Using Environmental Biotechnology Principles and Approaches to Target Problematic Compounds in Complex Systems
Gaspar, Jason T
Alvarez, Pedro J
Doctor of Philosophy
When put in practice, environmental engineering frequently requires the characterization and remediation of problematic compounds such as priority pollutants or other molecules that impair performance in complex systems. Here, we tackle two such complex systems: shale gas reservoirs and the human body. In shale gas wells, the presence of microbes is usually unwanted and can often have deleterious effects, including reservoir souring, plugging, equipment corrosion, and reduction in gas production volumes. Hydrocarbon souring represents the most significant financial and safety challenge to the oil and gas industry. H2S may originate from geochemical or biogenic sources, although its source is rarely discerned. Biocides are dogmatically utilized during hydraulic fracturing to prevent or inhibit H2S generation. Here we characterize whether souring in the Bakken shale play is from microbial or geological origins. We develop a regional temperature map showing that downhole temperatures in Bakken reservoir wells equal or exceed the upper known temperature limit for microbial life. Attempts to extract microbial DNA from produced water yielded little to no detectable quantities. Stable isotope analysis yielded 34Sδ values from 4.4 – 9.8‰, suggesting souring had a geochemical origin. In cases of geochemical souring, reevaluation of the need for biocide addition, based on first characterizing the H2S source as we describe here, would provide significant reductions in both operational costs and overall environmental footprint. Similar to a traditional environmental system, the human body also accumulates “garbage” (i.e., detrimental aggregates) over time. Lipofuscin (LF) is a brown-yellow, autofluorescent polymeric material that accumulates in a ceroid manner within postmitotic cells during aging. LF accumulation impairs proteosome and lysosome pathways critical to cell health and homeostasis. The ability to quickly generate LF in vitro, and identify drugs that mitigate the accumulation or clear LF would be of great benefit to aging research. Here, we developed a novel platform to quickly create LF-loaded (but otherwise healthy) cells and screen drugs for efficacy in LF bioremediation. The combination of leupeptin, iron (III) chloride and hydrogen peroxide generates significant amounts of LF within cells at a much faster rate and in a less labor-intensive manner than previous methods. We show that oxidative stress induced LF-loading is accompanied by cellular cholesterol increases, and that long-term administration of the small molecule 2-hydroxypropyl-beta-cyclodextrin (HPβCD) reduces LF accumulation (~-27%, p < 0.00001). LF-loading is associated with increases in LDLr and SREBP1 gene expression, which are mitigated by HPβCD addition. In the absence of oxidative stress, HPβCD addition induces a paradoxical response, increasing cholesterol accumulation (but not LF) via upregulation of cholesterol biosynthesis. These two distinct, but opposite effects highlight a previously overlooked therapeutic consideration: the cholesterol content of the treated cell determines which cholesterol pathways, either beneficial or harmful, are responsive to HPβCD. These results are particularly significant because they provide clarity to HPβCD’s mode of action; which until now has remained in dispute amongst the scientific community.