sources of ammonia have been elucidated, the microbial drivers of nitrate (NO3-) cycling in coastal Antarctica remains poorly understood. Here, we explore microbial N cycling in coastal Antarctica, unraveling the biological origin of NO3- via oxygen isotopes in soil and lake sediment, and through the reconstruction of 1968 metagenome-assembled genomes from 29 microbial phyla. Our analysis reveals the metabolic potential for microbial N2 fixation, nitrification, and denitrification, but not for anaerobic ammonium oxidation, signifying a unique microbial N-cycling dynamic. We identify the predominance of complete ammonia oxidizing (comammox) Nitrospira, capable of performing the entire nitrification process. Their adaptive strategies to the Antarctic environment likely include synthesis of trehalose for cold stress, high substrate affinity for resource utilization, and alternate metabolic pathways for nutrient-scarce conditions. We confirm the significant role of comammox Nitrospira in the autotrophic, nitrification process via 13C-DNA-based stable isotope probing. This research highlights the crucial contribution of nitrification to the N budget in coastal Antarctica, identifying comammox Nitrospira clade B as a nitrification driver.

Metabolic and phylogenetic diversity in the phylum Nitrospinota revealed by comparative genome analyses.

The most abundant known nitrite-oxidizing bacteria in the marine water column belong to the phylum Nitrospinota. Despite their importance in marine nitrogen cycling and primary production, there are only few cultured representatives that all belong to the class Nitrospinia. Moreover, although Nitrospinota were traditionally thought to be restricted to marine environments, metagenome-assembled genomes have also been recovered from groundwater. Over the recent years, metagenomic sequencing has led to the discovery of several novel classes of Nitrospinota (UBA9942, UBA7883, 2-12-FULL-45-22, JACRGO01, JADGAW01), which remain uncultivated and have not been analyzed in detail. Here, we analyzed a nonredundant set of 98 Nitrospinota genomes with focus on these understudied Nitrospinota classes and compared their metabolic profiles to get insights into their potential role in biogeochemical element cycling. Based on phylogenomic analysis and average amino acid identities, the highly diverse phylum Nitrospinota could be divided into at least 33 different genera, partly with quite distinct metabolic capacities. Our analysis shows that not all Nitrospinota are nitrite oxidizers and that members of this phylum have the genomic potential to use sulfide and hydrogen for energy conservation. This study expands our knowledge of the phylogeny and potential ecophysiology of the phylum Nitrospinota and offers new avenues for the isolation and cultivation of these elusive bacteria.

Synthesis and Performance of Biobased Surfactants Prepared by the One-Pot Reductive Amination of l-Arabinose and d-Galacturonic Acid.

Herein, we report a method for the synthesis of biobased surfactants derived from sugar beet pulp (SBP) monosaccharides, l-Ara and d-GalA. The surfactants were prepared via one-pot reductive amination, allowing the introduction of different alkyl chain lengths and methyl modifications. Optimal reaction conditions were established to achieve high yields and easy purification. The synthesized surfactants including the tertiary amines exhibited desirable properties, including solubility, foamability, and reduction of surface tension. Notably, the anionic surfactants derived from d-GalA demonstrated better solubility and foam performance compared to those derived from l-Ara. In addition, these surfactants exhibited surface tension and critical micelle concentration (CMC) comparable to those of the commercial surfactant sodium lauryl ether sulfate (SLES). Furthermore, the biodegradable surfactant GalA1.8 displayed excellent emulsifying properties and low skin irritation potential. On the l-Ara surfactant with a short chain, Ara1.6 has potential as a hydrotrope. These findings suggest that biobased surfactants derived from SBP monosaccharides have promising applications in various industries, including pharmaceuticals, cosmetics, detergents, and chemicals.

Drug discovery-based approach identifies new nitrification inhibitors.

Nitrogen (N) fertilization is crucial to sustain global food security, but fertilizer N production is energy-demanding and subsequent environmental N losses contribute to biodiversity loss and climate change. N losses can be mitigated be interfering with microbial nitrification, and therefore the use of nitrification inhibitors in enhanced efficiency fertilizers (EEFs) is an important N management strategy to increase N use efficiency and reduce N pollution. However, currently applied nitrification inhibitors have limitations and do not target all nitrifying microorganisms. Here, to identify broad-spectrum nitrification inhibitors, we adopted a drug discovery-based approach and screened 45,400 small molecules on different groups of nitrifying microorganisms. Although a high number of potential nitrification inhibitors were identified, none of them targeted all nitrifier groups. Moreover, a high number of new nitrification inhibitors were shown to be highly effective in culture but did not reduce ammonia consumption in soil. One archaea-targeting inhibitor was not only effective in soil, but even reduced - when co-applied with a bacteria-targeting inhibitor - ammonium consumption and greenhouse gas emissions beyond what is achieved with currently applied nitrification inhibitors. This advocates for combining different types of nitrification inhibitors in EEFs to optimize N management practices and make agriculture more sustainable.

Trichlorobacter ammonificans, a dedicated acetate-dependent ammonifier with a novel module for dissimilatory nitrate reduction to ammonia.

Dissimilatory nitrate reduction to ammonia (DNRA) is a common biochemical process in the nitrogen cycle in natural and man-made habitats, but its significance in wastewater treatment plants is not well understood. Several ammonifying Trichlorobacter strains (former Geobacter) were previously enriched from activated sludge in nitrate-limited chemostats with acetate as electron (e) donor, demonstrating their presence in these systems. Here, we isolated and characterized the new species Trichlorobacter ammonificans strain G1 using a combination of low redox potential and copper-depleted conditions. This allowed purification of this DNRA organism from competing denitrifiers. T. ammonificans is an extremely specialized ammonifier, actively growing only with acetate as e-donor and carbon source and nitrate as e-acceptor, but H2 can be used as an additional e-donor. The genome of G1 does not encode the classical ammonifying modules NrfAH/NrfABCD. Instead, we identified a locus encoding a periplasmic nitrate reductase immediately followed by an octaheme cytochrome c that is conserved in many Geobacteraceae species. We purified this octaheme cytochrome c protein (TaNiR), which is a highly active dissimilatory ammonifying nitrite reductase loosely associated with the cytoplasmic membrane. It presumably interacts with two ferredoxin subunits (NapGH) that donate electrons from the menaquinol pool to the periplasmic nitrate reductase (NapAB) and TaNiR. Thus, the Nap-TaNiR complex represents a novel type of highly functional DNRA module. Our results indicate that DNRA catalyzed by octaheme nitrite reductases is a metabolic feature of many Geobacteraceae, representing important community members in various anaerobic systems, such as rice paddy soil and wastewater treatment facilities.

Influence of filter age on Fe, Mn and NH4+ removal in dual media rapid sand filters used for drinking water production.

Rapid sand filtration is a common method for removal of iron (Fe), manganese (Mn) and ammonium (NH4+) from anoxic groundwaters used for drinking water production. In this study, we combine geochemical and microbiological data to assess how filter age influences Fe, Mn and NH4+ removal in dual media filters, consisting of anthracite overlying quartz sand, that have been in operation for between ∼2 months and ∼11 years. We show that the depth where dissolved Fe and Mn removal occurs is reflected in the filter medium coatings, with ferrihydrite forming in the anthracite in the top of the filters (< 1 m), while birnessite-type Mn oxides are mostly formed in the sand (> 1 m). Removal of NH4+ occurs through nitrification in both the anthracite and sand and is the key driver of oxygen loss. Removal of Fe is independent of filter age and is always efficient (> 97% removal). In contrast, for Mn, the removal efficiency varies with filter age, ranging from 9 to 28% at ∼2-3 months after filter replacement to 100% after 8 months. After 11 years, removal reduces to 60-80%. The lack of Mn removal in the youngest filters (at 2-3 months) is likely the result of a relatively low abundance of mineral coatings that adsorb Mn2+ and provide surfaces for the establishment of a microbial community. 16S rRNA gene amplicon sequencing shows that Gallionella, which are known Fe2+ oxidizers, are present after 2 months, yet Fe2+ removal is mostly chemical. Efficient NH4+ removal (> 90%) establishes within 3 months of operation but leakage occurs upon high NH4+loading (> 160 µM). Two-step nitrification by Nitrosomonas and Candidatus Nitrotoga is likely the most important NH4+ removal mechanism in younger filters during ripening (2 months), after which complete ammonia oxidation by Nitrospira and canonical two-step nitrification occur simultaneously in older filters. Our results highlight the strong effect of filter age on especially Mn2+but also NH4+ removal. We show that ageing of filter medium leads to the development of thick coatings, which we hypothesize leads to preferential flow, and breakthrough of Mn2+. Use of age-specific flow rates may increase the contact time with the filter medium in older filters and improve Mn2+ and NH4+ removal.

Comparison of the gill and gut microbiomes of common carp (Cyprinus carpio) and zebrafish (Danio rerio) and their RAS environment.

Recirculating aquaculture systems (RAS) are increasingly being used to grow fish, as intensive water reuse reduces water consumption and environmental impact. RAS use biofilters containing nitrogen-cycling microorganisms that remove ammonia from the aquaculture water. Knowledge of how RAS microbial communities relate to the fish-associated microbiome is limited, as is knowledge of fish-associated microbiota in general. Recently, nitrogen-cycling bacteria have been discovered in zebrafish and carp gills and shown to detoxify ammonia in a manner similar to the RAS biofilter. Here, we compared RAS water and biofilter microbiomes with fish-associated gut and gill microbial communities in laboratory RAS housing either zebrafish (Danio rerio) or common carp (Cyprinus carpio) using 16S rRNA gene amplicon sequencing. The phylogeny of ammonia-oxidizing bacteria in the gills and the RAS environment was investigated in more detail by phylogenetic analysis of the ammonia monooxygenase subunit A (amoA). The location from which the microbiome was sampled (RAS compartments and gills or gut) had a stronger effect on community composition than the fish species, but species-specific differences were also observed. We found that carp- and zebrafish-associated microbiomes were highly distinct from their respective RAS microbiomes, characterized by lower overall diversity and a small core microbiome consisting of taxa specifically adapted to the respective organ. The gill microbiome was also defined by a high proportion of unique taxa. Finally, we found that amoA sequences from the gills were distinct from those from the RAS biofilter and water. Our results showed that the gut and gill microbiomes of carp and zebrafish share a common and species-specific core microbiome that is distinct from the microbially-rich RAS environment.

Hot spring distribution and survival mechanisms of thermophilic comammox Nitrospira.

The recent discovery of Nitrospira species capable of complete ammonia oxidation (comammox) in non-marine natural and engineered ecosystems under mesothermal conditions has changed our understanding of microbial nitrification. However, little is known about the occurrence of comammox bacteria or their ability to survive in moderately thermal and/or hyperthermal habitats. Here, we report the wide distribution of comammox Nitrospira in five terrestrial hot springs at temperatures ranging from 36 to 80°C and provide metagenome-assembled genomes of 11 new comammox strains. Interestingly, the identification of dissimilatory nitrate reduction to ammonium (DNRA) in thermophilic comammox Nitrospira lineages suggests that they have versatile ecological functions as both sinks and sources of ammonia, in contrast to the described mesophilic comammox lineages, which lack the DNRA pathway. Furthermore, the in situ expression of key genes associated with nitrogen metabolism, thermal adaptation, and oxidative stress confirmed their ability to survive in the studied hot springs and their contribution to nitrification in these environments. Additionally, the smaller genome size and higher GC content, less polar and more charged amino acids in usage profiles, and the expression of a large number of heat shock proteins compared to mesophilic comammox strains presumably confer tolerance to thermal stress. These novel insights into the occurrence, metabolic activity, and adaptation of comammox Nitrospira in thermal habitats further expand our understanding of the global distribution of comammox Nitrospira and have significant implications for how these unique microorganisms have evolved thermal tolerance strategies.

A novel Nitrospira lineage isolated from activated sludge using elevated temperatures.

The genus Nitrospira represents the dominant nitrite-oxidizing clade in most wastewater treatment plants (WWTPs) globally, and several Nitrospira strains have been isolated from activated sludge. Using a pre-enrichment strategy with alternating nitrifying and denitrifying conditions, followed by incubation at elevated temperatures, we isolated a novel Nitrospira species, named Nitrospira tepida. This moderately thermophilic species with optimal growth between 37 and 45°C is only distantly related to other Nitrospira and forms a novel lineage VII within the genus, together with few environmental 16S rRNA gene sequences predominantly detected in thermal wastewater or oxygen-limited systems. Genomic and physiological analyses revealed remarkable differences between N. tepida and two other isolates previously obtained from the same WWTP, suggesting niche differentiation between these nitrite oxidizers. N. tepida grows in aggregates, and tolerates nitrite and nitrate concentrations of up to 20 mM and 40 mM, respectively. The Km value for nitrite of N. tepida is 77 ± 26 µM. In summary, this novel Nitrospira lineage seems to be well-adapted for wastewater treatment processes at elevated temperatures and limited aeration, conditions that potentially reduce operational costs of such systems.

Meta-omics profiling of full-scale groundwater rapid sand filters explains stratification of iron, ammonium and manganese removals.

Rapid sand filters (RSF) are an established and widely applied technology for groundwater treatment. Yet, the underlying interwoven biological and physical-chemical reactions controlling the sequential removal of iron, ammonia and manganese remain poorly understood. To resolve the contribution and interactions between the individual reactions, we studied two full-scale drinking water treatment plant configurations, namely (i) one dual-media (anthracite and quartz sand) filter and (ii) two single-media (quartz sand) filters in series. In situ and ex situ activity tests were combined with mineral coating characterization and metagenome-guided metaproteomics along the depth of each filter. Both plants exhibited comparable performances and process compartmentalization, with most of ammonium and manganese removal occurring only after complete iron depletion. The homogeneity of the media coating and genome-based microbial composition within each compartment highlighted the effect of backwashing, namely the complete vertical mixing of the filter media. In stark contrast to this homogeneity, the removal of the contaminants was strongly stratified within each compartment, and decreased along the filter height. This apparent and longstanding conflict was resolved by quantifying the expressed proteome at different filter heights, revealing a consistent stratification of proteins catalysing ammonia oxidation and protein-based relative abundances of nitrifying genera (up to 2 orders of magnitude difference between top and bottom samples). This implies that microorganisms adapt their protein pool to the available nutrient load at a faster rate than the backwash mixing frequency. Ultimately, these results show the unique and complementary potential of metaproteomics to understand metabolic adaptations and interactions in highly dynamic ecosystems.

Complete Genome Sequence of Nitrospina watsonii 347, Isolated from the Black Sea.

Here, we present the complete genome sequence of Nitrospina watsonii 347, a nitrite-oxidizing bacterium isolated from the Black Sea at a depth of 100 m. The genome has a length of 3,011,914 bp with 2,895 predicted coding sequences. Its predicted metabolism is similar to that of Nitrospina gracilis with differences in defense against reactive oxygen species.

Effects of demand-feeding and dietary protein level on nitrogen metabolism and symbiont dinitrogen gas production of common carp (Cyprinus carpio, L.).

Ammonia accumulation is a major challenge in intensive aquaculture, where fish are fed protein-rich diets in large rations, resulting in increased ammonia production when amino acids are metabolized as energy source. Ammonia is primarily excreted via the gills, which have been found to harbor nitrogen-cycle bacteria that convert ammonia into dinitrogen gas (N2) and therefore present a potential in situ detoxifying mechanism. Here, we determined the impact of feeding strategies (demand-feeding and batch-feeding) with two dietary protein levels on growth, nitrogen excretion, and nitrogen metabolism in common carp (Cyprinus carpio, L.) in a 3-week feeding experiment. Demand-fed fish exhibited significantly higher growth rates, though with lower feed efficiency. When corrected for feed intake, nitrogen excretion was not impacted by feeding strategy or dietary protein, but demand-fed fish had significantly more nitrogen unaccounted for in the nitrogen balance and less retained nitrogen. N2 production of individual fish was measured in all experimental groups, and production rates were in the same order of magnitude as the amount of nitrogen unaccounted for, thus potentially explaining the missing nitrogen in the balance. N2 production by carp was also observed when groups of fish were kept in metabolic chambers. Demand feeding furthermore caused a significant increase in hepatic glutamate dehydrogenase activities, indicating elevated ammonia production. However, branchial ammonia transporter expression levels in these animals were stable or decreased. Together, our results suggest that feeding strategy impacts fish growth and nitrogen metabolism, and that conversion of ammonia to N2 by nitrogen cycle bacteria in the gills may explain the unaccounted nitrogen in the balance.

Low diversity and microdiversity of comammox bacteria in wastewater systems suggest specific adaptations within the Ca. Nitrospira nitrosa cluster.

Studies have found Ca. Nitrospira nitrosa-like bacteria to be the principal or sole comammox bacteria in nitrogen removal systems for wastewater treatment. In contrast, multiple populations of strict ammonia and nitrite oxidizers co-exist in similar systems. This apparent lack of diversity is surprising and could impact the feasibility of leveraging comammox bacteria for nitrogen removal. We used full-length 16S rRNA gene sequencing and genome-resolved metagenomics to compare the species-level diversity of comammox bacteria with that of strict nitrifiers in full-scale wastewater treatment systems and assess whether this comparison is consistent or diverged at the strain-level. Full-length 16S rRNA gene sequencing indicated that Nitrosomonas-like bacteria exhibited higher species-level diversity in comparison with other nitrifying bacteria, while the strain-level diversity (also called microdiversity) of most Nitrospira-like bacteria were higher than Nitrosomonas-like bacteria with few exceptions (one Nitrospira lineage II population). Comammox bacterial metagenome assembled genomes (MAGs) were associated with Ca. Nitrospira nitrosa. The average amino acid identity between principal comammox bacterial MAGs (93% ± 3) across systems was significantly higher than that of the Nitrosomonas-like ammonia oxidizers (73% ± 8), the Nitrospira_A-like nitrite oxidizer (85% ± 4), and the Nitrospira_D-like nitrite oxidizer (83% ± 1). This demonstrated the low species-level diversity of comammox bacteria compared with strict nitrifiers and further suggests that the same comammox population was detected in all systems. Comammox bacteria (Nitrospira lineage II), Nitrosomonas and, Nitrospira_D (Nitrospira lineage II) MAGs were significantly less microdiverse than the Nitrospira_A (lineage I) MAGs. Interestingly, strain-resolved analysis also indicates that different nitrogen removal systems harbor different comammox bacterial strains within the Ca. Nitrospira nitrosa cluster. These results suggest that comammox bacteria associated with Ca. Nitrospira nitrosa have low species- and strain-level diversity in nitrogen removal systems and may thus harbor specific adaptations to the wastewater ecosystem.

Benzimidazole fungicide biotransformation by comammox Nitrospira bacteria: Transformation pathways and associated proteomic responses.

Benzimidazole fungicides are frequently detected in aquatic environments and pose a serious health risk. Here, we investigated the metabolic capacity of the recently discovered complete ammonia-oxidizing (comammox) Nitrospira inopinata and kreftii to transform a representative set of benzimidazole fungicides (i.e., benzimidazole, albendazole, carbendazim, fuberidazole, and thiabendazole). Ammonia-oxidizing bacteria and archaea, as well as the canonical nitrite-oxidizing Nitrospira exhibited no or minor biotransformation activity towards all the five benzimidazole fungicides. In contrast, the investigated comammox bacteria actively transformed all the five benzimidazole fungicides, except for thiabendazole. The identified transformation products indicated hydroxylation, S-oxidation, and glycosylation as the major biotransformation pathways of benzimidazole fungicides. We speculated that these reactions were catalyzed by comammox-specific ammonia monooxygenase, cytochrome P450 monooxygenases, and glycosylases, respectively. Interestingly, the exposure to albendazole enhanced the expression of the antibiotic resistance gene acrB of Nitrospira inopinata, suggesting that some benzimidazole fungicides could act as environmental stressors that trigger cellular defense mechanisms. Altogether, this study demonstrated the distinct substrate specificity of comammox bacteria towards benzimidazole fungicides and implies their significant roles in the biotransformation of these fungicides in nitrifying environments.

Metascan: METabolic Analysis, SCreening and ANnotation of Metagenomes.

Large scale next generation metagenomic sequencing of complex environmental samples paves the way for detailed analysis of nutrient cycles in ecosystems. For such an analysis, large scale unequivocal annotation is a prerequisite, which however is increasingly hampered by growing databases and analysis time. Hereto, we created a hidden Markov model (HMM) database by clustering proteins according to their KEGG indexing. HMM profiles for key genes of specific metabolic pathways and nutrient cycles were organized in subsets to be able to analyze each important elemental cycle separately. An important motivation behind the clustered database was to enable a high degree of resolution for annotation, while decreasing database size and analysis time. Here, we present Metascan, a new tool that can fully annotate and analyze deeply sequenced samples with an average analysis time of 11 min per genome for a publicly available dataset containing 2,537 genomes, and 1.1 min per genome for nutrient cycle analysis of the same sample. Metascan easily detected general proteins like cytochromes and ferredoxins, and additional pmoCAB operons were identified that were overlooked in previous analyses. For a mock community, the BEACON (F1) score was 0.72-0.93 compared to the information in NCBI GenBank. In combination with the accompanying database, Metascan provides a fast and useful annotation and analysis tool, as demonstrated by our proof-of-principle analysis of a complex mock community metagenome.

Some like it cold: the cellular organization and physiological limits of cold-tolerant nitrite-oxidizing Nitrotoga.

Chemolithoautotrophic production of nitrate is accomplished by the polyphyletic functional group of nitrite-oxidizing bacteria (NOB). A widely distributed and important NOB clade in nitrogen removal processes at low temperatures is Nitrotoga, which however remains understudied due to the scarcity of cultivated representatives. Here, we present physiological, ultrastructural and genomic features of Nitrotoga strains from various habitats, including the first marine species enriched from an aquaculture system. Immunocytochemical analyses localized the nitrite-oxidizing enzyme machinery in the wide irregularly shaped periplasm, apparently without contact to the cytoplasmic membrane, confirming previous genomic data suggesting a soluble nature. Interestingly, in two strains we also observed multicellular complexes with a shared periplasmic space, which seem to form through incomplete cell division and might enhance fitness or survival. Physiological tests revealed differing tolerance limits towards dissolved inorganic nitrogen concentrations and confirmed the generally psychrotolerant nature of the genus. Moreover, comparative analysis of 15 Nitrotoga genomes showed, e.g. a unique gene repertoire of the marine strain that could be advantageous in its natural habitat and confirmed the lack of genes for assimilatory nitrite reduction in a strain found to require ammonium for growth. Overall, these novel insights largely broaden our knowledge of Nitrotoga and elucidate the metabolic variability, physiological limits and thus potential ecological roles of this group of nitrite oxidizers.

Distinct comammox Nitrospira catalyze ammonia oxidation in a full-scale groundwater treatment bioreactor under copper limited conditions.

Microbial ammonia oxidation is the initial nitrification step used in biological nitrogen-removal during water treatment processes, and the discovery of complete ammonia-oxidizing (comammox) bacteria added a novel member to this functional group. It is important to identify and understand the predominant microorganisms responsible for ammonium removal in biotechnological process design and optimization. In this study, we used a full-scale bioreactor to treat ammonium in groundwater (9.3 ± 0.5 mg NH4+-N/L) and investigated the key ammonia-oxidizing prokaryotes present. The groundwater ammonium was stably and efficiently oxidized throughout ∼700 days of bioreactor operation. 16S rRNA gene amplicon sequencing of the bioreactor community showed a high abundance of Nitrospira (12.5-45.9%), with the dominant sequence variant (3.5-37.8%) most closely related to Candidatus Nitrospira nitrosa. Furthermore, analyses of amoA, the marker gene for ammonia oxidation, indicated the presence of two distinct comammox Nitrospira populations, however, the relative abundance of only one of these populations was strongly correlated to ammonia oxidation rates and was robustly expressed. After 380 days of operation copper wires were immersed into the reactor at 0.04-0.06 m2/m3 tank, which caused a gradual abundance increase of one discrete comammox Nitrospira population. However, further increase of the copper dosing (0.08 m2/m3 tank) inverted the most abundant ammonia-oxidizing population to Nitrosomonas sp. These results indicate that comammox Nitrospira were capable of efficient ammonium removal in groundwater without exogenous nutrients, but copper addition can stimulate comammox Nitrospira or lead to dominance of Nitrosomonas depending on dosage.