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Background and aims Nitrification is the first step in several pathways that lead to losses of nitrogen from agricultural systems. Biological nitrification inhibition (BNI) refers to the ability of some plant species to release chemicals from their roots that inhibit microbial ammonia oxidation thereby decreasing nitrification rates. BNI has been found in the wheat relative Leymus racemosus but not in Triticum aestivum. The aim of this work was to assess a number of landraces of Triticum aestivum for BNI ability. Methods Samples of root exudates and root tissue extracts, collected from hydroponically grown plants, were tested for their impact on nitrification rates when inoculated with pure cultures of two ammonia oxidising bacteria, Nitrosomonas europaea and Nitrosospira multiformis. Pot experiments were then conducted to confirm the results. Results The vast majority of the landraces tested caused some level of inhibition. However, of the 96 wheat landraces tested, 26 produced root exudates which caused a statistically significant reduction in nitrification rates of the two ammonia oxidising bacteria. Root exudates from four of the BNI positive landraces were shown to significantly inhibit nitrification rates in a sandy loam soil. Conclusions This is the first evidence of significant levels of BNI in Triticum aestivum. The discovery of landraces with BNI ability raises the potential for breeding this trait into modern, elite wheat cultivars.

Biological nitrification inhibition (BNI) refers to the plant-mediated process in which nitrification is inhibited through rhizospheric release of diverse metabolites. While it has been assumed that interactive effects of these metabolites shape rhizosphere processes, including BNI, there is scant evidence supporting this claim. Hence, it was a primary objective to assess the interactive effects of selected metabolites, including caffeic acid (CA), vanillic acid (VA), vanillin (VAN), syringic acid (SA), and phenylalanine (PHE), applied as single and combined compounds, against pure cultures of various ammonia-oxidising bacteria (AOB, Nitrosomonas europaea , Nitrosospira multiformis , Nitrosospira tenuis , Nitrosospira briensis ) and archaea (AOA, Nitrososphaera viennensis ), as well as soil nitrification. Additionally, benzoic acid (BA) was examined as a novel biological nitrification inhibitor. All metabolites, except SA, tested as single compounds, achieved varied levels of inhibition of microbial growth, with CA exhibiting the highest inhibitory potential. Similarly, all metabolites applied as single compounds, except PHE, inhibited soil nitrification by up to 62%, with BA being the most potent. Inhibition of tested nitrifying microbes was also observed when compounds were assessed in combination. The combinations VA + PH, VA + CA, and VA + VAN exhibited synergism against N. tenuis and N. briensis , while others showed antagonism against N. europaea , N. multiformis , and N. viennensis . Although all combinations suppressed soil nitrification, their interactions against soil nitrification revealed antagonism. Our findings indicate that both antagonism and synergism are possible in rhizospheric interactions involving BNI metabolites, resulting in growth inhibition of nitrifiers and suppression of soil nitrification.

Aim A simple, rapid, colourimetric method for screening biological nitrification inhibitors in plants is presented. Methods Our approach combines the use of the Griess assay to track the rate of nitrite NO₂ production by pure cultures of ammonia oxidising bacteria in the presence and absence of nitrification inhibitors with a simple method for collecting root exudates from plants. NO₂ formation was tracked colourimetically on a microplate reader over 9 h of incubation. The advantage of this method is that it provides a simple, high throughput means of measuring biological nitrification inhibition in root exudates, using wild-type bacterial cultures. Results NO₂ formation rates and inhibition levels measured using the high through-put method were highly correlated with those measured by tracking NO₂ formation using a segmented flow analyser. The method was able to quantify inhibition of Nitrosomonas europaea by the synthetic nitrification inhibitors allythiourea (AT), dicyandiamide (DCD) and 3,4,-dimethylpyrazole phosphate (DMPP) with IC50 values similar to those reported in the literature. The method detected biological nitrification inhibition (BNI) in root exudates from Brachiaria humidicola and the lack of BNI in root exudates from wheat cv. Janz with minimal alteration of the exudates prior to testing. The results also showed that the more common soil ammonia oxidising bacterium (AOB), Nitrosospira multiformis, was much less sensitive to AT and DCD than N europaea but had similar sensitivity to DMPP. Conclusions This method provides a potentially useful way of screening large numbers of root exudate samples allowing for phenotyping of the BNI trait in crop and pasture populations which will be required for the trait to be introduced into commercial varieties.

In this study, two new benzoazepines ( 1 and 2 ), together with five known analogs ( 3–7 ) were isolated from the cigar-tobacco-derived endophytic Aspergillus fumigatus. Their structures were determined by means of HR-ESI-MS and extensive 1D and 2D NMR spectroscopic studies. All compounds were evaluated for their antibacterial activities against five nitrosobacteria strains Nitrosomonas communis , Nitrosomonas nitrosa , Nitrosospira multiformis , Nitrobacter winogradskyi , and Nitrospira inopinata . Interestingly, compounds 1–7 exhibited notable antibacterial activity with bacteriostatic diameters within the range 13.8 ± 1.9–26.3 ± 2.5 mm against five nitrosobacteria strains, and most of the bacteriostatic diameters are higher than that of positive controls. Owing to the nitrosobacteria having the ability to convert nitrogen components into nitrite in tobacco, and then converting to tobacco-specific nitrosamines (TSNAs), the above compounds have the potential to reduce TSNAs in tobacco by inhibiting nitrosobacteria during the fermentation process of tobacco leaves.

In this study, two new ( 1 and 2 ), together with five known ( 3 – 4 ) naphthoquinones were isolated from the Nicotiana tabacum -derived endophytic Fusarium solani . Their structures were determined by means of HR-ESI-MS and extensive 1D and 2D NMR spectroscopic studies. All compounds were evaluated for their antibacterial activities against five nitrosobacteria strains ( Nitrosomonas communis , Nitrosomonas nitrosa , Nitrosospira multiformis , Nitrobacter winogradskyi , and Nitrospira inopinata ). Interestingly, compounds 1 – 7 exhibited notable antibacterial activity with the bacteriostatic diamater in the range of 3.2 ± 1.7~25.4 ± 2.3 mm against five nitrosobacteria strains, and most of the bacteriostatic diamaters were higher than that of positive control. Due to the nitrosobacteria having the ability to convert nitrogen components into nitrosamines in tobacco, and then convert them into tobacco specific nitrosamines (TSNAs), the above compounds have the potential to reduce TSNAs in tobacco by inhibiting nitrosobacteria during the fermentation process of tobacco leaves.

Abstract Nitrite is the highly toxic end product of ammonia oxidation that accumulates in the absence of a nitrite-consuming process and is inhibitory to nitrifying and other bacteria. The effects of nitrite on ammonia oxidation rates and regulation of a common gene set were compared in three ammonia-oxidizing bacteria (AOB) to determine whether responses to this toxic metabolite were uniform. Mid-exponential-phase cells of Nitrosomonas europaea ATCC 19718, Nitrosospira multiformis ATCC 25196, and Nitrosomonas eutropha C-91 were incubated for 6h in mineral medium supplemented with 0, 10, or 20mM NaNO2. The rates of ammonia oxidation (nitrite production) decreased significantly only in NaNO2-supplemented incubations of N. eutropha; no significant effect on the rates was observed for N. europaea or N. multiformis. The levels of norB (nitric oxide reductases), cytL (cytochrome P460), and cytS (cytochrome c′-β) mRNA were unaffected by nitrite in all strains. The levels of nirK (nitrite reductase) mRNA increased only in N. europaea in response to nitrite (10 and 20mM). Nitrite (20mM) significantly reduced the mRNA levels of amoA (ammonia monooxygenase) in N. multiformis and norS (nitric oxide reductase) in the two Nitrosomonas spp. Differences in response to nitrite indicated nonuniform adaptive and regulatory strategies of AOB, even between closely related species.

During and after the pandemic caused by the SARS-CoV-2 virus, the use of personal care products and disinfectants increased in universities worldwide. Among these, quaternary ammonium-based products stand out; these compounds and their intermediates caused substantial changes in the chemical composition of the wastewater produced by these institutions. For this reason, improvements and environmentally sustainable biological alternatives were introduced in the existing treatment systems so that these institutions could continue their research and teaching activities. For this reason, the objective of this study was to develop an improved culture medium to cultivate ammonium oxidising bacteria (AOB) to increase the biomass and use them in the treatment of wastewater produced in a faculty of sciences in Bogotá, D.C., Colombia. A Plackett Burman Experimental Design (PBED) and growth curves served for oligotrophic culture medium, and production conditions improved for the AOB. Finally, these bacteria were used with total heterotrophic bacteria (THB) for wastewater treatment in a pilot plant. Modification of base ammonium broth and culture conditions (6607 mg L−1 of (NH4)2SO4, 84 mg L−1 CaCO3, 40 mg L−1 MgSO4·7H2O, 40 mg L−1 CaCl2·2H2O and 200 mg L−1 KH2PO4, 10% (w/v) inoculum, no copper addition, pH 7.0 ± 0.2, 200 r.p.m., 30 days) favoured the growth of Nitrosomonas europea, Nitrosococcus oceani, and Nitrosospira multiformis with values of 8.23 ± 1.9, 7.56 ± 0.7 and 4.2 ± 0.4 Log10 CFU mL−1, respectively. NO2− production was 0.396 ± 0.0264, 0.247 ± 0.013 and 0.185 ± 0.003 mg L−1 for Nitrosomonas europea, Nitrosococcus oceani and Nitrosospira multiformis. After the 5-day wastewater treatment (WW) by co-inoculating the three studied bacteria in the wastewater (with their self-microorganisms), the concentrations of AOB and THB were 5.92 and 9.3 Log10 CFU mL−1, respectively. These values were related to the oxidative decrease of Chemical Oxygen Demand (COD), (39.5 mg L−1), Ammonium ion (NH4+), (6.5 mg L−1) Nitrite (NO2−), (2.0 mg L−1) and Nitrate (NO3−), (1.5 mg L−1), respectively in the five days of treatment. It was concluded, with the improvement of a culture medium and production conditions for three AOB through biotechnological strategies at the laboratory scale, being a promising alternative to bio-augment of the biomass of the studied bacteria under controlled conditions that allow the aerobic removal of COD and nitrogen cycle intermediates present in the studied wastewater.

An active pyrophosphate-dependent 6-phosphofructokinase (PPi-PFK) from the thermotolerant methanotroph Methylococcus capsulatus Bath, containing a six-residue polyhistidine tag, was characterized. The enzyme was homodimeric (2 x 45 kDa), nonallosteric and most active at pH 7.0. PPi-PFK catalyzed reactions of PPi-dependent phosphorylation of fructose-6-phosphate (F-6-P) (Km 2.27 mM and Vmax 7.6 U mg⁻¹ of protein), sedoheptulose-7-phosphate (Km 0.027 mM and Vmax 31 U mg⁻¹) and ribulose-5-phosphate. In the reaction with F-6-P, the apparent Km for PPi was 0.027 mM, while in the reverse reaction, Km for orthophosphate was 8.69 mM and that for fructose-1,6-bisphosphate 0.328 mM (Vmax 9.0 U mg⁻¹). Phylogenetically, M. capsulatus PPi-PFK was most similar to PPi-PFKs from the lithoautotrophic ammonia oxidizers Nitrosomonas europaea (74.0%), Nitrosospira multiformis (73.6%) and Betaproteobacterial methylotroph Methylibium petroleiphilum PM1 (71.6% identity). Genes coding PPi-PFK and a putative V-type H⁺-translocating pyrophosphatase (H⁺-PPi-ase) were cotranscribed as an operon. The potential significance of the PPi-PFK for regulation of carbon and energy fluxes in M. capsulatus Bath is discussed.

The lack of a universal method to extract RNA from soil hinders the progress of studies related to nitrification in soil, which is an important step in the nitrogen cycle. It is particularly difficult to extract RNA from certain types of soils such as Andosols (volcanic ash soils), which is the dominant agricultural soil in Japan, because of RNA adsorption by soil. To obtain RNA from these challenging soils to study the bacteria involved in nitrification, we developed a soil RNA extraction method for gene expression analysis. Autoclaved casein was added to an RNA extraction buffer to recover RNA from soil, and high-quality RNA was successfully extracted from eight types of agricultural soils that were significantly different in their physicochemical characteristics. To detect bacterial ammonia monooxygenase subunit A gene (amoA) transcripts, bacterial genomic DNA and messenger RNA were co-extracted from two different types of Andosols during incubation with ammonium sulfate. Polymerase chain reaction-denaturing gradient gel electrophoresis and reverse transcription polymerase chain reaction-denaturing gradient gel electrophoresis analyses of amoA in soil microcosms revealed that only few amoA, which had the highest similarities to those in Nitrosospira multiformis, were expressed in these soils after treatment with ammonium sulfate, although multiple amoA genes were present in the soil microcosms examined.[PUBLICATION ABSTRACT]