Back to the future: Comparing yeast as an outmoded artificial tracer for simulating microbial transport in karst aquifer systems to more modern approaches.

Bacterial contamination of karst groundwater is a major concern for public health. Artificial tracing studies are crucial for establishing links between locations where pollutants can rapidly reach the aquifer systems and subsequent receptors, as well as for enhanced understanding of pollutant transport. However, widely used solute artificial tracers do not always move through the subsurface in the same manner as particles and microorganisms, hence may not be ideal proxies for predicting movement of bacterial contaminants. This study evaluates whether a historically used microbial tracer (yeast) which is readily available, inexpensive, and environmentally friendly, but usually overlooked in modern karst hydrogeological studies due to challenges associated with its detection and quantification in the past, can reemerge as a valuable tracer using the latest technology for its detection. Two field-based studies on separate karst systems were carried out during low-flow conditions using a portable particle counter along with flow cytometry measurements to monitor the recovery of the yeast at the springs. Soluble fluorescent dyes were also injected simultaneously with the yeast for comparison of transport dynamics. On one tracer test, through a karst conduit of much higher velocities, the injected yeast and fluorescent dye arrived at the same time at the spring, in comparison to the tracer test on a conduit system with lower groundwater velocities in which the yeast particles were detected before the dye at the sampling site. Both a portable particle counter and flow cytometry successfully detected yeast during both tests, thereby demonstrating the applicability of this tracer with contemporary instrumentation. Even though no significant advantages of flow cytometry over the portable counter system can be reported on the basis of the presented results, this study has shown that flow cytometry can be successfully used to detect and quantify introduced microbial tracers in karst environments with extremely high precision.

Multiple fluorescence approaches to identify rapid changes in microbial indicators at karst springs.

Karst springs are globally important for drinking water supply but are often also exceptionally vulnerable to contamination. Such springs usually exhibit strong variation in microbial water quality in sharp response to rainfall events, thus, posing a health hazard to consumers of water supplied from these sources. The rapid detection of such changes is extremely important as well as being able to establish a link to the sources of such pollution, so that appropriate measures can be taken both in terms of immediate protection of human health and the management of karst aquifers. In this study, a fluorescence-based multi-parameter approach was trialed in order to evaluate which methods can be used to monitor rainfall-induced rapid changes in microbial water quality at karst springs, as well as determine whether such changes can be linked to sources of human effluent contamination. The results from three monitoring periods at two karst springs revealed marked responses to rainfall events for all of the microbial parameters measured. Total cell count (TCC) measurements using flow cytometry (FCM) showed very strong positive correlations with the more conventionally monitored faecal indicator bacteria (FIB) and total coliforms (TC), indicating that such a fluorescence-based and cultivation-independent technique can be very useful to indicate rapid changes in microbial water quality at karst springs. Furthermore, very strong positive correlations were also found between tryptophan-like fluorescence (TLF) measurements and concentrations of all monitored microbial parameters, again demonstrating that such a fluorescence-based approach can also be useful for detecting rapid changes in concentrations of traditional faecal indicators. Interestingly, it was found that fluorescent whitening compounds (FWCs) signals do not necessarily follow temporal variations of microbial indicators. However, the frequency of detection of positive FWCs signals may still reveal useful information about the overall magnitude of human wastewater effluent impacts on karst aquifer systems.

Flow Cytometry and Fecal Indicator Bacteria Analyses for Fingerprinting Microbial Pollution in Karst Aquifer Systems.

Microbial pollution of aquifers is a persistent water quality problem globally which poses significant risks to public health. Karst aquifer systems are exceptionally vulnerable to pollution from fecal contamination sources as a result of rapid recharge of water from the surface via discrete pathways linked to highly conductive, solutionally enlarged conduits alongside strong aquifer heterogeneity. Consequently, rapid changes in microbial water quality, which are difficult to monitor with expensive and time-consuming conventional microbiological methods, are a major concern in karst environments. This study examined flow cytometric (FCM) fingerprinting of bacterial cells in groundwater together with fecal indicator bacteria (FIB) at nine separate karst springs of varying catchment size over a 14 month period in order to assess whether such a technique can provide faster and more descriptive information about microbial pollution through such karst aquifer systems. Moreover, the data have also been evaluated with respect to the potential of using turbidity as an easy-to-measure proxy indicator of microbial pollution in a novel way. We argue that FCM provides additional data from which enhanced insights into fecal pollution sources and its fate and transport in such karst catchments can be gained. We also present valuable new information on the potential and limitations of turbidity as an indicator of fecal groundwater contamination in karst. FCM has the potential to become a more widely used tool in the field of contaminant hydrogeology.