Salinity Tolerance and Osmoadaptation Strategies in Four Genera of Anammox Bacteria: Brocadia, Jettenia, Kuenenia, and Scalindua.

The salinity tolerance and osmoadaptation strategies in four phylogenetically distant anammox species, Brocadia, Jettenia, Kuenenia, and Scalindua, were investigated by using highly enriched cell cultures. The first-emerged "Ca. Scalindua sp." showed optimum growth at 1.5-3% salinity and was tolerant to ∼10% salinity (a slight halophile). The second-emerged "Ca. Kuenenia stuttgartiensis" was tolerant to ∼6% salinity with optimum growth at 0.25-1.5% (a halotolerant). These early-emerged "Ca. Scalindua sp." and ″Ca. K. stuttgartiensis" rapidly accumulated K+ ions and simultaneously synthesized glutamate as a counterion. Subsequently, part of the glutamate was replaced by trehalose. In contrast, the late-emerged "Ca. B. sinica" and "Ca. J. caeni" were unable to accumulate sufficient amounts of K+─glutamate and trehalose, resulting in a significant decrease in activity even at 1-2% salinity (nonhalophiles). In addition, the external addition of glutamate may increase anammox activity at high salinity. The species-dependent salinity tolerance and osmoadaptation strategies were consistent with the genetic potential required for the biosynthesis and transport of these osmolytes and the evolutionary history of anammox bacteria: Scalindua first emerged in marine environments and then Kuenenia and other two species gradually expanded their habitat to estuaries, freshwater, and terrestrial environments, while Brocadia and Jettenia likely lost their ability to accumulate K+─glutamate.

Collaborative metabolisms of urea and cyanate degradation in marine anammox bacterial culture.

Anammox process greatly contributes to nitrogen loss occurring in oceanic oxygen minimum zones (OMZs), where the availability of NH4+ is scarce as compared with NO2-. Remineralization of organic nitrogen compounds including urea and cyanate (OCN-) into NH4+ has been believed as an NH4+ source of the anammox process in oxygen minimum zones. However, urea- or OCN-- dependent anammox has not been well examined due to the lack of marine anammox bacterial culture. In the present study, urea and OCN- degradation in a marine anammox bacterial consortium were investigated based on 15N-tracer experiments and metagenomic analysis. Although a marine anammox bacterium, Candidatus Scalindua sp., itself was incapable of urea and OCN- degradation, urea was anoxically decomposed to NH4+ by the coexisting ureolytic bacteria (Rhizobiaceae, Nitrosomonadaceae, and/or Thalassopiraceae bacteria), whereas OCN- was abiotically degraded to NH4+. The produced NH4+ was subsequently utilized in the anammox process. The activity of the urea degradation increased under microaerobic condition (ca. 32-42 µM dissolved O2, DO), and the contribution of the anammox process to the total nitrogen loss also increased up to 33.3% at 32 µM DO. Urea-dependent anammox activities were further examined in a fluid thioglycolate media with a vertical gradient of O2 concentration, and the active collaborative metabolism of the urea degradation and anammox was detected at the lower oxycline (21 µM DO).

Denitrification in low oxic environments increases the accumulation of nitrogen oxide intermediates and modulates the evolutionary potential of microbial populations.

Denitrification in oxic environments occurs when a microorganism uses nitrogen oxides as terminal electron acceptors even though oxygen is available. While this phenomenon is well-established, its consequences on ecological and evolutionary processes remain poorly understood. We hypothesize here that denitrification in oxic environments can modify the accumulation profiles of nitrogen oxide intermediates with cascading effects on the evolutionary potentials of denitrifying microorganisms. To test this, we performed laboratory experiments with Paracoccus denitrificans and complemented them with individual-based computational modelling. We found that denitrification in low oxic environments significantly increases the accumulation of nitrite and nitric oxide. We further found that the increased accumulation of these intermediates has a negative effect on growth at low pH. Finally, we found that the increased negative effect at low pH increases the number of individuals that contribute to surface-associated growth. This increases the amount of genetic diversity that is preserved from the initial population, thus increasing the number of genetic targets for natural selection to act upon and resulting in higher evolutionary potentials. Together, our data highlight that denitrification in low oxic environments can affect the ecological processes and evolutionary potentials of denitrifying microorganisms by modifying the accumulation of nitrogen oxide intermediates.

Growth of the Nitrosomonas europaea cells in the biofilm and planktonic growth mode: Responses of extracellular polymeric substances production and transcriptome.

Nitrosomonas europaea, an aerobic ammonia oxidizing bacterium, is responsible for the first and rate-limiting step of the nitrification process, and their ammonia oxidation activities are critical for the biogeochemical cycling and the biological nitrogen removal of wastewater treatment. In the present study, N. europaea cells were cultivated in the inorganic or organic media (the NBRC829 and the nutrient-rich, NR, media, respectively), and the cells proliferated in the form of planktonic and biofilm in those media, respectively. The N. europaea cells in the biofilm growth mode produced larger amounts of the extracellular polymeric substances (EPS), and the composition of the EPS was characterized by the chemical analyses including Fourier transform infrared spectroscopy (FT-IR) and 1H-nuclear magnetic resonance (NMR) measurements. The RNA-Seq analysis of N. europaea in the biofilm or planktonic growth mode revealed that the following gene transcripts involved in central nitrogen metabolisms were abundant in the biofilm growth mode; amo encoding ammonia monooxygenase, hao encoding hydroxylamine dehydrogenase, the gene encoding nitrosocyanine, nirK encoding copper-containing nitrite reductase. Additionally, the transcripts of the pepA and wza involved in the bacterial floc formation and the translocation of EPS, respectively, were also abundant in the biofilm-growth mode. Our study was first to characterize the EPS production and transcriptome of N. europaea in the biofilm and planktonic growth mode.

Oxygen tolerance and detoxification mechanisms of highly enriched planktonic anaerobic ammonium-oxidizing (anammox) bacteria.

Oxygen is a key regulatory factor of anaerobic ammonium oxidation (anammox). Although the inhibitory effect of oxygen is evident, a wide range of oxygen sensitivities of anammox bacteria have been reported so far, which makes it difficult to model the marine nitrogen loss and design anammox-based technologies. Here, oxygen tolerance and detoxification mechanisms of four genera of anammox bacteria; one marine species ("Ca. Scalindua sp.") and four freshwater anammox species ("Ca. Brocadia sinica", "Ca. Brocadia sapporoensis", "Ca. Jettenia caeni", and "Ca. Kuenenia stuttgartiensis") were determined and then related to the activities of anti-oxidative enzymes. Highly enriched planktonic anammox cells were exposed to various levels of oxygen, and oxygen inhibition kinetics (50% inhibitory concentration (IC50) and upper O2 limits (DOmax) of anammox activity) were quantitatively determined. A marine anammox species, "Ca. Scalindua sp.", exhibited much higher oxygen tolerance capability (IC50 = 18.0 µM and DOmax = 51.6 µM) than freshwater species (IC50 = 2.7-4.2 µM and DOmax = 10.9-26.6 µM). The upper DO limit of "Ca. Scalindua sp." was much higher than the values reported so far (~20 µM). Furthermore, the oxygen inhibition was reversible even after exposed to ambient air for 12-24 h. The comparative genome analysis confirmed that all anammox species commonly possess the genes considered to function for reduction of O2, superoxide anion (O2•-), and H2O2. However, the superoxide reductase (Sor)-peroxidase dependent detoxification system alone may not be sufficient for cell survival under microaerobic conditions. Despite the fact that anaerobes normally possess no or little superoxide dismutase (Sod) or catalase (Cat), only Scalindua exhibited high Sod activity of 22.6 ± 1.9 U/mg-protein with moderate Cat activity of 1.6 ± 0.7 U/mg-protein, which was consistent with the genome sequence analysis. This Sod-Cat dependent detoxification system could be responsible for the higher O2 tolerance of Scalindua than other freshwater anammox species lacking the Sod activity.

Metagenomic Analysis of Five Phylogenetically Distant Anammox Bacterial Enrichment Cultures.

Anaerobic ammonium-oxidizing (anammox) bacteria are slow-growing and fastidious bacteria, and limited numbers of enrichment cultures have been established. A metagenomic ana-lysis of our 5 established anammox bacterial enrichment cultures was performed in the present study. Fourteen high-quality metagenome-assembled genomes (MAGs) were obtained, including those of 5 anammox Planctomycetota (Candidatus Brocadia, Ca. Kuenenia, Ca. Jettenia, and Ca. Scalindua), 4 Bacteroidota, and 3 Chloroflexota. Based on the gene sets of metabolic pathways involved in the degradation of polymeric substances found in Chloroflexota and Bacteroidota MAGs, they are expected to be scavengers of extracellular polymeric substances and cell debris.

Growth of nitrite-oxidizing Nitrospira and ammonia-oxidizing Nitrosomonas in marine recirculating trickling biofilter reactors.

Aerobic ammonia and nitrite oxidation reactions are fundamental biogeochemical reactions contributing to the global nitrogen cycle. Although aerobic nitrite oxidation yields 4.8-folds less Gibbs free energy (∆Gr ) than aerobic ammonia oxidation in the NH4 + -feeding marine recirculating trickling biofilter reactors operated in the present study, nitrite-oxidizing and not ammonia-oxidizing Nitrospira (sublineage IV) outnumbered ammonia-oxidizing Nitrosomonas (relative abundance; 53.8% and 7.59% respectively). CO2 assimilation efficiencies during ammonia or nitrite oxidation were 0.077 µmol-14 CO2 /µmol-NH3 and 0.053-0.054 µmol-14 CO2 /µmol-NO2 - respectively, and the difference between ammonia and nitrite oxidation was much smaller than the difference of ∆Gr . Free-energy efficiency of nitrite oxidation was higher than ammonia oxidation (31%-32% and 13% respectively), and high CO2 assimilation and free-energy efficiencies were a determinant for the dominance of Nitrospira over Nitrosomonas. Washout of Nitrospira and Nitrosomonas from the trickling biofilter reactors was also examined by quantitative PCR assay. Normalized copy numbers of Nitrosomonas amoA were 1.5- to 1.7-folds greater than Nitrospira nxrB and 16S rRNA gene in the reactor effluents. Nitrosomonas was more susceptible for washout than Nitrospira in the trickling biofilter reactors, which was another determinant for the dominance of Nitrospira in the trickling biofilter reactors.

NH2OH Disproportionation Mediated by Anaerobic Ammonium-oxidizing (Anammox) Bacteria.

Anammox bacteria produce N2 gas by oxidizing NH4+ with NO2-, and hydroxylamine (NH2OH) is a potential intermediate of the anammox process. N2 gas production occurs when anammox bacteria are incubated with NH2OH only, indicating their capacity for NH2OH disproportionation with NH2OH serving as both the electron donor and acceptor. Limited information is currently available on NH2OH disproportionation by anammox bacteria; therefore, the stoichiometry of anammox bacterial NH2OH disproportionation was examined in the present study using 15N-tracing techniques. The anammox bacteria, Brocadia sinica, Jettenia caeni, and Scalindua sp. were incubated with the addition of 15NH2OH, and the production of 15N-labeled nitrogenous compounds was assessed. The anammox bacteria tested performed NH2OH disproportionation and produced 15-15N2 gas and NH4+ as reaction products. The addition of acetylene, an inhibitor of the anammox process, reduced the activity of NH2OH disproportionation, but not completely. The growth of B. sinica by NH2OH disproportionation (-240.3| |kJ mol NH2OH-1 under standard conditions) was also tested in 3 up-flow column anammox reactors fed with 1) 0.7| |mM NH2OH only, 2) 0.7| |mM NH2OH and 0.5| |mM NH4+, and 3) 0.7| |mM NH2OH and 0.5| |mM NO2-. NH2OH consumption activities were markedly reduced after 7| |d of operation, indicating that B. sinica was unable to maintain its activity or biomass by NH2OH disproportionation.

N2O Reduction by Gemmatimonas aurantiaca and Potential Involvement of Gemmatimonadetes Bacteria in N2O Reduction in Agricultural Soils.

Agricultural soil is the primary N2O sink limiting the emission of N2O gas into the atmosphere. Although Gemmatimonadetes bacteria are abundant in agricultural soils, limited information is currently available on N2O reduction by Gemmatimonadetes bacteria. Therefore, the effects of pH and temperature on N2O reduction activities and affinity constants for N2O reduction were examined by performing batch experiments using an isolate of Gemmatimonadetes bacteria, Gemmatimonas aurantiaca (NBRC100505T). G. aurantiaca reduced N2O at pH 5-9 and 4-50°C, with the highest activity being observed at pH 7 and 30°C. The affinity constant of G. aurantiaca cells for N2O was 4.4| |µM. The abundance and diversity of the Gemmatimonadetes 16S rRNA gene and nosZ encoding nitrous oxide reductase in agricultural soil samples were also investigated by quantitative PCR (qPCR) and amplicon sequencing ana-lyses. Four N2O-reducing agricultural soil samples were assessed, and the copy numbers of the Gemmatimonadetes 16S rRNA gene (clades G1 and G3), nosZ DNA, and nosZ mRNA were 8.62-9.65×108, 5.35-7.15×108, and 2.23-4.31×109 copies (g dry soil)-1, respectively. The abundance of the nosZ mRNA of Gemmatimonadetes bacteria and OTU91, OUT332, and OTU122 correlated with the N2O reduction rates of the soil samples tested, suggesting N2O reduction by Gemmatimonadetes bacteria. Gemmatimonadetes 16S rRNA gene reads affiliated with OTU4572 and OTU3759 were predominant among the soil samples examined, and these Gemmatimonadetes OTUs have been identified in various types of soil samples.

Determination of 15N/14N of Ammonium, Nitrite, Nitrate, Hydroxylamine, and Hydrazine Using Colorimetric Reagents and Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS).

In the nitrogen (N) cycle, nitrogenous compounds are chemically and biologically converted to various aqueous and gaseous N species. The 15N-labeling approach is a powerful culture-dependent technique to obtain insights into the complex nitrogen transformation reactions that occur in cultures. In the 15N-labeling approach, the fates of supplemented 15N- and/or unlabeled gaseous and aqueous compounds are tracked by mass spectrometry (MS) analysis, whereas MS analysis of aqueous N species requires laborious sample preparation steps and is performed using isotope-ratio mass spectrometry, which requires an expensive mass spectrometer. We developed a simple and high-throughput MS method for determining the 15N atoms percent of NH4+, NO2-, NO3-, NH2OH, and N2H4, where liquid samples (<0.5 mL) were mixed with colorimetric reagents (naphthylethylenediamine for NO2-, indophenol for NH4+, and p-aminobenzaldehyde for N2H4), and the mass spectra of the formed N complex dyes were obtained by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) MS. NH2OH and NO3- were chemically converted to NO2- by iodine oxidation and copper/hydrazine reduction reaction, respectively, prior to the above colorimetric reaction. The intensity of the isotope peak (M + 1 or M + 2) increased when the N complex dye was formed by coupling with a 15N-labeled compound, and a linear relationship was found between the determined 15N/14N peak ratio and 15N atom% for the tested N species. The developed method was applied to bacterial cultures to examine their N-transformation reactions, enabling us to observe the occurrence of NO2- oxidation and NO3- reduction in a hypoxic Nitrobacter winogradskyi culture. IMPORTANCE15N/14N analysis for aqueous N species is a powerful tool for obtaining insights into the global N cycle, but the procedure is cumbersome and laborious. The combined use of colorimetric reagents and MALDI-TOF MS, designated color MALDI-TOF MS, enabled us to determine the 15N atom% of common aqueous N species without laborious sample preparation and chromatographic separation steps; for instance, the 15N atom% of NO2- can be determined from >1,000 liquid samples daily at <$1 (U.S.) per 384 samples for routine analysis. This convenient MS method is a powerful tool that will advance our ability to explore the N-transformation reactions that occur in various environments and biological samples.

Endpoint Recombinase Polymerase Amplification (RPA) Assay for Enumeration of Thiocyanate-degrading Bacteria.

An endpoint recombination amplification reaction (RPA) assay for assessing the abundance of the gene encoding thiocyanate dehydrogenase (TcDH) in Thiohalobacter has been developed. The RPA reaction was performed at 37°C for 30| |min, terminated by the addition of sodium dodecyl sulfate (SDS) solution, and the DNA concentration of the RPA product was fluorometrically measured. The abundance of TcDH in 22 activated sludge samples and 7 thiocyanate-degrading enrichment cultures ranged between 2.5×103 and 1.5×106 copies µL-1, showing a linear relationship (R2=0.83) with those measured using a conventional quantitative PCR assay.

Full-scale application of a down-flow hanging sponge reactor combined with a primary sedimentation basin for domestic sewage treatment.

The down-flow hanging sponge (DHS) reactor is advantageous for sewage treatment since it produces an effluent quality that complies with the standards for reuse and there is little excess sludge. A full-scale DHS module was efficiently employed for the treatment of domestic sewage (200 m3 day-1) flowing from a primary sedimentation basin (PSB), which was used to reduce the suspended solids loading rate and enhance the oxidation of organics by heterotrophs. The combined PSB-DHS was successfully operated at a total hydraulic retention time of 3.4 h (2.4 h for PSB and 1.0 h for DHS) for the relatively long period of 600 days at sewage temperatures of 10 °C to 32 °C. The PSB-DHS consistently produced an effluent quality with minimum values of chemical oxygen demand, biochemical oxygen demand, and suspended solids of 59 ± 15, 12 ± 3.0, and 15 ± 7 mg L-1, respectively. The proposed system performed exceptionally well at removing organics and particulate matter over a short hydraulic retention time.

Complete Genome Sequence of Thiohalobacter sp. Strain COW1, Isolated from Activated Sludge Treating Coke Oven Wastewater.

A thiocyanate-degrading bacterium, Thiohalobacter sp. strain COW1, was isolated from activated sludge treating coke oven wastewater, and the complete genome sequence was determined. COW1 contained a single circular chromosome (3.23 Mb; G+C content, 63.4%) in which 2,788 protein-coding genes, 39 tRNA genes, and 3 rRNA genes were identified.

Draft Genome Sequence of Cytophagales sp. Strain WSM2-2, Isolated from Garden Soil.

We report the draft genome sequence of Cytophagales sp. strain WSM2-2, isolated from garden soil. A 5.5-Mb genome sequence comprising four contigs was successfully obtained using Illumina NovaSeq and MinION sequencers. This draft genome sequence will contribute to the genomic knowledge of the bacterial order Cytophagales.

Draft Genome Sequence of Enterobacter sp. AS-1, a Potential Eurytrophic Recombination Host.

Strain AS-1 was isolated from laboratory-scale activated sludge collected in Japan. This strain not only grows on rich medium, including R2A medium, but also forms colonies on medium lacking organic matter other than agar (water agar), indicating it could be used as a eurytrophic recombinant host in material production processes. Here, we present a draft genome sequence of Enterobacter sp. AS-1, which consists of a total of 24 contigs containing 5,207,146 bp, with a GC content of 55.64%, and comprising 4,921 predicted coding sequences. Based on 16S rRNA gene sequence analysis, strain AS-1 was designated as Enterobacter sp. AS-1.

Glycogen metabolism of the anammox bacterium "Candidatus Brocadia sinica".

Presence of glycogen granules in anaerobic ammonium-oxidizing (anammox) bacteria has been reported so far. However, very little is known about their glycogen metabolism and the exact roles. Here, we studied the glycogen metabolism in "Ca. Brocadia sinica" growing in continuous retentostat cultures with bicarbonate as a carbon source. The effect of the culture growth phase was investigated. During the growing phase, intracellular glycogen content increased up to 32.6 mg-glucose (g-biomass dry wt)-1 while the specific growth rate and ATP/ADP ratio decreased. The accumulated glycogen begun to decrease at the onset of entering the near-zero growth phase and was consumed rapidly when substrates were depleted. This clearly indicates that glycogen was synthesized and utilized as an energy storage. The proteomic analysis revealed that "Ca. B. sinica" synthesized glycogen via three known glycogen biosynthesis pathways and simultaneously degraded during the progress of active anammox, implying that glycogen is being continuously recycled. When cells were starved, a part of stored glycogen was converted to trehalose, a potential stress protectant. This suggests that glycogen serves at least as a primary carbon source of trehalose synthesis for survival. This study provides the first physiological evidence of glycogen metabolism in anammox bacteria and its significance in survival under natural substrate-limited habitat.

Cell Density-dependent Anammox Activity of Candidatus Brocadia sinica Regulated by N-acyl Homoserine Lactone-mediated Quorum Sensing.

The activity of anaerobic ammonia-oxidizing (anammox) bacteria is considered to depend on cell density; however, this has not yet been confirmed due to the fastidious nature of anammox bacteria (e.g., slow growth, oxygen sensitivity, and rigid aggregate formation). In the present study, the cell density-dependent occurrence of anammox activity (14-15N2 gas production rate) was investigated using planktonic enrichment cultures of Candidatus Brocadia sinica. This activity was detectable when the density of cells was higher than 107| |cells| |mL-1 and became stronger with increases in cell density. At the cell densities, the transcription of the BROSI_A1042 and BROSI_A3652 genes, which are potentially involved in the biosynthesis and reception of N-acyl homoserine lactone (AHL), was detectable in Brocadia sinica cells. The presence of AHL molecules in the MBR culture of B. sinica was confirmed by an AHL reporter assay and gas chromatography mass spectrometry analysis. The exogenous addition of the MBR culture extract and AHL molecules (a cocktail of C6, C8, C10, and C12-homoserine lactones) increased the specific 14-15N2 production rate of B. sinica. These results suggest that the specific anammox activity of B. sinica is regulated by AHL-mediated quorum sensing.

Food selectivity of anaerobic protists and direct evidence for methane production using carbon from prey bacteria by endosymbiotic methanogen.

Anaerobic protists are major predators of prokaryotes in anaerobic ecosystems. However, little is known about the predation behavior of anaerobic protists because almost none have been cultured. In particular, these characteristics of anaerobic protists in the phyla Metamonada and Cercozoa have not been reported previously. In this study, we isolated three anaerobic protists, Cyclidium sp., Trichomitus sp., and Paracercomonas sp., from anaerobic granular sludge in an up-flow anaerobic sludge blanket reactor used to treat domestic sewage. Ingestion and digestion of food bacteria by anaerobic protists with or without endosymbiotic methanogens were demonstrated using tracer experiments with green fluorescent protein and a stable carbon isotope. These tracer experiments also demonstrated that Cyclidium sp. supplied CO2 and hydrogen to endosymbiotic methanogens. While Cyclidium sp. and Trichomitus sp. ingested both Gram-negative and -positive bacteria, Paracercomonas sp. could only take up Gram-negative bacteria. Archaeal cells such as Methanobacterium beijingense and Methanospirillum hungatei did not support the growth of these protists. Metabolite patterns of all three protists differed and were influenced by food bacterial species. These reported growth rates, ingestion rates, food selectivity, and metabolite patterns provide important insights into the ecological roles of these protists in anaerobic ecosystems.

Biosynthesis of hydrazine from ammonium and hydroxylamine using an anaerobic ammonium oxidizing bacterium.

OBJECTIVES: To synthesize hydrazine (N2H4) from ammonium and hydroxylamine (NH2OH) using an anaerobic ammonium oxidation (anammox) bacterium, Candidatus Kuenenia stuttgartiensis. RESULTS: K. stuttgartiensis cells were anoxically cultivated with the addition of ammonium (2 mM) and NH2OH (1-100 mM) at pH 6-10.5, and 4-65 °C to examine the favorable cultivation conditions for N2H4 production. The influence of NH2OH concentration was more prominent than that of pH and temperature, and NH2OH concentration higher than 1 mM deteriorated N2H4 yields significantly. The following conditions were found to be favorable for N2H4 production using K. stuttgartiensis cells: pH 9, 38 °C, and < 1 mM NH2OH. In a continuous-feed system operated at these conditions, K. stuttgartiensis cells produced N2H4 with a maximum concentration of 0.65 mM, which is the highest N2H4 concentration previously reported in biological processes. CONCLUSIONS: Optimal cultivation conditions for K. stuttgartiensis for N2H4 production were successfully determined, and the present study is the first to document potential biological N2H4 production using anammox bacteria.