A 1998 study of interstitial fluid geochemistry within Charonis Cascade in the Echo River/River Styx area of the Mammoth Cave System found carbon dioxide pressures higher than that of the fluids of the cave stream itself. This was confirmed by a limestone weight loss experiment in which samples dissolved at various levels below the streambed despite the low fluid velocities. The high CO2 pressures appear to influence both conduit dissolution rates and geometry and presumably result from the microbial degradation of organics within the sediments. To explore the relationship between the geochemical environment of fluids and microbial ecology, additional samples were collected from the same location. Eight Coliform bacteria were identified to species level and inoculated in 65 milliliters of thioglycollate broth along with a calcite crystal of known weight and incubated at 12°C for 92 days. In the presence of five of the bacterial species, calcite dissolved more than the control, rangi...

The field of molecular fecal source tracking in the water environment has developed rapidly since the first PCR assays for general and host-­‐specific Bacteroides 16s rRNA markers were published. Numerous host-­‐specific molecular markers and PCR assays have been developed, adding greater specificity, sensitivity and quantitative methods to the array of options. The public demand for readying methods for transfer to the commercial lab, so that they may be used to generate data for public utilities, citizen action groups and regulatory agencies, has fueled the development of an entire new research community. These methods, however plentiful, have not found community agreement and there is no consensus concerning the appropriate implementation of molecular fecal source tracking in the field. Some issues plaguing the implementation include imperfect marker specificity, environmental variability, DNA extraction variability, PCR inhibition and high cost of molecular analysis. This thesis...

Numerous quantitative PCR assays for microbial fecal source tracking (MST) have been developed and evaluated in recent years. Widespread application has been hindered by a lack of knowledge regarding the geographical stability and hence applicability of such methods beyond the regional level. This study assessed the performance of five previously reported quantitative PCR assays targeting human-, cattle-, or ruminantassociated Bacteroidetes populations on 280 human and animal fecal samples from 16 countries across six continents. The tested cattle-associated markers were shown to be ruminantassociated. The quantitative distributions of marker concentrations in target and nontarget samples proved to be essential for the assessment of assay performance and were used to establish a new metric for quantitative source-specificity. In general, this study demonstrates that stable target populations required for marker-based MST occur around the globe. Ruminantassociated marker concentrations were strongly correlated with total intestinal Bacteroidetes populations and with each other, indicating that the detected ruminant-associated populations seem to be part of the intestinal core microbiome of ruminants worldwide. Consequently tested ruminant-targeted assays appear to be suitable quantitative MST tools beyond the regional level while the targeted human-associated populations seem to be less prevalent and stable, suggesting potential for improvements in human-targeted methods.

The field of molecular fecal source tracking in the water environment has developed rapidly since the first PCR assays for general and host-­‐specific Bacteroides 16s rRNA markers were published. Numerous host-­‐specific molecular markers and PCR assays have been developed, adding greater specificity, sensitivity and quantitative methods to the array of options. The public demand for readying methods for transfer to the commercial lab, so that they may be used to generate data for public utilities, citizen action groups and regulatory agencies, has fueled the development of an entire new research community. These methods, however plentiful, have not found community agreement and there is no consensus concerning the appropriate implementation of molecular fecal source tracking in the field. Some issues plaguing the implementation include imperfect marker specificity, environmental variability, DNA extraction variability, PCR inhibition and high cost of molecular analysis. This thesis...

A 1998 study of interstitial fluid geochemistry within Charonis Cascade in the Echo River/River Styx area of the Mammoth Cave System found carbon dioxide pressures higher than that of the fluids of the cave stream itself. This was confirmed by a limestone weight loss experiment in which samples dissolved at various levels below the streambed despite the low fluid velocities. The high CO2 pressures appear to influence both conduit dissolution rates and geometry and presumably result from the microbial degradation of organics within the sediments. To explore the relationship between the geochemical environment of fluids and microbial ecology, additional samples were collected from the same location. Eight Coliform bacteria were identified to species level and inoculated in 65 milliliters of thioglycollate broth along with a calcite crystal of known weight and incubated at 12°C for 92 days. In the presence of five of the bacterial species, calcite dissolved more than the control, rangi...