An Experimental Investigation of Nitrogen Gas Produced during Denitrification by J.D. Istok 1 , M. M. Park 2 , A. D. Peacock 3 , M. Oostrom 4 , and T. W. Wietsma 4 Abstract In situ denitrification relies on indigenous microorganisms to reduce nitrate to N 2 gas. However, when initial nitrate concentrations are large, produced gas volumes also can be very large, potentially resulting in reduced water saturation and hydraulic conductivity in the treatment zone. In this study, we investigated the fate of N 2 and other gases produced during denitrification in a laboratory flow cell containing packed sediment. Denitrifying activity was stimulated by additions of nitrate and ethanol. Microbial activity was monitored by measuring nitrate, nitrite, and ethanol concentrations; gas saturations were measured during the experiment using a gamma imaging system. Biomass was measured using phospholipid fatty acid analysis of sediment samples. Bioenergetic calculations calibrated to measured nitrate consumed and biomass produced predicted that 1.2 L N 2 gas/L water should have been produced following the addition of 100 mM nitrate. However, the maximum measured gas saturation was only 23%, indicating substantial gas loss from the sediment pack. Temporal gamma images and visual observations confirm that small gas bubbles formed in the sediment pack coalesced into larger bubbles and migrated upward through gas-filled channels to the sediment pack surface. Although gas saturations increased, there was no signifi- cant change in sediment pack hydraulic conductivity. These results suggest that in permeable reactive barriers used for in situ denitrification, gas production will not necessarily lead to unlimited gas accumulation in the pore space and that the effects of gas production on water saturation and hydraulic conductivity may be relatively minor. Introduction Nitrate is one of the most widespread inorganic ground water contaminants in the world (Hallberg and Keeney 1993). In situ denitrification is one way to re- move nitrate from ground water (Mecado et al. 1988). In this approach, organic substrates are added to stimulate the growth and activity of denitrifying bacteria that reduce nitrate to nitrite, nitrous oxide, nitric oxide, and ultimately to nitrogen gas (Soares et al. 1991; Robertson et al. 2000; Schipper and Vojvodic-Vukovic 2001). For example, the addition of ethanol to contaminated ground water has been used to stimulate in situ denitrification at the U.S. Department of Energy’s (DOE) Field Research Center (FRC) at Oak Ridge National Laboratory (Istok et al. 2004). At that site, disposal of uranium processing waste resulted in extensive nitrate contamination of the subsurface, with nitrate concentrations exceeding 100 mM. The use of in situ denitrification to remove such large quantities of nitrate will inevitably lead to the production of large amounts of biomass, N 2 , and other gases. For example, bioenergetic calculations using well- established procedures detailed in Rittmann and McCarty (2001) suggest that a plausible overall growth reaction for microorganisms that couple denitrification to ethanol oxidation is as follows: 4:9CH 3 CH 2 OHðethanolÞ 12:4H 1 17:2NO 2 3 ¼ C 5 H 7 O 2 NðcellsÞ 13:1N 2 ðaqÞ 110H 2 O14:8HCO 2 3 ð1Þ Based on this stoichiometry, we can estimate that the complete removal of 100 mM nitrate will result in the 1 Corresponding author: Department of Civil Engineering, Oregon State University, Corvallis, OR 97331; (541) 737-8547; fax (541) 737-9090; jack.istok@oregonstate.edu 2 Department of Civil Engineering, Oregon State University, Corvallis, OR 97331. 3 Center for Biomarker Analysis, The University of Tennessee, Rm 319 OSB, West Call St., Knoxville, TN 37932. 4 William R. Wiley Environmental Molecular Sciences Labora- tory, Pacific Northwest National Laboratory, Richland, WA 99352. Received June 2006, accepted February 2007. Journal compilation ª 2007 National Ground Water Association. No claim to original US government works. doi: 10.1111/j.1745-6584.2007.00319.x Vol. 45, No. 4—GROUND WATER—July–August 2007 (pages 461–467) 461