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Contamination by 2,4,6-trinitrotoluene (TNT), an explosive extensively used by the military, represents a serious environmental problem. In this study, whey has been selected as the most technologically and economically suitable primary substrate for anaerobic in situ biodegradation of TNT. Under laboratory conditions, various additions of whey, molasses, acetate and activated sludge as an inoculant were tested and the process was monitored using numerous chemical analyses including phospholipid fatty acid analysis. The addition of whey resulted in the removal of more than 90% of the TNT in real contaminated soil (7 mg kg(-1) and 12 mg kg(-1) of TNT). The final bioremediation strategy was suggested on the basis of the laboratory results and tested under real conditions at a TNT contaminated site in the Czech Republic. During the pilot test, three repeated injections of whey suspension into the sandy aquifer were performed over a 10-month period. In total, approximately 5m(3) of whey were used. A substantial decrease in the TNT groundwater concentration from the original levels (equalling 1.49 mg l(-1) to 8.58 mg l(-1)) was observed in most of the injection wells, while the concentrations of the TNT biotransformation products were found to be elevated. Pilot-scale application results showed that the anoxic and/or anaerobic conditions in the aquifer were sufficient for TNT bio-reduction by autochthonous microorganisms. Whey application was not accompanied by undesirable effects such as a substantial decrease in the pH or clogging of the wells. The results of the study document the suitability of application of whey to bioremediate TNT contaminated sites in situ., (Copyright © 2015 Elsevier B.V. All rights reserved.)