Podcast Session Series:‘Looking up for novel nitrification inhibitors: New stories with old compounds’
Part I: Νitrification inhibitors used in agriculture: A fresh look to an old issue. Dr Evangelia Papadopoulou discusses late breakthroughs in the microbiology of nitrification and highlights raising issues on the effectiveness of the established nitrification inhibition strategies.
https://www.youtube.com/watch?v=zmfN-mlGynU
Agricultural Magazine Article
“The role of nitrification inhibitors in modern agriculture: Challenges, problems, and potential solutions” by Evangelia S. Papadopoulou and Dimitrios G. Karpouzas
Published in Agriculture CROP &ANIMAL HUSBANDRY, July 2020
Videos on NITRIC research protocols
I. Ammonium determination
In this video, soil ammonium content was quantified in 1 M KCl extracts by a modified indophenol method based on the classical Berthelot reaction (Kandeler and Gerber, 1988)1. During this process, NH4+ is oxidized to monochloroamine by sodium dichloroisocyanuric acid and subsequently forms a green indophenol compound in the presence of phenolic compounds in an alkaline medium. Nitroprusside serves as a catalyst. The color reagent was prepared by mixing equal volumes (1:1:1 v/v/v) of a combined 1.06 M sodium salicylate and 4.29 mM sodium nitroprusside solution with 0.3 M NaOH solution and deionized water. The salicylate– nitroprusside solution was prepared fresh daily by dissolving 8.5 g sodium salicylate and 63.9 mg sodium nitroprusside dihydrate in 50 mL of deionized water. The oxidation reagent (3.9 mM) was prepared by dissolving 0.1 g of dichloroisocyanuric acid sodium salt dihydrate in 100 mL of deionized water. For color reaction, a 600-μL of blank, standards or samples (extraction solution) were pipetted into an 1.5mL eppendorf, followed by 300 μL of color reagent and 120 μL of oxidation reagent. Mixtures were shaken at a horizontal shaker at 300 rpm at room temperature for 30 min for color development, and the absorbance was subsequently measured photometrically at 660 nm on a microtiter plate reader. Standards were prepared fresh daily from an NH4Cl stock solution (1000 mg N L−1, stable at 4°C for several weeks) in the respective extractant by serial dilution ranging from 5 to 0,02mg N L−1.
1Kandeler, E., Gerber, H. (1988). Short-term assay of soil urease activity using colorimetric determination of ammonium. Biol Fertil Soils 6, 68–72.
II. Nitrate determination
In this video, soil NO3− concentration was quantified in 1 M KCl extracts based on the VCl3/Griess method, as described by Miranda et al. (2001)2 for biological samples. Nitrate is converted to NO2− in an acidic VCl3 medium, and the NO2− concentration is measured by direct coupling with the Griess reaction. Absorbance is then measured photometrically at 540 nm. A saturated VCl3 reagent solution (50.9 mM VCl3) was prepared fresh daily by dissolving 400 mg VCl3 in 50 mL of 1M HCl, and excess solids were filtered through Whatmann filter paper. Griess reagent 1 (0.77 mM) was prepared by dissolving 50 mg of N-napthylethylenediamine dihydrochloride in 250 mL of deionized water. Griess reagent 2 (58 mM sulfanilamide) was prepared by dissolving 5 g of sulfanilamide in 500 mL of 3M HCl. For color reaction, each 100-μL of blank, standards or samples (extraction solution) were pipetted into the microtiter plates and 100 μL of VCl3 was added, rapidly followed by 50 μL each of Griess reagents 1 and 2 to give a total volume of 300 μL. The plates were incubated at 37°C for 60 min and measured in a multimode microplate reader (Varioskan™ LUX) at 540 nm. Nitrate standards were prepared fresh daily from a stock solution of KNO3 (1000 mg N L−1) in the range of 20 to 0.02 mg N L−1 by serial 1:2 dilution.
2Miranda, K.M., Espey, M.G., Wink, D.A. (2001). A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite. Nitric Oxide 5, 62–71.
III. Determination of Potential Nitrification
In this video soil potential nitrification was determined by the method of Kandeler (1995)3. Briefly, 5-g soil samples were amended with 20 ml of 1 mM (NH4)2SO4 (as ammonium substrate) and 0.1 ml of 1.5 M NaClO3 (for inhibition of NO2- oxidation) and incubated under constant agitation at 20°C for 5 h, while triplicate control samples were treated in the same way and incubated at -20°C for the same period. At the end of the incubation period, NO2- was extracted from all samples with 2 M KCl. The extract (5 ml) was amended with 3 ml of 0.19 M NH4Cl and 2 ml of a colorimetric indicator prior to final determination of its adsorption at 520 nm. The potential nitrification in the soil samples was then determined with an external calibration curve prepared by adsorption determination of a series of NaNO2 solutions, in the range of 0 – 0.5 μmol NO2- -N ml-1.
3Kandeler E. 1995. Potential nitrification, p 146–149. In Schinner F, Ohlinger R, Kandeler E, Margesin R (ed), Methods in soil biology. Springer, Heidelberg, Germany.
IV. Culture maintenance of soil nitrifying isolates
In this video, cultivation methods of a diverse range of soil nitrifying isolates including (i) the ammonia-oxidizing bacterial strains Nitrosomonas europaea and Nitrosospira multiformis, (ii) the ammonia-oxidizing archaeal strains “Candidatus Nitrosocosmicus franklandus” and “Candidatus Nitrosotalea sinensis”, and (iii) one nitrite-oxidizing bacterium, Nitrobacter sp. NHB1 are presented.
All nitrifying strains were kindly provided by the culture collection of Prof. G. Nicol (Ecole Centrale de Lyon, France).