Nitrosopumilus zosterae sp. nov., an autotrophic ammonia-oxidizing archaeon of phylum Thaumarchaeota isolated from coastal eelgrass sediments of Japan.

A novel mesophilic and aerobic ammonia-oxidizing archaeon of the phylum Thaumarchaeota, strain NM25T, was isolated from coastal eelgrass zone sediment sampled in Shimoda (Japan). The cells were rod-shaped with an S-layer cell wall. The temperature range for growth was 20-37 °C, with an optimum at 30 °C. The pH range for growth was pH 6.1-7.7, with an optimum at pH 7.1. The salinity range for growth was 5-40 %, with an optimum range of 15-32 %. Cells obtained energy from ammonia oxidation and used bicarbonate as a carbon source. Utilization of urea was not observed for energy generation and growth. Strain NM25T required a hydrogen peroxide scavenger, such as α-ketoglutarate, pyruvate or catalase, for sustained growth on ammonia. Growth of strain NM25T was inhibited by addition of low concentrations of some organic compounds and organic mixtures, including complete inhibition by glycerol, peptone and yeast extract. Phylogenetic analysis of four concatenated housekeeping genes (16S rRNA, rpoB, rpsI and atpD) and concatenated AmoA, AmoB, AmoC amino acid sequences indicated that the isolate is similar to members of the genus Nitrosopumilus. The closest relative is Nitrosopumilus ureiphilus PS0T with sequence similarities of 99.5 % for the 16S rRNA gene and 97.2 % for the amoA gene. Genome relatedness between strain NM25T and N. ureiphilus PS0T was assessed by average nucleotide identity and digital DNA-DNA hybridization, giving results of 85.4 and 40.2 %, respectively. On the basis of phenotypic, genotypic and phylogenetic data, strain NM25T represents a novel species of the genus Nitrosopumilus, for which the name sp. nov, is proposed. The type strain is NM25T (=NBRC 111181T=ATCC TSD-147T).

Enrichment and Physiological Characterization of a Cold-Adapted Nitrite-Oxidizing Nitrotoga sp. from an Eelgrass Sediment.

Nitrite-oxidizing bacteria (NOB) are responsible for the second step of nitrification in natural and engineered ecosystems. The recently discovered genus Nitrotoga belongs to the Betaproteobacteria and potentially has high environmental importance. Although environmental clones affiliated with Nitrotoga are widely distributed, the limited number of cultivated Nitrotoga spp. results in a poor understanding of their ecophysiological features. In this study, we successfully enriched the nonmarine cold-adapted Nitrotoga sp. strain AM1 from coastal sand in an eelgrass zone and investigated its physiological characteristics. Multistep-enrichment approaches led to an increase in the abundance of AM1 to approximately 80% of the total bacterial population. AM1 was the only detectable NOB in the bacterial community. The 16S rRNA gene sequence of AM1 was 99.6% identical to that of "Candidatus Nitrotoga arctica," which was enriched from permafrost-affected soil. The highest nitrogen oxidation rate of AM1 was observed at 16°C. The half-saturation constant (Km ) and the generation time were determined to be 25 µM NO2- and 54 h, respectively. The nitrite oxidation rate of AM1 was stimulated at concentrations of <30 mM NH4Cl but completely inhibited at 50 mM NH4Cl. AM1 can grow well under specific environmental conditions, such as low temperature and in the presence of a relatively high concentration of free ammonia. These results help improve our comprehension of the functional importance of NitrotogaIMPORTANCE Nitrite-oxidizing bacteria (NOB) are key players in the second step of nitrification, which is an important process of the nitrogen cycle. Recent studies have suggested that the organisms of the novel NOB genus Nitrotoga were widely distributed and played a functional role in natural and engineered ecosystems. However, only a few Nitrotoga enrichments have been obtained, and little is known about their ecology and physiology. In this study, we successfully enriched a Nitrotoga sp. from sand in a shallow coastal marine ecosystem and undertook a physiological characterization. The laboratory experiments showed that the Nitrotoga enrichment culture could adapt not only to low temperature but also to relatively high concentrations of free ammonia. The determination of as-yet-unknown unique characteristics of Nitrotoga contributes to the improvement of our insights into the microbiology of nitrification.

Complete Genome Sequence of the Ammonia-Oxidizing Bacterium Nitrosospira sp. Strain NRS527, Isolated from the Rhizoplane of Paddy Rice.

This work reports the complete genome sequence of the chemoautotrophic ammonia-oxidizing bacterium Nitrosospira sp. strain NRS527. The assembled genome is composed of a circular chromosome and two plasmids (80,750 bp and 41,389 bp, respectively).

Quantification and Phylogenetic Analysis of Ammonia Oxidizers on Biofilm Carriers in a Full-Scale Wastewater Treatment Plant.

Biofilm carriers have been used to remove ammonia in several wastewater treatment plants (WWTPs) in Japan. However, the abundance and species of ammonia oxidizers in the biofilms formed on the surface of carriers in full-scale operational WWTP tanks remain unclear. In the present study, we conducted quantitative PCR and PCR cloning of the amoA genes of ammonia-oxidizing bacteria and archaea (AOB and AOA) and a complete ammonia oxidizer (comammox) in the biofilm formed on the carriers in a full-scale WWTP. The quantification of amoA genes showed that the abundance of AOB and comammox was markedly greater in the biofilm than in the activated sludge suspended in a tank solution of the WWTP, while AOA was not detected in the biofilm or the activated sludge. A phylogenetic analysis of amoA genes revealed that as-yet-uncultivated comammox Nitrospira and uncultured AOB Nitrosomonas were predominant in the biofilm. The present results suggest that the biofilm formed on the surface of carriers enable comammox Nitrospira and AOB Nitrosomonas to co-exist and remain in the full-scale WWTP tank surveyed in this study.

Alternative strategies of nutrient acquisition and energy conservation map to the biogeography of marine ammonia-oxidizing archaea.

Ammonia-oxidizing archaea (AOA) are among the most abundant and ubiquitous microorganisms in the ocean, exerting primary control on nitrification and nitrogen oxides emission. Although united by a common physiology of chemoautotrophic growth on ammonia, a corresponding high genomic and habitat variability suggests tremendous adaptive capacity. Here, we compared 44 diverse AOA genomes, 37 from species cultivated from samples collected across diverse geographic locations and seven assembled from metagenomic sequences from the mesopelagic to hadopelagic zones of the deep ocean. Comparative analysis identified seven major marine AOA genotypic groups having gene content correlated with their distinctive biogeographies. Phosphorus and ammonia availabilities as well as hydrostatic pressure were identified as selective forces driving marine AOA genotypic and gene content variability in different oceanic regions. Notably, AOA methylphosphonate biosynthetic genes span diverse oceanic provinces, reinforcing their importance for methane production in the ocean. Together, our combined comparative physiological, genomic, and metagenomic analyses provide a comprehensive view of the biogeography of globally abundant AOA and their adaptive radiation into a vast range of marine and terrestrial habitats.

Whole-Genome Sequence of the Ammonia-Oxidizing Bacterium Nitrosomonas stercoris Type Strain KYUHI-S, Isolated from Composted Cattle Manure.

This work reports the complete genome sequence of a chemoautotrophic ammonia-oxidizing bacterium, Nitrosomonas stercoris strain KYUHI-ST (= ATCC BAA-2718T and NBRC 110753T). The assembled genome is composed of a circular chromosome and a large plasmid.

Eelgrass Sediment Microbiome as a Nitrous Oxide Sink in Brackish Lake Akkeshi, Japan.

Nitrous oxide (N2O) is a powerful greenhouse gas; however, limited information is currently available on the microbiomes involved in its sink and source in seagrass meadow sediments. Using laboratory incubations, a quantitative PCR (qPCR) analysis of N2O reductase (nosZ) and ammonia monooxygenase subunit A (amoA) genes, and a metagenome analysis based on the nosZ gene, we investigated the abundance of N2O-reducing microorganisms and ammonia-oxidizing prokaryotes as well as the community compositions of N2O-reducing microorganisms in in situ and cultivated sediments in the non-eelgrass and eelgrass zones of Lake Akkeshi, Japan. Laboratory incubations showed that N2O was reduced by eelgrass sediments and emitted by non-eelgrass sediments. qPCR analyses revealed that the abundance of nosZ gene clade II in both sediments before and after the incubation as higher in the eelgrass zone than in the non-eelgrass zone. In contrast, the abundance of ammonia-oxidizing archaeal amoA genes increased after incubations in the non-eelgrass zone only. Metagenome analyses of nosZ genes revealed that the lineages Dechloromonas-Magnetospirillum-Thiocapsa and Bacteroidetes (Flavobacteriia) within nosZ gene clade II were the main populations in the N2O-reducing microbiome in the in situ sediments of eelgrass zones. Sulfur-oxidizing Gammaproteobacteria within nosZ gene clade II dominated in the lineage Dechloromonas-Magnetospirillum-Thiocapsa. Alphaproteobacteria within nosZ gene clade I were predominant in both zones. The proportions of Epsilonproteobacteria within nosZ gene clade II increased after incubations in the eelgrass zone microcosm supplemented with N2O only. Collectively, these results suggest that the N2O-reducing microbiome in eelgrass meadows is largely responsible for coastal N2O mitigation.

Phylogenetic relationships among ammonia-oxidizing bacteria as revealed by gene sequences of glyceraldehyde 3-phosphate dehydrogenase and phosphoglycerate kinase.

The three previously recognized genera of 'Nitrosolobus', Nitrosospira and 'Nitrosovibrio' were combined into one genus, Nitrosospira, on the basis of 16S rDNA sequence similarities. However, this classification has been controversial for some time, since the marked differences in their shapes suggest that they are not closely related. In this study, the phylogenetic analyses of the three groups using two genotypical markers, glyceraldehyde-3-phosphate dehydrogenase (GAP, gap), and 3-phosphoglycerate kinase (PGK, pgk), were performed. In the phylogenetic tree inferred from gap and pgk, the three genera appeared as clearly separated clusters. This is the first study of markers that are able to reveal the precise phylogenetic relationship among 'Nitrosolobus', Nitrosospira and 'Nitrosovibrio'.

Nitrosomonas stercoris sp. nov., a Chemoautotrophic Ammonia-Oxidizing Bacterium Tolerant of High Ammonium Isolated from Composted Cattle Manure.

Among ammonia-oxidizing bacteria, Nitrosomonas eutropha-like microbes are distributed in strongly eutrophic environments such as wastewater treatment plants and animal manure. In the present study, we isolated an ammonia-oxidizing bacterium tolerant of high ammonium levels, designated strain KYUHI-S(T), from composted cattle manure. Unlike the other known Nitrosomonas species, this isolate grew at 1,000 mM ammonium. Phylogenetic analyses based on 16S rRNA and amoA genes indicated that the isolate belonged to the genus Nitrosomonas and formed a unique cluster with the uncultured ammonia oxidizers found in wastewater systems and animal manure composts, suggesting that these ammonia oxidizers contributed to removing higher concentrations of ammonia in strongly eutrophic environments. Based on the physiological and phylogenetic data presented here, we propose and call for the validation of the provisional taxonomic assignment Nitrosomonas stercoris, with strain KYUHI-S as the type strain (type strain KYUHI-S(T) = NBRC 110753(T) = ATCC BAA-2718(T)).

Identification of genus Nitrosovibrio, ammonia-oxidizing bacteria, by comparison of N-terminal amino acid sequences of phosphoglycerate kinase.

Nitrosospira, Nitrosolobus, and Nitrosovibrio, three genera of ammonia-oxidizing bacteria (AOB), are morphologically distinct from each other (Nitrosospira: spiral, Nitrosolobus: lobate, Nitrosovibrio: slender curved rods). However, these three genera cannot be classified using the phylogenetic tree based on 16S rDNA sequences. Phosphoglycerate kinase (PGK) was purified from 10 AOB strains as an index for identification, and its enzymatic properties were investigated. No distinction could be made between these three genera of AOB, because there was no notable difference in the molecular weight, K(m), or other properties of their PGKs. However, the N-terminal amino acid residues of PGK of Pro(P)-1, Ile(I)-6 and Asp(D)-12 have sequences characteristic of genus Nitrosovibrio (three strains) in four AOB genera. Therefore, it is proved possible to use the differences in the sequence to identify individual strains of the genus Nitrosovibrio.

Two kinds of ammonia-oxidizing bacteria isolated from biologically deodorizing plants in cold district.

Two kinds of ammonia-oxidizing bacteria isolated from biologically deodorizing plants in cold districts in Japan were identified as Nitrosomonas sp. IWT202 and Nitrosomonas sp. IWT514. The optimum pHs for growth were 8.0 (IWT202) and 7.5 (IWT514). Although rockwool samples for isolation were collected from the same plants, the optimum temperature for growth of strain IWT202 (37 degrees C) differed from that of strain IWT514 (30 degrees C). The bacteria had a higher (IWT202, 37 degrees C) and lower (IWT514, 20 degrees C) growth temperature than is usually the case. Both strains were shown to differ completely in regard to the effect of the ammonium sulfate concentration in the medium for a 20 degrees C culture and 30 degrees C culture. The inoculation of these bacteria provides the possibility of recovering ammonia-oxidizing activity, when the ammonia-oxidizing activity is lowered in biological deodorizing plants in cold districts. It seems that these strains are suitable for application to deodorization.

Nitrosomonas communis strain YNSRA, an ammonia-oxidizing bacterium, isolated from the reed rhizoplane in an aquaponics plant.

An ammonia-oxidizing bacterium (strain YNSRA) was isolated from the rhizoplane of the reed (Phragmites communis) used in an aquaponics plant which is a wastewater treatment plant. Strain YNSRA was identified as Nitrosomonas communis by taxonomic studies. The hydroxylamine-cytochrome c reductase (HCR) of strain YNSRA was found to have a higher activity (25.60 u/mg) than that of Nitrosomonas europaea ATCC25978T (8.94 u/mg). Ribulose-1,5-bisphosphate carboxylase (RubisCO) activity was detected at very low levels in strain YNSRA, whereas strain ATCC25978T had definite activity.

Susceptibility of ammonia-oxidizing bacteria to nitrification inhibitors.

Activity of nitrification inhibitors to several typical ammonia-oxidizing bacteria isolated recently, i. e. Nitrosococcus, Nitrosolobus, Nitrosomonas, Nitrosospira and Nitrosovibrio species was assayed using 2-amino-4-methyl-trichloromethyl-1,3,5-triazine (MAST), 2-amino-4-tribromomethyl-6-trichloromethyl-1,3,5-triazine (Br-MAST), 2-chloro-6-trichloromethylpyridine (nitrapyrin) and others, and compared to confirm the adequate control of ammonia-oxidizing bacteria by the inhibitors. The order of activity of the inhibitors to 13 species of ammonia-oxidizing bacteria examined was approximately summarized as Br-MAST > or = nitrapyrin > or = MAST > other inhibitors. Two Nitrosomonas strains, N. europaea ATCC25978 and N. sp. B2, were extremely susceptible to Br-MAST, exhibiting a pI50 > or = 6.40. These values are the position logarithms of the molar half-inhibition concentration. The 16S rRNA gene sequence similarity for the highly susceptible 4 strains of genus Nitrosomonas was 94% to 100% of Nitrosomonas europaea, although those of the less susceptible 3 strains of ammonia-oxidizing bacteria, Nitrosococcus oceanus C-107 ATCC19707, Nitrosolobus sp. PJA1 and Nitrosolobus multiformis ATCC25196, were 77.85, 91.53 and 90.29, respectively. However, no clear correlation has been found yet between pI50-values and percent similarity of 16S rRNA gene sequence among ammonia-oxidizing bacteria.

Enrichment of a novel marine ammonia-oxidizing archaeon obtained from sand of an eelgrass zone.

Ammonia-oxidizing archaea (AOA) are generally cultivated at ammonium concentrations of less than 2 mM. The physiology and abundance in the environment of AOA suggest an important role in the nitrogen cycle. We report here a novel marine ammonia-oxidizing crenarchaeote, strain NM25 belonged to 'Candidatus Nitrosopumilus', that was enriched from coastal sand of an eelgrass zone and grew in a medium containing 15 mM ammonium at 30°C. A phylogenetic analysis based on the 16S rRNA gene revealed this crenarchaeote was related to the ammonia-oxidizing archaeon 'Candidatus Nitrosopumilus maritimus' strain SCM1, with 98.5% identity. The ammonia monooxygenase subunit A (amoA) gene of strain NM25 was less closely related to that of known cultivable AOA (>95%) and environmental clones (>97%). This finding suggests the existence of AOA adapted to high ammonium-containing environments.

Seasonal change in vertical distribution of ammonia-oxidizing archaea and bacteria and their nitrification in temperate forest soil.

Seasonal change in the vertical distribution of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in temperate forest soil was examined from March 2008 to January 2009 by quantitative PCR of the amoA genes. Abundances of AOA amoA genes (ranging from 2.0×10(8) to 1.2×10(9) copies per gram dry soil) were significantly higher than those of AOB amoA genes (1.9×10(5) to 1.7×10(7) copies). A significant increase in AOB was observed at a depth of 0-5 cm in July when net nitrification was also high in the top soil, while AOA increased significantly at depths of 5-10 cm, 10-15 cm, and over 15 cm in July. Sequencing of the crenarchaeotal amoA gene revealed shifts in major AOA components along the soil depth profile and among sampling dates. Betaproteobacterial amoA clone libraries at 0-5 cm in March, May, and July were dominated by Nitrosospira clusters 1 and 4. A microcosm experiment at 0-5 cm in July revealed a decrease in the ratio of AOA/AOB amoA genes in microcosms. These results suggest that AOB play an important role in net nitrification in the top layer in temperate forest soil.

Seasonal changes in abundance of ammonia-oxidizing archaea and ammonia-oxidizing bacteria and their nitrification in sand of an eelgrass zone.

Seasonal changes in the abundance of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) within the sand of an eelgrass (Zostera marina) zone were examined by a quantitative PCR of both crenarchaeotal and betaproteobacterial ammonia monooxygenase alpha subunit (amoA) genes together with temperature and concentrations of ammonium, nitrite, and nitrate from May 2007 to June 2008 at Tanoura Bay, Shizuoka, Japan. The abundance of both amoAs in the sand between May and June 2007 and between January and March 2008 was 1.5 to 2 orders of magnitude higher than the 10(4) copies g(-1) of estimated amoA between September and December. Archaeal amoA was more diverse than betaproteobacterial amoA. Betaproteobacterial amoA clone libraries were dominated by Nitrosospira-like sequence types. An incubation experiment was conducted with sands collected in February 2008 and community structure was analyzed based on reverse-transcribed amoAs. RNA was extracted from sand incubated for 12 days at 30°C, 17 days at 20°C, and 80 days at 10°C. Different amoA clones were detected from in situ sand and incubated sand. This study reveals clear evidence of seasonal change in the abundance of AOA and AOB within the sand of an eelgrass zone.

Analysis of ammonia monooxygenase and archaeal 16S rRNA gene fragments in nitrifying acid-sulfate soil microcosms.

The present study describes the occurrence of a unique archaeal ammonia monooxygenase alpha subunit (amoA) gene in nitrifying acid-sulfate soil microcosms at pH 3.5. The soil was collected from an abandoned paddy field in Thailand. Microcosms were incubated in the dark at 30°C for 372 days with the following three treatments: addition of ammonium sulfate solution once a month (I) or once a week (II), and addition of only sterilized water (III). A quantitative PCR analysis revealed an increase in abundance of the archaeal amoA gene in microcosm soils in which nitrate concentrations increased after incubation. A phylogenetic analysis indicated a predominance of the novel gene, and a predominance of a betaproteobacterial amoA gene affiliated with the genus Nitrosospira. A 16S rRNA gene-based PCR assay revealed that crenarchaeotic Group I.1d was predominant among the Crenarchaeota in microcosms. These results suggest the presence of ammonia-oxidizing archaea corresponding to the unique amoA lineage in nitrifying acid-sulfate soil microcosms at pH 3.5.