Growth Of Ammonia-oxidizing Bacteria Research Articles

Sealing solid agar in serum bottles for rapid isolation and long-term preservation of chemoautotrophic ammonia-oxidizing bacteria

Ammonia-oxidizing bacteria (AOB) are ubiquitous on the earth and have broad applications in bioremediation. However, the number of their species with standing in nomenclature and deposited in Microbial Culture Collections still remains low. Moreover, only a few novel species have been reported over the last decades. In this study, we sealed agar in serum bottles to develop a kind of solid agar plate with the oxygen concentration in the headspace maintained at low levels. By using these plates, eight AOB isolates including two novel species were obtained. When AOB cells were grown on the sealed solid agar plates, the time to form visible colonies was largely reduced and the maximum diameter of colonies reached 2 mm, which makes the process of AOB isolation rapid and efficient. Based on five AOB isolates, the headspace oxygen concentration had a significant influence on AOB growth either on solid plate or in liquid culture. Especially, when grown under 21 % O2, the number of colonies formed on solid agar plates was very low and sometimes no visible colony formed. Besides the application on AOB isolation, the sealed solid agar plate was also effective for the enumeration and preservation of AOB cells. When preserved under room temperature for more than ten months, the AOB colonies on the plate could still be recovered. This method provides a feasible way to isolate more novel AOB species from the environment and deposit more species in Microbial Culture Collections.

Insights into the effects of exogenous folate on the recovery of SPNA system after starvation: Performance, microbial community and metabolism

To investigate the effects of folate addition on the recovery process of the single-stage partial nitritation-anammox (SPNA) system, two continuous stirred tank reactors (CSTRs), named R1 (adding folate) and R2 (control group), were operated for 105 days at room temperatures. The highest nitrogen removal rate (NRR) observed in R1 was 0.47 kg N/(m3·d) with the addition of 3.0 mg/L folate. Additionally, the activities of HZO and HZS in R1 were higher than those in R2. The relative abundance of Candidatus Brocadia reached 23.7% in R1 and 18.2% in R2. Moreover, the relative abundance of nitrogen metabolism genes and carbon metabolism genes increased with folate addition. However, 3.0 mg/L folate addition reduced the relative abundance of nirK/S and the activity and growth of ammonia-oxidizing bacteria, which was not conducive to continually improving the NRR. This study provided a novel perspective on improving the activity and abundance of anammox bacteria during the restoration process.

Developing antibiotics-based strategies to efficiently enrich ammonia-oxidizing archaea from wastewater treatment plants

The effects of five antibiotics (i.e., ampicillin, streptomycin, carbenicillin, kanamycin and tetracycline) on ammonia-oxidizing archaea (AOA) enrichment from anoxic activated sludge were investigated. The combined use of five antibiotics during 90-day cultivation could selectively inhibit nitrite-oxidizing bacteria (NOB) and ammonia-oxidizing bacteria (AOB) with AOA unaffected, as evidenced by the nitrite accumulation ratio of 100 % and the proportion of AOA in ammonia-oxidizing microbes over 91 %. The alternative use of five antibiotics was the optimal approach to screening for AOA during 348-day cultivation, which inhibited AOB growth at a level equivalent to the combined use of five antibiotics (the AOB-amoA gene decreased by over 99.90 %), further promoted AOA abundance (the much higher AOA-amoA to AOB-amoA gene copy number ratio (1453.30) than that in the groups with the combined use of five antibiotics (192.94)), eliminated bacterial adaptation to antibiotics and reduced antibiotic-resistant bacteria to form Nitrocosmicus-dominant community (42.35 % in abundance).

Partial nitrification response to dissolved oxygen variation and aerobic starvation: Kinetics and microbial community analyses

Partial nitrification (PN) has great significance in promoting both anaerobic ammonium oxidation (anammox) and advanced nitrogen removal. However, the kinetics of PN have not been sufficiently studied. Deterioration of PN often occurs due to nitrite-oxidizing bacteria (NOB) outgrowth, for which the dissolved oxygen (DO) is a critical factor. This study investigated the kinetic parameters of ammonia-oxidizing bacteria (AOB) and NOB by varying the DO concentration. The partial nitrification-anammox (PNA) process was operated under sequentially low (0.3–0.8 mg/L) and high (4.0–5.0 mg/L) DO. The results show that AOB could not adapt to low DO, resulting in a significant decrease in the maximum AOB activity (Vmax,AOB) and a slightly altered oxygen half-saturation constant of AOB (KO,AOB). Conversely, KO,NOB decreased and Vmax,NOB maintained, indicating that the main NOB genus, Nitrospira, was more adaptable to DO changes. Under high DO, AOB growth was weaker than NOB, leading to a low Vmax,AOB – Vmax,NOB value; KO,AOB increased and was larger than KO,NOB and PNA was eventually destroyed. The aerobic starvation was then adopted to restore PN. AOB recovered faster and dominated, with Vmax,AOB greatly exceeding Vmax,NOB. Finally, NOB were inhibited and PN was recovered. During this process, KO,NOB increased and was higher than KO,AOB. Changes in the kinetic parameters, especially the Vmax,AOB -Vmax,NOB value, have specific guiding significance for DO regulation during PNA. This work helps better understand the effect of DO variations on the nitrifying microbial community, which influences oxygen affinity and maximum activity.

Influence of Light Irradiation on Nitrification in Microalgal–Bacterial Systems for Treating Wastewater

The use of bacterial and microalgal consortia to remove nitrogen from wastewater has garnered attention as a potential alternative to conventional systems. This approach not only reduces energy consumption but also aids in nutrient recovery. Light is essential for algae photosynthesis; however, nitrifying bacteria are also influenced by light radiation. This mini-review summarizes the current knowledge concerning photoinhibition, the light stimulation of ammonia-oxidizing bacteria (AOB), resistance to light radiation, the implementation of microalgal–bacterial systems, and the possible mechanisms involved. Nitrosomonadaceae AOB and Nitrospiraceae nitrite-oxidizing bacteria (NOB) often coexist in a microalgal–bacterial system. Studies have suggested that AOB can tolerate light radiation at 200 μmol m−2·s−1 in microalgal–bacterial systems, whereas NOB are almost completely suppressed, which can result in partial nitrification in the bioreactor. An appropriate light level can stimulate AOB growth in microalgal–bacterial granular reactors and may improve algae metabolic activity. Granular sludges or artificial “light-shielding hydrogel” could effectively protect nitrifying bacteria from light intensities up to 1600 μmol m−2·s−1 in wastewater treatment reactors. Microalgal–bacterial systems along with the associated “algal shading effect” have been widely used in pond aquaculture. This approach minimizes the need for costly mechanical aeration through photo-oxygenation and facilitates nutrient recovery by filter-feeding fish.

Contribution of ammonia-oxidizing archaea and bacteria to nitrogen transformation in a soil fertilized with urea and organic amendments

The contribution of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) is crucial for nitrogen transformation. The effects of four organic amendments (OAs) plus urea on soil nitrogen transformation and the contribution of the ammonia-oxidizing microbial community were investigated using an incubation experiment. The OAs plus urea treatments included pig manure plus urea (PM + U), wheat straw plus urea (WS + U), compost plus urea (CP + U) and improved-compost plus urea (IC + U), while no OAs and urea amended control was noted as CK. The abundance and composition of AOA and AOB were determined using high through-put sequencing. Compared with CK, the OA plus urea treatments significantly enhanced the amount of total mineralized nitrogen released during the incubation process. After incubation, the highest mineralized nitrogen and net nitrogen mineralization was under the PM + U treatment and the lowest was in the WS + U treatment. In conclusion, among all OA plus urea treatments, the microbial biomass nitrogen content was the highest in WS + U treatment and dissolved organic nitrogen content was the highest with the PM + U treatment. Additionally, the abundance of AOB was inhibited in comparison to that of AOA; however, AOB contributed more to nitrification than AOA. Soil NO3−-N and dissolved organic nitrogen were the principal components influencing the distribution of AOA and AOB. The result illustrated that the OAs plus urea, especially PM plus urea promoted mineralization to produce more dissolved organic nitrogen and NH4+-N, thus accelerating the growth of AOB to strengthen nitrification in soil.

Achieving partial nitrification: A strategy for washing NOB out under high DO condition

This study investigated the effect of high DO concentrations on PN. The experimental setup involved operating at high DO concentrations (1.5–2.5 mg/L) and environmental temperatures (15–20 °C) over a period of 180 days. Through a sludge enrichment process, the kinetic parameters of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were determined. Surprisingly, contrary to conventional reports, it was observed that NOB exhibited a stronger affinity for DO compared to AOB. As a result, high DO concentrations were necessary to provide favorable conditions for the growth of AOB. In order to establish PN, strategies including intermittent aeration, free ammonia (FA), and controlled sludge retention time (SRT) were employed. The successful PN was achieved with a specific ammonia oxidation rate of 24 mg N/g MLVSS/h and a specific nitrite oxidation rate below 0.10 mg N/g MLVSS/h. The nitrite accumulation rate (NAR) was maintained at 100% during stable operation. The abundance of Nitrosomonas, a typical genus of AOB, was found to be 68.62%, which surpasses previous studies in similar research. A slightly higher DO concentrations may increase energy consumption, but achieve higher efficiency and stability in PN. This study provided new insights into the application of PN in wastewater treatment.

Reassembly of active ammonia oxidizing bacteria following invasion of exogenous microbiome in an acidic soil

Ammonia oxidation is the rate-limiting step of the global nitrogen cycle, and it is mainly catalyzed by ammonia-oxidizing bacteria (AOB) and archaea (AOA). AOA can carry out nitrification even in acidic soils, whereas AOB activity was not consistently observed in acidic soils. In this study, we manipulated two environmental variables, i.e., the microbial composition and ammonium source (urea and ammonium sulphate), in an acidic forest soil and tested their impacts on AOB abundance, diversity and activity. Soil microcosms were established either using the native forest soil (FS) or after amending the forest soil with 5% (w/w) agricultural soil (AFS) to inoculate new, exogenous microbes. Under the same microcosm incubation condition for 28 days, AOB did not grow in FS but they grew in AFS, suggesting that exogenous microbial invasion could stimulate AOB activity in the acidic soil. Urea amendment led to higher growth of AOB than ammonium sulphate amended in AFS microcosms. DNA-based stable isotope probing revealed that the active AOB lineages were dominated by Nitrosospira cluster 3a.2 and Nitrosospira cluster 10 in the ammonium sulphate supplied soil, but shifted to co-dominance of Nitrosospira cluster 3a.2 and Nitrosospira cluster 1 after urea amendment, demonstrating differential preferences of ammonia sources of phylogenetically distinct AOB lineages. Additionally, AOB abundance showed lower stability than AOA in soils subjected to these two environmental changes, suggesting AOB are more important in determining the fate of soil nitrification following short-term environmental disturbances.

Predominance of comammox bacteria among ammonia oxidizers under low dissolved oxygen condition

Although low-oxygen nitrification can significantly cut down the aeration demand in wastewater treatment plants, little is known about the community dynamics of relevant microorganisms under different oxygen concentrations. Here, by conducting a series of bioreactors with oxygen concentrations of 0%, 2%, 5%, 10%, 20%, 40%, and 70%, we provided a comprehensive investigation on the behaviors and performances of comammox bacteria (CMX), ammonia-oxidizing bacteria (AOB) and archaea (AOA) during the nitrification process. Quantitative PCR analysis demonstrated that CMX was the dominant ammonia-oxidizer under low oxygen condition (10%) after the four-month operation with the abundance increased by 8.65 times higher than the initial operation, whereas the growth of AOA and AOB was inhibited. Moreover, Nitrospira nitrosa dominated the CMX species (relative abundance >96%) in low dissolved oxygen concentrations, while Nitrospira nitrificans (3.39%) seemed to prefer high oxygen conditions. Our study indicates the long-term effects of oxygen concentrations on the niche differentiation of ammonia oxidizers, and highlights the significance of CMX in low-oxygen nitrification for reducing global carbon emission and energy consumption.

Dynamic calibration of process-wide partial-nitritation modeling with airlift granular for nitrogen removal in a full-scale wastewater treatment plant

A main challenge in rapid nitrogen removal from rejected water in wastewater treatment plants (WWTPs) is growth of biomass by nitrite-oxidizing bacteria (NOB) and ammonia-oxidizing bacteria (AOB). In this study, partial nitritation (PN) coupled with air-lift granular unit (AGU) technology was applied to enhance nitrogen-removal efficiency in WWTPs. For successful PN process at high-nitrogen-influent conditions, a pH of 7.5–8 for high free-ammonia concentrations and AOB for growth of total bacterial populations are required. The PN process in a sequential batch reactor (SBR) with AGU was modeled as an activated sludge model (ASM), and dynamic calibration using full-scale plant data was performed to enhance aeration in the reactor and improve the nitrite-to-ammonia ratio in the PN effluent. In steady-state and dynamic calibrations, the measured and modeled values of the output were in close agreement. Sensitivity analysis revealed that the kinetic and stoichiometric parameters are associated with growth and decay of heterotrophs, AOB, and NOB microorganisms. Overall, 80% of the calibrated data fit the measured data. Stage 1 of the dynamic calibration showed NO2 and NO3 values close to 240 mg/L and 100 mg/L, respectively. Stage 2 showed NH4 values of 200 mg/L at day 30 with the calibrated effluent NO2 and NO3 value of 250 mg/L. In stage 3, effluent NH4 concentration was 200 mg/L at day 60.

Effects of flow mode on the performance and microbial community in tidal flow constructed wetland treating mariculture wastewater

This study aimed to investigate the influence of flow mode (down-flow and up-flow) on the removal of organic matter and nitrogen, and their transformation processes in two tidal flow constructed wetlands (TFCWs) fed with simulated mariculture wastewater with 2.4% salinity and wastewater without adding salt (0.0% salinity). Down-flow mode was beneficial for COD removal (98.28–99.62%), while up-flow mode improved the denitrification capacity and benefited the removal of total inorganic nitrogen (TIN) (81.46–88.26%). High salinity decreased the NH4+-N removal (70.42–73.05%) compared with 0.0% salinity (80.57–86.04%), and lead to higher NO2−-N accumulation. High-thought sequencing indicated flow mode, salinity and depth along the TFCWs together alter the composition and abundance of functional bacteria in the wetlands. Higher abundance of ammonia oxidizing bacteria (AOB) was observed under down-flow mode (0.3–3.7%) compared with up-flow mode (0.2–0.9%) in two TFCWs. Denitrifying bacteria (DNB) enriched in 38–43 cm depth layer in saline system and 0–5 cm depth layer in freshwater system, with the relative abundance of 18.3–63.8%. High salinity inhibited the growth of AOB and nitrite oxidizing bacteria (NOB), while DNB could adapt to salinity stress and maintained high abundance in saline system. The enrichment of halotolerant/halophilic bacteria and diverse nitrogen removal pathways provided a solid foundation for the efficient nitrogen removal in TFCW treating mariculture wastewater.

Effects of Nitrification Inhibitors on Nitrogen Dynamics and Ammonia Oxidizers in Three Black Agricultural Soils

The application of nitrification inhibitors (NIs) based on ammonium (NH4+) is considered to be an efficient way to reduce nitrogen (N) loss by delaying the nitrification process through influencing ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). However, the inhibition mechanisms of NIs on AOA and AOB in different soils remain unclear. Hence, we explored the two commonly used NIs (3, 4-dimethylepyrazole phosphate (DMPP) and dicyandiamide (DCD) and their combination (DMPP + DCD) on the soil nitrification and abundance of ammonia oxidizers (AOA and AOB) in three black soils (HLJ, NA, and DA) with different physicochemical properties using a 120-day incubation experiment. The results demonstrated that NIs significantly increased NH4+-N concentrations and decreased NO3−-N concentrations in all three tested soils. There was no significant difference in inhibiting nitrification in HLJ among all NI treatments, while DCD was more efficient in NA, DMPP + DCD had better efficiency in DA. The potential nitrification rate (PNR) was greatly decreased by NIs addition, and PNR was significantly positively correlated with AOB (p < 0.05). AOA was dominant in the acid soil. All NI treatments significantly inhibited soil nitrification through inhibiting the growth of AOB in the two soils with higher pH. The abundance of AOA and AOB was significantly correlated with different soil types (positively correlated with soil pH, and negatively correlated with organic matter). Moreover, soil pH and soil organic matter were considered to be the most important factors influencing the inhibition efficiency of NIs and the abundance of AOA and AOB. The application of the NIs combination (DMPP + DCD) was considered to be the most cost-effective way to inhibit soil nitrification in soil with higher pH and lower SOM, which provides a theoretical basis for a field experiment.

Light Irradiation Enables Rapid Start-Up of Nitritation through Suppressing nxrB Gene Expression and Stimulating Ammonia-Oxidizing Bacteria.

Nitritation facilitates the application of anaerobic ammonium oxidation (Anammox)-based processes for cost-efficient nitrogen removal from wastewater. This study proposed light irradiation as a novel strategy to rapidly start up nitritation by stimulating both the activities and growth of ammonia-oxidizing bacteria (AOB) while suppressing that of nitrite-oxidizing bacteria (NOB). Batch assays and kinetic model jointly suggested that AOB activity presented an initial increase followed by a decline while NOB decreased continuously throughout the light energy densities applied. Under optimal light energy densities (0.03-0.08 kJ/mg VSS), the highest nitrite accumulation ratio of 70.0% was achieved in sequencing batch reactors with both mainstream online and sidestream offline light treatments when treating real or synthetic municipal wastewater. Light irradiation induced different responses of AOB and NOB, leading to microbial structure optimization. Specifically, the expression of nxrB was downregulated, while the expression of amoA was upregulated under appropriate light irradiation. Moreover, although Nitrosomonas as typical AOB disappeared, the family Nitrosomonadaceae was doubled with enrichment of Ellin6067 and another four Nitrosomonadaceae genera that were only identified in light-treated reactors, thus ensuring AOB predominance and stable nitritation. These findings offer a new approach to rapidly establishing nitritation using light irradiation in municipal wastewater, especially for nitritation/microalgae system.

Ammonia Nitrogen Removal Performance with Parallel Operation of Conventional and Inverted A2/O Sewage Treatment Processes in Winter

Ammonia nitrogen (NH4+-N) removal capacities of the A2/O and inverted A2/O processes were analyzed with the same inlet and parallel operation during winter. When the operating water temperature was 14℃, the inverted A2/O process exhibited lower NH4+-N removal from the volumetric load[0.13 kg ·(m3 ·d)-1vs. 0.29 kg ·(m3 ·d)-1] and a lower ammonia oxidation rate (AOR)[0.07 kg ·(kg ·d)-1 vs. 0.11 kg ·(kg ·d)-1] than the A2/O process, whereas the two processes exhibited similar performance at 26℃.The quantitative results for the ammonia oxidizing bacteria (AOB) population were almost the same in the two parallel processes (3.2%±0.24% for the inverted A2/O process and 3.4%±0.31% for the A2/O process). Clone library analysis showed that at low temperatures, the inverted A2/O process had a lower capacity for ammonia nitrogen removal than A2/O process. This is because the particular AOB species[spirillum (Nitrosospira)] facilitated the slower AOR type (K-growth strategy) of nitrosation in the inverted A2/O process, whereas in the A2/O process, the faster AOR type (r-growth strategy) of nitrosation was facilitated by bacterium (Nitrosomonas). At 26℃, the dominant species in the two processes were Nitrosomonas. Through comprehensive analysis of the pollutants during the removal process, we found that although temperature is the leading cause of AOB advantage in species succession, the changes in the inverted A2/O process structure, caused by the aerobic unit, resulted in high COD load and high NH4+-N concentration, which were unfavorable for the growth of AOB. This shows that under conventional sewage conditions, the K-growth strategy is advantageous for the AOB species. Therefore, the structure of the inverted A2/O process for heterotrophic bacteria (phosphorus accumulating bacteria and denitrifying bacteria) indirectly affects the population distribution and succession of autotrophic ammonia-oxidizing bacteria, through COD load and other factors, thereby leading to weakened nitrification capacity at low temperatures.

Application of Anammox-Based Processes in Urban WWTPs: Are We on the Right Track?

The application of partial nitritation and anammox processes (PN/A) to remove nitrogen can improve the energy efficiency of wastewater treatment plants (WWTPs) as well as diminish their operational costs. However, there are still several limitations that are preventing the widespread application of PN/A processes in urban WWTPs such as: (a) the loss of performance stability of the PN/A units operated at the sludge line, when the sludge is thermally pretreated to increase biogas production; (b) the proliferation of nitrite-oxidizing bacteria (NOB) in the mainstream; and (c) the maintenance of a suitable effluent quality in the mainstream. In this work, different operational strategies to overcome these limitations were modelled and analyzed. In WWTPs whose sludge is thermically hydrolyzed, the implementation of an anerobic treatment before the PN/A unit is the best alternative, from an economic point of view, to maintain the stable performance of this unit. In order to apply the PN/A process in the mainstream, the growth of ammonia-oxidizing bacteria (AOB) should be promoted in the sludge line by supplying extra sludge to the anaerobic digesters. The AOB generated would be applied to the water line to partially oxidize ammonia, and the anammox process would then be carried out. Excess nitrate generated by anammox bacteria and/or NOB can be removed by recycling a fraction of the WWTP effluent to the biological reactor to promote its denitrification.

Molecular analysis of microbial nitrogen transformation and removal potential in mangrove wetlands under anthropogenic nitrogen input

Mangrove ecosystems are natural nitrogen removal systems that are primarily mediated by nitrogen cycle microorganisms, but their relative contributions to nitrogen transformation and removal in mangrove sediments under anthropogenic nitrogen input needs further resolution and characterization. Here, we investigated the responses and the relative contributions of ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), anaerobic ammonium oxidizing (anammox) bacteria and denitrifying bacteria after spiking urea into mangrove sediments incubated in a laboratory microcosm experiment for four weeks. During incubation, the diversity, abundances and transcription levels of the hzo genes for anammox bacteria, amoA genes for AOA and AOB, and nirS genes for denitrifying bacteria were monitored using targeted gene clone library analyses and quantitative PCR assays at the DNA and RNA levels. The results showed that mangrove sediments harbour habitat-specific anammox bacteria which related to Candidatus Scalindua and Candidatus Kuenenia clades. Mangrove specific AOA related to deep branched clades within Candidatus Nitrososphaera and Candidatus Nitrosotalea, and AOB related to Nitrosomonas and Nitrosospira were also detected in the collected sediment samples. Growth and activity of AOA were detected at all levels of amendment of nitrogen input, whereas AOB growth was detectable only at the high-level nitrogen input (1.5 mg urea per gram of dry sediment) with no amoA transcripts and lower abundance than AOA. The abundance and transcription levels of the nirS gene were higher (~1000 times) than those of the hzo gene in all groups. Pearson correlation analysis demonstrated that the abundance of both AOA and AOB amoA genes had a significant positive correlation with the nirS gene (p < 0.01). These results indicated that nitrification (primarily mediated by the AOA)-denitrification process played the most important role in nitrogen removal from the amendment of nitrogen short-term input in the mangrove sediments.

Comparison of Novel and Established Nitrification Inhibitors Relevant to Agriculture on Soil Ammonia- and Nitrite-Oxidizing Isolates.

Nitrification inhibitors (NIs) applied to soil reduce nitrogen fertilizer losses from agro-ecosystems. NIs that are currently registered for use in agriculture appear to selectively inhibit ammonia-oxidizing bacteria (AOB), while their impact on other nitrifiers is limited or unknown. Ethoxyquin (EQ), a fruit preservative shown to inhibit ammonia-oxidizers (AO) in soil, is rapidly transformed to 2,6-dihydro-2,2,4-trimethyl-6-quinone imine (QI), and 2,4-dimethyl-6-ethoxy-quinoline (EQNL). We compared the inhibitory potential of EQ and its derivatives with that of dicyandiamide (DCD), nitrapyrin (NP), and 3,4-dimethylpyrazole-phosphate (DMPP), NIs that have been used in agricultural settings. The effect of each compound on the growth of AOB (Nitrosomonas europaea, Nitrosospira multiformis), ammonia-oxidizing archaea (AOA; “Candidatus Nitrosocosmicus franklandus,” “Candidatus Nitrosotalea sinensis”), and a nitrite-oxidizing bacterium (NOB; Nitrobacter sp. NHB1), all being soil isolates, were determined in liquid culture over a range of concentrations by measuring nitrite production or consumption and qPCR of amoA and nxrB genes, respectively. The degradation of NIs in the liquid cultures was also determined. In all cultures, EQ was transformed to the short-lived QI (major derivative) and the persistent EQNL (minor derivative). They all showed significantly higher inhibition activity of AOA compared to AOB and NOB isolates. QI was the most potent AOA inhibitor (EC50 = 0.3–0.7 μM) compared to EQ (EC50 = 1–1.4 μM) and EQNL (EC50 = 26.6–129.5 μM). The formation and concentration of QI in EQ-amended cultures correlated with the inhibition patterns for all isolates suggesting that it was primarily responsible for inhibition after application of EQ. DCD and DMPP showed greater inhibition of AOB compared to AOA or NOB, with DMPP being more potent (EC50 = 221.9–248.7 μM vs EC50 = 0.6–2.1 μM). NP was the only NI to which both AOA and AOB were equally sensitive with EC50s of 0.8–2.1 and 1.0–6.7 μM, respectively. Overall, EQ, QI, and NP were the most potent NIs against AOA, NP, and DMPP were the most effective against AOB, while NP, EQ and its derivatives showed the highest activity against the NOB isolate. Our findings benchmark the activity range of known and novel NIs with practical implications for their use in agriculture and the development of NIs with broad or complementary activity against all AO.

Effects of commonly used nitrification inhibitors—dicyandiamide (DCD), 3,4-dimethylpyrazole phosphate (DMPP), and nitrapyrin—on soil nitrogen dynamics and nitrifiers in three typical paddy soils

The use of nitrification inhibitors (NIs) with ammonium (NH4+) -based fertilizers is an efficient strategy for reducing nitrogen (N) loss by affecting ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in agricultural soils. The inhibition mechanisms of NIs on AOA and AOB are still debated, as some studies have demonstrated that NIs inhibited functionally dominant groups, while others have reported selective inhibition of AOB rather than AOA. Here, we identified the impacts of the most commonly used NIs (dicyandiamide (DCD), 3,4-dimethylpyrazole phosphate (DMPP) and nitrapyrin) on the nitrification activity, N2O emission, and abundance and metabolic activity of ammonia oxidizers (AOA, AOB, and comammox (detected only in HL)) in three paddy soils (HL, JX, and SC) with distinct properties using microcosm incubation combined with the 13CO2-DNA-stable isotope probing (SIP) technique. Most of the NIs treatments effectively inhibited the N2O emission with efficacy suppressed the metabolic activity of AOB, regardless of soil type and NI type, whereas the nitrification process and AOA activity could not be inhibited. The efficacy of NIs was also dependent on soil chemical properties, as nitrapyrin did not inhibit AOB growth at the beginning of microcosm incubation in HL, and DCD successfully inhibited the activity of both AOA and AOB in JX. In HL, DCD and DMPP effectively inhibited the activity of comammox, while nitrapyrin could not inhibit its activity. This study is the first to identify the response pattern of comammox to DCD, DMPP, and nitrapyrin in paddy soil by DNA-SIP microcosm incubation. This study also suggested that NIs might selectively inhibit AOB rather than AOA.

Nitrification and urease inhibitors improve rice nitrogen uptake and prevent denitrification in alkaline paddy soil

Increasing evidence indicates that nitrification is a vital factor in crop growth and nitrous oxide emission. Nitrification and urease inhibitors have been demonstrated to be effective in inhibiting the nitrification process and are widely used as fertilizer additives in agricultural soils. However, the effects of these inhibitors on rice N uptake and N2O production through denitrification in paddy soils remain unclear. In the present work, we compared the influences of nitrification inhibitors dicyandiamide (DCD), nitrapyrin (2-chloro-6-(trichloromethyl) pyridine; NP) and N-(n-butyl) thiophosphoric triamide (NBPT) on rice growth, the fate of urea nitrogen (N), and the abundances and activities of ammonia oxidizers and denitrifiers. The fate of urea N was determined by the 15N isotope labeling technique, and the abundances of ammonia oxidizers and denitrifiers were determined using qPCR. All three inhibitors improved rice growth mainly due to the increase in urea N use efficiency. Urea N uptake was negatively correlated with nitrification. The growth of ammonia-oxidizing bacteria (AOB), important in nitrification, was directly blocked by DCD. Additionally, NP and NBPT impeded the growth of ammonia-oxidizing archaea (AOA). In addition, NP significantly increased the microbial biomass to promote more residual urea N in soil and increased soil N transformation. NBPT significantly inhibited urea hydrolysis indirectly affecting nitrification. All three inhibitors decreased the potential denitrification rate (PDR) at the rice heading stage but had little effect on the denitrifier gene abundance except for nitrapyrin, which decreased the nirK gene abundance. DCD and NBPT may reduce the denitrification activity by decreasing the denitrification substrate (NO3−) concentration. These results suggest that DCD, NP and NBPT have a beneficial effect on improving rice N uptake and have the potential to reduce N2O generation through denitrification.

Small Sample Stress: Probing Oxygen-Deprived Ammonia-Oxidizing Bacteria with Raman Spectroscopy In Vivo.

The stress response of ammonia-oxidizing bacteria (AOB) to oxygen deprivation limits AOB growth and leads to different nitrification pathways that cause the release of greenhouse gases. Measuring the stress response of AOB has proven to be a challenge due to the low growth rates of stressed AOB, making the sample volumes required to monitor the internal stress response of AOB prohibitive to repeated analysis. In a proof-of-concept study, confocal Raman microscopy with excitation resonant to the heme c moiety of cytochrome c was used to compare the cytochrome c content and activity of stressed and unstressed Nitrosomonas europaea (Nm 50), Nitrosomonas eutropha (Nm 57), Nitrosospira briensis (Nsp 10), and Nitrosospira sp. (Nsp 02) in vivo. Each analysis required no more than 1000 individual cells per sampling; thus, the monitoring of cultures with low cell concentrations was possible. The identified spectral marker delivered reproducible results within the signal-to-noise ratio of the underlying Raman spectra. Cytochrome c content was found to be elevated in oxygen-deprived and previously oxygen-deprived samples. In addition, cells with predominantly ferrous cytochrome c content were found in deprived Nitrosomonas eutropha and Nitrosospira samples, which may be indicative of ongoing electron storage at the time of measurement.