Second Step Of Nitrification Research Articles

An investigation of Nitrospira bacteria in coastal wetlands of China: distribution pattern and related environmental driving factors

Nitrate is mainly converted via aerobic nitrite oxidation during the second step of nitrification, which is catalyzed by nitrite-oxidizing bacteria (NOB) and the recently discovery complete ammonia oxidizers (comammox). Members of the genus Nitrospira are the most diverse and widespread known NOB and comammox. However, the community assembly of Nitrospira in estuary and coastal wetland and the major environmental shaping factors remain understudied. Here in this study, we investigated the geographical distribution pattern of Nitrospira along the large-scale coastal wetlands of China. The results showed that the abundance of Nitrospira ranged from 4.96×106 - 3.88×107 copies/g dry sediment, significantly (more than one order of magnitude) higher than amoA gene of ammonia-oxidizers. The identified Nitrospira belong to Nitrospira lineage IV (50%), lineage I and II. The adaptability of the three lineages to environmental factors (such as temperature, pH, salinity and particle size) are different, which leads to the diversity of its distribution composition in different estuaries. Network analysis showed that the cooperation takes greater portion than competition in the relationship of Nitrospira population. This study revealed the abundance and community composition of Nitrospira bacteria, as well as the major environmental driving factors in coastal wetland ecosystems, which deepens our understanding of the niche separation of Nitrospira with the nitrogen cycling.

Diversity of the Hydroxylamine Oxidoreductase (HAO) Gene and Its Enzyme Active Site in Agricultural Field Soils.

Nitrification is a key process in the biogeochemical nitrogen cycle and a major emission source of the greenhouse gas nitrous oxide (N2O). The periplasmic enzyme hydroxylamine oxidoreductase (HAO) is involved in the oxidation of hydroxylamine to nitric oxide in the second step of nitrification, producing N2O as a byproduct. Its three-dimensional structure demonstrates that slight differences in HAO active site residues have inhibitor effects. Therefore, a more detailed understanding of the diversity of HAO active site residues in soil microorganisms is important for the development of novel nitrification inhibitors using structure-guided drug design. However, this has not yet been examined. In the present study, we investigated hao gene diversity in beta-proteobacterial ammonia-oxidizing bacteria (β-AOB) and complete ammonia-oxidizing (comammox; Nitrospira spp.) bacteria in agricultural fields using a clone library ana-lysis. A total of 1,949 hao gene sequences revealed that hao gene diversity in β-AOB and comammox bacteria was affected by the fertilizer treatment and field type, respectively. Moreover, hao sequences showed the almost complete conservation of the six HAO active site residues in both β-AOB and comammox bacteria. The diversity of nitrifying bacteria showed similarity between hao and amoA genes. The nxrB amplicon sequence revealed the dominance of Nitrospira cluster II in tea field soils. The present study is the first to reveal hao gene diversity in agricultural soils, which will accelerate the efficient screening of HAO inhibitors and evaluations of their suppressive effects on nitrification in agricultural soils.

The coexistence and competition of canonical and comammox nitrite oxidizing bacteria in a nitrifying activated sludge system – Experimental observations and simulation studies

The second step of nitrification can be mediated by nitrite oxidizing bacteria (NOB), i.e. Nitrospira and Nitrobacter, with different characteristics in terms of the r/K theory. In this study, an activated sludge model was developed to account for competition between two groups of canonical NOB and comammox bacteria. Heterotrophic denitrification on soluble microbial products was also incorporated into the model. Four 5-week washout trials were carried out at dissolved oxygen-limited conditions for different temperatures (12 °C vs. 20 °C) and main substrates (NH4+-N vs. NO2−-N). Due to the aggressive reduction of solids retention time (from 4 to 1 d), the biomass concentrations were continuously decreased and stabilized after two weeks at a level below 400 mg/L. The collected experimental data (N species, biomass concentrations, and microbiological analyses) were used for model calibration and validation. In addition to the standard predictions (N species and biomass), the newly developed model also accurately predicted two microbiological indicators, including the relative abundance of comammox bacteria as well as nitrifiers to heterotrophs ratio. Sankey diagrams revealed that the relative contributions of specific microbial groups to N conversion pathways were significantly shifted during the trial. The contribution of comammox did not exceed 5 % in the experiments with both NH4+-N and NO2−-N substrates. This study contributes to a better understanding of the novel autotrophic N removal processes (e.g. deammonification) with nitrite as a central intermediate product.

Distribution, Diversity, and Abundance of Nitrite Oxidizing Bacteria in the Subterranean Estuary of the Daya Bay

The oxidation of nitrite to nitrate as the second step of nitrification is vital for the global nitrogen cycle, and the genus Nitrospira is the most widespread nitrite-oxidizing bacteria (NOB) in diverse natural ecosystems. However, our understanding of Nitrospira distribution and their response to dynamic environmental parameters is still limited in the subterranean estuary (STE), a special environment of the mixed zone between land and sea. In this study, Nitrospira communities were collected within 5–10 cm depth intervals of sediments with the physiochemical gradients along the transections of the Daya Bay (DYB) from the landward member to the seaward member. The abundance, community structure of Nitrospira, and their potentially influencing environmental factors were investigated using Q-PCR, the high throughput sequencing targeting Nitrospira nxrB gene, and biostatistics analyses. The abundance of Nitrospira showed uniform distribution along the transections, but significantly decreasing variations were observed from the surface (<20 cm) to the bottom (>20 cm) of sediments. The community diversity and structure of Nitrospira also displayed a remarkable vertical distribution in the DYB (STE), although no significant differences were observed along the transections of STE. Nitrospira marina (N. marina) predominated in the sediments, Candidatus Nitrospira bockiana (Ca. N. bockiana), and Candidatus Nitrospira defluvii (Ca. N. defluvii) were the main Nitrospira species, and Candidatus Nitrospira lenta (Ca. N. lenta) and Nitrospira japonica (N. japonica) also existed with relatively low abundance in the DYB (STE). These findings revealed that Nitrospira species (Ca. N. defluvii, Ca. N. lenta, and N. japonica) derived from activated sludge was also widespread in natural habitats and deduced that the STE may be affected by the pollution derived from terrigenous human activities. The statistical analysis combined with the STE dynamic variation indicated that dissolved organic carbon (DOC), salinity, and ammonium along the sediment depths attributed to the vertical community distribution of the Nitrospira species. In summary, the vertical distribution of Nitrospira and their response to the dynamic physicochemical parameters imply their important role in the nitrite oxidation of the STE and provide insights into the niche differentiation and diversely physiologic metabolism of NOB.

Response of activity and community composition of nitrite-oxidizing bacteria to partial substitution of chemical fertilizer by organic fertilizer.

Nitrite oxidation as the second step of nitrification can become the determining step in disturbed soil systems. As a beneficial fertilization practice to maintain high crop yield and soil fertility, partial substitution of chemical fertilizer (CF) by organic fertilizer (OF) may exert a notable disturbance to soil systems. However, how nitrite oxidation responds to different proportions of CF to OF is still unclear. We sampled soils from a 4-year field experiment subject to a gradient of increasing proportions of OF to CF application. Activity, size, and structure of Nitrospira-like and Nitrobacter-like nitrite-oxidizing bacteria (NOB) community were measured. The results revealed that with increasing proportion of OF to CF application, potential nitrite oxidation activity (PNO) showed a marked decreasing trend. PNO was significantly correlated with the abundance of Nitrobacter-like but not Nitrospira-like NOB. The abundance of Nitrobacter-like was significantly influenced by soil organic matter, organic nitrogen (N), and available N. In addition, PNO was also affected by the structure of Nitrobacter-like NOB. The relative abundance of Nitrobacter hamburgensis, alkalicus, winogradskyi, and vulgaris responded differently to the proportions of OF to CF application. Organic N, organic matter, and available N were the main factor shaping their community structure. Overall, Nitrobacter-like NOB is more sensitive and plays a more important role than Nitrospira-like NOB in responding to different proportions of OF to CF application.

Community assembly mechanisms and co-occurrence patterns of nitrite-oxidizing bacteria communities in saline soils

Nitrite-oxidizing bacteria (NOB) catalyze the second step of nitrification by oxidizing nitrite to nitrate, which is a key process in the biogeochemical nitrogen cycling. However, little is known about the co-occurrence patterns and assembly processes of NOB communities in agricultural soils with different salinities. Here, we explored the effects of salinity on Nitrobacter and Nitrospira community using high-throughput sequencing and multivariate statistical analyses. Our results showed that high salinity significantly inhibited the nitrite oxidation rates and decreased the abundance of Nitrobacter and Nitrospira. Extreme salty conditions significantly altered the diversity and composition of Nitrospira community but had little effect on Nitrobacter community. Nitrobacter network in high salinity soils was more closely connected while the connectivity of Nitrospira network became weak. Nitrobacter and Nitrospira community exhibited distinct assembly processes at different salinity levels. Stochastic processes were dominant in the Nitrobacter community assembly in both low and high salinity soils. Interestingly, the assembly of Nitrospira community was governed by stochastic and deterministic processes in low and high salinity soils, respectively. To our knowledge, our study provides the first description of the co-occurrence patterns and assembly processes of NOB communities in agricultural soils with different salinities. These results can help us understand the NOB ecological roles and improve the nitrite oxidation activity in a high salinity environment.

Effects of Biodegradable Insecticides on Biofilter Bacteria: Implications for Aquaponics

Even though aquaponics is the production system that is increasingly gaining focus, there is an absence of publications studying the use of pesticides in the aquaponics. Therefore, the aim of this study was to assess negative effects of one synthetic (chlorpyrifos) and two botanical insecticides (azadirachtin and pyrethrin) to matured biofilter in plastic buckets. The effectiveness of biofilter bacteria is determined indirectly, as concentrations of ammonia, nitrites and nitrates in the water were measured at 0h, 5h, 13h, 21h, 29h, 37h, 45h and 53h after insecticides application. The results showed negative effects of azadirachtin on the first step of nitrification, as concentration of ammonia was higher comparing to other groups for almost every sample point. Negative effects on second step of nitrification and higher concentrations of nitrites in the water were also detected, but no statistical differences were observed in the present study, due to the large variation between buckets. Nitrates were lower in water treated with azadirachtin from 29 hours from start to the end of the experiment. Application of other two insecticides to biofilter did not cause any effects and showed no difference comparing to the control group.

Genomic and Physiological Characteristics of a Novel Nitrite-Oxidizing Nitrospira Strain Isolated From a Drinking Water Treatment Plant.

Nitrite-oxidizing bacteria (NOB) catalyze the second step of nitrification, which is an important process of the biogeochemical nitrogen cycle and is exploited extensively as a biological nitrogen removal process. Members of the genus Nitrospira are often identified as the dominant NOB in a diverse range of natural and artificial environments. Additionally, a number of studies examining the distribution, abundance, and characterization of complete ammonia oxidation (comammox) Nitrospira support the ecological importance of the genus Nitrospira. However, niche differentiation between nitrite-oxidizing Nitrospira and comammox Nitrospira remains unknown due to a lack of pure cultures. In this study, we report the isolation, physiology, and genome of a novel nitrite-oxidizing Nitrospira strain isolated from a fixed-bed column at a drinking water treatment plant. Continuous feeding of ammonia led to the enrichment of Nitrospira-like cells, as well as members of ammonia-oxidizing genus Nitrosomonas. Subsequently, a microcolony sorting technique was used to isolate a novel nitrite-oxidizing Nitrospira strain. Sequences of strains showing the growth of microcolonies in microtiter plates were checked. Consequently, the most abundant operational taxonomic unit (OTU) exhibited high sequence similarity with Nitrospira japonica (98%) at the 16S rRNA gene level. The two other Nitrospira OTUs shared over 99% sequence similarities with N. japonica and Nitrospira sp. strain GC86. Only one strain identified as Nitrospira was successfully subcultivated and designated as Nitrospira sp. strain KM1 with high sequence similarity with N. japonica (98%). The half saturation constant for nitrite and the maximum nitrite oxidation rate of strain KM1 were orders of magnitude lower than the published data of other known Nitrospira strains; moreover, strain KM1 was more sensitive to free ammonia compared with previously isolated Nitrospira strains. Therefore, the new Nitrospira strain appears to be better adapted to oligotrophic environments compared with other known non-marine nitrite oxidizers. The complete genome of strain KM1 was 4,509,223 bp in length and contained 4,318 predicted coding sequences. Average nucleotide identities between strain KM1 and known cultured Nitrospira genome sequences are 76.7–78.4%, suggesting at least species-level novelty of the strain in the Nitrospira lineage II. These findings broaden knowledge of the ecophysiological diversity of nitrite-oxidizing Nitrospira.

Defining Culture Conditions for the Hidden Nitrite-Oxidizing Bacterium Nitrolancea.

Nitrification is a key process for N-removal in engineered and natural environments, but recent findings of novel nitrifying microorganisms with surprising features revealed that our knowledge of this functional guild is still incomplete. Especially nitrite oxidation – the second step of nitrification – is catalyzed by a phylogenetically diverse bacterial group, and only recently bacteria of the phylum Chloroflexi have been identified as thermophilic nitrite-oxidizing bacteria (NOB). Among these, Nitrolancea hollandica was isolated from a laboratory-scale nitrifying bioreactor operated at 35°C with a high load of ammonium bicarbonate. However, its distribution remains cryptic as very few closely related environmental 16S rRNA gene sequences have been retrieved so far. In this study, we demonstrate how such thermophilic NOB can be enriched using modified mineral media inoculated with samples from a wastewater side-stream reactor operated at 39.5°C. Distinct cultivation conditions resulted in quick and reproducible high enrichment of two different strains of Nitrolancea, closely related to Nl. hollandica. The same cultivation approach was applied to a complex nitrite-oxidizing pre-enrichment at 42°C inoculated with biomass from a geothermal spring in the Copahue volcano area in Neuquen, Argentina. Here, an additional distinct representative of the genus Nitrolancea was obtained. This novel species had 16S rRNA and nitrite oxidoreductase alpha subunit (nxrA) gene sequence identities to Nl. hollandica of 98.5% and 97.2%, respectively. A genomic average nucleotide identity between the Argentinian strain and Nl. hollandica of 91.9% indicates that it indeed represents a distinct species. All Nitrolancea cultures formed lancet-shaped cells identical to Nl. hollandica and revealed similar physiological features, including the capability to grow at high nitrite concentrations. Growth was optimal at temperatures of 35–37°C and was strongly enhanced by ammonium supplementation. Genomic comparisons revealed that the four Nitrolancea strains share 2399 out of 3387 orthologous gene clusters and encode similar key functions. Our results define general growth conditions that enable the selective enrichment of Nitrolancea from artificial and natural environments. In most natural habitats these NOB apparently are of low abundance and their proliferation depends on the balanced presence of nitrite and ammonium, with an optimal incubation temperature of 37°C.

Inhibitory mechanisms and fate of the analgesic drug acetaminophen in nitrifying activated sludge

This work investigated the inhibitory effects and fate of acetaminophen (N-acetyl-p-aminophenol, APAP) on activated sludge (AS) under nitrifying and aerobic conditions. APAP disrupted the two-step biological nitrification process in a dose-dependent manner. 100 mg/L APAP inhibited ammonia oxidation (the first step of nitrification) accompanied by a significant reduction (> 80 %) of Nitrosomonas oligotropha in relative abundance. 50 mg/L of APAP had no significant effects on ammonia oxidation but interrupted nitrite oxidation (the second step of nitrification) with more than 90 % reduction of Candidatus Nitrospira defluvii. APAP was removed in nitrifying activated sludge via largely the biotransformation route. Both nitrifiers and heterotrophic microorganisms contributed to overall APAP removal. An AS bioreactor was acclimated to 100 mg/L APAP as the sole source of carbon, nitrogen, and energy to enrich the microbial community with APAP-metabolizing heterotrophs. During acclimation, dynamic changes in community phenotypes occurred with significant reduction in species richness and diversity. Community acclimation significantly increased APAP biotransformation rates. 16S rRNA gene-based community profiling showed selective enrichment for Pseudomonas and Sphingomonas, both with demonstrated APAP metabolic capacity.

Cultivation and Transcriptional Analysis of a Canonical Nitrospira Under Stable Growth Conditions.

Nitrite-oxidizing bacteria (NOB) are vital players in the global nitrogen cycle that convert nitrite to nitrate during the second step of nitrification. Within this functional guild, members of the genus Nitrospira are most widespread, phylogenetically diverse, and physiologically versatile, and they drive nitrite oxidation in many natural and engineered ecosystems. Despite their ecological and biotechnological importance, our understanding of their energy metabolism is still limited. A major bottleneck for a detailed biochemical characterization of Nitrospira is biomass production, since they are slow-growing and fastidious microorganisms. In this study, we cultivated Nitrospira moscoviensis under nitrite-oxidizing conditions in a continuous stirred tank reactor (CSTR) system. This cultivation setup enabled accurate control of physicochemical parameters and avoided fluctuating levels of their energy substrate nitrite, thus ensuring constant growth conditions and furthermore allowing continuous biomass harvesting. Transcriptomic analyses under these conditions supported the predicted core metabolism of N. moscoviensis, including expression of all proteins required for carbon fixation via the reductive tricarboxylic acid cycle, assimilatory nitrite reduction, and the complete respiratory chain. Here, simultaneous expression of multiple copies of respiratory complexes I and III suggested functional differentiation. The transcriptome also indicated that the previously assumed membrane-bound nitrite oxidoreductase (NXR), the enzyme catalyzing nitrite oxidation, is formed by three soluble subunits. Overall, the transcriptomic data greatly refined our understanding of the metabolism of Nitrospira. Moreover, the application of a CSTR to cultivate Nitrospira is an important foundation for future proteomic and biochemical characterizations, which are crucial for a better understanding of these fascinating microorganisms.

Anthropogenic impact on nitrification dynamics in coastal waters of the Mediterranean Sea

The anthropogenic alteration of the nitrogen cycle results in the modification of the whole food web. And yet, the impact caused on nitrogen dynamics in marine systems is still very uncertain. We propose a workflow to evaluate changes to coastal nitrification by modelling nitrite dynamics, the intermediary compound. Nitrite concentrations were estimated with a simple steady state nitrification model, which was calibrated in 9 NW Mediterranean coastal sites with different anthropogenic pressures, located within 250 km. The results obtained indicate that nitrite peaks are observed in winter and explained by nitrification response to temperature, but these dynamics are altered in impacted coastal waters. We found the second step of nitrification to be more sensitive to temperature, which entails a significant impact of climate change on the decoupling of the two steps of nitrification. The results could be extrapolated to numerous coastal regions of the Mediterranean Sea with similar characteristics.

Optimization of the medium for the growth of Nitrobacter winogradskyi by statistical method.

Due to the extremely slow growth rate and the low growth yield of ammonia-oxidizing bacteria and NOB (nitrite-oxidizing bacteria), nitrification is still the rate-limiting step of nitrogen cycle in the current research. Nitrobacter winogradskyi, an important NOB, participates in the second step of nitrification in water and soil. This study reported an optimized culture condition for N.winogradskyi, which increased the growth yield by 5·06 times than that in the basal medium and uncovered the potential mechanism. We expect our study will contribute to the research on water and soil nitrogen cycle. In addition, the optimized culture conditions have the potential to be suitable for the chemoorganotrophic growth of other nitrifiers.

Shifts in Nitrobacter- and Nitrospira-like nitrite-oxidizing bacterial communities under long-term fertilization practices

Nitrite-oxidizing bacteria (NOB) are key players in the second step of nitrification, which is an important process in the soil nitrogen (N) cycle. However, the ecology of nitrite oxidizers and their response to disturbances such as long-term fertilization practices are scarcely known in agricultural ecosystems. We used samples from a Red soil subject to a long-term chemical and organic fertilization experiment, including control without fertilizer (CK), swine manure (M), chemical fertilization (NPK), and chemical/manure combined fertilization (MNPK) treatment, to explore how agricultural practices impact the community structure, abundance, and potential activity of nitrite oxidizers (PNO). The abundance of Nitrobacter was significantly increased in the M and MNPK plots, whereas the abundance of Nitrospira was significantly reduced in the M and NPK treatment plots and less inhibited in the MNPK treatment. The PNO showed a similar trend to that for Nitrobacter abundance. The diversity of Nitrobacter increased in the M-treated plots, while that of Nitrospira increased in the M and MNPK plots and decreased in the NPK plots. Non-metric multidimensional scaling (NMDS) revealed that the Nitrobacter- and Nitrospira-like NOB community was shift in these four fertilization treatments. Redundancy analysis showed that pH+SOC (soil organic carbon) and pH+TN (total nitrogen) significantly explained the variation in the composition of Nitrobacter and Nitrospira, respectively. In addition, the Nitrospira/Nitrobacter abundance ratio and community structure of Nitrobacter- and Nitrospira-like NOB are responsible for the changes of soil PNO. Collectively, these data suggest that the nitrite-oxidation process in the red soil is possibly controlled by both Nitrospira and Nitrobacter-like NOB, which were shaped by pH+TN and pH+SOC, respectively.

Nitrite-Oxidizing Bacteria Community Composition and Diversity Are Influenced by Fertilizer Regimes, but Are Independent of the Soil Aggregate in Acidic Subtropical Red Soil.

Nitrification is the two-step aerobic oxidation of ammonia to nitrate via nitrite in the nitrogen-cycle on earth. However, very limited information is available on how fertilizer regimes affect the distribution of nitrite oxidizers, which are involved in the second step of nitrification, across aggregate size classes in soil. In this study, the community compositions of nitrite oxidizers (Nitrobacter and Nitrospira) were characterized from a red soil amended with four types of fertilizer regimes over a 26-year fertilization experiment, including control without fertilizer (CK), swine manure (M), chemical fertilization (NPK), and chemical/organic combined fertilization (MNPK). Our results showed that the addition of M and NPK significantly decreased Nitrobacter Shannon and Chao1 index, while M and MNPK remarkably increased Nitrospira Shannon and Chao1 index, and NPK considerably decreased Nitrospira Shannon and Chao1 index, with the greatest diversity achieved in soils amended with MNPK. However, the soil aggregate fractions had no impact on that alpha-diversity of Nitrobacter and Nitrospira under the fertilizer treatment. Soil carbon, nitrogen and phosphorus in the soil had a significant correlation with Nitrospira Shannon and Chao1 diversity index, while total potassium only had a significant correlation with Nitrospira Shannon diversity index. However, all of them had no significant correlation with Nitrobacter Shannon and Chao1 diversity index. The resistance indices for alpha-diversity indexes (Shannon and Chao1) of Nitrobacter were higher than those of Nitrospira in response to the fertilization regimes. Manure fertilizer is important in enhancing the Nitrospira Shannon and Chao1 index resistance. Principal co-ordinate analysis revealed that Nitrobacter- and Nitrospira-like NOB communities under four fertilizer regimes were differentiated from each other, but soil aggregate fractions had less effect on the nitrite oxidizers community. Redundancy analysis and Mantel test indicated that soil nitrogen, carbon, phosphorus, and available potassium content were important environmental attributes that control the Nitrobacter- and Nitrospira-like NOB community structure across different fertilization treatments under aggregate levels in the red soil. In general, nitrite-oxidizing bacteria community composition and alpha-diversity are depending on fertilizer regimes, but independent of the soil aggregate.

Oxidation kinetics and inverse isotope effect of marine nitrite-oxidizing isolates

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 80:289-300 (2017) - DOI: https://doi.org/10.3354/ame01859 Oxidation kinetics and inverse isotope effect of marine nitrite-oxidizing isolates Juliane Jacob1,2,*, Boris Nowka3, Véronique Merten4, Tina Sanders1, Eva Spieck3, Kirstin Dähnke1 1Helmholtz Center Geesthacht, Institute of Coastal Research, Max-Planck-Str. 1, 21502 Geesthacht, Germany 2Institute of Biogeochemistry and Marine Chemistry, University of Hamburg, Bundesstr. 55, 20146 Hamburg, Germany 3Biozentrum Klein Flottbek, Department of Microbiology and Biotechnology, University of Hamburg, Ohnhorststr. 18, 22609 Hamburg, Germany 4GEOMAR Helmholtz Center for Ocean Research Kiel, Wischhofstr. 1-3, 24148 Kiel, Germany *Corresponding author: juliane.jacob@uni-hamburg.de ABSTRACT: Nitrification, the step-wise oxidation of ammonium to nitrite and nitrate, is important in the marine environment because it produces nitrate, the most abundant marine dissolved inorganic nitrogen (DIN) component and N-source for phytoplankton and microbes. This study focused on the second step of nitrification, which is carried out by a distinct group of organisms, nitrite-oxidizing bacteria (NOB). The growth of NOB is characterized by nitrite oxidation kinetics, which we investigated for 4 pure cultures of marine NOB (Nitrospina watsonii 347, Nitrospira sp. Ecomares 2.1, Nitrococcus mobilis 231, and Nitrobacter sp. 311). We further compared the kinetics to those of non-marine species because substrate concentrations in marine environments are comparatively low, which likely influences kinetics and highlights the importance of this study. We also determined the isotope effect during nitrite oxidation of a pure culture of Nitrospina (Nitrospina watsonii 347) belonging to one of the most abundant marine NOB genera, and for a Nitrospira strain (Nitrospira sp. Ecomares 2.1). The enzyme kinetics of nitrite oxidation, described by Michaelis-Menten kinetics, of 4 marine genera are rather narrow and fall in the low end of half-saturation constant (Km) values reported so far, which span over 3 orders of magnitude between 9 and >1000 µM NO2-. Nitrospina has the lowest Km (19 µM NO2-), followed by Nitrobacter (28 µM NO2-), Nitrospira (54 µM NO2-), and Nitrococcus (120 µM NO2-). The isotope effects during nitrite oxidation by Nitrospina watsonii 347 and Nitrospira sp. Ecomares 2.1 were 9.7 ± 0.8 and 10.2 ± 0.9‰, respectively. This confirms the inverse isotope effect of NOB described in other studies; however, it is at the lower end of reported isotope effects. We speculate that differences in isotope effects reflect distinct nitrite oxidoreductase (NXR) enzyme orientations. KEY WORDS: Nitrification · Nitrite-oxidizing bacteria · Isotope effect · Enzyme kinetics · Marine environment Full text in pdf format PreviousCite this article as: Jacob J, Nowka B, Merten V, Sanders T, Spieck E, Dähnke K (2017) Oxidation kinetics and inverse isotope effect of marine nitrite-oxidizing isolates. Aquat Microb Ecol 80:289-300. https://doi.org/10.3354/ame01859 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 80, No. 3. Online publication date: December 13, 2017 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2017 Inter-Research.

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.

Improved isolation strategies allowed the phenotypic differentiation of two Nitrospira strains from widespread phylogenetic lineages.

The second step of nitrification, the oxidation of nitrite to nitrate, is vital for the functioning of the nitrogen cycle, but our understanding of the ecological roles of the involved microorganisms is still limited. The known diversity of Nitrospira, the most widely distributed nitrite-oxidizing bacteria, has increased remarkably by analyses of 16S rRNA and functional gene sequences. However, only few representatives could be brought into laboratory cultures so far. In this study, two Nitrospira from activated sludge were isolated using novel approaches together with established methods. Highly enriched 'Candidatus Nitrospira defluvii' was separated from concomitant heterotrophs by taking advantage of its resistance against ampicillin and acriflavine. Beside this member of lineage I, a novel species of lineage II, named N. lenta, was initially enriched at 10°C and finally purified by using optical tweezers. The tolerance to elevated nitrite levels was much higher in N. defluvii than in the more fastidious N. lenta and was accompanied by pronounced biofilm formation. Phylogenetic classification of 12 additional enrichments indicated that Nitrospira lineage I is common in extreme and moderate ecosystems like lineage II. The new cultures will help to explore physiological and genomic differences affecting niche separation between members of this highly diverse genus.

Functionally relevant diversity of closely related Nitrospira in activated sludge.

Nitrospira are chemolithoautotrophic nitrite-oxidizing bacteria that catalyze the second step of nitrification in most oxic habitats and are important for excess nitrogen removal from sewage in wastewater treatment plants (WWTPs). To date, little is known about their diversity and ecological niche partitioning within complex communities. In this study, the fine-scale community structure and function of Nitrospira was analyzed in two full-scale WWTPs as model ecosystems. In Nitrospira-specific 16S rRNA clone libraries retrieved from each plant, closely related phylogenetic clusters (16S rRNA identities between clusters ranged from 95.8% to 99.6%) within Nitrospira lineages I and II were found. Newly designed probes for fluorescence in situ hybridization (FISH) allowed the specific detection of several of these clusters, whose coexistence in the WWTPs was shown for prolonged periods of several years. In situ ecophysiological analyses based on FISH, relative abundance and spatial arrangement quantification, as well as microautoradiography revealed functional differences of these Nitrospira clusters regarding the preferred nitrite concentration, the utilization of formate as substrate and the spatial coaggregation with ammonia-oxidizing bacteria as symbiotic partners. Amplicon pyrosequencing of the nxrB gene, which encodes subunit beta of nitrite oxidoreductase of Nitrospira, revealed in one of the WWTPs as many as 121 species-level nxrB operational taxonomic units with highly uneven relative abundances in the amplicon library. These results show a previously unrecognized high diversity of Nitrospira in engineered systems, which is at least partially linked to niche differentiation and may have important implications for process stability.

Nitrogen removal via the nitrite pathway during wastewater co-treatment with ammonia-rich landfill leachates in a sequencing batch reactor

The biological treatment of ammonia-rich landfill leachates due to an inadequate C to N ratio requires expensive supplementation of carbon from an external carbon source. In an effort to reduce treatment costs, the objective of the study was to determine the feasibility of nitrogen removal via the nitrite pathway during landfill leachate co-treatment with municipal wastewater. Initially, the laboratory-scale sequencing batch reactor (SBR) was inoculated with nitrifying activated sludge and fed only raw municipal wastewater (RWW) during a start-up period of 9 weeks. Then, in the co-treatment period, consisting of the next 17 weeks, the system was fed a mixture of RWW and an increasing quantity of landfill leachates (from 1 to 10 % by volume). The results indicate that landfill leachate addition of up to 10 % (by volume) influenced the effluent quality, except for BOD5. During the experiment, a positive correlation (r 2 = 0.908) between ammonia load in the influent and nitrite in the effluent was observed, suggesting that the second step of nitrification was partially inhibited. The partial nitrification (PN) was also confirmed by fluorescence in situ hybridisation (FISH) analysis of nitrifying bacteria. Nitrogen removal via the nitrite pathway was observed when the oxygen concentration ranged from 0.5 to 1.5 mg O2/dm3 and free ammonia (FA) ranged from 2.01 to 35.86 mg N-NH3/dm3 in the aerobic phase. Increasing ammonia load in wastewater influent was also correlated with an increasing amount of total nitrogen (TN) in the effluent, which suggested insufficient amounts of assimilable organic carbon to complete denitrification. Because nitrogen removal via the nitrite pathway is beneficial for carbon-limited and highly ammonia-loaded mixtures, obtaining PN can lead to a reduction in the external carbon source needed to support denitrification.