AN ABSTRACT OF THE THESIS OF

Margaret C. Schneider for the degree of Honors Baccalaureate of Science in Chemical Engineering presented on June 3rd, 2014. Title: Influence of Corexit 9500A and Alaska North Slope Crude oil toxicity to the -oxidizing , europaea, and the ammonia-oxidizing archaea, Nitrosopumilus maritimus

Abstract Approved: ______Lewis Semprini

The inhibitory effects of Corexit 9500A, Alaska North Slope Crude oil (ANSC), and mixtures of the two on the ammonia-oxidizing bacteria, Nitrosomonas europaea, and the ammonia-oxidizing archaea, Nitrosopumilus maritimus, were investigated. Corexit 9500A was found to be minimally toxic to both microorganisms with concentrations of 2000 and 3000 ppm yielding a 50% reduction in activity. The water-associated fraction (WAF) of ANSC was found to be reasonably toxic to N. europaea and N. maritimus with concentrations of 6 and 7 mL per 50 mL media resulting in 50% inhibition. Corexit 9500A:ANSC dispersant enhanced water-associated fraction (DEWAF) mixtures were found to be more toxic to each species as the ratio increased and as larger volumes were added per 50 mL media. ANSC WAF was found to be the most toxic, compared to the DEWAF mixtures, to both N. europaea and N. maritimus when compared on a per COD basis as most of the COD without dispersant present is attributed to low molecular weight aromatic hydrocarbons. These hydrocarbons are, most likely, the cause of the majority of inhibition caused by crude oil. Using dispersants, such as Corexit 9500A, may have a significant effect on microorganism activity and population due to the toxicity of hydrocarbons present in crude oil. In general, similar responses were observed in the different exposures of both microorganisms.

Key Words: Nitrosomonas europaea, Nitrosopumilus maritimus, Corexit 9500A, Alaska North

Slope Crude oil, Deepwater Horizon, ammonia-oxidizing bacteria, ammonia-oxidizing archaea

Corresponding email address: [email protected]

© Copyright Margaret C. Schneider June 3, 2014 All Rights Reserved

Influence of Corexit 9500A and Alaska North Slope Crude oil toxicity to the ammonia- oxidizing bacteria, Nitrosomonas europaea, and the ammonia-oxidizing archaea, Nitrosopumilus maritimus

By

Margaret C. Schneider

A PROJECT

Submitted to

Oregon State University

University Honors College

in partial fulfillment of the requirements for the degree of

Honors Baccalaureate of Science in Chemical Engineering (Honors Scholar)

Presented June 3, 2014

Commencement June 2014

Honors Baccalaureate of Chemical Engineering project of Margaret C. Schneider presented on June 3, 2014.

APPROVED:

______Mentor, representing Environmental Engineering

______Committee Member, representing Environmental Engineering

______Committee Member, representing Botany and Plant Pathology

______School Head, Chemical, Biological, and Environmental Engineering

______Dean, University Honors College

I understand that my project will become part of the permanent collection of Oregon State University, University Honors College. My signature below authorizes release of my project to any reader upon request.

______Margaret C. Schneider, Author

ACKNOWLEDGMENTS

First, thank you to Dr. Lewis Semprini for giving me a space in his lab to foster my interest in research as an undergraduate and for providing guidance during the thesis writing process and throughout my five years as an undergraduate. The experiences I have had working in the AOB lab were extremely valuable in helping me decide to pursue graduate studies.

Thank you to Dr. Tyler Radniecki for mentoring me during my first experience in academic research and teaching me how to synthesize results to build a bigger picture. I will forever compare every mentor I have to you, and they will have a lot to live up to.

Thank you to Dr. Neeraja Vajrala for teaching me how to work with N. maritimus, for serving on my thesis committee, and for agreeing to take on a random Honors College student on top of all your other work.

Thank you to my parents, Rosemarie and Bernard Schneider, for providing me with a quality education and a desire to learn. Thank you for always supporting me and giving me the freedom to explore my own interests. I owe all of my successes and opportunities to you.

Thank you to my grandpa, Dr. Ed Kos, for encouraging my love of science and helping with my college education. Not many people have grandfathers who pull out their old metabolic charts when their granddaughter is taking biochemistry. I am grateful that I do.

Thank you to Joe Anderson and Anees Sardari for being great lab mates and for helping with inhibition tests.

Funding was provided by the Pete and Rosalie Johnson Scholarship Fund.

TABLE OF CONTENTS

INTRODUCTION ...... 1

METHODS AND MATERIALS ...... 7

N. europaea culturing methods ...... 7 N. maritimus culturing methods ...... 7 Corexit 9500A:oil mixture preparation ...... 7 Acute N. europaea inhibition studies ...... 8 Acute N. maritimus inhibition studies...... 9 Chemical oxygen demand ...... 10

RESULTS AND DISCUSSION ...... 11

N. europaea and N. maritimus acute inhibition studies ...... 11 Acute Corexit 9500A inhibition ...... 12 Acute ANSC WAF inhibition ...... 13 Acute Corexit 9500A:ANSC mixture toxicity ...... 14 Toluene inhibition to N. maritimus ...... 18 Comparison of growth rates and nitrification activity ...... 19

FUTURE RESEARCH ...... 21

Method for normalizing N. maritimus results to total cell mass ...... 21 N. maritimus response to individual compounds (, toluene, phenol) ...... 21 Chronic inhibition of N. maritimus in the presence of Corexit 9500A and ANSC...... 21 Effect of growth media on inhibition ...... 22 Application of molecular methods to N. maritimus ...... 22 Comparison of N. maritimus activity as biofilm and suspended cells ...... 23

APPENDIX ...... 29

LIST OF FIGURES

1. Figure 1: The two step mechanism utilized by ammonia-oxidizing bacteria to convert ammonia to nitrite...... 4

2. Figure 2: Bottles containing ANSC and Corexit 9500A with ratios ranging from 0 to 1:5 Corexit 9500A:ANSC ...... 8

3. Figure 3: (A) Nitrite production per cell mass over time for N. europaea control reactors and N. europaea reactors treated with 2, 4, and 8 mL 1:5 DEWAF per 50 mL test media. (B) Nitrite production over time for N. maritimus control reactors and N. maritimus reactors treated with 2000, 3000, and 4000 ppm Corexit 9500A ...... 11

4. Figure 4: Percent nitrification activity of N. europaea and N. maritimus when exposed to various concentrations of Corexit 9500A...... 12

5. Figure 5: Percent nitrification activity of N. europaea and N. maritimus when exposed to various concentrations of SDS ...... 13

6. Figure 6: Percent nitrification activity of N. europaea and N. maritimus when exposed to various volumes of WAF per 50 mL test media...... 14

7. Figure 7: (A) Percent nitrification activity of N. europaea and (B) percent nitrification of N. maritimus in the presence of various volumes of ANSC WAF, 1:40 DEWAF, 1:20 DEWAF, and 1:5 DEWAF per 50 mL test media...... 15

8. Figure 8: (A) Percent nitrification activity of N. europaea and (B) percent nitrification of N. maritimus compared to the COD contribution from various volumes of ANSC WAF, 1:40 DEWAF, 1:20 DEWAF, and 1:5 DEWAF per 50 mL test media ...... 16

9. Figure 9: Percent nitrification activity of N. maritimus when exposed to various concentrations of toluene ...... 19

- 10. Figure 10: (A) NO2 production over time for N. europaea and N. maritimus in control - batch reactors containing only test media. (B) NO2 production over time for N. europaea and N. maritimus in batch reactors containing 1 mL WAF per 50 mL test media ...... 20

11. Figure 11: Percent nitrification activity of N. europaea when exposed to 4 mL of various DEWAF ratios per 50 mL media for 4 d and 3 h ...... 22

LIST OF TABLES

1. Table 1: The average COD in ANSC WAF and DEWAF serum bottles. The ± represents 95% confidence intervals for the COD measurements based on triplicate samples...... 16

2. Table 2: Two-factor ANOVA with replication analyzing the volume of DEWAF added to 50 mL media and the Corexit 9500A to ANSC ratio of the DEWAF mixture...... 30

This thesis is dedicated to my parents, who taught me to be an independent and

self-motivated learner. 1

Influence of Corexit 9500A and Alaska North Slope Crude oil toxicity to the ammonia-oxidizing bacteria, Nitrosomonas europaea, and the ammonia-oxidizing archaea, Nitrosopumilus maritimus

INTRODUCTION

Millions of gallons of South Louisiana sweet crude (LSC) began spilling into the Gulf of Mexico on April 20, 2010 when the Deepwater Horizon oil rig on the BP-operated Macondo Prospect exploded. The oil gusher was capped on July 15, 2010 after releasing 4.9 million barrels of oil into the environment (United States Coast Guard National Response Team (USCG NRT), 2011).

Several strategies were used to clean up and contain the oil spill, including in situ burning, use of containment booms, surface skimming, direct capture from the source, and the use of chemical oil dispersants (USCG NRT, 2011).

Crude oil composition depends largely on its geographic origin. Hydrocarbons, ranging from light, gasoline-like fractions to heavier tars and wax-like compounds, are the main component comprising 50-98% of the total volume of crude blends (National Oceanic and Atmospheric

Administration (NOAA), 2014). The oil from the Deepwater Horizon spill, Louisiana Sweet crude, is considered a light crude oil as it has an American Petroleum Institute (API) gravity of

350, which indicates a high gasoline potential of LSC (high naptha content), and a sweet crude because of low sulfur content (API Petroleum HPV Testing Group, 2003; Cleveland, 2011). LSC is high in alkanes and relatively low in polyaromatic hydrocarbons (PAHs), generally making the oil less toxic to the environment and easily biodegradable. LSC also contains volatile organic compounds including toluene, benzene, and xylene (Cleveland, 2011). LSC as a water-associated fraction (WAF) caused a median lethal dose (LC50) in mysid shrimp of 2.7 (2.5-3.0) milligrams 2

total petroleum hydrocarbons per liter (mg TPH/L) and LC50 of 3.5 (3.4-3.7) mg TPH/L in inland silversides, a small estuarine fish (Hemmer et al., 2011).

A model crude oil, Alaska North Slope crude (ANSC), was used in this study. ANSC has considerably less parafinnic hydrocarbons and more aromatic compounds than LSC and a smaller

API gravity of 280 (API, 2003). Crude composition is a consideration in determining toxicity to the environment, specifically when mixed with dispersants. ANSC has been shown to have detrimental effects on the development of fish embryos after brief intervals in the environment.

Juvenile pink salmon exhibited less mobility, melanosis, erratic swimming, and loss of equilibrium when exposed to the water associated fraction of ANSC in combination with UV sun exposure (Barron et al., 2004).

Oil dispersants are chemicals that can be used to break up oil slicks and increase their degradation rates by bringing oil into the water column when conditions, such as rough seas, or location prevent the use of burning or skimming (United State Environmental Protection Agency (US

EPA), 2010). Dispersants were used in multiple ways throughout the Deepwater Horizon cleanup effort. Dispersants were applied at the source of the spill nearly one mile below the ocean surface, on the surface to control volatile organic compounds, and aerially to control slicks more than 5 nautical miles from the source (USCG NRT, 2011). The United States Environmental Protection

Agency (US EPA) limited BP to 15,000 gallons per day for undersea dispersant application (US

EPA, 2010). An estimated 1.5-1.9 million gallons of dispersant were applied during the 87 day oil release (Hayworth and Clement, 2011; Judson, et al., 2010).

The main dispersant used in the Deepwater Horizon cleanup was Corexit 9500A, which is on the

U.S. Environmental Protection Agency’s National Contingency Plan (NCP) Product Schedule

(US EPA, 2014). The NCP Product Schedule lists all products relevant to oil spill cleanups that 3

have been submitted to the EPA with supporting data. Not all products on the NCP Product

Schedule have been approved or recommended by the EPA.

Corexit 9500A is a mixture of two nonionic surfactants, ethoxylated sorbitan monooleate (Tween

80) and sorbitan monooleate (Span 80) and an anionic surfactant, dioctyl sodium sulfosuccinate

(AOT) (Thibodeaux et al., 2011). The surfactants act to bring oil into the water column making the oil components chemically available to microorganisms and more accessible to other aquatic species. Tests conducted by BP prior to the oil spill indicated that Corexit 9500A was less toxic than other dispersants (US EPA, 2010). An independent EPA study comparing eight chemical dispersants, including Corexit 9500A, concluded that Corexit 9500A had generally similar toxicity to many other dispersants when tested alone but a lower toxicity when mixed with LSC.

Corexit 9500A had a LC50 of 42 ± 5 μl/L and 130 ± 8 μl/L for mysid shrimp and inland silversides, respectively, which fall into the slightly toxic and practically nontoxic EPA defined toxicity classifications. Mixing LSC and Corexit 9500A resulted in LC50s of

5.4 (4.9-6.7) mg TPH/L and 7.6 (6.2-8.5) mg TPH/L for mysid shrimp and inland silversides, respectively, which was less toxic than most of the other dispersants tested (Hemmer et al., 2011).

Toxicity evaluations of dispersant and dispersant-oil mixtures have mainly been associated with eukaryotic life forms. More investigation is required to assess the effects of dispersant and dispersant-crude oil mixtures on the activity of microorganisms that play vital roles in marine ecosystems and global biogeochemical cycles, including the cycle.

The major steps of the nitrogen cycle are nitrogen fixation, ammonification, nitrification, and denitrification, which are all performed largely by microorganisms. Nitrogen cycles from

- - atmospheric nitrogen (N2) to ammonia (NH3) to nitrite (NO2 ) to nitrate (NO3 ) and back to N2

(Sprent, 1987). The supply of nitrogen in all its forms is a key factor controlling the nature and diversity of plant life, population dynamics of animals, and other ecological processes, such as 4

carbon and mineral cycling (Vitousek et al., 1997). Human activities have put increased demand on nitrogen cycling by accelerating the rate of industrial nitrogen fixation to produce synthetic fertilizers, cultivation of nitrogen-fixing crops, and fossil fuel burning, among others.

This puts added importance on the role of microorganisms in nitrogen cycling. Aquatic microorganisms are especially important as the concentration of nitrogen species increases in water systems due to human activities (Vitousek et al., 1997).

Two microorganisms important to ammonia oxidation in aquatic environments are Nitrosomonas europaea (N. europaea), an ammonia-oxidizing bacterium (AOB), and Nitrosopumilus maritimus

(N. maritimus), an ammonia-oxidizing archaeon (AOA). Both microorganisms convert NH3 to

- NO2 for the first and rate-limiting step of the nitrogen cycle, with N. europaea functioning in both freshwater and coastal systems and N. maritimus in marine ecosystems. While both microorganisms serve similar environmental functions, differences in cell membrane and cell wall construction between archaea and bacteria may lead to differences in response to pollutants.

N. europaea is the model AOB and has been studied extensively. AOB have been shown to be sensitive to the presence of hydrocarbons including aromatics, aliphatics, alkanes, and alkenes

(Juliastuti et al., 2011; Radniecki et al., 2008, 2011; Keener and Arp, 1993; Chang et al., 2002), which are the main components of crude oil. Hydrocarbons act to disrupt the two step mechanism

N. europaea uses to oxidize ammonia. In the first step, NH3 is oxidized to hydroxylamine

(NH2OH) by an ammonia monooxygenase (AMO) enzyme, followed by NH2OH oxidation to

- NO2 by a hydroxylamine oxidoreductase (HAO) enzyme (Figure 1).

퐴푀푂 + − 푁퐻3 + 푂2 + 2퐻 + 2푒 → 푁퐻2푂퐻 + 퐻2푂 퐻퐴푂 − + − 푁퐻2푂퐻 + 퐻2푂 → 푁푂2 + 5퐻 + 4푒 Figure 1: The two step mechanism utilized by ammonia-oxidizing bacteria to convert ammonia to nitrite. 5

The initial AMO reaction consumes two electrons, while the HAO reaction produces four electrons. Two of the produced electrons are used to reduce AMO and the other two are used for cell energy, growth, and maintenance (Arp et al., 2002). AMO is non-specific allowing the enzyme to oxidize available hydrocarbons, which cannot be further oxidized by HAO (Chang et al., 2002; Keener and Arp, 1994). Hydrocarbon oxidation leads to a co-metabolic energy drain on the AOB cells as electrons are consumed by AMO without being replenished by HAO.

Less is known regarding the effect of different environmental factors on AOA as their discovery is relatively recent. N. maritimus strain SCM1 was the first chemolithoautotrophic nitrifier in the domain Archaea and the first reported mesophilic isolate within the phylum Crenarchaeota. The pure culture was isolated from gravel in a marine tropical fish tank at the Seattle Aquarium

(Könneke et al., 2005). Archaea were formerly thought to play a limited role in ecosystems as all previously cultivated Crenarcheota were sulphur-metabolizing thermophiles (Burggraf et al.,

1997). However, more recently Archaea have been estimated at an abundance of 1028 cells in the world’s oceans. Pelagic Crenarchaeota, such as N. maritimus, were reported to be one of the ocean’s most abundant cell types, suggesting that the microorganisms play a significant role in the global biogeochemical cycles (Karner et al., 2001).

N. maritimus was the first Crenarchaea found to aerobically oxidize ammonium to nitrite

(Könneke et al., 2005) in, most likely, a similar metabolic pathway as N. europaea (Vajrala et al.,

2013). N. maritimus oxidizes ammonia in the absence of organic carbon, demonstrating that the organism fixes inorganic carbon (Könneke et al., 2005), a trait common to all nitrifiers (Bock and

Wagner, 2006). N. maritimus cell growth was found to correlate with near-stoichiometric conversion of ammonium to nitrite of 1:1.52 similar to that of AOB (Martens-Habbena et al.,

2009). AMO-encoding genes were also found to coincide with nitrification activity, and growth was limited in the presence of unspecified organic compounds (Könneke et al., 2005), further suggesting similarities to AOB. However, N. maritimus exhibited high oxygen and ammonium 6

uptake at levels as low as 0.2 μM ammonium chloride, whereas AOB showed no activity. This indicates that AOA, such as N. maritimus, are likely responsible for much of the nitrite production in oligotrophic environments, including open oceans which contain low concentrations of ammonia (Martens-Habbena et al., 2009).

Understanding how crude oil and oil dispersants affect the nitrification activity of ammonia- oxidizing archaea and bacteria is important in assessing the toxicity of crude oil and dispersant on the environment. This information can then be used to better understand the overall environmental impact of oil spills and better direct cleanup efforts to minimize damage to ecosystems surrounding a spill site. The current study examines the acute effects of Corexit

9500A, Alaska North Slope crude oil, and Corexit 9500A:ANSC mixtures to both N. europaea and N. maritimus.

The following list details the specific objectives of this study.

 Test acute (3 h) toxicity of Corexit 9500A concentrations ranging from 0-8000 ppm to

N. europaea and N. maritimus

 Test acute toxicity of ANSC water-associated fraction (WAF) concentrations of 0-10 mL

per 50 mL test media to N. europaea and N. maritimus

 Test acute toxicity of 1:40, 1:20, and 1:5 Corexit 9500A:ANSC dispersant enhanced

water-associated fraction (DEWAF) mixtures at concentrations of 0-8 mL per 50 mL test

media to N. europaea and N. maritimus

 Quantify the amount of hydrocarbons introduced into the water column for the WAF and

DEWAF mixtures used for toxicity tests

7

METHODS AND MATERIALS

N. europaea culturing methods

N. europaea ATCC 19718 cells were cultured in 2 L of AOB growth media (pH ~7.8) containing

(NH4)2SO4, KH2PO4, MgSO4, CaCl2, EDTA free acid, FeSO4, CuSO4, NaH2PO4, and Na2CO3

(Radniecki et al., 2011) at 30 °C in the dark, while shaking at 100 RPM on a rotary shaker table for a 3 d growth period. The cells were harvested in the late-exponential growth phase after reaching an OD600 of ~0.070 and centrifuged at 9000 RPM for 1 h. Cells were resuspended in

30 mL of 30 mM HEPES buffer (pH 7.8) and centrifuged for 20 min at 9000 RPM. Cells were resuspended in an equal amount of HEPES buffer and stored at 10 °C until used for an inhibition study.

N. maritimus culturing methods

N. maritimus strain SCM1 cells were cultured in 1 L of HEPES-buffered (pH ~7.6) modified synthetic crenarchaeota media with a basal salt solution (NaCl, MgSO4, MgCl, CaCl2, and KBr) with additions of KH2PO4, FeNaEDTA, NaHCO3, and a trace element mixture with 1mM NH4Cl

(Könneke et al., 2005). Cultures were incubated in the dark at 30 °C until they reached the mid- exponential growth phase, which was indicated by the nitrite concentration reaching 400-600 µM.

Cells were harvested on a 0.2 μm filter paper using a vacuum manifold system as described in

Vajrala et al. (2013).

Corexit 9500A:oil mixture preparation

Corexit 9500A:ANSC oil mixtures (Figure 2) were prepared based on a 3 mL standard volume of

ANSC. ANSC (3 mL) was added to DI water (100 mL) in a 125 mL serum bottle. Corresponding volumes of Corexit 9500A were added to the ANSC/DI water mixture to make ratios of Corexit

9500A to ANSC (by volume) of 1:5, 1:20, and 1:40. Once capped, the bottles were inverted and 8

shaken at 150 RPM on a rotary shaker table in the dark at 30 °C for a day (~18 h). Bottles were stored inverted without shaking for a minimum of 24 h before use to allow the aqueous and non- aqueous phases to separate. The aqueous phase of either the ANSC water-associated fraction

(WAF) or dispersant enhanced water-associated fraction (DEWAF) of the Corexit 9500A:ANSC mixtures was used for treatment in inhibition studies.

Figure 2: Bottles containing ANSC and Corexit 9500A with ratios ranging from 0 to 1:5 Corexit 9500A:ANSC. Ratios of 0, 1:40, 1:20, and 1:5 were used for characterization with N. maritimus and N. europaea. Further characterization of N. europaea nitrification activity with 1:80 and 1:10 ratios can be found in Radniecki et al. (2013).

Acute N. europaea inhibition studies

N. europaea cells were added to 50 mL of fresh growth medium with a reduced (NH4)2SO4 concentration of 2.5 mM in a 155 mL batch reactor to reach a final cell concentration of 5 mg/L

(OD600 ~ 0.070). Treatments of either Corexit 9500A, WAF, or DEWAF were added in varying amounts to quantify their inhibitory effects on the nitrification activity of N. europaea. An 80% concentrated growth medium was used with a balance of WAF or DEWAF and DI water for tests requiring large treatment volumes. Controls consisted of batch reactors with cells and (NH4)2SO4, but without the dispersant or dispersant:oil mixtures.

Tests were conducted over a 3 h period with nitrite production measured every 45 min while the batch reactors were shaken at 250 RPM in the dark at 25 °C. Nitrite concentration was monitored 9

using a colorimetric assay (Hageman and Hucklesby, 1971). The assay reagents, 1% w/v sulfanilamide in 1 M HCl (890 µL) and 0.2% w/v n-1-napthyl-ethyldiamine (n-1-NED, 100 µL), were mixed with 10 µL of experimental sample. Absorbance at 540 nm wavelength was measured after 10 min using a Beckman Coulter DU 530 Life Science UV/Vis

Spectrophotometer. Nitrite concentration was determined from absorbance using a linear standard curve.

Nitrification activity of the treatment was compared to the control activity after the end of the 3 h exposure test. When possible, nitrite production was normalized to cell density of the batch reactor. Many treatments affected the optical density measurements, so the average optical density of the control batch reactors was used as the optical density for all treatment batch reactors. Nitrification activity for N. europaea inhibition tests determined using Equation 1, where the protein concentration of the treatment batch reactors was determined using the average optical density of the control batch reactors.

[푁푂−] 푚푚표푙 2 ( )| [푝푟표푡푒푖푛] 푚푔 % 푁푖푡푟푖푓푖푐푎푡푖표푛 퐴푐푡푖푣푖푡푦 = 푡=180 푚푖푛,푡푟푒푎푡푚푒푛푡 × 100% (1) [푁푂−] 푚푚표푙 2 ( )| [푝푟표푡푒푖푛] 푚푔 ( 푡=180 푚푖푛,푐표푛푡푟표푙 )

Acute N. maritimus inhibition studies

N. maritimus batch reactors were prepared similarly to N. europaea batch reactors. Crenarchaeota media was used with 1 mM NH4Cl and treatment doses. N. maritimus cells attached to filter paper were added to the batch reactor after filtering 500 mL of cell culture through the vacuum manifold system.

Inhibition tests were conducted over a 3 h period with nitrite samples collected at 45 min intervals while the batch reactors incubated in a 30 °C dark room. The nitrite colorimetric assay (Hageman and Hucklesby, 1971) was prepared by adding 450 µL of DI water, 250 µL of 0.02% w/v 10

n-1-NED, and 250 µL of 1% w/v sulfanilamide in 1.5 M HCl to 50 µL of sample. Absorbance at

540 nm wavelength was measured after a 30 min incubation using a Beckman DU 640

Spectrophotometer. Nitrite concentration was determined from absorbance using a linear standard curve. Because the cell mass added to each batch reactor was not determined, nitrification activity was reported as the ratio of nitrite produced by the treatment to the nitrite produce by the control at the 3 h time point (Equation 2).

− [푁푂2 ] (휇푀)|푡=180 푚푖푛,푡푟푒푎푡푚푒푛푡 % 푁푖푡푟푖푓푖푐푎푡푖표푛 퐴푐푡푖푣푖푡푦 = ( − ) × 100% (2) [푁푂2 ] (휇푀)|푡=180 푚푖푛,푐표푛푡푟표푙

Chemical oxygen demand

Chemical oxygen (COD) assays were performed to quantify the concentration of hydrocarbon mixture in the aqueous phase of the ANSC WAF and DEWAF treatments. No attempt was made to measure the individual hydrocarbon components in the mixture. 11

RESULTS AND DISCUSSION

N. europaea and N. maritimus acute inhibition studies

Inhibition to nitrification activity of N. europaea and N. maritimus in the presence of different concentrations of Corexit 9500A, ANSC WAF, and various Corexit 9500A:ANSC DEWAF mixtures was measured by determining the nitrite production of the cells over a 3 h period.

Figure 3 shows sample inhibition tests completed for this study. Figure 3A is the nitrification rate of N. europaea with 2, 4, and 8 mL of 1:5 DEWAF added per 50 mL media. Figure 3B is the nitrification activity of N. maritimus with 2000, 3000, and 4000 ppm Corexit 9500A. Nitrification activity indicates the ratio of nitrite produced by the treatment to nitrite produced by the control.

A

B

Figure 3: (A) Nitrite production per cell mass over time for N. europaea control reactors (◊) and N. europaea reactors treated with 2 mL 1:5 Corexit 9500A:ANSC DEWAF (■), 4 mL 1:5 DEWAF (∆), and 8 mL 1:5 DEWAF (●) per 50 mL test media. (B) Nitrite production over time for N. maritimus control reactors (◊) and N. maritimus reactors treated with 2000 ppm (■), 3000 ppm (∆), and 4000 ppm Corexit 9500A (●). Error bars represent 95% confidence intervals from triplicate experiments. 12

Acute Corexit 9500A inhibition

Neither N. europaea nor N. maritimus displayed high sensitivity to Corexit 9500A.

Concentrations of 2000 and 3000 ppm Corexit 9500A resulted in a ~50% reduction in nitrification activity compared to the controls (Figure 4). N. europaea and N. maritimus showed similar levels of inhibition at both high and low Corexit 9500A concentrations. The data suggests that Corexit 9500A causes minimal inhibition to both N. europaea and N. maritimus when examined alone.

Figure 4: Percent nitrification activity of N. europaea (■) and N. maritimus (◊) when exposed to various concentrations of Corexit 9500A. Error bars represent 95% confidence intervals from triplicate experiments.

Other studies have found similar results when examining the response of the bacterium Vibrio fisheri (Fuller et al., 2004) and deep-sea microbial microcosms from the Gulf of Mexico at environmentally relevant concentrations of 1-100 ppm Corexit 9500A (Baelum et al., 2012;

Chakroborty et al., 2012). Mixed soil microbial microcosms showed contradictory effects when exposed to Corexit 9500A. Some heterotrophic bacterial populations showed inhibitory effects of

Corexit 9500A (< 50 ppm), while others were able to use Corexit 9500A as a nutrient and carbon source and grow to significant populations as indicated by a considerable change in bacterial community over several days of incubation (Lindstrom and Braddock, 2002; Yoshida et al.,

2006). Corexit 9500A has also been shown to cause a significant reduction in mammalian cell viability, depending on nutrient availability to the cells, at concentrations ranging from 20-200 13

ppm (Zheng et al., 2014). Mammalian cell response would be most relevant once oil slicks and dispersant reach the shoreline and coastal ecosystems.

In comparison, exposure to sodium dodecyl sulfate (SDS), a model surfactant, resulted in greater inhibition with concentrations of 200 ppm for N. europaea and 50 ppm for N. maritimus causing a ~50% reduction in nitrification activity (Figure 5). SDS, an anionic surfactant, interacts with the cell membranes of the microorganisms to disrupt cell structure. Differences in membrane structure between archaea and bacteria are a likely cause of the differences in degree of inhibition caused by SDS. In general, N. europaea and N. maritimus were shown to respond similarly to each of the different surfactants tested.

Figure 5: Percent nitrification activity of N. europaea (■) and N. maritimus (◊) when exposed to various concentrations of SDS. Error bars represent 95% confidence intervals from triplicate experiments.

Acute ANSC WAF inhibition

N. europaea and N. maritimus showed similar responses to ANSC WAF. Greater added volumes of WAF caused increased inhibition. Concentrations of 6 to 7 mL WAF per 50 mL media resulted in a ~50% reduction in nitrification activity (Figure 6). 14

Figure 6: Percent nitrification activity of N. europaea (■) and N. maritimus (◊) when exposed to varying volumes of WAF per 50 mL test media. Error bars represent 95% confidence intervals from triplicate experiments.

N. europaea displayed a more consistent trend with increasing volumes of WAF added, while

N. maritimus showed a significant decrease in nitrification activity from 4 to 10 mL of WAF added per 50 mL media. N. maritimus activity was consistent between 1 and 4 mL of added WAF

(~75% activity). The nitrification activity of N. maritimus was 83 ± 9% and 14 ± 4% when exposed to 4 mL and 10 mL of WAF, respectively, whereas N. europaea had activities of

70 ± 5% and 41 ± 4%, respectively. Reproducibility for each microorganism was relatively consistent with average 95% confidence intervals less than ±10% nitrification activity, despite not normalizing N. maritimus activity to cell mass and increased difficulty harvesting N. maritimus cells.

Acute Corexit 9500A:ANSC mixture toxicity

Both microorganisms showed increased inhibition when exposed to different ratios of DEWAF.

Higher ratios of Corexit 9500A to ANSC resulted in increased inhibition as did larger added volumes per 50 mL test media (Figure 7). N. maritimus activity exhibited considerable variability when tested with different DEWAF ratios and volumes, but followed similar trends as

N. europaea. A two-factor ANOVA run on the N. maritimus data (Table 2, Appendix) indicated that volume added, DEWAF ratio, and the interaction between the two were statistically 15

significant. Factors contributing to the variability among N. maritimus data includes time frame in which inhibition tests were run, not normalizing activity to cell mass, and difficulty harvesting cells.

A

B

Figure 7: (A) Percent nitrification activity of N. europaea and (B) percent nitrification activity of N. maritimus in the presence of various volumes of ANSC WAF (◊), 1:40 Corexit 9500A:ANSC DEWAF (▲), 1:20 DEWAF (□), and 1:5 DEWAF (●) per 50 mL test media. Error bars represent 95% confidence intervals from triplicate experiments.

The COD of the aqueous phase increased from the WAF to the higher ratios of Corexit 9500A to

ANSC with the WAF having a COD of 200 ± 118 ppm and 1:5 DEWAF having a COD of

16090 ± 1965 ppm (Table 1). A visual depiction of the greater COD is illustrated by the darker colors in Figure 1. COD indicates the hydrocarbon content of the aqueous phase for the mixtures used in the treatments. The hydrocarbons are suspected to be the main cause of inhibition for microorganisms. 16

Table 1: The average COD in ANSC WAF and DEWAF serum bottles. The ± represents 95% confidence intervals for the COD measurements based on triplicate samples. Treatment Bottle COD (ppm) ANSC WAF 200 ± 118 1:40 DEWAF 2273 ± 111 1:20 DEWAF 4332 ± 232 1:5 DEWAF 16090 ± 1965

The relationship between nitrification activity and COD had the opposite trend as shown in

Figure 7 with WAF causing the most significant inhibition on a per COD basis (Figure 8). Both microorganisms were most inhibited by WAF followed by 1:40 DEWAF, 1:20 DEWAF, and 1:5

DEWAF when compared on a per COD basis.

A

B

Figure 8: (A) Percent nitrification activity of N. europaea and (B) percent nitrification activity of N. maritimus compared to the COD contribution from various volumes of ANSC WAF (◊), 1:40 Corexit 9500A:ANSC DEWAF (▲), 1:20 DEWAF (□), and 1:5 DEWAF (●) per 50 mL test media. Error bars represent 95% confidence intervals from triplicate experiments. 17

Increasing Corexit 9500A:ANSC ratios lead to decreasing nitrification activity when compared to the COD in the reactor. This indicates that Corexit 9500A brings more of the larger, less inhibitory hydrocarbons into solution and only a small amount of the smaller, more soluble hydrocarbons. It appears that the more soluble hydrocarbons in the WAF are responsible for the inhibition.

Without the presence of dispersant, the main hydrocarbons brought into the WAF are generally the smaller and more water soluble compounds including the BTEX compounds (benzene, toluene, ethylbenzene, and xylene) and semi-volatiles (naphthalenes and phenols). Larger polycyclic aromatic hydrocarbons (PAHs) with 4 or more rings contribute very little to the WAF due to their low solubility (Faksness et al., 2008). Corexit 9500A solubilizes more of the larger and hydrophobic hydrocarbons into the aqueous phase (Couillard et al., 2005; Venosa and

Holder, 2007), resulting in a higher COD.

ANSC consists mainly of large organic molecules including paraffins, asphaltenes, resins, and waxes, which are hydrophobic, have low solubility, and contribute little to WAF (Fingas, 2010).

Only a portion of the excess COD brought into the DEWAF by Corexit 9500A was likely responsible for the increased nitrification inhibition to N. europaea and N. maritimus. The low molecular weight, aromatic hydrocarbons that have been shown to be highly toxic to N. europaea

(Chang et al., 2002; Keener and Arp, 1994; Radniecki et al., 2008) likely constitute a larger portion of the COD for the WAF than for the DEWAF mixtures, thus causing more inhibition on a per COD basis.

Attributing inhibition to the more soluble and aromatic components of the crude oil is further supported by decreased inhibition when N. europaea was tested using weathered ANSC.

Weathering is a natural process that occurs after oil spills as the churning action of water transfers nearly all the volatile fractions of the oil to the atmosphere (Galt et al., 1991). Weathering has 18

been shown to decrease the inhibition caused by WAF and DEWAF mixtures with N. europaea without significantly changing the COD (Radniecki et al., 2013). This further indicates that the volatile hydrocarbons are likely responsible for any observed inhibition. In oil spills these compounds would not be present in weathered-plume exposed to the atmosphere resulting in limited inhibition from the presence of crude oil. More research needs to be performed with

N. maritimus to study the effect of weathering.

NE1545, a putative membrane protein, is over-expressed in N. europaea cells exposed to aromatic hydrocarbons (Radniecki et al., 2011). Overexpression of the NE1545 gene in

N. europaea after exposure to WAF and DEWAF mixtures showed that aromatic hydrocarbons were bioavailable to the cells (Radniecki et al., 2013). This suggests that aromatic hydrocarbons in both the WAF and the DEWAF mixtures are partially responsible for the reduced nitrification activity. The similarity between the nitrification activity of N. europaea and N. maritimus when exposed to the same concentrations of WAF and DEWAF suggest that aromatic hydrocarbons have a similar effect on the nitrification activity of N. maritimus as has been shown with N. europaea. However, an NE1545 gene homolog has not been identified in N. maritimus.

Toluene inhibition to N. maritimus

Toluene is one of the water soluble compounds present in crude oils and has been shown to cause significant inhibition to N. europaea. A 20 µM toluene concentration has been shown to cause a

50% inhibition in N. europaea nitrification activity after 3 h exposure. This inhibition was attributed to the activity of the AMO enzyme, which also showed a 50% reduction in activity, while HAO activity showed no change. Oxidized forms of toluene, benzyl alcohol and benzaldehyde, were produced during the 3 h exposure, which was likely a result of AMO activity

(Radniecki et al., 2008). 19

N. maritimus displayed more resistance to toluene than N. europaea with exposure to 20 μM toluene resulting in 85.7 ± 7.6% nitrification activity. Nitrification activity decreased as toluene concentration increased with 40 μM toluene resulting in 67.5 ± 8.4% nitrification activity

(Figure 9). This variation in response to toluene between N. europaea and N. maritimus may be a result of structural differences of the AMO type enzymes for each microorganism. However, the narrow range of toluene concentrations tested does not allow for definitive conclusions to be made regarding the differences between the effect of toluene on N. europaea and N. maritimus.

Figure 9: Percent nitrification activity of N. maritimus (◊) when exposed to various concentrations of toluene. Error bars represent 95% confidence intervals from triplicate experiments.

Comparison of growth rates and nitrification activity

N. maritimus grows and oxidizes ammonia at a significantly slower rate than N. europaea. In

- - these tests, uninhibited N. europaea produced NO2 at a rate of 10.1 ± 1.2 μM NO2 /min compared

- to N. maritimus which had a production rate of 0.70 ± 0.06 μM NO2 /min (Figure 10A). Cells treated with 1 mL WAF resulted in a percent nitrification activity of 78.5 ± 6.3% and

76.7 ± 12.3% for N. europaea and N. maritimus, respectively (Figure 10B) when compared to the

- activity presented in Figure 10A. The NO2 production rates for the 1 mL WAF treatments were

- - 9.3 ± 1.6 μM NO2 /min and 0.55 ± 0.22 μM NO2 /min for N. europaea and N. maritimus, respectively. The slow growth rate of N. maritimus may have contributed to the high variability 20

among observed inhibition levels for the Corexit 9500A, WAF, and DEWAF treatments. A longer time period for acute inhibition tests for N. maritimus may have resulted in more consistent activity levels as the archaea would have had a similar amount of time to process ammonia as N. europaea relative to their respective growth rates.

A B - Figure 10: (A) NO2 production over time for N. europaea (■) and N. maritimus (♦) in control - batch reactors containing only test media. (B) NO2 production over time for N. europaea (□) and N. maritimus (◊) in batch reactors containing 1 mL WAF per 50 mL test media. Error bars represent 95% confidence intervals from triplicate experiments.

Previous research has shown that N. europaea growth rate has a positive correlation with inhibition rate in the presence of phenol (Lauchnor and Semprini, 2013). More inhibition was observed with faster growing cells. The current study suggests that inhibition rate is not purely a function of nitrification rate, as N. maritimus oxidized ammonia an order of magnitude slower than N. europaea, but rather a microorganism specific effect.

21

FUTURE RESEARCH

Method for normalizing N. maritimus results to total cell mass

N. maritimus nitrification activity consistently displayed more variability than results with

N. europaea. This is possibly a result of the lack of normalization to cell mass with the N. maritimus results. Applying a method for determining the total cell mass of the batch reactors for

N. maritimus would reduce variation between triplicate samples and increase confidence in the reported activity. Possible methods for determining total cell mass are measuring the weight change of the filter paper when vacuum filtering the cells or developing a standard curve of nitrite production of the cell culture to total cell mass of the culture. Inhibition tests might be applied where the nitrite production rate of an individual batch reactor is first measured prior to addition of the treatment.

N. maritimus response to individual compounds (benzene, toluene, phenol)

Further work characterizing inhibition induced by individual hydrocarbon compounds (benzene, phenol, toluene, acetylene) would illustrate better the effect of small, aromatic hydrocarbons on

N. maritimus. Concentrations of 40 µM benzene, 20 µM toluene, and 10 µM phenol result in approximately 50% inhibition for N. europaea in 3 h tests as the non-specific AMO enzyme oxidizes the hydrocarbons rather than ammonia (Radniecki et al., 2008). Determining whether

N. maritimus also oxidizes compounds into daughter products would illustrate the mechanism that N. maritimus uses to oxidize ammonium and how the aromatics affect enzymatic activity and specificity.

Chronic inhibition of N. maritimus in the presence of Corexit 9500A and ANSC

N. europaea showed significantly increased inhibition when exposed to Corexit 9500A:ANSC

DEWAF mixtures for 4 d over the 3 h tests (Figure 11) suggesting that long term effects of 22

Corexit 9500A and ANSC may be much greater than shown with acute 3 h tests (Radniecki et al.,

2013). Because N. maritimus is a slow-growing archaea it may exhibit even greater inhibition than N. europaea when comparing chronic growth tests to acute 3 h toxicity tests. Oil spills may have a greater impact on microorganism activity when considering the long term effects on overall growth and population.

Figure 11: Percent nitrification activity of N. europaea when exposed to 4 mL of various DEWAF ratios per 50 mL media for 4 d ( ) and 3 h (□). Error bars represent 95% confidence intervals from triplicate experiments. Originally presented in Radniecki et al. (2013).

Effect of growth media on inhibition

N. maritimus is grown in a saltwater based media whereas N. europaea is grown in a freshwater based media. This difference in media composition may have an effect on the interaction of

Corexit 9500A and hydrocarbons from the oil with the microorganisms. Growing and performing

N. europaea tests with a saltwater based media may provide a better comparison of inhibition between the two species.

Application of molecular methods to N. maritimus

Further research is required to determine if there is specific gene expression in N. maritimus that results from the exposure to aromatic hydrocarbons. Tests need to be performed to investigate whether specific N. maritimus genes are up-regulated upon exposure to aromatic hydrocarbons, as 23

were similarly performed to identify over expression of N. europaea gene NE1545 (Radniecki et al., 2011).

Comparison of N. maritimus activity as biofilm and suspended cells

N. maritimus cells used for the inhibition tests were in one mass on a filter paper, which essentially formed a biofilm. A previous study found that N. europaea biofilms were less inhibited than cells suspended in media when exposed to phenol (Lauchnor and Semprini, 2013).

Further tests are needed to compare the activity of suspended N. maritimus cells and the cells attached to the filter paper to determine whether the filter paper biofilm protected the cells from the presence of inhibitory hydrocarbons. 24

CONCLUSIONS

N. europaea and N. maritimus exhibited similar responses to the presence of Corexit 9500A

(Figure 4), ANSC (Figure 6), and mixtures of the two (Figure 7). Neither species showed significant inhibition in the presence of Corexit 9500A. Both microorganisms were considerably more sensitive to another surfactant, SDS (Figure 5). ANSC WAF and Corexit 9500A:ANSC

DEWAF mixtures caused considerable inhibition. The majority of the observed inhibition is thought to be attributed to the volatile, aromatic hydrocarbons present in the oil as shown by the correlation between total COD of the batch reactor and nitrification activity (Figure 8). Cells treated with ANSC WAF showed the greatest inhibition on a per COD basis with cells exposed to the 1:5 DEWAF showing the least inhibition on a per COD basis. Overall, mixtures of Corexit

9500A and ANSC were shown to be the most inhibiting to nitrification activity because of the increased amount of low molecular weight, soluble hydrocarbons brought into the aqueous phase.

These findings indicate that using dispersants to treat oil spills may have a significant effect on microorganism activity and population in coastal ecosystems as a greater fraction of aromatic hydrocarbons are brought in to solution. 25

BIBLIOGRAPHY

American Petroleum Institute Petroleum HPV Testing Group (API). (2003). Test Plan: Crude Oil Category. Submitted to the United States Environmental Protection Agency. Retrieved from http://www.epa.gov/hpv/pubs/summaries/crdoilct/c14858tp.pdf

Arp, D. J., Sayavedro-Soto, L. A., Hommes, N. G. (2002). Molecular biology and biochemistry of ammonia oxidation by Nitrosomonas europaea. Archives of Microbiology 178, 250-255. Barron, M. G., Carls, M. G., Short, J. W., Rice, S. D., Heintz, R. A., Rau, M., DiGiulio, R. (2005). Assessment of the phototoxicity of weathered Alaska North Slope crude oil to juvenile pink salmon. Chemosphere 60, 105-110. Bock., E., Wagner, M. (2006). Oxidation of inorganic nitrogen compounds as an energy source. In: Dworkin, M., Falkow, S., Rosenberg, E., Schleifer, K. H., Stackebrandt, E. (Eds.), The Prokaryotes—Volume 2: Ecophysiology and Biochemistry, 457-495. Burggraf, S., Huber, H., Stetter, K. O. (1997). Reclassification of the crenarchaeal orders and families in accordance with 16S rRNA sequence data. International Journal of Systematic and Evolutionary Microbiology 47, 657-660. Chakroborty, R., Borglin, S. E., Dubinsky, E. A., Andersen, G. L., Hazen, T. C. (2012). Microbial response to the MC-252 oil and Corexit 9500 in the Gulf of Mexico. Frontiers in Microbiology 3, 1-6. Chang, S. W., Hyman, M. R., Williamson, K. J. (2002). Cooxidation of naphthalene and other polycyclic aromatic hydrocarbons by the nitrifiying bacterium, Nitrosomonas europaea. Biodegradation 13, 373-381. Cleveland, C. J. (Ed.). (2011). Characteristics of Deepwater Horizon oil. In Encyclopedia of Earth. Retrieved from http://www.eoearth.org/view/article/162270/

Couillard, C. M., Lee, K., Légaré, B., King, T. I. (2005). Effect of dispersant on the water- accommodated fraction of crude oil and its toxicity to larval marine fish. Environmental Toxicology and Chemistry 24, 1496-1504. Faksness, L. G., Brandvik, P. J., Sydnes, L. K. (2008). Composition of the water accommodated fractions as a function of exposure times and temperatures. Marine Pollution Bulletin 56, 1746-1754. Fingas, M. (2010). Review of the North Slope oil properties relevant to environmental assessment and prediction. Prince William Sound Regional Citizens’ Advisory Council. Fuller, C., Bonner, J., Page, C., Ernest, A., McDonald, T., McDonald, S. (2004). Comparative toxicity of oil, dispersant, and oil plus dispersant to several marine species. Environmental Toxicology and Chemistry 23, 2941-2949. Galt, J. A., Lehr, W. J., Payton, D. L. (1991). Fate and transport of the Exxon Valdez oil spill (Part 4). Environmental Science and Technology 25, 202-209. 26

Hageman, R. H., Hucklesby, D. P. (1971). Nitrate reductase from higher plants. Methods in Enzymology 23, 491-503. Hayworth, J. S., Clement, T. P. (2011). BP’s Operation Deep Clean—Could Dilution be the Solution to Beach Pollution? Environmental Science and Technology 45, 4201-4202. Hemmer, M. J., Barron, M. G., Greene, R. M. (2011). Comparative toxicity of eight oil dispersants, Louisiana Sweet Crude oil (LSC), and chemically dispersed LSC to two aquatic test species. Environmental Toxicology and Chemistry 30, 2244-2252. Judson, R. S., Martin, M. T., Reif, D. M., Houck, K. A., Knudsen, T. B., Rotroff, D. M., Xia, M. Sakamuru, S., Huang, R., Shinn, P., Austin, C. P., Kavlock, R. J., Dix, D. J. (2010). Analysis of eight oil spill dispersants using rapid, in vitro tests for endocrine and other biological activity. Environmental Science and Technology 44, 5979-5985. Juliastuti, S. R., Baeyens, J., Creemers, C., Bixio, D., Lodewyckx, E. (2003). The inhibitory effects of heavy metals and organic compounds on the net maximum growth rate of the autotrophic biomass in activated sludge. Journal of Hazardous Materials 100, 271-283. Karner, M. B., DeLong, E. F., Karl, D. M. (2001). Archaeal dominance in the mesopelagic zone of the Pacific Ocean. Nature 409, 507-510. Keener, W. K., Arp, D. J., (1993). Kinetic studies of ammonia monooxygenase inhibition in Nitrosomonas europaea by hydrocarbons and halogenated hydrocarbons in an optimized whole-cell assay. Applied and Environmental Microbiology 59, 2501-2510. Keener, W. K., Arp, D. J. (1994). Transformations of aromatic compounds by Nitrosomonas europaea. Applied and Environmental Microbiology 60, 1914-1920. Könneke, M., Bernhard, A. E., de la Torre, J. R., Walker, C. B., Waterbury, J. B., Stahl, D. A. (2005). Isolation of an autotrophic ammonia-oxidizing marine archaeon. Nature 437, 543-546. Lauchnor, E. G., Semprini, L. (2013). Inhibition of phenol on the rates of ammonia oxidation by Nitrosomonas europaea grown under batch, continuous fed, and biofilm conditions. Water Research 47, 4692-4700. Lindstrom, J. E., Braddock, J. F. (2002). Biodegradation of petroleum hydrocarbons at low temperature in the presence of the dispersant Corexit 9500. Marine Pollution Bulletin 44, 739-747. Martens-Habbena, W., Berube, P. M., Urakawa, H., de la Torre, J. R., Stahl, D. A. (2009). Ammonia oxidation kinetics determine niche separation of nitrifying archaea and bacteria. Nature 461, 976-981. National Oceanic and Atmospheric Administration (NOAA). (2010). Deepwater Oil Spill: Natural Resource Damage Assessment. Retrieved from http://www.restorethegulf.gov/sites/default/files/documents/pdf/OilSpillsAssessRest_11_ 10.pdf

National Oceanic and Atmospheric Administration (NOAA). (2014). Alaska North Slope Crude Blends. Retrieved from http://response.restoration.noaa.gov/oil-and-chemical-spills/oil- spills/resources/alaska-north-slope-crude-blends.html 27

Radniecki, T. S., Dolan, M. E., Semprini, L. (2008). Physiological and transcriptional responses of Nitrosomonas europaea to toluene and benzene inhibition. Environmental Science and Technology 42, 4093-4098. Radniecki, T. S., Gilroy, C. A., Semprini, L. (2011). Linking NE1545 gene expression with cell volume changes in Nitrosomonas europaea cells exposed to aromatic hydrocarbons. Chemosphere 82, 514-520. Radniecki, T. S., Lauchnor, E. G. (2011). Investigating Nitrosomonas europaea stress biomarkers in batch, continuous culture, and biofilm reactors. In: Klotz, M. G., Stein, L. Y. (Eds.), Methods in Enzymology – research on nitrification and related processes Part B. Academic Press, San Diego, CA, pp. 219-247. Radniecki, T. S., Schneider, M. C., Semprini, L. (2013). The influence of Corexit 9500A and weathering on Alaska North Slope crude oil toxicity to the ammonia oxidizing bacterium, Nitrosomonas europaea. Marine Pollution Bulletin 68, 64-70. Sprent, J. I. (1987). The Ecology of the Nitrogen Cycle. New York: Cambridge University Press. Thibodeaux, L. J., Valsaraj, K. T., John, V. T., Papadopoulos, K. D., Pratt, L. R., Pesika, N. S. (2011). Marine Oil Fate: Knowledge Gaps, Basic Research, and Development Needs; A perspective based on the Deepwater Horizon spill. Environmental Engineering Science 28, 87-93. United States Coast Guard National Response Team (USCG NRT). (2011). On Scene Coordinator Report: Deepwater Horizon Oil Spill. Department of Homeland Security, United State Coast Guard. Retrieved from http://www.uscg.mil/foia/docs/dwh/fosc_dwh_report.pdf

United States Environmental Protection Agency (US EPA). (2010). The Federal Government Response: Dispersant Use in BP Oil Spill. Retrieved from http://www.restorethegulf.gov/sites/default/files/imported_pdfs/external/content/documen t/2931/835583/1/Fact%20Sheet%20Dispersants%20july%2029%202010-1.pdf

United States Environmental Protection Agency (US EPA). (2014). National Contingency Plan Product Schedule. Retrieved from http://www2.epa.gov/sites/production/files/2013- 08/documents/schedule.pdf

Vajrala, N., Martens-Habbena, W., Sayavedro-Soto, L. A., Schauer, A., Bottomley, P. J., Stahl, D. A., Arp, D. J. (2013). Hydroxylamine as an intermediate in ammonia oxidation by globally abundant marine archaea. Proceedings of the National Academy of Sciences 110, 1006-1011. Venosa, A. D., Holder, E. L. (2007). Biodegradability of dispersed crude oil at two different temperatures. Marine Pollution Bulletin 54, 545-553. Vitousek, P. M., Aber, J., Howarth, R. W., Likens, G. E., Matson, P. A., Schindler, D. W., Schlesinger, W. H., Tilman, G. D. (1997). Human alteration of the global nitrogen cycle: causes and consequences. Issues in Ecology 1.

28

Yoshida, A., Nomura, H., Toyoda, K., Nishino, T., Seo, Y., Yamada, M., Nishimura, M., Wada, M., Okamoto, K., Shibata, A., Takada, H., Kogure, K., Ohwada, K. (2006). Microbial responses using denaturing gradient gel electrophoresis to oil and chemical dispersant in enclosed ecosystems. Marine Pollution Bulletin 52, 89-95. Zheng, M., Ahuja, M., Bhattacharya, D., Clement, T. P., Hayworth, J. S., Dhanasekaran, M. (2014). Evaluation of differential cytotoxic effects of the oil spill dispersant Corexit 9500. Marine Pollution Bulletin 95, 108-117.

29

APPENDIX

A two factor with replication analysis of variance (ANOVA) was run on the N. maritimus data for the Corexit 9500A:ANSC DEWAF mixtures (presented in Figure 7B). Interactions between the different added volumes, the DEWAF ratio, and the combination of the two factors was analyzed to determine whether the factors were statistically significant. Both factors and the interaction of the two factors was concluded to be statistically significant (p < 0.05). This led to the conclusion that both added volume and Corexit 9500A to ANSC ratio were important factors effecting the activity of N. maritimus.

30

Table 2: Two-factor ANOVA with replication analyzing the volume of DEWAF added to 50 mL media and the Corexit 9500A to ANSC ratio of the DEWAF mixture. SUMMARY WAF 1:40 DEWAF 1:20 DEWAF 1:5 DEWAF Total 2 mL Count 3 3 3 3 12 Sum 2.347 2.133 2.257 2.291 9.028 Average 0.782 0.711 0.752 0.764 0.752 Variance 0.004 0.020 0.005 0.001 0.006

4 mL Count 3 3 3 3 12 Sum 2.334 1.285 1.217 0.867 5.702 Average 0.778 0.428 0.406 0.289 0.475 Variance 0.007 0.011 0.002 0.001 0.040

6 mL Count 3 3 3 3 12 Sum 1.632 1.082 0.989 0.674 4.376 Average 0.544 0.361 0.330 0.225 0.365 Variance 0.017 0.001 0.001 0.000 0.018

8 mL Count 3 3 3 3 12 Sum 0.606 1.015 0.662 1.132 3.415 Average 0.202 0.338 0.221 0.377 0.285 Variance 0.001 0.000 0.000 0.000 0.006

Total Count 12 12 12 12 Sum 6.918 5.514 5.124 4.964 Average 0.576 0.460 0.427 0.414 Variance 0.067 0.030 0.045 0.048

ANOVA Source of Variation SS df MS F P-value F crit Sample 1.502 3 0.501 111.388 5.12E-17 2.901 Columns 0.198 3 0.066 14.652 3.53E-06 2.901 Interaction 0.437 9 0.049 10.795 1.77E-07 2.189 Within 0.144 32 0.004

Total 2.281 47