Rapid and unbiased colorimetric quantification of nitrite and ammonium ions released from 2,4,6-trinitrotoluene during biodegradation studies: Eliminating interferences

Worldwide contamination of soils and groundwater by 2,4,6-trinitrotoluene (TNT) has drawn considerable attention to bioremediation solutions. To evaluate the environmental relevance of a biodegradation process or to discover new TNT degrader microorganisms, effective and reliable monitoring strategies are essential, including the sensitive detection of inorganic nitrogen species released from TNT. In this study we describe improved colorimetric methods for a rapid and unbiased monitoring of nitrite and/or ammonium ions produced in TNT-biodegradation experiments. Considerable interferences in the colorimetric detection of nitrite by the Griess reaction were observed in the presence of various chemicals used in biodegradation assays including reduced pyridine nucleotides (NAD(P)H) and commonly used biological buffers and buffer constituents. In the colorimetric quantification of ammonium through the Berthelot reaction, significant interferences were generated by TNT itself and some reduced TNT metabolites. We highlight these pitfalls and give solutions to overcome these problems, including buffer selection, appropriate dilutions, oxidation of NAD(P)H and selective removal of TNT and some of its metabolites.