Interaction of Alcanivorax borkumensis with a Surfactant Decorated Oil

Article pubs.acs.org/Langmuir

Interaction of Alcanivorax borkumensis with a Surfactant Decorated Oil−Water Interface Michelle Bookstaver,† Arijit Bose,‡ and Anubhav Tripathi*,† †

Center for Biomedical Engineering, School of Engineering, Brown University, Providence, Rhode Island 02912, United States Department of Chemical Engineering, University of Rhode Island, Kingston, Rhode Island 02881, United States

‡

S Supporting Information *

ABSTRACT: Alcanivorax borkumensis is a hydrocarbon degrading bacterium linked to oil degradation around oil spill sites. It is known to be a surface bacterium leading to substantial interaction with the oil−water interface. Because of its abundance in oil spill regions, it has great potential to be used actively in oil spill remediation. Dispersants are thought to be important in the creation of oil-in-water emulsions that are meant to aid in the biodegradation process by bacteria. Although it is likely that some sort of dispersant will be used again in the case of another oil spill, to date, no studies have shown the impact of dispersants on the bacteria population. Corexit 9500 was the main dispersant used during the Deepwater Horizon oil spill, but little is known about its effect on the bacteria community. We built an experimental platform to quantitatively measure the transient growth of Alcanivorax borkumensis at the interface of oil and water. To our knowledge, this is the first study of how A. borkumensis interacts with a surfactant decorated oil−water interface. We use COREXIT EC9500A, cetylytrimethylamonium bromide, dioctyl sulfosuccinate sodium salt, L-α-phosphatidylcholine, sodium dodecyl sulfate, and Tween 20 to investigate the impact of dispersants on Alcanivorax borkumensis. We assess the impact of these dispersants on the growth rate, lag time, and maximum concentration of Alcanivorax borkumensis. We show that the charge, structure, and surface activity of these surfactants greatly impact the growth of A. borkumensis. Our results indicated that out of the surfactants tested only Tween 20 assists Acanivorax borkumensis growth. The results of this study will be important in the decision of dispersant use in the future.

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INTRODUCTION In the energy market today, oil is still the top traded commodity available for a variety of uses. American consumption is estimated at a rate of approximately 18.7 million barrels a day,1 making it the most widely used fossil fuel. With such a large industry comes a substantial amount of risks. The last major catastrophic event was the Deepwater Horizon oil spill in 2010.2 Four million barrels of oil were released into the Gulf of Mexico during the spill,1 and it claimed 11 lives.3 The ecological effects, some of which are still being felt today,4−6 have raised the bar on the motivation and necessity to understand ways in which we can better remediate oil spills. As oil becomes more rare on continental terrain, deep-water drilling has grown in use.1 The Deepwater Horizon oil spill occurred at a deep-water drilling rig and resulted in the release of hydrocarbons into the water column. While the short saturated hydrocarbon and aromatic components of the crude oil remained in the deep-water column, insoluble hydrocarbon components made their way to the surface eventually where they could reside for long periods.7 These surface hydrocarbons are mostly degraded by naturally occurring bacteria and sunlight.8 These marine bacteria most often reside near oil rigs and feed off of natural seepage, but in the presence of oil spills, their population explodes.9−11 They have hydrocarbon degradation capabilities, and many produce biosurfactants and © 2015 American Chemical Society

biofilms that serve to emulsify the oil into discrete droplets or lower the surface tension of the oil−water interface to assist the bacteria’s natural hydrocarbon degradation process.12−14 Dispersants are generally used to emulsify the oil into discrete oil-in-water droplets that increase the available surface area for bacteria adhesion therefore increasing their biodegradation rate.15 Additionally, dispersants lower the surface tension of the oil slick enough so that it will disperse into the water column, which is thought to decrease the harmful effects to beach life.16 It has been shown in the past that the bacteria’s ability to access the hydrocarbon inside the water droplet is dependent on the droplet size.17 Different bacteria species have different droplet size preference.18 Furthermore, the interfacial electrostatic properties that result from the added dispersant can affect the bacteria’s ability to adhere to the interface.19,20 Since adherence is a vital step for many bacteria to degrade the oil, this is an important consideration when choosing a dispersant. The viability of certain bacteria upon exposure to a commonly used dispersant COREXIT EC9500A has been studied.21 We present a first look at the way in which Alcanivorax borkumensis interacts with an oil−water interface that has been Received: February 20, 2015 Revised: May 1, 2015 Published: May 12, 2015 5875

DOI: 10.1021/acs.langmuir.5b00688 Langmuir 2015, 31, 5875−5881

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the live cell population only. Once this correlation was determined, the live cell intensity was found for all future experiments and converted to the optical density based on the calibration curve, not vice versa, so that values should accurately represent the live cell concentration. Interfacial Tension Measurements. Interfacial tensions of the surfactant solutions in octane surrounded by marine broth supplemented with potassium nitrate with were measured using a pendant drop technique along with a drop shape analyzer (Easy Drop FM40, Krüss GmbH Germany). Emulsion Images. Images of the oil−water emulsion were taken at various time points on a Leica DM5500 B microscope using 63× magnification and analyzed using ImageJ.

decorated with surfactant. Alcanivorax borkumensis was discovered in 1998 as a hydrocarbon degrading bacteria, part of the γ-proteobacteria family.22 It dominates microbial communities in the weeks after an oil spill as it works to degrade n-alkanes and branched alkanes.23 A. borkumensis is known to produce a glucolipid biosurfactant and a separate biofilm that aids in the degradation of alkanes.24,25 The biosurfactant is only produced when a cell signaling pathway, involving AlkB genes, is induced by bacteria contact with hydrocarbons.22 The biosurfactant is then formed as part of its cell membrane.14 The growth of A. borkumensis is extremely dependent on nitrogen and phosphorus concentrations.23 We investigated the change in growth rate, lag time, and maximum concentration of Alcanivorax borkumensis as a result of changing surfactants in the oil layer. We use the structure, surface tension, and emulsion characteristics to interpret how these bacteria grow and thus affect biodegradation of oil following spills.

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RESULTS AND DISCUSSION Figure 1 shows the experimental setup we used to test the impact of the various surfactants on the growth of bacteria. Surfactant was first added to octane at the appropriate concentration. This mixture was vortexed to evenly distribute the surfactant into the oil before adding 3 mL of the mixture to the bacteria solution. The surfactant concentrations listed are the concentrations in the oil layer and not the aqueous phase. The bacteria suspension was composed of the desired concentration of bacteria in marine broth supplemented with potassium nitrate. 3 mL of surfactant−oil mixture was added to 7 mL of the bacteria suspension and then grown for 72 h. At certain time points, a LIVE/DEAD stain was executed in order to quantify the live cell concentration of bacteria. These concentrations were normalized to their starting concentration to yield the growth curves shown in Figure 2. The dashed black line represents the normal growth curve of Alcanivorax borkumensis in octane but no surfactants. The growth curve for the octane control exhibits a clear lag phase for ∼18 h, after which the bacteria enters an exponential growth phase. The bacteria continue their growth for ∼36 h when they enter a brief stationary phase or maximum growth cmax. This is followed by a steady decline in the live cell concentration as a result of the buildup of octanol within the bacteria, an intermediate of the bacteria’s degradation of octane, which is toxic to A. borkumensis.26 The overall growth of bacteria can be defined by27

EXPERIMENTAL SECTION

Bacteria Inoculation. Alcanivorax borkumensis (ATCC 700651) were grown in a 7 mL marine broth solution (BD Difco 2216) supplemented with 2 g/L potassium nitrate. 3 mL of a surfactant solution in octane was added to the marine broth suspension containing bacteria, as shown in Figure 1. Solutions were grown on a

Figure 1. Introduction of surfactant into the oil−water system. shake rack to simulate the effect of waves in the ocean on both the bacteria growth and the emulsions. The surfactants studied were cetylytrimethylamonium bromide (CTAB), COREXIT EC9500A, L-α-phosphatidylcholine (Lecithin), sodium dodecyl sulfate (SDS), dioctyl sulfosuccinate sodium salt (AOT) and Tween 20. Surfactant concentrations were chosen to be either at critical micelle concentration (CMC), slightly below, or slightly higher than CMC in order to formulate emulsions and keep the emulsions fairly consistent across all tests. Although the solubility in the bulk oil varied across the surfactants, the intention was to decorate the interface. Any emulsion formation next to the interface was noted. However, no quantitative measurements of these emulsions were possible. Bacteria Growth. At various time points during the experiment 500 μL of bacterial solution was removed and stained for a live cell intensity reading. Live/dead stain (LIVE/DEAD BacLight Bacterial viability Kit, Life Sciences) was mixed into the solution before the signals were read using a PHERAstar Plus microplate reader (BMG labtech). Live cell intensities were correlated to OD600 readings using the curve below where I is the live cell intensity.

dc = β (c )c dt

where c(0) = c0 and 0 ≤ c ≤ cmax

(1)

β(c), the linearized specific growth rate during the exponential phase, is a function of the bacteria type, concentration, and the surrounding environment. c0 is the initial concentration of bacteria. From Figure 2A, it is clear that 1:20 COREXIT and concentrations of CTAB around the CMC completely prevent the growth of the bacteria. Figure 2B shows the effects of lecithin on the growth of A. borkumensis. There was a significant increase in lag time for concentrations ≤ CMC as well as a substantial decrease in both growth rate and cmax. At concentrations corresponding to 10 × CMC the bacteria showed no growth. In Figure 2C, the effects of AOT at CMC (0.18 mM in oil) and 1 order of magnitude above and below CMC were tested.28 At concentrations ≥ CMC there was no growth of bacteria, but at 1/10 × CMC a decrease in lag time can be seen. The curve of 0.018 mM AOT closely follows that of octane but is just shifted to the left by ∼6 h. Figure 2D shows that at low concentrations of SDS (CMC = 8 mM)29 the growth curve is remarkably similar to the control without surfactant up until around ∼24 h. At that point the bacteria reach a stationary phase and their growth plateaus.

OD600 = 0.95 exp(0.002I ) Once the OD600 readings were obtained, they were converted to a live cell concentration using a previously determined calibration (OD600 of 1 = 8 × 108 cells/mL). Note that the correlation was obtained by finding the live and dead cell intensity and then finding the optical density of the same sample. The ratio of live to dead cells was very high so it can be assumed that the optical density is a good representation of 5876


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Figure 2. Growth curves of Alcanivorax borkumensis under various surfactant solutions in octane (A−E). The time it takes various concentrations of Tween 20 to reach C/C0 = 5 (F).

Higher concentrations of SDS demonstrated no growth. SDS is soluble in oil but not entirely at the CMC reported so it is possible that at a lower concentration more growth might be seen. The most compelling effect of any surfactant on the growth of A. borkumensis was seen when Tween 20 was added to the oil, shown in Figure 2E. The CMC of Tween 20 in water is 0.06 mM.30 Since Tween 20 comes as a liquid, we first mixed the required amount of Tween 20 into the octane using vortexing. This resulted in a homogeneous emulsion of Tween 20 and octane. Subsequently, this Tween 20 emulsion was placed above the bacteria solution which allowed it to come in contact with the oil−water interface. Hence, it can be assumed that there is an even distribution of Tween 20 at the oil interface. At this concentration, similar to tests with 0.018 mM AOT, the growth curve was shifted left by ∼6 h compared to octane. Otherwise, the curve appears to follow similar trends to octane. The time it takes for bacteria to grow 5 times its starting concentration is shown in Figure 1F. The curves show how this time increases initially with increasing concentration. At a certain critical point though, this time

reaches a maximum at which point it starts to decline with increasing concentration. This closely follows the trend for the lag time and can therefore be explained in the same way as the change in lag time. At 10 × CMC, the lag time actually increases significantly to ∼36 h, but then the A. borkumensis begins exponential growth at a similar rate to that when octane only is used. In contrast to the control case, the bacteria continued to grow exponentially for at least double the time as octane. Experiments were stopped at this point. Because of these results, concentrations in between 0.06 and 0.6 mM were tested. At 0.2 mM Tween 20 in octane, a similar curve was seen as with 0.6 mM Tween 20. The lag time was slightly longer, and the growth rate and cmax were both slightly decreased relative to 0.6 mM Tween 20. Relative to octane, there was a significant increase in lag time and cmax. Figure 3 shows comparison charts for the growth rate, lag time, and maximum concentration of Alcanivorax borkumensis in relation to the values calculated for the octane control. All numbers were normalized by results from the control. Figure 3A shows the results for various concentrations of AOT. There was no growth for the two higher concentrations. 5877


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Figure 3. Calculated growth rate (cells/h), lag time (h), and cmax (cells/mL) of Alcanivorax borkumensis in relation to octane control (A−D).

likely because the growth had not completed by the 72 h end of the experiment. To better understand why certain surfactants enhanced the growth of Alcanivorax borkumensis while others stunted it, we examined the structures and interfacial tension of some of the more interesting surfactants. Table 1 shows the measured IFT values as well as the structures for select surfactants. The interfacial tension of the octane in marine broth is ∼36 mN/m. The IFT of SDS could not be calculated using our method because the SDS was not completely soluble in octane at the concentrations studied. The same was true with the lecithin, but both remained evenly distributed in the oil layer throughout the experiments. The IFT of COREXIT EC9500 is so low that it is not possible to measure it with a pendant drop. At 0.018 mM AOT the IFT of 33.6 mN/m was not lowered significantly compared to that of octane. At 0.18 mM AOT the IFT is 10.2 mN/m, and the oil−water interface is likely to be saturated with AOT, resulting in no or marginal access to oil for approaching bacterial cells. Hence, too low of a surface tension is not conducive to bacteria adhesion to the oil layer. This is supported further by the lack of growth with COREXIT given its extremely low surface tension. To probe this further, we measured the IFT of Tween 20 at two concentrations. At 0.06 mM Tween 20, the surface tension was ∼29 mN/m. This is lower than octane but still relatively close. At such low concentrations of surfactants, it is unlikely that any noticeable droplet formation could occur next to the interface. Indeed, at least visually, we did not observe any emulsion next to interface at these low concentrations. Therefore, a slightly lower surface tension than octane allows for faster adherence of bacteria to the oil layer and therefore a shorter lag time prior to bacteria growth. Because oil is highly hydrophobic, it is not conducive to bacteria adhesion. A limited amount of bacteria initially come in contact with the oil layer. Then an upregulation of genes leads to biosurfactant and biofilms formation and alkane degradation. The biosurfactant serves to lower the surface

At the lowest concentration of AOT, 0.018 mM, which was 1/10 × CMC, the lag time was calculated to be roughly half of octane and the growth rate was about 1.5 times that of octane. These results were promising because it showed the potential to use AOT as a dispersant in the future. On the other hand, the maximum concentration was about the same as octane so even though AOT helped the bacteria grow sooner and faster, it reached a maximum at the same concentration. This would result in the same final degradation rate as the case without surfactant. AOT by itself would not serve as the best option for use as a dispersant. The results of the lecithin and SDS tests are shown in Figures 3B and 3C. At the highest concentration of lecithin there was no growth, as seen in Figure 3B. The lowest concentration increased the lag time, decreased the growth rate by more than half, and decreased the maximum concentration significantly. At the CMC, the lag time was increased compared to the control, but once again there was such a decrease in growth rate and maximum concentration that lecithin is not viable as a potential dispersant at any concentration. At higher concentration of SDS (around CMC), no growth was seen (Figure 3C). The lowest concentration did allow for some growth of A. borkumensis, but both the growth rate and maximum concentration only reached about half of those of the octane control. Tween 20 showed very promising results at all concentrations tested (Figure 3D). At CMC, 0.06 mM, the lag time was cut by about half and the growth rate was close to doubled. cmax stayed about the same. The higher concentrations of Tween 20 yielded the most interesting possibilities. At both 0.2 mM Tween 20 and 0.6 mM Tween 20 there was a significant increase in cmax. This is important because the concentration of bacteria controls the rate of biodegradation. The lag time also more than doubled for both concentrations, but the growth rates were roughly equal to those of octane. At 0.1 mM Tween 20 and 0.15 mM Tween 20, the maximum concentrations were less than that of octane, but it is most 5878


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Langmuir Table 1. Physical and Chemical Properties of Select Surfactants

centrations of lecithin. The small amount of growth of bacteria at lower concentration of lecithin is likely because of a lower IFT of the oil interface. Bacteria most likely failed to grow beyond this small amount though because it likely could not produce its own biosurfactant in these conditions. SDS’s structure has one long carbon chain attached to a sulfate group and is an anionic surfactant. It has few similarities if any to the natural biosurfactant, other than its negative charge. It is possibly because of this lack of similarity that no bacteria grew around the CMC of SDS. It is also possible that due to SDS’s anionic charge, it repelled the Gram-negative bacteria with the anionic biosurfactant, which is why the interface affected by SDS was unusable to the bacteria. At the lowest concentration of AOT, the lag time was decreased, but the bacteria growth curve was virtually the same otherwise. Looking at the structure of AOT, there are two carbon chains, each containing one ester, attached to a sulfate group. AOT is also an anionic surfactant. At such a low concentration, the AOT does not affect the octane interface to a significant degree but is able to lower the surface tension enough for the bacteria to adhere to the interface and begin growing prior to the formation of its own biosurfactant. At higher concentrations, though, the anionic AOT most likely repels the bacteria away from the interface impeding its growth. Tween 20’s structure shows a similar cyclic sugar unit but has no glycine-terminating end and has a long carbon chain with only one ester. The similarity in these structures and its nonionic properties could explain how Tween 20 works with A. borkumensis to promote adherence and degradation. This bacterium is known to only be able to metabolize aliphatic

tension to allow for better adhesion, and the biofilms form emulsions to increase the surface area of oil available for degradation.31 Too low of a surface tension though, as is the case with COREXIT, 0.18 mM AOT (10.16 mN/m) and 0.6 mM Tween 20 (14.84 mN/m), may prevent bacteria adherence. At high concentrations of surfactants on the interface (hence low interfacial tensions), the bacteria has limited access to oil; however, at low concentrations of surfactants on the interface (high interfacial tension), more surface area is available to bacteria. We believe biofilm formation depends on the degree of coverage of surfactant on the oil−water interface. Biofilm formation was visually observed to have formed in almost every experiment where bacteria grew. However, quantitative measurements of biofilm growth were not performed in this study. The IFT for 0.6 mM Tween 20 was 14.8 mN/m. This value is very similar to that observed with 0.18 mM AOT. Contrary to AOT, at this concentration of Tween 20, there was an exceptional amount of growth of A. borkumensis with lag time doubled. Table 1 also shows the structures of certain surfactants. The structure of the natural biosurfactant produced by Alcanivorax borkumensis is shown in the bottom row. Some of the important characteristics are a cyclic sugar unit and a long chain of esters terminating in a glycine. Additionally, it is an anionic surfactant attached to a Gram-negative bacterium. The structure of lecithin, on the other hand, shows two long carbon chains, one containing an ester, attached to a phosphorus molecule. This structure has few similarities to that of the bacteria’s own biosurfactant. Although the IFT could not be calculated, inoculations showed extremely stable emulsions at all con5879


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Langmuir hydrocarbons and a limited amount of short chain fatty acids.14 There is no literature available that suggests these bacteria are capable of partially or fully metabolizing Tween 20. At low concentrations, it decreases the lag time prior to growth because it can allow the bacteria to interact with the interface sooner by serving as its natural biosurfactant. At the higher concentrations, it lowers the surface tension of the octane too much so the bacteria have a harder time beginning degradation. This is why the lag time is increases. Eventually, enough bacteria overcome this barrier to begin degradation and therefore start producing their own biosurfactant. The bacteria’s biosurfactant most likely works synergistically with the Tween 20 to form individual oil droplets. These droplets increase the available surface area for bacteria adhesion and therefore allow for more overall bacteria growth. Examining the structure of the surfactants provided invaluable information on how charges may affect the bacteria’s own ability to adhere to the interface. It was clear that anionic surfactants typically repel the bacteria away from the interface decreasing its overall growth ability of degrade. At very low concentrations, where not the entire interface is affected by surfactant, some of these anionic surfactants can lower the surface tension slightly and decrease the lag time prior to bacteria growth, but it is not suggested that any of them be used. Tween 20 yielded outstanding results and warranted further study to see how it may help the bacteria. For these reasons, images were taken of the emulsions made with Tween 20 at time 0 to investigate the droplet size and emulsion stability. Figure 4A illustrates the emulsion of 0.6 mM Tween 20. From this image, as well as several in Supporting Information

the Tween 20 emulsion droplets and begin degradation. In this case, the Tween 20 increased the available surface area by creating emulsions, and then once the bacteria produced its own biosurfactant it was able to utilize this increase in surface area to increase its overall cmax.

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CONCLUSION After looking at the growth curves, surface tension measurements, surfactant structure, and emulsions, it is clear that not every surfactant will necessarily aid the bacteria’s natural degradation abilities. When the oil−water interface is decorated with anionic surfactant, this works against the bacteria’s own anionic biosurfactant and repels the bacteria away from the interface, prohibiting adherence and growth. At low concentration, these surfactants can lower the surface tension and decrease the lag time, but this will not ultimately result in more growth and therefore more degradation. Using a nonionic surfactant, such as Tween 20, is a possibility that should be investigated further. At low concentration, the lag time is decreased due to a decrease in surface tension. At higher concentrations, Tween 20 works synergistically with the bacteria’s own natural biosurfactant to increase the available oil interface as well as the bioavailability of the oil to allow for more growth of bacteria and thereby more degradation.

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ASSOCIATED CONTENT

* Supporting Information S

A schematic of decorated oil−water interface and additional images of the oil−water emulsion droplets created using 0.06 mM Tween 20 and 0.6 mM Tween 20. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b00688.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; Tel (401)-863-3063 (A.T.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was made possible by a grant from BP/The Gulf of Mexico Research Initiative.

Figure 4. Emulsion droplet images for 0.6 mM Tween 20 (A) and 0.06 mM Tween 20 (B).

Figure 1A−C, it is most important to note that droplets are typically distinct with an average size of 44 ± 20 μm. Emulsions were stable for well over 10 min at this concentration. Emulsions at 0.06 mM Tween 20 are seen in Figure 4B and in Supporting Information Figure 1D−F. At this concentration, droplets were almost entirely coalesced after about 5 min. Images were taken within these 5 min to get an idea of what the remaining droplets at the interface would look like. The average droplet size at 0.06 mM Tween 20 was 125 ± 80 μm. There is such a distribution in droplet size due to the coalescing of droplets. It was clear from the images though that the few individual droplets were roughly the same size as those at the larger concentration. At the higher concentration of Tween 20, the stable emulsion prevented the bacteria from initially adhering to the interface efficiently. Once some of the bacteria were able to adhere it was able to produce its own biosurfactant, which then most likely was able to adhere to

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Interaction of Alcanivorax borkumensis with a Surfactant Decorated Oil

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