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 ( N) as a novel bacterial tracer. Nonpathogenic E. coli from two springs in central Kentucky were grown on 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 δ 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, δ N values increased in T. pyriformis while bacterial populations decreased. In a field test, following a 2.1-cm rainfall, 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 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.

Bacterial contamination of karst aquifers is a global concern as water quality deteriorates in the face of decreasing water security. Traditional abiotic groundwater tracers, which do not exhibit surface properties similar to bacteria, may not be good proxies for risk assessment of bacterial transport in karst environments. This study examined the transport and attenuation of two isolates of in relation to traditional groundwater tracers (rhodamine WT dye and 1-μm-diam. latex microspheres) through ∼30 m of epikarst in western Kentucky. Differential movement of the four tracers was observed, with tracer behavior dependent on flow conditions. Dye arrived at the sampling site prior to particulates. Molecular biology techniques successfully detected bacteria in the cave and showed attenuation was greater for a bacterial isolate with high attachment efficiency compared with an isolate known to have low attachment efficiency. Microspheres were first detected simultaneously with the low-attachment isolate but attained maximum concentrations during increases in discharge >11 d post-injection. Bacteria were remobilized by storm events >60 d after injection, illustrating the storage capacity of epikarst with regard to potential contaminants. The two bacterial strains were not transported at the same rate within the epikarst, showing breakthroughs during differing storm events and illustrating the importance of cell surface chemistry in the prediction of microorganism movement. Moreover, this study has shown that molecular analysis can be successfully used to target, quantify, and track introduced microbial tracers in karst terrains.

Lack of filtration and rapid transport of groundwater and particulate matter make karst aquifers susceptible to bacterial contamination. This study utilized quantitative polymerase chain reaction (qPCR) to examine the transport and attenuation of two nonvirulent isolates of Escherichia coli (E. coli) in relation to traditional groundwater tracers (rhodamine WT dye and 1-μm diameter latex microspheres) in a karst-conduit aquifer in central Kentucky. Bacterial isolates were labeled with stable isotopes ( 15 N and 13 C). All tracers were detected more than 6 km downstream from the injection site and demonstrated overlapping breakthrough curves, with differential transport observed between the two bacterial strains. The E. coli isolate containing the kps gene (low attachment) arrived at sampling sites 1.25 to 36 h prior to the bacterial isolate containing the iha gene (high attachment) and was detected in samples collected following storm events in which the iha isolate was not detected. The storage potential of contaminants within karst systems was demonstrated by the remobilization of all tracers during storm events more than 1 month after injection. Bacteria-sized microspheres were more easily remobilized during periods of increased discharge compared to other tracers. The study demonstrated that molecular biology techniques such as qPCR can be utilized as a sensitive analysis of bacterial tracers in karst aquifers and may prove to be a more sensitive analytical technique than stable isotope analysis for field-scale traces.

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