Date of Award

Spring 2024

Project Type

Dissertation

Program or Major

Earth and Environmental Sciences

Degree Name

Doctor of Philosophy

First Advisor

Paula J Mouser

Second Advisor

Serita Frey

Third Advisor

Stuart Grandy

Abstract

During hydraulic fracturing, a water-based fluid containing proppants and several chemical additives is injected under high pressure into a ~2 km deep wellbore, to extend fractures in the underlying rock. This process introduces a host of microorganisms into the reservoir, some of which survive, persist for long periods of time and cause several problems including production of H2S, a corrosive toxicant and biofilm formation which clogs rock fractures. Over time, the bacterial genus Halanaerobium dominates the microbial community composition in many geologically distinct fractured formations. Our understanding of shale reservoir microbial dynamics remains limited despite extensive studies in the past decade. These unknowns include drivers of community shifts, energetics, nutrient cycling, stress tolerance and biofilm physiologies. My dissertation aims to advance our understanding of stress tolerance and biofilm formation in fractured shale bacteria using a multi-omic approach that integrates laboratory microcosms with field-based investigations. It queries plasma membrane physicochemical remodeling and metabolic regulations in H. congolense WG10 and mixed microbial consortia enriched from shale produced fluids, in context with salinity and under varying growth conditions. The plasma membrane responds to intracellular cues and external disturbances through lipidomic modifications that modulate its biophysical features, with consequences for microbial survival and processes.

In one approach, H. congolense WG10 and mixed consortia in shale produced fluids were incubated/enriched in chemostat bioreactors (continuous culture/planktonic) and drip flow biofilm reactors under different conditions. Salinity and HRT (only for planktonic) gradients for the isolate cultures were 7%, 13% and 20% NaCl, and 19.2 h, 24 h and 48 h, respectively. For the mixed enrichment cultures, only HRT was evaluated in planktonic mode at three gradients, 19.2 h, 48 h and 72 h. In the second approach, produced water samples were collected from a shale natural gas well at four timepoints between December 2019 and May 2021, filtered and preserved for analyses. Cell pellets from all samples were analyzed using GC-MS for fatty acids and LC-MS/MS for proteins and lipids. Cell-free chemostat media and produced water filtrates were analyzed for exometabolite composition using nuclear magnetic resonance (NMR).

Chapter 1 (objective 1) describes changes in plasma membrane phospholipid fatty acid (PLFA) composition in H. congolense WG10 and the mixed consortia across the salinity and HRT gradients. It shows that H. congolense WG10 makes its membrane more fluid and thicker through increasing its polyunsaturated fatty acid (PUFA) composition under non-optimal salinities (7% [too low] and 20% [too high]) compared to the optimum (13% NaCl).

Chapter 2 (objective 2) characterizes changes in plasma membrane intact polar lipid (IPL) chemistry in H. congolense WG10 and the mixed consortia under varying growth modes, salinities and HRT. It demonstrates that anionic phospholipids are important for high salinity tolerance in H. congolense WG10 while zwitterionic species modulate biofilm physiologies in both isolate and mixed cultures. Using machine learning-based modeling, this chapter presents a panel of lipid biomarkers whose levels in produced water could be useful for monitoring reservoir biofouling.

Finally, chapter 3 (objective 3) elucidates biochemical regulations in H. congolense WG10 and the mixed consortia under varying growth modes, salinities and HRTs using an integrative proteomics and exometabolomics approach. It shows that tyrosine accumulation and overexpression of Na+/H+ antiporters are essential for osmoprotection in H. congolense WG10 under high salinity. Upregulation of aromatic amino acid synthesis and cell envelope maintenance underpin biofilm formation. In addition, coping with metabolic stress induced by longer microbial retention in the reservoir requires increased protein metabolism and active transport regulation; the latter involves expression of more efflux transporters on the membrane interface.

In conclusion, bacterial persistence in fractured shale reservoirs under environmental and engineered perturbations requires strategic plasma membrane lipid chemistry adjustments and metabolic restructuring. Insights provided by this dissertation will inform better shale microbiology management for more efficient hydrocarbon extraction.

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