Nitrogen fertilization in agriculture has serious environmental consequences, including production of the greenhouse gas nitrous oxide (N2O), pollution of groundwater with nitrate (NO3-), and river eutrophication. Nitrogen use efficiency can be increased by amending fertilizers with inhibitors to slow microbial nitrification processes, which transform ammonia to NO3-. Unfortunately, commercial inhibitors have failed to perform reliably across various agroecosystems for reasons not well understood. Using a combination of bacterial studies and soil incubations, we demonstrate here that 4-methyl-1-(prop-2-yn-1-yl)-1H-1,2,3-triazole (MPT) exhibits superior nitrification inhibitory properties. Unlike the commercial reversible inhibitors, MPT acts as a mechanistic, irreversible inhibitor of the key enzyme ammonia monooxygenase, enabling effective retention of ammonium (NH4+) and suppression of NO3- and N2O production over 21 days in several agricultural soils with pH values ranging from 4.7 to 7.5. A bacterial viability stain and a suite of freshwater and terrestrial ecotoxicity tests did not indicate any acute or chronic toxicity. Real-time quantitative polymerase chain reaction (qPCR) analysis revealed an enhanced inhibitory effect of MPT on both ammonia-oxidizing bacteria and archaea. Thus, MPT outperforms currently available nitrification inhibitors and has great potential for broad application in various agricultural settings.

Recently, 1,4-disubstituted 1,2,3-triazoles were reported by us as a new class of nitrification inhibitors, which can outperform the commercial compound 3,4-dimethylpyrazole phosphate (DMPP) in soil incubations. In this work, the mechanism of inhibition of five 1,2,3-triazoles with different substitution patterns was explored using a bacterial assay based on the measurement of nitrite (NO2-) production by pure cell cultures of Nitrosomonas europaea and Nitrosospira multiformis. While polar functional groups, such as amines, esters, and alkoxy residues, were detrimental to inhibiting production of NO2-, triazoles carrying only aliphatic substituents showed the highest inhibition of up to 98%. The observed correlation between lipophilicity and inhibitory activity suggests that more lipophilic compounds could more easily access the membrane-bound ammonia monooxygenase (AMO), which catalyzes the first step of the nitrification process. Measurement of the Michaelis-Menten kinetics suggests that the disubstituted 1,2,3-triazoles studied in this work act as reversible, noncompetitive inhibitors. Real-time measurements of the oxygen (O-2) consumption showed that the O-2 uptake rate by AMO follows zero-order kinetics in the presence of the triazoles, confirming the nonmechanistic mode of inhibition.

The microbial conversion of ammonia to nitrite in soils involves three enzymatic steps. Nitrification inhibitors (NIs) are designed to inhibit ammonia monooxygenase (AMO), the enzyme performing the initial oxidation of ammonia to hydroxylamine, to mitigate excessive nitrogen fertilizer losses in agricultural systems. Because the efficiency of the current commercial NIs is highly unreliable, novel, better performing compounds need to be developed. Previously, time-consuming soil incubation studies were required as the first step to test new potential NIs. We present here a simple and cost-efficient colorimetric assay that has been developed for the rapid assessment of the efficiency of new synthetic NIs to identify the most promising compounds for subsequent soil studies. This protocol enables screening of the inhibitor activity of multiple compounds at the same time with high reproducibility and can be manipulated to determine pH and temperature-dependent effects on NIs.

Nitrification inhibitors have been coformulated with nitrogen fertilizers since the 1970s to modulate the microbiological conversion of nitrogen in agricultural soils. 3,4-Dimethyl-1H-pyrazole (DMP) and dicyandiamide (DCD) are currently the most used commercial nitrification inhibitors, but their mode of action is not well understood. This work seeks to fill this void by assessing for the first time in detail their mechanism of inhibition, efficacy, and acute toxicity with pure cell cultures of Nitrosomonas europaea. Bacterial assays based on the quantification of the nitrite (NO2-) production showed that both inhibitors reversibly target ammonia monooxygenase (AMO), which catalyzes the first step of the nitrification process. Michaelis-Menten kinetics suggest that both DMP and DCD act as uncompetitive inhibitors. Real-time measurements of the oxygen (O2) consumption confirmed the nonmechanistic mode of inhibition and showed that DMP reduced the O2 uptake rate by AMO much more at considerably lower concentrations than DCD, in line with the lower inhibitory efficiency of the latter. Acute toxicity tests revealed that DCD has a 10% higher toxicity than DMP when comparing treatments at the same inhibition efficacy (i.e., DMP at 10 ppm, DCD at 100 ppm), indicating that the inhibition of the nitrification process cannot simply be achieved by increasing the inhibitor concentration. The methods presented in this study could assist the development of more reliable nitrification inhibitors in the future.

We present here the draft genome sequences of two Janthinobacterium lividum strains, GW456P and GW458P, isolated from groundwater samples collected from a background site at the Oak Ridge Field Research Center. Production of a purple pigment by these two strains was observed when grown on diluted (1/10) LB agar plates.

Complex intrinsic and extrinsic mechanisms determine neural cell fate during development of the nervous system. Using Id4 deficient mice, we show that Id4 is required for normal development of the central nervous system (CNS), timing neural differentiation in the developing forebrain. In the absence of Id4, the ventricular zone of the neocortex, future hippocampus as well as lateral and medial ganglionic eminences exhibited a 20–30% reduction in mitotic neural precursor cells (NPCs). Although the number of apoptotic cells was significantly increased, the neocortex of Id4−/− embryos was consistently thicker due to premature neuronal differentiation, which resulted in an increase in early-born neurons in the adult Id4−/− cortex. Late-born cortical neurons and astrocytes in the cortex, septum, hippocampus and caudate putamen of Id4−/− adult brains were decreased, however, likely due to the depletion of the NPC pool. Consequently, adult Id4−/− brains were smaller and exhibited enlarged ventricles. In vitro analysis of neurosphere cultures revealed that proliferation of Id4-deficient NPCs was impaired and that BMP2-mediated astrocyte differentiation was accelerated in the absence of Id4. Together, these in vivo and in vitro data suggest a crucial role for Id4 in regulating NPC proliferation and differentiation.