Use of Nitrogen-15-Enriched Escherichia coli as a Bacterial Tracer in Karst Aquifers

Karst aquifers are susceptible to contamination by microorganisms, but relatively few studies have used bacteria as tracers. We demonstrate the utility of Escherichia coli enriched in the stable isotope nitrogen-15 (¹⁵ N) as a novel bacterial tracer. Nonpathogenic E. coli from two springs in central Kentucky were grown on ¹⁵ N-enriched media. Survival of E. coli and persistence of the isotopic signal were assessed in two sets of laboratory experiments conducted with sterilized spring water in dark microcosms at 14 °C. First, isotopically labeled bacteria survived for 130 d at concentrations within one log unit of the average initial value, and there was no significant difference in δ¹⁵ N values from Day 1 to Day 130. Second, water samples with E. coli were inoculated with either of two different species of protozoa (Tetrahymena pyriformis or Colpoda steinii). During 7 d, δ¹⁵ N values increased in T. pyriformis while bacterial populations decreased. In a field test, following a 2.1-cm rainfall, ¹⁵ N-labeled E. coli, solutes (rhodamine WT dye and bromide), and latex microspheres were injected into a sinkhole approximately 530 m upgradient of a spring. Breakthrough of all tracers coincided, but microspheres were remobilized by subsequent storms, unlike other tracers. Enriched E. coli exhibited more tailing than solute tracers during the initial storm-flow recession. These results indicate that ¹⁵ N-enriched E. coli is a viable tracer of bacterial transport in karst aquifers, although predation may attenuate the isotopic signal in systems that are not rapidly flushed.