100 • FINE FOCUS, VOL. 3 (2) HALOPHILIC BACTERIA • 101 ISOLATION OF HALOPHILIC BACTERIA FROM INLAND PETROLEUM- PRODUCING WELLS MAEDGEN Q. LINDSEY & JENNIFER R. HUDDLESTON* BIOLOGY DEPARTMENT, ABILENE CHRISTIAN UNIVERSITY, ABILENE, TX 79699 MANUSCRIPT RECEIVED 28 MARCH 2017; ACCEPTED 20 MAY 2017 Copyright 2017, Fine Focus. All rights reserved. 102 • FINE FOCUS, VOL. 3 (2) HALOPHILIC BACTERIA • 103 a Pennsylvanian era channel-sand deposition organic osmotic solutes to remain isotonic located in Central Northern Texas (5), and thus relative to the environment (24). slightly older and deeper than the Flippen ABSTRACT Limestone, measuring at a depth of 2000 feet Due to the range of salinities, temperatures, at the oil well location. Saltwater produced at and possible carbon sources, oil felds and The goals of this study were to isolate microorganisms these sites is pumped to the surface with oil their related infrastructure pose a potential from oil well-produced water, identify the and gas, where the components are separated treasure trove of microbial diversity. microorganisms, and test the microorganisms’ salt and eventually utilized or discarded. Previously identifed microorganisms isolated tolerance. Saltwater collected from two well locations from oil well associated environments producing from different zones in Jones County, Texas, A consequence of the prevalence of oil include Marinobacter aquaeolei, which was was spread onto Mannitol Salt Agar (MSA). Isolates drilling is hydrocarbon contamination of described after being isolated from the head showed a 16S rDNA gene sequence identity of 99% soils and groundwater. Microbial-mediated of an offshore oil rig off the coast of Vung with Idiomarina baltica and Marinobacter persicus. bioremediation of hydrocarbon contaminated Tau, Vietnam, but which also occurs in the Salt tolerance assays indicated an optimal growth environments is an effective treatment, water column of the same area (17). From the concentration of 10-12.5% NaCl for the Idiomarina however the effcacy is limited by the same oil feld, Desulfovibri vietnamensis was isolate and a decrease in growth beyond 5% NaCl for conditions necessary for the microorganisms isolated from oil storage tanks, as well as oil the Marinobacter isolate. In conclusion, organisms that to optimally biodegrade hydrocarbons (9). well produced saltwater (8). Members of the are phylogenetically similar to marine microorganisms Salinity of the environment, either indigenous genus Desulfovibri have been implicated in the are present in oil well environments, and have variable or elevated by the contamination event, is corrosion of oil infrastructure via reduction salt tolerances, which may prove useful in microbial- an inhibitory condition for microorganisms of iron (13). The genus Petrotoga, named for its mediated hydrocarbon bioremediation of high salinity responsible for the bioremediation (25). outer toga-like sheath, is associated exclusively environments. Studies of optimum salinities for halophilic, with oil production, having been found in hydrocarbon degrading microorganisms could offshore wells and inland oil reservoirs (23). lead to targeted bioremediation efforts. The diversity of microorganisms in these salt rich, petroleum associated environments led INTRODUCTION Halophilic microorganisms are a type of us to look for variable halophilicity in microbe CORRESPONDING extremophile which require a high salt content populations by sampling saltwater produced AUTHOR As of 2015, there were an estimated 1.7 million oil in order to survive. The extent of halotolerance from different geological formations. In this and gas wells currently active in the United States (18). is categorized by the percent salt concentration work, we isolated two microorganisms from Saltwater is produced as a byproduct of drilling as it Jennifer R. Huddleston within the environment required for optimum saltwater produced as a byproduct of oil is released from the geological formation along with Biology Department growth. Slight halophiles exhibit optimum drilling, belonging to the genera Idiomarina oil and gas. The saltwater was originally sequestered as Abilene Christian University growth at 1-5% NaCl, moderate halophiles at and Marinobacter, and characterized their the respective formations were being laid down at the ACU Box 27868 5-20% NaCl, and extreme halophiles from respective salt tolerances. bottom of shallow oceans in past geological eras (11). In Abilene, TX 79699 20-30% NaCl (10). Halotolerant organisms this study, saltwater samples were obtained from the [email protected] are resistant to the deleterious effects a high Flippen Limestone and the King Sand formations. The salt content environment poses, but do not Flippen Limestone is located at a depth of 1850 feet at require a high salt content for survival. An the drilling location, and is localized to Fisher and Jones environment high in salt is inhibitory for County, Texas. The limestone deposition process started most microorganisms, as water from inside a in the late Pennsylvanian and continued through the cell will diffuse through the semipermeable KEYWORDS Permian geological time periods (16), approximately cell membrane into the environment via • Idiomarina 300 million years ago. Sequestered saltwater was osmosis and ultimately result in plasmolysis • Marinobacter released from the limestone upon hydraulic fracturing, (3). Halotolerant microorganisms utilize • Halophile a process in which highly pressurized water is used to haloadaptation to overcome this challenge, • Saltwater open fssures within the formation (12). The King Sand excluding salt from the cytoplasm where • Petroleum was the second formation sampled in this study, and is possible and biosynthesizing or accumulating 104 • FINE FOCUS, VOL. 3 (2) HALOPHILIC BACTERIA • 105 IDENTIFICATION while the Marinobacter isolate was tested in METHODS To identify the microorganisms, the 16S 0.5-12.5% NaCl. Culture tubes containing 2 rDNA genes were amplifed and sequenced. ml of TSB with concentrations of NaCl from SAMPLING DNA was extracted using a Zymo DNA 0.5-25.0% were inoculated with 30 μl of an Permission to sample, access to well sites, extraction kit (Zymo Research Corporation, overnight culture and incubated at 37°C for and instructions on equipment use were Irvine, CA) according to the manufacturer’s in- 48 hours with aeration by a shaker at 200 provided by the lease holder, JQL ENERGY, structions. Recombinant Vent DNA polymerase rpm. The optical density at 600 nm (OD600) LLC. Saltwater, produced as a byproduct of (New England BioLabs Inc., Ipswich, MA) was of four replicates at each NaCl concentration oil drilling, was collected aseptically from used in PCR to amplify a portion of the 16S were measured using a Hewlett Packard 8453 two oil well locations in Jones county, Texas. rDNA gene. PCR was executed utilizing the UV-Visible Spectrophotometer. Cultures were Locations, shown in Figure 1 were chosen due primers 5′-AGAGTTTGATCCTGGCTCAG-3′ diluted 2-fold with sterile water to obtain to contrasting geological characteristics, as the (F’-27m) and 5′-TACCTTGTTACGACTT-3′ accurate OD600 readings as necessary. frst oil well pumps from a solid limestone (R’-1492) (Positions 11–27 and 1489–1506, re- formation, while the second pumps from a spectively, according to the Escherichia coli 16S STATISTICAL ANALYSIS course-grain sand formation. The Flippen rRNA numbering system of the International The error bars in Figure 2 indicate standard Limestone sample was obtained from a Union of Biochemistry) (14). Primers were deviation of measured OD600 between four fberglass saltwater storage tank, while the designed to our specifcations and synthesized replicates. Statistical analysis of the OD600 for King Sand sample was obtained from a water by Invitrogen. The thermocycling conditions both data sets was calculated using Microsoft knockout, a horizontal tank which separates for amplifcation were as follows: initial dena- Excel’s Data Analysis add-in. An alpha of oil from saltwater based on varying density. turation: 95˚C for 5 minutes, 30 cycles of 95˚C 0.05 was used as the cutoff for statistical Four samples, two from each well site, were for 30 seconds, 55˚C for 30 seconds, 72˚C for signifcance. The Idiomarina isolate data set has collected using 118 mL sterile glass jars and 1 minute 30 seconds followed by a fnal ex- a p-value of 0.43238 and is therefore beyond stored overnight at 4°C. The King Sand tension at 72˚C for 10 minutes. The products the cutoff for statistical signifcance, while the samples were collected via release valve located were then confrmed using gel electrophoresis Marinobacter isolate data set has a p-value of on the bottom of the water knockout tank. Figure 1. Map of sampled locations in Jones of a 0.8% agarose (Invitrogen; Carlsbad, CA) 0.00017 and is therefore statistically signifcant. The Flippen Limstone samples were collected County, Texas. The Muehlstein lease, located at gel containing ethidium bromide under a 70 to via an access port on top of the saltwater 32°50’06.29”N, 99°43’51.26 W, elevation 1550 feet, 90 voltage for 30 to 45 minutes. The resulting tank, directly from the separated saltwater is the location of the sampled saltwater storage tank amplicons of 1.5 kb were further purifed using stored within. Initial sampling took place in from which the Idiomarina isolate was isolated, and DNA Clean & Concentrator (Zymo Research RESULTS November, 2015. The Idiomarina isolate has is used to store saltwater