Denitrifier Method for Nitrite Removal in Electrochemical Analysis of the Electron Accepting Capacity of Humic Substances

Humic substances (HSs) are important electron acceptors and donors in soils and aquifers. The coupling of anoxic nitrogen (N) cycling to the function of HSs as a redox battery, however, remains poorly understood. Mediated electrochemical analysis is an emerging tool to determine the redox properties (i.e., electron donating capacity (EDC), electron accepting capacity (EAC), and redox state) of HS. However, the presence of nitrite (NO2-), a central intermediate of the N-cycle, interferes with the electrochemical determination of the EAC. To eliminate this interference, we developed a bioassay to remove nitrite in HS samples using the denitrifying bacterium Pseudomonas nitroreducens. Cell suspensions of P. nitroreducens completely removed NO2- at various concentrations (1, 2, and 5 mM) from humic acid samples (1 g HA/L) of different redox states. As P. nitroreducens is not able to exchange electrons with dissolved humic acids, the procedure allows an accurate and reliable determination of the EAC of humic acid samples. The proposed method thus opens new perspectives in biogeochemistry to study interactions between HSs and N cycling.