Narrow host range phages infect essential bacteria for water purification reactions in groundwater-fed rapid sand filters.

Biofiltration is used worldwide to provide safe potable water due to its low energy demand and excellent treatment performance. For instance, in Denmark, over 95% of drinking water is supplied through groundwater-fed rapid sand filters (RSF). Bacteriophages, viruses that infect bacteria, have been shown to shape the taxonomic and functional composition of microbial communities across a range of natural and engineering systems. However, phages in the biofiltration systems are rarely studied, despite the central role microbes play in water purification. To probe this, metagenomic data from surface water, groundwater and mixed source water biofiltration units (n = 26 from China, Europe and USA) for drinking water production were analysed to characterize prokaryotic viruses and to identify their potential microbial hosts. The source water type and geographical location are found to exert influence on the composition of the phageome in biofilters. Although the viral abundance (71,676 ± 17,841 RPKM) in biofilters is only 14.4% and 17.0% lower than those of the nutrient-rich wastewater treatment plants and fresh surface waters, the richness (1,441 ± 1,046) and diversity (Inverse Simpson: 91 ± 61) in biofiltration units are significantly less by a factor of 2-5 and 3-4, respectively. In depth analysis of data from 24 groundwater-fed RSFs in Denmark revealed a core phageome shared by most RSFs, which was consistently linked to dominant microbial hosts involved in key biological reactions for water purification. Finally, the high number of specific links detected between phages and bacterial species and the large proportion of lytic phages (77%) led to the conjecture that phages regulate bacterial populations through predation, preventing the proliferation of dominant species and contributing to the established functional redundancy among the dominant microbial groups. In conclusion, bacteriophages are likely to play a significant role in water treatment within biofilters, particularly through interactions with key bacterial species.

Biodegradation of trace sulfonamide antibiotics accelerated by substrates across oxic to anoxic conditions during column infiltration experiments.

Frequent occurrence of trace organic contaminants in aquatic environments, such as sulfonamide antibiotics in rivers receiving reclaimed water, is concerning. Natural attenuation by soil and sediment is increasingly relied upon. In the case of riverbank filtration for water purification, the reliability of antibiotic attenuation has been called into question due to incomplete understanding of their degradation processes. This study investigated influence of substrates and redox evolution along infiltration path on biotransformation of sulfonamides. Eight sand columns (length: 28 cm) with a riverbed sediment layer at 3-8 cm were fed by groundwater-sourced tap water spiked with 1 µg/L of sulfadiazine (SDZ), sulfamethazine (SMZ), and sulfamethoxazole (SMX) each, with or without amendments of dissolved organic carbon (5 mg-C/L of 1:1 yeast and humics) or ammonium (5 mg-N/L). Two flow rates were tested over 120 days (0.5 mL/min and 0.1 mL/min). Iron-reducing conditions persisted in all columns for 27 days during the initial high flow period due to respiration of sediment organics, evolving to less reducing conditions until the subsequent low flow period to resume more reducing conditions. With surplus substrates, the spatial and temporal patterns of redox conditions differentiated among columns. The removal of SDZ and SMZ in effluents was usually low (15 ± 11%) even with carbon addition (14 ± 9%), increasing to 33 ± 23% with ammonium addition. By contrast, SMX removal was higher and more consistent among columns (46 ± 21%), with the maximum of 64 ± 9% under iron-reducing conditions. When sulfonamide removal was compared between columns for the same redox zones during infiltration, their enhancements were always associated with the availability of dissolved or particulate substrates, suggesting co-metabolism. Manipulation of the exposure time to optimal redox conditions with substrate amendments, rather than to simply prolong the overall residence time, is recommended for nature-based solutions to tackle target antibiotics.

Virus impacted community adaptation in oligotrophic groundwater environment revealed by Hi-C coupled metagenomic and viromic study.

Viruses play a crucial role in microbial mortality, diversity and biogeochemical cycles. Groundwater is the largest global freshwater and one of the most oligotrophic aquatic systems on Earth, but how microbial and viral communities are shaped in this special habitat is largely unexplored. In this study, we collected groundwater samples from 23 to 60 m aquifers at Yinchuan Plain, China. In total, 1920 non-reductant viral contigs were retrieved from metagenomes and viromes constructed by Illumina and Nanopore hybrid sequencing. Only 3% of them could be clustered with known viruses, most of which were Caudoviricetes. Coupling 1.2 Tb Hi-C sequencing with CRISPR matching and homology search, we connected 469 viruses with their hosts while some viral clusters presented a broad-host-range trait. Meanwhile, a large proportion of biosynthesis related auxiliary metabolism genes were identified. Those characteristics might benefit viruses for a better survival in this special oligotrophic environment. Additionally, the groundwater virome showed genomic features distinct from those of the open ocean and wastewater treatment facilities in GC distribution and unannotated gene compositions. This paper expands the current knowledge of the global viromic records and serves as a foundation for a more thorough understanding of viruses in groundwater.

Prevalence of Methylated Arsenic and Microbial Arsenic Methylation Genes in Paddy Soils of the Mekong Delta.

Microbially mediated inorganic-methylated arsenic (As) transformation in paddy soil is crucial to rice safety; however, the linkages between the microbial As methylation process and methylated As species remain elusive. Here, 62 paddy soils were collected from the Mekong River delta of Cambodia to profile As-related functional gene composition involved in the As cycle. The soil As concentration ranged from <1 to 16.6 mg kg-1, with average As contents of approximately 81% as methylated As and 54% as monomethylarsenate (MMAs(V)) in the phosphate- and oxalate-extractable fractions based on As sequential extraction analysis. Quantitative PCR revealed high arsenite-methylating gene (arsM) copy numbers, and metagenomics identified consistently high arsM gene abundance. The abundance of As-related genes was the highest in bacteria, followed by archaea and fungi. Pseudomonas, Bradyrhizobium, Burkholderia, and Anaeromyxobacter were identified as bacteria harboring the most genes related to As biotransformation. Moreover, arsM and arsI (As demethylation) gene-containing operons were identified in the metagenome-assembled genomes (MAGs), implying that arsM and arsI could be transcribed together. The prevalence of methylated As and arsM genes may have been overlooked in tropical paddy fields. The As methylation-demethylation cycle should be considered when manipulating the methylated As pool in paddy fields for rice safety.

Genomic profiling of Nitrospira species reveals ecological success of comammox Nitrospira.

BACKGROUND: The discovery of microorganisms capable of complete ammonia oxidation to nitrate (comammox) has prompted a paradigm shift in our understanding of nitrification, an essential process in N cycling, hitherto considered to require both ammonia oxidizing and nitrite oxidizing microorganisms. This intriguing metabolism is unique to the genus Nitrospira, a diverse taxon previously known to only contain canonical nitrite oxidizers. Comammox Nitrospira have been detected in diverse environments; however, a global view of the distribution, abundance, and diversity of Nitrospira species is still incomplete. RESULTS: In this study, we retrieved 55 metagenome-assembled Nitrospira genomes (MAGs) from newly obtained and publicly available metagenomes. Combined with publicly available MAGs, this constitutes the largest Nitrospira genome database to date with 205 MAGs, representing 132 putative species, most without cultivated representatives. Mapping of metagenomic sequencing reads from various environments against this database enabled an analysis of the distribution and habitat preferences of Nitrospira species. Comammox Nitrospira's ecological success is evident as they outnumber and present higher species-level richness than canonical Nitrospira in all environments examined, except for marine and wastewaters samples. The type of environment governs Nitrospira species distribution, without large-scale biogeographical signal. We found that closely related Nitrospira species tend to occupy the same habitats, and that this phylogenetic signal in habitat preference is stronger for canonical Nitrospira species. Comammox Nitrospira eco-evolutionary history is more complex, with subclades achieving rapid niche divergence via horizontal transfer of genes, including the gene encoding hydroxylamine oxidoreductase, a key enzyme in nitrification. CONCLUSIONS: Our study expands the genomic inventory of the Nitrospira genus, exposes the ecological success of complete ammonia oxidizers within a wide range of habitats, identifies the habitat preferences of (sub)lineages of canonical and comammox Nitrospira species, and proposes that horizontal transfer of genes involved in nitrification is linked to niche separation within a sublineage of comammox Nitrospira. Video Abstract.

Efficient management of the nitritation-anammox microbiome through intermittent aeration: absence of the NOB guild and expansion and diversity of the NOx reducing guild suggests a highly reticulated nitrogen cycle.

Obtaining efficient autotrophic ammonia removal (aka partial nitritation-anammox, or PNA) requires a balanced microbiome with abundant aerobic and anaerobic ammonia oxidizing bacteria and scarce nitrite oxidizing bacteria. Here, we analyzed the microbiome of an efficient PNA process that was obtained by sequential feeding and periodic aeration. The genomes of the dominant community members were inferred from metagenomes obtained over a 6 month period. Three Brocadia spp. genomes and three Nitrosomonas spp. genomes dominated the autotrophic community; no NOB genomes were retrieved. Two of the Brocadia spp. genomes lacked the genomic potential for nitrite reduction. A diverse set of heterotrophic genomes was retrieved, each with genomic potential for only a fraction of the denitrification pathway. A mutual dependency in amino acid and vitamin synthesis was noted between autotrophic and heterotrophic community members. Our analysis suggests a highly-reticulated nitrogen cycle in the examined PNA microbiome with nitric oxide exchange between the heterotrophs and the anammox guild.

Micron-scale biogeography reveals conservative intra anammox bacteria spatial co-associations.

Micron-scale resolution can help to reliably identify true taxon-taxon interactions in complex microbial communities. Despite widespread recognition of the critical role of metabolic interactions in anaerobic ammonium oxidation (anammox) system performance, no studies have examined microbial interactions at the micron-scale in anammox consortia. To fill this gap, we extensively sampled (totally 242 samples) the consortia of a lab-scale anammox reactor at different length scales, including bulk-scale (∼cm), macro-scale (300-500 µm) and micron-scale (70-100 µm). We firstly observed evident micron-scale heterogeneity in anammox consortia, with the relative abundance of anammox bacteria fluctuated greatly across individual clusters (2.0%-79.3%), indicating that the biotic interactions play a significant role in the assembly of anammox communities under well-controlled and well-mixed condition. Importantly, by mapping the spatial associations in anammox consortia at micron-scale, we demonstrated that the conserved co-associations for anammox bacteria were restricted to three different Brocadia species over time, and their co-associations with heterotrophs were random, implying that there was no statistically significant symbiotic interaction between anammox bacteria and other heterotrophic populations. Further metagenomic binning revealed that the quorum sensing with secondary messenger c-di-GMP potentially holding on the conservative metabolic cooperation among Brocadia species. These results shed new light on the social behavior of the anammox community. Overall, delineating of biological structures at micron-scale opens a new way of monitoring the microbial spatial structure and interactions, paving the way for improved community engineering of biotreatment systems.

Evolutionary Ecology of Natural Comammox Nitrospira Populations.

Microbes commonly exist in diverse and complex communities where species interact, and their genomic repertoires evolve over time. Our understanding of species interaction and evolution has increased during the last decades, but most studies of evolutionary dynamics are based on single species in isolation or in experimental systems composed of few interacting species. Here, we use the microbial ecosystem found in groundwater-fed sand filter as a model to avoid this limitation. In these open systems, diverse microbial communities experience relatively stable conditions, and the coupling between chemical and biological processes is generally well defined. Metagenomic analysis of 12 sand filters communities revealed systematic co-occurrence of at least five comammox Nitrospira species, likely promoted by low ammonium concentrations. These Nitrospira species showed intrapopulation sequence diversity, although possible clonal expansion was detected in a few abundant local comammox populations. Nitrospira species showed low homologous recombination and strong purifying selection, the latter process being especially strong in genes essential in energy metabolism. Positive selection was detected for genes related to resistance to foreign DNA and phages. We found that, compared to other habitats, groundwater-fed sand filters impose strong purifying selection and low recombination on comammox Nitrospira populations. These results suggest that evolutionary processes are more affected by habitat type than by species identity. Together, this study improves our understanding of species interaction and evolution in complex microbial communities and sheds light on the environmental dependency of evolutionary processes. IMPORTANCE Microbial species interact with each other and their environment (ecological processes) and undergo changes in their genomic repertoire over time (evolutionary processes). How these two classes of processes interact is largely unknown, especially for complex communities, as most studies of microbial evolutionary dynamics consider single species in isolation or a few interacting species in simplified experimental systems. In this study, these limitations are circumvented by examining the microbial communities found in stable and well-described groundwater-fed sand filters. Combining metagenomics and strain-level analyses, we identified the microbial interactions and evolutionary processes affecting comammox Nitrospira, a recently discovered bacterial type capable of performing the whole nitrification process. We found that abundant and co-occurrent Nitrospira populations in groundwater-fed sand filters are characterized by low recombination and strong purifying selection. In addition, by comparing these observations with those obtained from Nitrospira species inhabiting other environments, we revealed that evolutionary processes are more affected by habitat type than by species identity.

Survival strategy of comammox bacteria in a wastewater nutrient removal system with sludge fermentation liquid as additional carbon source.

Complete ammonia oxidizing (comammox) bacteria are frequently detected in wastewater biological nutrient removal (BNR) systems. This study identified "Candidatus Nitrospira nitrosa"-like comammox bacteria as the predominant ammonia oxidizers (97.5-99.4%) in a lab-scale BNR system with acetate and sludge fermentation liquid as external carbon sources. The total nitrogen and phosphorus removals of the system were 75.9% and 86.9% with minimal N2O emission (0.27%). Low ammonia concentration, mixotrophic growth potentials and metabolic interactions with diverse heterotrophs collectively contributed to the survival of comammox bacteria in the system. The recovered draft genomes of comammox bacteria indicated their potentials in using acetate and propionate but not butyrate. Acetate and propionate indeed stimulated the transcription of comammox amoA genes (up-regulated by 4.1 folds compared with no organic addition), which was positively correlated with the ammonia oxidation rate of the community (r = 0.75, p < 0.05). Comammox bacteria could provide vitamins/cofactors (e.g., cobalamin and biotin) to heterotrophs (e.g., Burkholderiaceae), and in return receive amino acids (e.g., phenylalanine and tyrosine) from heterotrophs, which they cannot synthesize. Compared with comammox bacteria, ammonia oxidizing bacteria (AOB) exhibited lower metabolic versatility, and lacked more pathways for the synthesis of amino acids and vitamin/cofactors, leading to their washout in the studied system. BNRs with comammox bacteria as the major nitrifiers hold great potentials in achieving superior performance at low aeration cost and low N2O emission and at full-scale plants.

Pathogenic and Indigenous Denitrifying Bacteria are Transcriptionally Active and Key Multi-Antibiotic-Resistant Players in Wastewater Treatment Plants.

The global rise and spread of antibiotic resistance greatly challenge the treatment of bacterial infections. Wastewater treatment plants (WWTPs) harbor and discharge antibiotic resistance genes (ARGs) as environmental contaminants. However, the knowledge gap on the host identity, activity, and functionality of ARGs limits transmission and health risk assessment of the WWTP resistome. Hereby, a genome-centric quantitative metatranscriptomic approach was exploited to realize high-resolution qualitative and quantitative analyses of bacterial hosts of ARGs (i.e., multiresistance, pathogenicity, activity, and niches) in the 12 urban WWTPs. We found that ∼45% of 248 recovered genomes expressed ARGs against multiple classes of antibiotics, among which bacitracin and aminoglycoside resistance genes in Proteobacteria were the most prevalent scenario. Both potential pathogens and indigenous denitrifying bacteria were transcriptionally active hosts of ARGs. The almost unchanged relative expression levels of ARGs in the most resistant populations (66.9%) and the surviving ARG hosts including globally emerging pathogens (e.g., Aliarcobacter cryaerophilus) in treated WWTP effluent prioritize future examination on the health risks related to resistance propagation and human exposure in the receiving environment.

Temperature modulates stress response in mainstream anammox reactors.

Autotrophic nitrogen removal by anaerobic ammonium oxidizing (anammox) bacteria is an energy-efficient nitrogen removal process in wastewater treatment. However, full-scale deployment under mainstream conditions remains challenging for practitioners due to the high stress susceptibility of anammox bacteria towards fluctuations in dissolved oxygen (DO) and temperature. Here, we investigated the response of microbial biofilms with verified anammox activity to DO shocks under 20 °C and 14 °C. While pulse disturbances of 0.3 mg L-1 DO prompted only moderate declines in the NH4+ removal rates, 1.0 mg L-1 DO led to complete but reversible inhibition of the NH4+ removal activity in all reactors. Genome-centric metagenomics and metatranscriptomics were used to investigate the stress response on various biological levels. We show that temperature regime and strength of DO perturbations induced divergent responses from the process level down to the transcriptional profile of individual taxa. Community-wide gene expression differed significantly depending on the temperature regime in all reactors, and we found a noticeable impact of DO disturbances on genes involved in transcription, translation, replication and posttranslational modification at 20 °C but not 14 °C. Genome-centric analysis revealed that different anammox species and other key biofilm taxa differed in their transcriptional responses to distinct temperature regimes and DO disturbances.

Stable nitrogen removal by anammox process after rapid temperature drops: Insights from metagenomics and metaproteomics.

This study investigated the impacts of rapid temperature drops on anammox process performance and the metabolism of its core microbial populations through proteomic analysis. Over a 50-day period, the temperature of an up-flow granular bed anammox reactor was stepwise decreased from 35 °C to 15 °C and resulted in repeated transient increases in effluent nitrite concentrations. At 15 °C, a nitrogen removal rate of 2.71 ± 0.23 gN/(L·d) was maintained over 100 days operation. Total AnAOB population abundance (20.9%±4.9%) and AnAOB protein abundances (75.7% ± 3.3%) remained stable with decreased temperature. Key proteins of Ca. Brocadia for nitrogen metabolism, as well as for carbohydrate metabolism and primary metabolite biosynthesis were less expressed at 15 °C than 35 °C, while several proteins of heterotrophic Chloroflexi spp. involved in carbohydrate and metabolites metabolisms were expressed to a higher degree at 15 °C. Overall, metabolism of AnAOB responded at a higher degree to low temperatures than that of heterotrophs.

Repeated Pulses of Methyl-Prednisolone in Adults Hospitalized With COVID-19 Pneumonia and Acute Respiratory Distress Syndrome: A Preliminary Before-After Study (CortiCOVID Study).

Introduction: The use of systemic corticosteroids in severely ill patients with coronavirus disease 2019 (COVID-19) is controversial. We aimed to evaluate the efficacy and safety of corticosteroid pulses in patients with COVID-19 pneumonia. Methods: A quasi-experimental study, before and after, was performed in a tertiary referral hospital, including admitted patients showing COVID-19-associated pneumonia. The standard treatment protocol included targeted COVID-19 antiviral therapy from 23rd March 2020, and additionally pulses of methylprednisolone from 30th March 2020. The primary outcome was a composite endpoint combining oro-tracheal intubation (OTI) and death within 7 days. Results: A total of 24 patients were included. Standard of care (SOC) (before intervention) was prescribed in 14 patients, while 10 received SOC plus pulses of methylprednisolone (after intervention). The median age of patients was 64.5 years and 83.3% of the patients were men. The primary composite endpoint occurred in 13 patients (92.9%) who received SOC vs. 2 patients (20%) that received pulses of methylprednisolone (odds ratio, 0.02; 95% confidence interval, 0.001 to 0.25; p = 0.019). Length of hospitalization in survivors was shorter in the corticosteroids group (median, 14.5 [8.5-21.8] days vs. 29 [23-31] days, p = 0.003). There were no differences in the development of infections between both groups. There were 3 deaths, none of them in the corticosteroids group. Conclusions: In patients with severe pneumonia due to COVID-19, the administration of methylprednisolone pulses was associated with a lower rate of OTI and/or death and a shorter hospitalization episode.

Introducción: El uso de corticosteroides sistémicos en pacientes gravemente enfermos por enfermedad coronavírica de 2019 (covid-19) es controvertido. Nuestro objetivo fue evaluar la eficacia y la seguridad de los pulsos de corticoesteroides en los pacientes con neumonía por covid-19. Métodos: Se realizó un ensayo cuasiexperimental, tipo antes y después, en un hospital terciario de referencia que incluyó a pacientes ingresados por neumonía asociada a covid-19. El protocolo de tratamiento estándar incluía un tratamiento antiviral dirigido contra el virus de la covid-19 desde el 23 de marzo de 2020 y añadió pulsos de metilprednisolona desde el 30 de marzo de 2020. El resultado primario fue un criterio combinado compuesto por la intubación orotraqueal y el fallecimiento durante los siguientes siete días. Resultados: Se incluyó un total de 24 pacientes. El protocolo de tratamiento (antes de la intervención) se prescribió en 14 pacientes, mientras que 10 recibieron el protocolo de tratamiento además de los pulsos de metilprednisolona (después de la intervención). La edad media de los pacientes fue de 64,5 años y el 83,3% de los pacientes eran hombres. El resultado combinado primario tuvo lugar en 13 pacientes (92,9%) que recibieron el protocolo de tratamiento frente a 2 pacientes (20%) que recibieron los pulsos de metilprednisolona (odds ratio = 0,02; intervalo de confianza del 95% = 0,001-0,25; p = 0,019). La duración de la hospitalización en los supervivientes fue más corta en el grupo que recibió corticoesteroides (media = 14,5 [8,5-21,8] días frente a 29 [23-31] días, p = 0,003). No hubo diferencias en el desarrollo de infecciones entre ambos grupos. Hubo tres fallecimientos, ninguno de ellos en el grupo que recibió corticoesteroides. Conclusiones: En los pacientes con neumonía grave por covid-19, la administración de pulsos de metilprednisolona se asoció a unas tasas menores de intubación orotraqueal y/o muerte y a episodios de hospitalización más cortos.

Cultivation of methanotrophic bacteria in a novel bubble-free membrane bioreactor for microbial protein production.

Microbial protein is proposed as an alternative protein source with low environmental impact. Methane oxidizing bacteria are already produced at commercial scale from natural gas. However, their productivity is limited because of the creation of explosive atmospheres in the fermenters during production. This work demonstrates the applicability of bioreactors with a membrane-based gas supply via diffusion. Methanotrophic bacteria were successfully cultivated, with growth yields from 0.26 to 0.43 g-VSS g-CH4-1, slightly below those observed in analogous fermenters relying on bubbling. However, ammonia yields ranged from 5.2 to 6.9 g-VSS g-NH3-1, indicating higher nitrogen assimilation than in conventional fermenters. Indeed, protein content increased during the operational period reaching up to 51% of dry weight. The amino acid profile included the majority of the essential amino acids, demonstrating suitability as feed ingredient. Never during the operational period was an explosive atmosphere established in the reactor. Thus, bubble-free membrane bioreactors are a promising technology for microbial protein production relying on explosive gas mixtures.

Minimum influent concentrations of oxytetracycline, streptomycin and spiramycin in selecting antibiotic resistance in biofilm type wastewater treatment systems.

It has been demonstrated that antibiotic resistance could be induced and selected under high antibiotic concentrations in biological wastewater treatment systems. However, little is available regarding the minimum concentrations of antibiotics for selecting antibiotic resistance during wastewater treatment. Herein, the minimum influent concentrations of oxytetracycline, streptomycin, and spiramycin in selecting antibiotic resistance in biofilm type wastewater treatment systems were investigated by spiking respective antibiotic into wastewater with an antibiotic dose increasing from 0 to 0.1, 1, 5, 25, 50 mg/L stepwise over a period of 606 days. Significant increase (p < .01) in the total abundance of antibiotic resistance genes was observed for both streptomycin and oxytetracycline at a dose of 0.1 mg/L according to metagenomic sequencing, while the concentration levels leading to significant increases (p < .05) in resistant bacteria ratio were higher: 5 mg/L for streptomycin and 25 mg/L for oxytetracycline. Although resistome abundance increased with the increase of spiramycin dose, neither the corresponding Macrolide-Lincosamide-Streptogramin (MLS) resistance genes nor the resistant bacteria ratio showed perceptible increase. Partial canonical correspondence analysis showed that both bacterial community shift and mobile genetic elements alteration contributed to the enrichment of resistomes under the presence of streptomycin and oxytetracycline. Regarding spiramycin which is mainly targeting on Gram-positive bacteria, the dominance of the intrinsically resisting Gram-negative bacteria in the biofilm microbiota might be responsible for the vague change of MLS resistant determinants under the spiramycin stress. The results demonstrated that it is possible to prevent the development of antibiotic resistance during wastewater treatment by controlling the influent streptomycin and oxytetracyline concentrations below 0.1 mg/L. This work proposed an actionable approach for the management of antibiotic production wastewater.

Comparative genomics sheds light on niche differentiation and the evolutionary history of comammox Nitrospira.

The description of comammox Nitrospira spp., performing complete ammonia-to-nitrate oxidation, and their co-occurrence with canonical ß-proteobacterial ammonia oxidizing bacteria (ß-AOB) in the environment, calls into question the metabolic potential of comammox Nitrospira and the evolutionary history of their ammonia oxidation pathway. We report four new comammox Nitrospira genomes, constituting two novel species, and the first comparative genomic analysis on comammox Nitrospira. Unlike canonical Nitrospira, comammox Nitrospira genomes lack genes for assimilatory nitrite reduction, suggesting that they have lost the potential to use external nitrite nitrogen sources. By contrast, compared to canonical Nitrospira, comammox Nitrospira harbor a higher diversity of urea transporters and copper homeostasis genes and lack cyanate hydratase genes. Additionally, the two comammox clades differ in their ammonium uptake systems. Contrary to ß-AOB, comammox Nitrospira genomes have single copies of the two central ammonia oxidation pathway operons. Similar to ammonia oxidizing archaea and some oligotrophic AOB strains, they lack genes involved in nitric oxide reduction. Furthermore, comammox Nitrospira genomes encode genes that might allow efficient growth at low oxygen concentrations. Regarding the evolutionary history of comammox Nitrospira, our analyses indicate that several genes belonging to the ammonia oxidation pathway could have been laterally transferred from ß-AOB to comammox Nitrospira. We postulate that the absence of comammox genes in other sublineage II Nitrospira genomes is the result of subsequent loss.

Comammox Nitrospira are abundant ammonia oxidizers in diverse groundwater-fed rapid sand filter communities.

The recent discovery of completely nitrifying Nitrospira demands a re-examination of nitrifying environments to evaluate their contribution to nitrogen cycling. To approach this challenge, tools are needed to detect and quantify comammox Nitrospira. We present primers for the simultaneous quantification and diversity assessement of both comammox Nitrospira clades. The primers cover a wide range of comammox diversity, spanning all available high quality sequences. We applied these primers to 12 groundwater-fed rapid sand filters, and found comammox Nitrospira to be abundant in all filters. Clade B comammox comprise the majority (∼75%) of comammox abundance in all filters. Nitrosomonadaceae were present in all filters, although at low abundance (mean = 1.8%). Ordination suggests that temperature impacts the structure of nitrifying communities, and in particular that increasing temperature favours Nitrospira. The nitrogen content of the filter material, sulfate concentration and surface ammonium loading rates shape the structure of the comammox guild in the filters. This work provides an assay for simultaneous detection and diversity assessment of clades A and B comammox Nitrospira, expands our current knowledge of comammox Nitrospira diversity and demonstrates a key role for comammox Nitrospira in nitrification in groundwater-fed biofilters.

Nematode distributions as spatial null models for macroinvertebrate species richness across environmental gradients: A case from mountain lakes.

Nematode species are widely tolerant of environmental conditions and disperse passively. Therefore, the species richness distribution in this group might largely depend on the topological distribution of the habitats and main aerial and aquatic dispersal pathways connecting them. If so, the nematode species richness distributions may serve as null models for evaluating that of other groups more affected by environmental gradients. We investigated this hypothesis in lakes across an altitudinal gradient in the Pyrenees. We compared the altitudinal distribution, environmental tolerance, and species richness, of nematodes with that of three other invertebrate groups collected during the same sampling: oligochaetes, chironomids, and nonchironomid insects. We tested the altitudinal bias in distributions with t-tests and the significance of narrow-ranging altitudinal distributions with randomizations. We compared results between groups with Fisher's exact tests. We then explored the influence of environmental factors on species assemblages in all groups with redundancy analysis (RDA), using 28 environmental variables. And, finally, we analyzed species richness patterns across altitude with simple linear and quadratic regressions. Nematode species were rarely biased from random distributions (5% of species) in contrast with other groups (35%, 47%, and 50%, respectively). The altitudinal bias most often shifted toward low altitudes (85% of biased species). Nematodes showed a lower portion of narrow-ranging species than any other group, and differed significantly from nonchironomid insects (10% and 43%, respectively). Environmental variables barely explained nematode assemblages (RDA adjusted R2 = 0.02), in contrast with other groups (0.13, 0.19 and 0.24). Despite these substantial differences in the response to environmental factors, species richness across altitude was unimodal, peaking at mid elevations, in all groups. This similarity indicates that the spatial distribution of lakes across altitude is a primary driver of invertebrate richness. Provided that nematodes are ubiquitous, their distribution offers potential null models to investigate species richness across environmental gradients in other ecosystem types and biogeographic regions.

Underestimation of ammonia-oxidizing bacteria abundance by amplification bias in amoA-targeted qPCR.

Molecular methods to investigate functional groups in microbial communities rely on the specificity and selectivity of the primer set towards the target. Here, using rapid sand filters for drinking water production as model environment, we investigated the consistency of two commonly used quantitative PCR methods to enumerate ammonia-oxidizing bacteria (AOB): one targeting the phylogenetic gene 16S rRNA and the other, the functional gene amoA. Cloning-sequencing with both primer sets on DNA from two waterworks revealed contrasting images of AOB diversity. The amoA-based approach preferentially recovered sequences belonging to Nitrosomonas Cluster 7 over Cluster 6A ones, while the 16S rRNA one yielded more diverse sequences belonging to three AOB clusters, but also a few non-AOB sequences, suggesting broader, but partly unspecific, primer coverage. This was confirmed by an in silico coverage analysis against sequences of AOB (both isolates and high-quality environmental sequences). The difference in primer coverage significantly impacted the estimation of AOB abundance at the waterworks with high Cluster 6A prevalence, with estimates up to 50-fold smaller for amoA than for 16S rRNA. In contrast, both approaches performed very similarly at waterworks with high Cluster 7 prevalence. Our results highlight that caution is warranted when comparing AOB abundances obtained using different qPCR primer sets.

Metagenomic analysis of rapid gravity sand filter microbial communities suggests novel physiology of Nitrospira spp.

Rapid gravity sand filtration is a drinking water production technology widely used around the world. Microbially catalyzed processes dominate the oxidative transformation of ammonia, reduced manganese and iron, methane and hydrogen sulfide, which may all be present at millimolar concentrations when groundwater is the source water. In this study, six metagenomes from various locations within a groundwater-fed rapid sand filter (RSF) were analyzed. The community gene catalog contained most genes of the nitrogen cycle, with particular abundance in genes of the nitrification pathway. Genes involved in different carbon fixation pathways were also abundant, with the reverse tricarboxylic acid cycle pathway most abundant, consistent with an observed Nitrospira dominance. From the metagenomic data set, 14 near-complete genomes were reconstructed and functionally characterized. On the basis of their genetic content, a metabolic and geochemical model was proposed. The organisms represented by draft genomes had the capability to oxidize ammonium, nitrite, hydrogen sulfide, methane, potentially iron and manganese as well as to assimilate organic compounds. A composite Nitrospira genome was recovered, and amo-containing Nitrospira genome contigs were identified. This finding, together with the high Nitrospira abundance, and the abundance of atypical amo and hao genes, suggests the potential for complete ammonium oxidation by Nitrospira, and a major role of Nitrospira in the investigated RSFs and potentially other nitrifying environments.