Optimizing Conditions for the Measurement of Soil Inorganic Nitrogen with a Micro-Plate Reader


Nitrate N, Extraction, Ammonium N, Well microplate

How to Cite

Qihong Tu, Fugen Dou, Haytham M. Salem. Optimizing Conditions for the Measurement of Soil Inorganic Nitrogen with a Micro-Plate Reader. Glob. J. Agric. Innov. Res. Dev [Internet]. 2022 Jan. 22 [cited 2022 Nov. 27];8:22-31. Available from: https://avantipublishers.com/index.php/gjaird/article/view/1033


There are numerous methods for measuring inorganic nitrogen (N) in soils, but many of these are complex or require expensive equipment. In order to seek an accurate and rapid measurement method, we optimized the conditions for soil inorganic nitrogen (N) (NH4+-N and NO3–N) determination with a micro-plate reader; a systematic study was carried out. The effects of extraction regent concentrations, ratios of extraction solution to the soil, extraction time, extracted solution storage methods and time, and reaction time on soil inorganic N measurement were investigated. Results showed that all tested conditions except the storage method had a significant effect on the determination of soil NH4+-N and NO3–N. Compared with the non-stored treatment, the value of the measured soil nitrate-nitrogen increased after being stored in a refrigerator for seven weeks, while that for ammonium-nitrogen content was reduced. The appropriate protocol was using extracting solution directly to determine the content of NH4+-N and NO3–N in soil (otherwise, keep it at room temperature), 1.0 mol/L KCl as the extraction regent, solution/soil ratio with 10:1, extraction for 45 min, and reaction for 25 min (only for NH4+-N). The recovery rate of adding standard solution was above 99% as it met the detection requirements.



Li, S. X., Wang, Z. H., Malhi, S. S., Li, S. Q., Gao, Y. J., and Tian, X. H. 2009. Nutrient and water management effects on crop production, and nutrient and water use efficiency in dryland areas of China. Advances in Agronomy 102:221–262. Doi:10.1016/S0065- 2113(09)01007-4.

Zhu, Z. L., Wen, Q. X. 1994. Soil nitrogen of China. Jiangsu Science and Technology Press 1-303.

Zhu, Z. L., Chen, D. L. 2002. Nitrogen fertilizer use in China-Contributions to food production, impacts on the environment and best management strategies. Nutrient Cycling in Agroecosystems 63(2):117-127. doi:10.1023/A:1021107026067.

Harmsen, G. W., and Van Schreven, D. A. 1955. Mineralization of organic nitrogen in soil. Advances in Agronomy 7:299-398. doi:10.1016/S0065-2113(08)60341-7.

Livens, J. 1959. Contribution à l'étude de l'azote minéralisable du sol. Agr. Louvain 7: 27-44.

Carlson, R. M. 1978. Automated separation and conductimetric determination of ammonia and carbon dioxide. Analytical Chemistry 50:1528-31. doi:10.1021/ac50033a035.

Carlson, R. M. 1986. Continuous flow reduction of nitrate to ammonia by granular zinc. Analytical Chemistry 50:1590-91. doi:10.1021/ac00298a077.

Saha, U. K., L. Sonon., and D. Kissel. 2012. Comparison of conductimetric and colorimetric methods with distillation-Titration method of analyzing ammonium nitrogen in total kjeldahl digests. Communications in Soil Science and Plant Analysis 43:2323-41. doi:10.1080/00103624.2012.708081.

Uttam K. Saha, Leticia Sonon., and Bipul K. Biswas. 2018. A Comparison of Diffusion-Conductimetric and Distillation-Titration Methods in Analyzing Ammonium- and Nitrate-Nitrogen in the KCl-Extracts of Georgia Soils. Communications in Soil Science and Plant Analysis. 49:1, 63-75, doi: 10.1080/00103624.2017.1421647.

Dou, H., A. K. Alva., and T. Appel. 2000. An evaluation of plant-available soil N in selected sandy soils by electro-ultrafiltration, KCl, and CaCl2 extraction methods. Biology and Fertility of Soils. 30:328–332. doi:10.1007/s003740050011.

Stephanie, R., Lara, S., and Zackary I. J. 2011. A suite of microplate reader-based colorimetric methods to quantify ammonium, nitrate, orthophosphate and silicate concentrations for aquatic nutrient monitoring. J. Environ. Monit. 13:370–376. doi: 10.1039/c0em00290a.

Tu, X. H., Xiao, B. D., Xiong, J., and Chen, X. D. 2010. A simple miniaturised photometrical method for rapid determination of nitrate and nitrite in freshwater. Talanta 82: 976–983. doi:10.1016/j.talanta.2010.06.002.

Bremner, J. M., and Keeney, D. R. 1965. Steam distillation method for determination of ammonium, nitrite, and nitrate. Analytica Chimica Acta 32:485-95. doi:10.1016/S0003-2670(00)88973-4.

James, D., and John, H. G. 2011. A new cadmium reduction device for the microplate determination of nitrate in water, soil, plant tissue, and physiological fluids. Journal of AOAC International 94(6): 1896-1905. doi:10.5740/jaoacint.10-454.

Ye, X. S., and Zhao, Z. Q. 2011. Comparison between flow injection analyzer method and national standard method in determining soil nitrate-N concentration. Hubei Agricultural Science. 50(4): 698-707.

D’Angelo, E., Crutchfield, J., and Vandiviere, M. 2001. Rapid, sensitive, microscale determination of phosphate in water and soil. Journal of Environmental Quality 30(6): 2206-2209. doi:10.2134/jeq2001.2206.

Wu, J. P., Hong, Y. G., Guan, F. J., Wang, Y., Tan, Y. H., Yue, W. Z., Wu, M. L., Bin, L. Y., Wang, J. P., Wen, J. L. 2016. A rapid and high- throughput microplate spectrophotometric method for field measurement of nitrate in seawater and freshwater. Scientific reports, 6:1- 9. doi: 10.1038/srep20165.

Wang, Y. H., Salatasa, A., Minesa, P. D., Jakobsenc, M. H., and Andersen, H. R. 2016. Graduated characterization method using a multi- well microplate forreducing reactivity of nanoscale zero valent iron materials. Applied Catalysis B: Environmental 181:314–320. doi: 10.1016/j.apcatb.2015.07.041.

Rebecca, H. N., Nina, H. N. U., Erich, I., Petra, O. L., Wolfgang, W. 2010. Alternative Methods for Measuring Inorganic, Organic, and Total Dissolved Nitrogen in Soil. Nutrient Management & Soil & Plant Analysis. 74(3): 1018-1027. Doi: 10.2136/sssaj2009.0389.

Crutchfield, J. D., Grove, J. H. 2011. A New Cadmium Reduction Device for the Microplate Determination of Nitrate in Water, Soil, Plant Tissue, and Physiological Fluids. Journal of AOAC International. 94(6): 1896–1905. doi: 10.5740/jaoacint.10-454.

Smith, K. A., and Li, S. X. 1993. Estimation of potentially mineralisable N in soil by KCl extraction. Plant and Soil 157:167–174. doi: 10.1007/BF00011046.

Gianello, C., and Bremner, J. M. 1986. A simple chemical method of assessing potentially available organic nitrogen in soil. Communications in Soil Science and Plant Analysis 17:195-214. doi:10.1080/00103628609367708.

Westfall, D. G., Henson, M.A., Evans, E. P. 1978. The effect of soil sample handling between collection and drying on nitrate concentration. Communications in Soil Science and Plant Analysis 9(2):169-185. doi:10.1080/00103627809366797.

Lin, S., Miihling, K. H., Sattelmacher, B. 1997. Soil nitrogen fraction as influenced by sample preparation and extraction. Communications in Soil Science and Plant Analysis 28(6):551-559. doi:10.1080/00103629709369810.

Vaughan, B., J. Denning., and H. Frank. 1995. Bray and Kurts phosphorous (PI), 1M potassium chloride, and saturated calcium oxide extraction of soil nitrate comparison to 2M potassium chloride. Communications in Soil Science and Plant Analysis 26:453-57. doi:10.1080/00103629509369310.

Houba, V. J. G., Temminghoff, E. J. M., Gaikhorst, G. A., and Vark, W. van. 2008. Soil analysis procedures using 0.01 M calcium chloride as extraction reagent. Communications in Soil Science and Plant Analysis. 31(9): 1299-1396. doi: 10.1080/00103620009370514.

Kachurina, O. M., H. Zhang, W. R. Raun., and E. G. Krenzer. 2000. Simultaneous determination of soil aluminum, ammonium- and nitrate-nitrogen using 1M potassium chloride extraction. Communications in Soil Science and Plant Analysis 31:893-903. doi:10.1080/00103620009370485.

Zhao, R. F., Yu, Z. Y., Cheng, B., Zhang, Q. 2009. Effects of pretreatment on content of soil NO3--N, NH4+-N. Chinese Agricultural Science Bulletin 25(10):174-177.

McTaggart, I. P., Smith, K.A. 1993. Estimation of potentially mineralisable nitrogen in soil by KCl extraction. Plant and Soil 157:175-184. doi:10.1007/BF00011046.

Murphy, D. V., Macdonald, A. J., Stockdale, E. A, Goulding, K. W. T., Fortune, S., Gaunt, J. L., Poulton, P. R., Wakefield, J. A., Webster, C. P., and Wilmer, W. S. 2000. Soluble organic nitrogen in agricultural soils. Biology and Fertility of Soils 30(5/6): 374-387. doi:10.1007/s003740050018.

Kachurina, O. M., Zhang, H., Raun, W. R., and Krenzer, E. G. 2000. Simultaneous determination of soil aluminum, ammonium- and nitrate-nitrogen using 1M potassium chloride extraction. Communications in Soil Science and Plant Analysis 31: 893-903. doi:10.1080/00103620009370485.

Li, K. Y., Zhao, Y. Y., Yuan, X. L., Zhao, H. B., Wang, Z. H., Li, S. X., and Malhi, S. S. 2012. Comparison of factors affecting soil nitrate nitrogen and ammonium nitrogen extraction. Communications in Soil Science and Plant Analysis 43: 571-588. doi:10.1080/00103624.2012.639108.

Tao, S. U., Ru, S. M., Wang, Z. H., Li, S. X. 2005. Effects of pretreatment, shaking and conserving method and extracting solution on results for soil mineral nitrogen. Journal of Agro-Environment Science 24(6): 1238-1242.

Houba, V. J. G., Temminghoff, E. J. M., Gaikhorst, G. A. 2000. Soil analysis procedures using 0.01 M calcium chloride as extraction regent. Communications in Soil Science and Plant Analysis 31:1299-1396. doi:10.1080/00103620009370514.

Ma, B. L., Ying, J., Balchin, D. 2005. Impact of sample preservation methods on the extraction of inorganic nitrogen by potassium chloride. Journal of Plant Nutrition 28(5):785-796. doi:10.1081/PLN-200055536.

Lei, W. Q., Zhang, Z. X., Zhang, H. M., Wang, J. L., Ren, X. S. 2019. Comparative research on determination of ammonium nitrogen in soil which extracted by potassium chloride and sodium chloride. Forum of South China 43-44. doi:10.1080/00103624.2012.639108.

Zhang, L.,Yang, S.S.,Cao, B.J.,Hu, B.,Peng. H.W., Chen, X.,Liu, S.B.,Liu, Y.D.,Zhu, Y. 2018. Optimization of nitrate-N extraction technology from Tobacco planting soil. Guizhou Agricultural Sciences. 46(2): 66-69.

Creative Commons License

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.