Aerobic and anaerobic microbial degradation of weathered and fresh oil from the BP Deepwater Horizon Oil Spill

Transcription

1 Aerobic and anaerobic microbial degradation of weathered and fresh oil from the BP Deepwater Horizon Oil Spill Kelsey Gosselin 1, Joe Vallino 2 The New School 1, 65 W. 12 th Street, New York, NY Ecosystems Center 2,Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA Semester in Environmental Science, 2013 Abstract This study compared the bacterial degradation rates of two varieties of Macondo Oil from the BP Deepwater Horizon well under both aerobic and anaerobic conditions. The oil types used were fresh oil directly from the drilling well and weathered oil collected from the shore after the 2010 well blowout in the Gulf of Mexico. I set up 120 ml serum bottles, which incubated for a total of 20 days and were sampled at 4,10, and 20 days. The anaerobic conditions were enhanced with nitrate and iron as electron acceptors to aid the degradation process, while the aerobic treatments were maintained with only oxygen as the electron acceptor. I analyzed the amount of oil degradation by: bacterial productivity with 14 C-Leucine, CO2 respiration with gas chromatography, and ammonium uptake levels with colorimetric analyses on a spectrophotometer. Through these analyses I found highest oil degradation with oxygen, followed by nitrate and iron. I found higher rates of bacterial degradation in the weathered oil treatment under aerobic conditions. Alternatively under anaerobic conditions the bacteria more successfully degraded new oil.

2 Key phrases and words Hydrocarbon; Degradation; Anaerobic; BP; microcosm Microbial degradation of hydrocarbons; BP Deepwater Horizon oil spill Introduction- The sinking of the Deepwater Horizon oil-drilling platform released several million barrels of Arabian light crude oil into the Gulf of Mexico between April and July of The oil was composed of 86.6% carbon, 12.6% hydrogen, 0.38% nitrogen, and 0.39% sulfur. The carbon and hydrogen fractions were comprised of 74% saturated hydrocarbons and 16% aromatic hydrocarbons which Widdel and Rabus (2001), found to be the most toxic portions of the oil (Reddy, et al., 2011). Unlike other historical spills that emitted oil along the water surface, the oil escaped from the well 1.5 km deep in the water column (Reddy et al., 2011). Although the spill occurred near the sea floor, some of the oil moved through the water column wherein some remained suspended because of the small drop size and near neutral buoyancy. The remaining oil eventually moved up to the surface of the water forming a visible slick on the water, where some of the gasses could evaporate off (Thibodeaux et al., 2011). The oil that remained along the sea floor was in an anaerobic zone with little ability to evaporate or degrade. The potential for bacterial degradation of oil along the water surface has been recognized and exploited through bioremediation techniques. Under aerobic conditions, bacteria use oxygen to carry out oxidation-reduction reactions and change the structure of the hydrocarbons, a process from which they can gain energy. Recently, the potential for bacterial degradation was recognized in anaerobic conditions with the use of nitrate, sulfate, iron, or methane as electron acceptors. Although anaerobic degradation typically occurs at a lesser rate, it has the potential to break down the hydrocarbons (Widdel and Rabus, 2001). While aerobic degradation breaks down more oil, typically degrading 90-95% of the alkanes, bacterial use of other electron acceptors benefits cases where oil is in anaerobic areas such as in the BP oil spill or older spills where the oil has become deposited deep in sediments (Grischenkov et al., 2000). This study looks at the use of oxygen, nitrogen, and ferric iron as electron acceptors for bacterial degradation of oil to measure breakdown of both fresh and weathered oil. Furthermore, I sought to find the different levels of breakdown between aerobic and 2

3 anaerobic treatments. Three different oil treatments were used: fresh un-weathered Macondo oil from before the blowout, weathered Macondo oil collected from the water column after the blowout, and no oil as a control. The weathered oil was collected from the shore after the spill, having lost most of the lighter more degradable compounds including the gasses, alkenes, n-alkanes, and potentially some of the lighter aromatic hydrocarbons (Leahy and Colwell, 1990). The new oil was pre-blowout Macondo oil containing all the components of crude oil including the gasses and lighter compounds. The water was not inoculated with oil degrading bacteria, but instead studied using bacteria already present in a medium of pond water. Methods Microcosm setup I prepared 120 ml serum bottles with either nitrate, iron, or oxygen, and either fresh oil, weathered oil, or no oil in a medium of freshwater from John s Pond in Mashpee, Massachusetts. I added 331 μm carbon to all oil treatments based on previous studies of oil degradation which added between μm C (Meckenstock et al. 1999,; Barbaro, et al., 1992). In the weathered oil treatments the carbon was added in the form of 100 ul of Macondo Crude oil from the Deepwater Horizon Oil Spill made less viscose with the addition of methylene chloride. For the fresh oil treatments I added.56 μl of Macondo Crude Oil with an 86.6% carbon content by mass and a density of 820 μg/μl. All oil was from British Petroleum but sourced from Robert Nelson at Woods Hole Oceanographic Institute. Aerobic treatments contained 100 ml of water, while the nitrate and iron treatments were composed of 95 ml of water and 5 ml of either nitrate or iron stock respectively for a final concentration of 3 mm based on the work of Meckenstock, et al., (1999). All treatments were then brought up to 30 μm NH4 + to prevent nitrogen limitation. I sealed the bottles with rubber Viton stoppers and crimped them to prevent the loss or entrance of oxygen. There was a total volume of ml of liquid, leaving approximately 17 ml of headspace in the 120 ml sealed bottles. Since the sampling procedures were destructive, I carried out the treatments in triplicate to allot for three sampling times, and duplicates of the triplicate treatments for accuracy. 3

4 I sparged the anaerobic treatments with nitrogen gas and used a secondary needle open to the air to release oxygen. Sparging was carried out for 3 minutes per bottle, during which time the bottles were swirled to release oxygen from the water as well. The bottles were incubated in the dark at 22.5 C and sampled at 4 days, 10 days, and 20 days. Bacterial Productivity I measured bacterial productivity to determine how much carbon uptake occurred across the various treatments. I determined bacterial productivity using radio labeled 12 μm Leucine with a specific activity of Ci mmol -1. To maintain the anaerobic conditions of the treatments, I held the micro centrifuge tubes under nitrogen throughout the entire process and filled the tubes to maximum capacity so that oxygen would not be present in the headspace. 2 ml of sample were added to 2.2 ml micro centrifuge tubes containing 27 μl of Leucine and 133μL of 100% TCA for time zero samples. Another 2 ml of sample was added to 27 μl of Leucine in a separate tube and incubated for 45 minutes. This process was carried out for all treatments. Following the 45 minute incubation period the treatments were killed with 133 μl of 100% TCA. Both the zero and 45 minute samples were centrifuged on high for 15 minutes. After centrifuging I removed the tubes and poured out all liquid, leaving only an invisible cell pellet. I then added 1 ml of 5% TCA, recentrifuged the tubes on high for 5 minutes, repeated the pouring process, rinsed with 1 ml of 80% ethanol, spun again on high for 5 minutes, poured liquid and allowed to dry with caps open. After drying I added 1 ml of 30% ScintiSafe scintillation cocktail and vortexed the samples. I placed the samples in scintillation vials and measured destructions per minute on a Multipurpose Scintillation Counter (Beckman Coulter LS 6500, Brea, CA) set up to measure C 14. Bacterial Productivity Calculation I calculated productivity from the slope of regression line attained from plotting the zero and 45 minute incubation time DPMs. Using 1 Ci in 2.2 x DPM and.318 Ci in a mmol of Leucine, I converted DPMs to mmol of Leucine. Then using pseudo-constants I converted.073 mol protein in 1 mol of Leucine, and grams of protein in 1 mol protein. Then assuming.63 g of cell dry weight in 1 gram of protein, I calculated carbon assuming.54 g of carbon in 1 gram of cell dry weight. 4

5 Using the grams of carbon I reported my numbers in μmol of carbon per litre per day. CO2 I measured carbon dioxide in the headspace to determine how much hydrocarbon degradation occurred. I acidified the 100 ml water samples with 1 ml of 25% H3PO 4 to lower the ph of the water to 2 so that all of the carbonate in the water would be driven to carbon dioxide which could then be measured with gas chromatography. Once acidified I pulled up 10 ml of CO2 free air using a scrubber and pushed the air through the syringe and needle to remove all CO2 before pulling up the sample. I then pulled up 10 ml of headspace from my samples, which was injected into a Shimadzu GC-8A gas chromatograph (Shimadzu, Kyoto, Japan) equipped to measure CO2 from which I found the area of the peak at 0.88 minutes. Ammonium After the water was acidified for CO2 measurements I sampled and filtered the water through 47 mm GF/F filters (Fisher Scientific, Fair Lawn, NJ). I then measured NH4 + concentrations following the methods of Strickland and Parsons (1972) based on the phenol-hypochloric method from Solarazano (1969), using a 5:1 dilution, and measured absorbance on spectrophotometer (Shimadzu, Kyoto, Japan). Results CO2 After the full incubation period of 20 days, the CO2 in the headspace of the bottles showed the trend of oxygen as the best electron acceptor (Figure 1), followed by nitrate then iron. Most of the oil treatments had higher values than the control conditions, particularly in the aerobic treatments, wherein CO2 production was higher in the weathered oil treatments than the new oil (Figure 2). The amounts shown represent CO2 production solely from the breakdown of hydrocarbons since the respective control conditions (Table 1) have been taken into account. CO2 production in the aerobic treatments increased over time in the weathered treatment. In the new oil treatment, CO2 production began at levels lower than the control at 4 days but increased over time to levels higher than the control at 20 days. 5

6 In the nitrate conditions, CO2 production increased consistently over time in the weathered oil treatment, but only increased towards the end in the new oil treatment (Figure 3). Unlike the aerobic treatment which had higher CO2 production in the weathered oil, the nitrate treatment led to higher CO2 production in the new oil treatment. After 20 days the CO2 in the new oil was 0.5 μmol CO2 (+/-.04) compared to 0.36 μmol CO2 in the weathered oil. The iron treatments experienced little change over time by oil type (Figure 4). Similar to nitrate, there was higher production in the new oil treatments rather than the weathered. Bacterial Productivity In the no oil/control treatment, the rate of bacterial productivity decreased over time, most noticeably in the nitrate conditions decreasing by 1.46 μmol C L -1 day -1 (Figure 5). The aerobic and iron treatments decreased as well. The iron treatments which started low initially (0.37 as opposed to 1.46 and 2.26 in the other treatments), decreased to nearly 0 after 6 additional days. In the weathered oil treatment, productivity decreased slightly in the nitrate and iron conditions, but increased in the aerobic (Figure 6). In the new oil treatment, the rate decreased across all treatments (Figure 7). Although the rate of carbon uptake started at initial rates higher than the weathered treatment, (~2.5 μmol C for aerobic and nitrate in the new, compared with 1.6 and 2 μmol C in the weathered), the productivity dropped rapidly in the new oil to levels below the productivity in the weathered oil. Taking into account the control conditions reveals the higher productivity in the aerobic treatment in both oil types. (Figure 8). However, in the new oil the nitrate is also more productive than the control. Although nearly all treatments decreased in productivity over time, the control conditions decreased as well, allowing for productivity in the oil treatments to remain higher than control conditions across all treatments after 10 days (Figure 9). Although the new oil was more productive than the weathered after 4 days, the weathered became more productive than the new oil at 10 days most noticeably in the aerobic treatment. NH4 + Initially treatments began at 30 μm + NH4. In the control conditions, after 4 days, ammonium levels dropped to 10 μm and continued to decrease (Figure 10). Oxygen, 6

7 nitrate, and iron followed the same trend despite different levels of productivity. In the weathered oil treatment, NH4 + concentration decreased from 30 μm initially to 20 μm after 4 days and continued to decrease (Figure 11). Despite a decrease early on, the weathered oil ammonium concentrations did not drop as quickly as the control conditions. Again, despite different levels of degradation, all treatments followed the same rates of uptake. In the new oil treatment, the NH4 + concentration in followed a very similar pattern to the the control conditions (Figure 12). Discussion Over the course of my incubations I found as a consistent trend in almost all measurements, oxygen was the best electron acceptor followed by nitrate, and then iron. Oil was most degraded with the presence of oxygen. While high degradation in aerobic conditions benefits remediation in surface spills, it does not provide much benefit for cases like the Deepwater Horizon spill wherein the oil lingered in anaerobic conditions. However, degradation under anaerobic conditions provides potential for remediation without oxygen. Furthermore, the more difficult to degrade weathered oil composed mostly of polycyclic aromatic hydrocarbons, still degraded. Thus bacteria were able to use the less energetically efficient electron acceptors: nitrate and ferric iron to break down these very difficult to degrade carbon compounds. An additional benefit to the bacteria was the composition of the Macondo light sweet crude oil. The lighter crude contains a higher proportion of simpler lower molecular weight hydrocarbons that are more readily biodegraded (Atlas, 2010). Thus, even though the oil was weathered, there were still some degradable compounds because of the higher ratio of lower molecular weight hydrocarbons. In addition to the composition of the weathered oil, the increased surface area from the method of the oil addition likely contributed to the increased degradation rates seen in those treatments. The weathered oil was made less viscose with the addition of methylene chloride. The concentration added was still 331 μm C, however the volume added to each bottle was 100 μl as opposed to 0.56 μl of the fresh oil. The 100 μl created a thin film along the bottom of the bottle rather than a small droplet as seen in the new oil. The increased surface area benefited bacterial mineralization rates because biodegradation 7

8 occurs where the hydrocarbons and water meet (Atlas, 2010). Typically weathered oil in the water forms tar balls or other large masses with low surface area. However, in these incubations the weathered oil was spread out with increased surface area similar to the affect of dispersant addition in an oil spill. While the weathered oil was degraded more readily in the aerobic treatments, the bacteria degraded the new oil more rapidly in the anaerobic conditions (Figures 3 and 4). The trends in CO2 production show the compounds in the fresh oil to be more difficult to break down under aerobic conditions than in anaerobic conditions. Or, these trends may suggest that the presence of lighter compounds outweighs the benefits of increased surface area. Despite the bacterial degradation of oil towards the end of the incubations, the bacteria initially began with lower levels of bacterial productivity and CO2 production than the control in most cases. The novelty of the bacterial exposure to oil could explain the lag time in bacterial activity. Although the water was collected from a site with motorboat activity, the type of fuel the bacteria in the pond water encountered resembled a much different composition than the crude oil introduced through the incubations. The initial days of incubation could have represented time when the bacteria were developing genetic changes resulting in new metabolic capabilities for degrading the oil (Leahy and Colwell, 1990). Furthermore, the population of hydrocarbon degrading bacteria likely increased over time due to the presence of the oil, eventually decreasing microbial diversity (Nyman, 1999). Over the incubation period, the amount of hydrocarbon tolerant bacteria likely increased, causing degradation of the hydrocarbons to increase over the incubation period leading to the bacterial productivity higher than control conditions seen at 10 days as well as the higher C02 respiration rates. Although bacterial productivity in the oil treatments did increase to levels higher than control conditions over time, the levels of respiration did not correspond to the bacterial productivity levels. For example in the aerobic weathered oil treatment, at 10 days the bacteria were taking up 2.8 μmol C/L/day, however after 20 days the amount of CO2 in the headspace was 1.7 μmol CO2. Bacterial growth efficiency usually ranges from 0.05 to 0.6 (Giorgio and Cole, 1998). Assuming the bacteria have a growth efficiency of.4 and knowing the bacterial uptake 2.8 μmol of carbon daily from bacterial productivity measurements, 8

9 then they should be respiring 1.68 μmol C per day, and using the other 1.12 μmol C to produce new biomass. After 20 days the amount respired would be 1.68 (μmol C)x 20 (days)= 33.6 μmol C respired after 20 days, however the measurements only showed 1.7 μmol CO2 respired. The discrepancy in scale is likely due to the pseudo constants used to calculate carbon uptake from the readings of destructions per minute on the scintillation counter. As the degradation of the hydrocarbons increased, the amount of ammonium taken up increased as well. Despite different carbon uptake trends among the treatments, the levels of ammonium uptake followed the same pattern across the oil types (Figures 10, 11, and 12). Based on previous studies and an understanding of bioremediation techniques, the amount of nutrients taken up should reflect the amount of oil being degraded (Bragg, et al., 1994). The addition of nitrogen should have spurred the breakdown of the oil due to the availability of nutrients, allowing the bacteria to remain normal C:N levels rather than very high levels due to higher carbon content in the water. Although the ammonium uptake levels were consistent across the treatments, the weathered oil ammonium uptake did not drop as rapidly as the new oil and control treatments. These two treatments experienced higher productivity originally reflecting the higher ammonium uptake. The weathered oil treatment decreased by around 10 um less at the 4 day point but decreased more rapidly from there out as productivity and respiration increased on the longer time scale in this incubation. While these changes can be seen across the different oil treatment types, not much change can be seen among the different electron acceptors within oil treatments likely due to the similarity in amounts of hydrocarbon degradation among the different electron acceptors. Conclusion I saw aerobic and anaerobic degradation of both the new and weathered oil. The rates of anaerobic degradation were not largely different from the aerobic, although lower across all treatments. The continuing degradation of the weathered oil shows the potential for oil in the Gulf to continue to be degraded. It appears that the weathered oil can be broken down better than the new oil under aerobic conditions, while the new oil is further degraded than the weathered under the anaerobic conditions. 9

10 Future projects on oil degradation rates should consider using higher amounts of oil. Although high concentrations of hydrocarbons can cause inhibition of biodegradation by causing nutrient or oxygen limitation (Leahy and Colwell, 2010), these problems could be avoided by adding nutrients to match the added carbon content and by using anaerobic treatments. Furthermore, the issue of surface area could be explored by comparing the effects of different surface area and measuring bacterial degradation rates on the same oil type but dispersed in different ways. In addition to using different electron acceptors such as sulfate, the use of zero valence iron could provide another interesting result as this form of iron might provide more surface area for bacteria as well as better reducing capacities. Other beneficial measurements would be gas chromatography/mass spectrometry to determine the quantity of compounds present before incubating and then measuring what compounds were left after bacterial degradation. The GC/MS would be particularly interesting for understanding why new oil would be degraded more rapidly in the anaerobic conditions rather than the aerobic. Acknowledgments I would like to thank Ken Foreman for making this research opportunity possible. I also thank Joe Vallino for giving me guidance through this entire process. Thanks also to Julie Huber and Robert Nelson for lending me supplies, and Rich McHorney, Sarah Nalven, Alice Carter, Fiona Jevon, and Lauren Wind for their willingness to help and moral support. 10

11 Literature Cited Atlas, R Oil Biodegradation and Bioremediation: A Tale of the Two Worst Spills in U.S. History. Environmental Science and Technology, 45(16): Barbaro, J., Barker, J., Lemon, L., Mayfield., C Biotransformation of BTEX under anaerobic, denitrifying conditions: Field and laboratory observations. Journal of Contaminant Hydrology, 11: Bragg, J., Prince, R., Harner, J., and Atlas, R Effectiveness of bioremediation for the Exxon Valdez oil spill. Nature, 368: Giorgio,P., and Cole, J Bacterial Growth Efficiency in Natural Aquatic Systems. Annual Review of Ecology and Systematics, 29: Grishchenkov, V., Townsend, R., McDonals. T., Autenrieth, R., Bonner, J., Boronin, A Degradation of petroleum hydrocarbons by facultative anaerobic bacteria under aerobic and anaerobic conditions. Process biochemistry, 35: Leahy, J., and Colwell, R Microbial degradation of hydrocarbons in the environment. Microbiology and Molecular Biology Reviews, 54 (3): Meckenstock, R., Morasch, B., Warthmann, R., Schink, B., Annweiler, E., Michaelis, W., Richnow., H C/ 12 C isotope fractionation of aromatic hydrocarbons during microbial degradation. Environmental Microbiology, 1 (5): Nyman, J Effect of Crude Oil and Chemical Additives on Metabolic Activity of Mixed Microbial Populations in Fresh Marsh Soils. Microbial Ecology, 37: Reddy, C., Arey, S., Seewald, J., Sylva, S., Lemkau, K., Nelson, R., Carmichael, C., McIntyre, C., Fenwick, J., Ventura, G, Mooy, B., Camilli, R Composition and fate of gas and oil released to the water column during the Deepwater Horizon oil spill. Proceedings of the National Academy of Sciences of the United States of America, 109 (50): Thibodeaux, L., Valsaraj, K., John, V.,m Papadopoulos, K., Pratt, L., Pesika, N Marine Oil Fate: Knowledge Gaps, Basic Research, and Development Needs; A Perspective Based on the Deepwater Horizon Spill. Environmental Engineering Science, 28 (2): Widdel, F., Rabus, R, Anaerobic biodegradation of saturated and aromatic 11

12 Hydrocarbons. Current Opinion in Biotechnology. 12: Tables and Figures Table 1: CO2 values across weathered, fresh, and no oil treatments over three measurement times. Figure 1: Amount of CO2 in the headspace after 20 days of incubation. Figure 2: Difference in CO2 production in control treatments from aerobic treatments over period of incubation. Figure 3: Difference in CO2 production in control treatments from nitrate treatments over period of incubation. Figure 4: Difference in CO2 production in control treatments from iron treatments over period of incubation. Figure 5: Bacterial productivity in no oil/control treatment. Figure 6: Bacterial productivity in weathered oil treatment. Figure 7: Bacterial productivity in new oil treatment. Figure 8: Bacterial productivity from the uptake of hydrocarbons as the difference between treatments and control values at 4 days. Figure 9: Bacterial productivity from the uptake of hydrocarbons as the difference between treatments and control values at 10 days. Figure 10: NH4 + concentration in control treatment over full incubation period. Figure 11: NH4 + concentration in weathered oil treatment over full incubation period. Figure 12: NH4 + concentration in new oil treatment over full incubation period. 12

13 Units μmol CO2 μmol CO2 μmol CO2 μmol CO2 μmol CO2 μmol CO2 Oil type Treatment 4 days 4 days (2) 10 days 10 days (2) 20 days 20 days (2) Weathered Aerobic Nitrate Iron New Aerobic Nitrate Iron No oil Aerobic Nitrate Iron Table 1- CO2 values across weathered, fresh, and no oil treatments over three measurement times. 13

14 μmol CO CO2 in headspace- 20 days Aerobic Nitrate Iron Weathered New No oil Figure 1- Amount of CO2 in the headspace after 20 days of incubation. 14

15 μmol CO CO2 production: Aerobic difference from control conditions Weathered New days 10 days 20 days Figure 2- Difference in CO2 production in control treatments from aerobic treatments over period of incubation. 15

16 μmol CO CO2 production: Nitrate difference from control conditions Weathered New days 10 days 20 days Figure 3- Difference in CO2 production in control treatments from nitrate treatments over period of incubation. 16

17 μmol CO CO2 production: Iron difference from control conditions Weathered New days 10 days 20 days Figure 4- Difference in CO2 production in control treatments from iron treatments over period of incubation. 17

18 μmol C/L/day Bacterial productivity-control Aerobic NO3 FeCl Figure 5- Bacterial productivity in no oil/control treatment. 18

19 μmol C/L/day Bacterial Productivity-Weathered oil Time (days) Aerobic NO3 FeCl Figure 6- Bacterial productivity in weathered oil treatment. 19

20 μmol C/L/day 3.00 Bacterial Productivity- New oil Aerobic NO3 FeCl Time (days) Figure 7- Bacterial productivity in new oil treatment. 20

21 μmol C/L/day Bacterial productivity difference from control, 4 days Aerobic Weathered New Nitrate Iron Figure 8-Bacterial productivity from the uptake of hydrocarbons as the difference between treatments and control values at 4 days. 21

22 μmol C/L/day Bacterial Productivity difference from control, 10 days Aerobic Nitrate Iron Weathered New Figure 9- Bacterial productivity from the uptake of hydrocarbons as the difference between treatments and control values at 10 days. 22

23 35 NH 4 + Concentration - Control μm NH Aerobic Nitrate Iron Time (days) Figure 10- NH4 + concentration in control treatment over full incubation period. 23

24 NH 4 + Concentration-Weathered oil μm NH Aerobic Nitrate Iron Time (days) Figure 11- NH4 + concentration in weathered oil treatment over full incubation period. 24

25 35 NH 4 + Concentration-New oil μm NH Aerobic Nitrate Iron Time (days) Figure 12- NH4 + concentration in new oil treatment over full incubation period. 25