Cultivation-Dependent Analysis of Microorganisms Associated with Various Hydraulic Fracturing Fluids
Creators: name: Harrington, Anthony 
Publisher: name: University of Nevada, Las Vegas
Year: 2015

description: The overarching goal of this study is to gain more insight to the microbial interactions associated with hydraulic fracturing by studying the culturable microorganisms present in various types of hydraulic fracturing fluids. Extraction of natural gas and other unconventional resources using hydraulic fracturing has increased in the last decade and very few studies have been conducted on the microorganisms associated with the various water types used in the process. From the very few published studies, the only cultivation-dependent method used involved determining the most probable number (MPN) of various metabolic groups, however the researchers failed to ask more in-depth questions regarding whether the microorganisms found are capable of metabolizing hydrocarbons or if they produce biosurfactants/bioemulsifiers. This study we diverge from the previous work by using a culture-dependent approach to characterize unique colony types of culturable aerobic-heterotrophic microorganisms associated with hydraulic fracturing fluids and to answer more in-depth questions, which can only be answered by growth of the microbes under various conditions. 
The first objective of this research focuses on isolating and identifying unique colony types of culturable microorganisms from several hydraulic fracturing fluid samples from different oil-bearing formations. Identifying the microorganisms present in the various fluids can help uncover potential microbiological problems that affect the efficiency of oil and natural gas extraction. Currently, deleterious microorganisms affecting the petroleum industry include sulfate-reducing bacteria (SRB), acid-producing bacteria (APB) and microorganisms that make extracellular polymeric substances (EPS). Sulfate-reducing bacteria are well-known for their role in corrosion, well fouling, and toxicity to human health. APB also contribute to corrosion of steel and concrete while EPS producing bacteria promote the formation of biofilms that further contributes to biocorrosion. Comparing the microorganisms present in the different hydraulic fracturing water samples will help identify potential microbiological problems from the study sites and define where the most potential for microbial problems exist. 
In order to address the other objectives of this study, culturable microorganisms are required; therefore, limitations due to isolation and low isolate number must be acknowledged. Cultivation-independent analysis describe the bacterial community associated with a sample whether living or dead and without concern for the origin of the bacteria within, whereas cultivation-dependent analysis provides isolates with which to further explore potential interactions of microbes and the petroleum. 
In the second objective of this research we characterize the culturable isolates found from the various fluid samples by investigating their response to various ranges of salinity, pH, and temperature, including those understood to be present in the subsurface environment. Data from previous published studies have inferred that the microorganisms found in flowback and produce waters are more adapted to harsh conditions based on geochemical data collected from the samples. A recently published study by Kelvin Gregory at Carnegie Mellon University (2014) found more genes associated with stress tolerance in produce water compared to source water using a metagenomic survey. This study will determine if the inferences from the previous metagenomic survey are valid from the perspective of viable and culturable isolates. 
The third objective will determine if the isolated microorganisms can create emulsions with petroleum oils and if they can use petroleum oils as their sole source of carbon and energy. Many microorganisms can use hydrocarbons as a source of carbon and energy by utilizing various metabolic pathways with beta-oxidation being the primary pathway (Alvarez, 2003; Atlas, 1981). Some research has shown that certain microbes produce biosurfactants (BS) or bioemulsifiers (BE) in the presence of hydrocarbons to aid in their utilization of hydrocarbons (Atlas, 1981). The purpose of this research objective will be to further expand on biosurfactant or bioemulsifier production and its relationship to hydrocarbon utilization, along with determining whether wastewaters associated with hydraulic fracturing could serve as a source to find novel BS/BE-producing isolates.