Behavior of Marine Bacteria in Clean Environment and Oil-Spill

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Biological and Environmental Phenomena at the Interface

Behavior of Marine Bacteria in Clean Environment and Oil-Spill Conditions Michael P Godfrin, Maswazi Sihlabela, Arijit Bose, and Anubhav Tripathi Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01319 • Publication Date (Web): 05 Jul 2018 Downloaded from http://pubs.acs.org on July 7, 2018

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Behavior of Marine Bacteria in Clean Environment and Oil-Spill Conditions 1

Michael P. Godfrin1, Maswazi Sihlabela1, Arijit Bose2, Anubhav Tripathi1† Center for Biomedical Engineering, School of Engineering, Brown University, Providence, RI; 2 Department of Chemical Engineering, University of Rhode Island, Kingston, RI

Abstract Alcanivorax Borkumensis is a hydrocarbon-degrading bacterium that dominates hydrocarbondegrading communities around many oil spills. The physicochemical conditions that prompt bacterial binding to oil-water interfaces are not well understood. To provide key insights into this process, A.borkumensis cells were cultured either with a clean environment condition (dissolved organic carbon) or with an oil spill condition (hexadecane as sole energy source). The ability of these bacteria to bind to the oil/water interface was monitored through interfacial tension measurements, bacteria cell hydrophobicity and fluorescence microscopy.

Our experiments

show that A.borkumensis cultured in clean environment conditions remain hydrophilic and do not show significant transport or binding to the oil/water interface. In sharp contrast, bacteria cultured in oil spill conditions become partially hydrophobic, and their amphiphilicity drives them to oil/water interfaces, where they reduce interfacial tension and form the early stages of a biofilm. We show that it is A.borkumensis cells that attach to the oil/water interface, and not a synthesized biosurfactant that is released into solution that reduced interfacial tension. This study provides key insights into the physicochemical properties that allow A.borkumensis to adhere to oil/water interfaces.

Keywords: Alcanivorax borkumensis, physicochemical binding mechanisms, oil-degrading marine bacteria, oil/water interface

†

Corresponding author: [email protected]

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TOC Figure

Introduction Naturally occurring marine bacteria are a major component of the carbon cycle in ocean water column.1-3 Many bacteria are able to metabolize hydrocarbons as an energy source.4-6 Hence, the population of these oil-degrading bacteria explodes around oil spill sites.7-9 A diverse set of bacteria thrive in these sites, with each bacterial strain capable of degrading particular types of hydrocarbons, including alkanes or aromatics.10-11 To support their access and utilization of hydrocarbons, oil-degrading bacteria produce biosurfactants of diverse chemical nature and molecular size.12 Alcanivorax borkumensis (A.borkumensis ) is a Gram negative bacterium that is capable of degrading n- and branched- alkanes.4,

13

This bacterium dominates bacterial communities

around oil spills, and it synthesizes two forms of an anionic glycoprotein surfactant.14-15 One form is glycine-free, and is released in the death phase of bacterial growth, leads to bacterial detachment from the interface. The second, cell membrane-bound form contains glycine and reports suggest that it increases cell hydrophobicity.16-17 A.borkumensis genes for glucolipid synthesis and transport to the cell membrane have been found to be up-regulated in the presence of hydrocarbons, and down-regulated in their absence.18 A.borkumensis cells access hydrocarbons by adhering to oil/water interfaces; cells are able to either uptake oil as small 2 ACS Paragon Plus Environment

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droplets, through active transport of the droplet across the cell membrane, or locate at the oil/water interface, and oil molecules are transported across the interface and into the cell. 19-20 As A.borkumensis is a dominant bacterium in oil degradation, a variety of studies have been performed to understand the effect of oil dispersants on bacterial utilization of hydrocarbons21-22, as well as trying to develop new mechanisms to support bacterial transport23. Ultimately, it is imperative to understand how A. borkumensis cells transport to and adhere to the interface. In this work, we focus on the physicochemical aspects of the migration and binding of these bacteria to oil-water interfaces, and provide key new insights into these processes. Binding of A.borkumensis to the oil/water interface is a critical step in the bacterium gaining access to hydrocarbons, whether that interface is an emulsion droplet or a bulk interface, which provides understanding of how these naturally occurring bacteria degrade oil after oil spills in the marine environment.

The importance of this binding step is the drive for this investigation into

physicochemical properties effects on A.borkumensis binding to oil/water interfaces. Our insights are based on confocal and fluorescence microscopy, UV-spectroscopy and measurements of the dynamic oil/water interfacial tension. Materials and Methods 1. Bacteria growth A.borkumensis (ATCC) was routinely maintained on Difco™ Marine Agar 2216 plates and cultured as described in our earlier work24. Cells were then allowed to grow in two different solution conditions: a marine broth (BD Difco™ 2216) with sodium pyruvate as an energy source (clean environment condition, Figure 1a) or a minimalistic broth with a hexadecane oil layer as an energy source (oil spill condition, Figure 1d). Marine broth was prepared by dissolving 37.4 g/L of BD Difco 2216 in deionized water, along with 2 g/L supplemented KNO3, and then


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mixed and boiled for 1 minute and autoclaved at 121 ⁰C for 40 min. The minimalistic broth was synthesized by dissolving 19.45 g/L NaCl, 5.9 g/L MgCl2, 3.24 g/L MgSO4, 1.8 g/L CaCl2, 0.55 g/L KCl, 2 g/L KNO3 and 0.126 g/L HK2O4P into deionized water and mixed and autoclaved with the same procedure as the marine broth. KNO3 was supplemented into the broth solutions as it has been reported as a limiting factor in culture25-26 arine broth suspensions were supplemented with 10 g/L sodium pyruvate as an energy source. Minimalistic broth suspensions were supplemented with 2 mL of hexadecane as a carbon source on top of 6 mL of the aqueous phase containing ~106 cells/mL. The bacteria suspensions were gently stirred and incubated at 30°C for various growth times. Note that the oxygen transport was secured through diffusion from the ambient air through the hexadecane layer into the aqueous phase. This is in addition to the dissolved oxygen in the culture media. The oxygen diffusion coefficients for water and hexadecane are reported as 2.11 x 10-5 cm2/s and 2.49 x 10-5 cm2/s, respectively. Hence, it is assumed that oxygen supply for bacteria was similar for both clean environment and oil-spill cases. 2. Optical Density and fluorescence microscopy OD600 values were obtained for the bacteria suspension to calculate bacteria concentration. Live/dead stains were carried out with a LIVE/DEAD® BacLight™ Bacterial Viability Kit.

Absorbance values were measured on a 96 well microtiter plate using a

PHERAstar Plus microplate reader (BMG labtech). Live/dead images were obtained using a Leica DM5500B fluorescence microscope and a Zeiss Observer A1 inverted fluorescence microscope. To ascertain where possible biosurfactant was located (free in solution or bound to cells), bacteria suspensions were centrifuged for 10 minutes at 7000rpm and the supernatant was


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removed. The supernatant and the cells, re-suspended in broth, were used to analyze their effect on an oil/water interface.

3. Single Droplet Interfacial Tension Measurements A 1x1x3 cm cuvette was filled with the bacteria suspension after its growth time. A hexadecane droplet was dispensed using a mechanical syringe pump controlled by the Drop Shape Analyzer (DSA 100 Krüss, Hamburg, Germany). For the interfacial tension measurements, t0 is considered the time at which the hexadecane reaches the desired droplet volume of 4.5 μL. The droplet shapes were monitored and recorded27. For 4.5 μL droplets, the Bond number Bo=ΔρgR2/γ ~ 0.1, where Δρ is the difference in densities between hexadecane and water, g is the acceleration due to gravity, R is the average radius of the droplet and γ is the interfacial tension. The Bond number fits well within the applicability’s of the fitting with the Young-Laplace equation for estimating best value of interfacial tension. 4. BATH Optical Density Measurements In addition to the single droplet measurements, the bacterial adhesion to hydrocarbon (BATH) test (Rosenberg et al.28) was also conducted using hexadecane as the hydrocarbon phase. The OD600 of a bacterial suspension grown in media with dissolved organic carbon (nospill condition) or with hexadecane layer (oil spill condition) for 92 hours is measured; 1mL of cultured AB was then vortexed at 3000 rpm with 0.5 mL of hexadecane for 2 minutes. The OD600 of this aqueous phase was measured, ensuring that bacteria from the oil/water interface were not perturbed. The ratio of the OD600 values describes the hydrophobicity of the bacterial cells, as hydrophobic cells will move to the oil/water interface reducing the absorbance of the aqueous phase. OD600 of the bacterial suspension was measured with a PHERAstar Plus


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microplate reader prior to and after vortexing with hexadecane. Droplets were imaged using a Leica DM5500B upright fluorescence microscope. 5. Confocal Microscopy 1 mL of bacteria (OD600 ~ 0.15) is incubated with 0.75 µL each of the live and dead stain of Life Sciences BacLight Live/Dead stain kit for approximately 15 minutes.

Cells are

centrifuged at 8000 rpm for 10 minutes and separated from the supernatant. The cells are then resuspended in custom carbon-free broth. Approximately 50 µL of bacterial suspension is pipetted into a well of a glass-bottomed 96 well plate (MatTek Corporation and Invitro Scientific). 50 µL of hexadecane is pipetted on top of the aqueous phase. The 96 well plate is then incubated at 30°C for 24 hours and the interface is imaged using a Nikon confocal fluorescence microscope with an Argon ion 488 nm laser. A 40X/1.15 WI Apo LWD DIC N2 objective was used with an immersion fluid, Immersol W (Zeiss). A z-scan is conducted, starting at approximately 25-50 µm below the oil/water interface, in the aqueous phase. It is run to approximately 25 µm above interface, in the oil phase. Results and Discussion 1. A.borkumensis growth in clean environment and oil spill conditions Cultured in a clean environment i.e. in marine broth containing only sodium pyruvate as an energy source (Figure 1a), the concentration of A.borkumensis cells increased by 3 orders of magnitude in 55 hours. Figure 1b shows the concentration of bacterial cells at various growth times, tg. The growth curve shows three typical regimes: an initial lag period λ of about 30 hours, an exponential growth phase of about 20 hours and the stationary phase. Bacteria in the stationary phase were imaged using fluorescence microscopy. Figure 1c shows a suspension of A.borkumensis with very few agglomerates containing < 5 bacteria.


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In contrast, when A.borkumensis was cultured under oil spill conditions i.e. under a hexadecane layer as the sole energy source (Figure 1d), bacteria concentration again increased 3 orders of magnitude, however in 75 hours (see figure 1e). Observed maximum concentrations, as shown in figures 1(b) and 1(e), were similar in both clean environment and oil spill conditions. As discussed in our previous work23, the amounts of KNO3, which served as limiting nutrient co-factor, were kept the same in both cases. Although observed maximum concentrations were similar, the different energy sources led to different lag times (30 hours with organic carbon vs. 75 hours with hexadecane).

Figure 1e shows the concentration of bacterial

cells for various growth times, tg. The curves show a longer lag period of λ ~55 hours. The qualitative behavior of the growth curve is similar to cell cultured in clean environment conditions. A key difference however is revealed in the fluorescence microscopy images of bacteria in the stationary phase (Figure 1f). Only agglomerates of approximately 25-50 µm are observed. Agglomeration of A.borkumensis cells corroborates reports that the cells become partially hydrophobic after the exponential growth phase, when grown under a hydrocarbon layer, due to the production of biosurfactant at the cell membrane29. Although the exact cause or kinetics analysis of bacteria growth is out of the scope of this study, it is not likely that limited oxygen supply resulted in the differences in lag times. Based in diffusion coefficients of oxygen through water and hexadecane, it is safe to assume that oxygen supply was similar for both clean environment and oil-spill cases. We note that genomic studies of A.borkumensis that have found an up-regulation of glycoprotein regulation and transport genes in the presence of hydrocarbons.15, 30. A ~25 hour increase in lag time during cell culture may stem from the time required for gene regulation leading to biosurfactant production and access to oil as an energy source31. Note that cells cultured with hexadecane are smaller than those cultured with organic


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carbon, which has been reported previously32 The differences in growth curves of A.borkumensis under clean environment and oil-spill condition are important for understanding complex marine environments with respect to fate, transport, and transformation.

Figure 1. a) Schematic of the experiment showing A.borkumensis grown in clean environment conditon, (b) Concentration of bacteria versus growth time, tg. c) Fluorescence image of the bacteria suspension at tg = 92 hours (Scale bar is 10 μm). d) Schematic of the experiment showing A.borkumensis grown in an oil spill condition. e) Concentration of bacteria versus growth time, tg, f) Fluorescence image of the bacteria suspension at tg = 92 hours (Scale bar is 10μm).

2. Biosurfactant renders A.borkumensis partial hydrophobic and leads to oil droplet adhesion A.borkumensis cells cultured either under a clean environment condition or oil spill condition were exposed to an oil droplet (figure 2). The droplet was imaged and dynamic interfacial tension  was calculated, the change of which was used to infer bacteria binding to the oil/water

interface.


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a)

b)

Figure 2. Schematic of bacterial growth and single droplet experiments. After being grown for tg , the bacterial suspension is placed around a single droplet of hexadecane. The shape of the droplet is analyzed from which the interfacial tension is extracted. A.borkumensis is grown in a) Clean Environment (marine broth with sodium pyruvate) and b) oil spill condition (carbon-free broth under a hexadecane layer).

When hexadecane oil droplets were exposed to A.borkumensis cells grown for tg = 0 and 94 hours with clean environment condition, the interfacial tension was reduced by 10-20% in 1500 seconds as seen in Figure 3a. The interfacial tension was also reduced by 10-20% when a hexadecane oil droplet was exposed to the marine broth with dissolved organic carbon and without bacteria, as a control (not shown). Hence, A.borkumensis grown in marine broth without exposure to hydrocarbons had essentially no effect on interfacial tension, suggesting that cells not exposed to hydrocarbons during culture are not able to adhere to the oil/water interface within 1500sec.

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Figure 3. Interfacial tension vs. time for bacteria grown at different tg. (a) with clean environment condition (b) with oil spill conditions. Results represent the average of at least 5 repeats. The error bars are the standard deviation, and are shown only at one point for each tg for clarity. Measured interfacial tension values normalized by the interfacial tension at t0, γ0. The difference in measured interfacial tension between the control runs at tg stem from differences in electrolyte contents.


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When A.borkumensis cells were cultured under oil spill conditions, the interfacial tension changes by approximately 10-15% in 1500 seconds for A.borkumensis cells grown for tg = 0 and 53 hours, suggesting no interfacial effects through interface attachment. As shown in figure 1e, the concentration of bacteria at tg = 53 hours is small and before the exponential growth phase. However, the interfacial tension is reduced to 53% and 39% of γ0 in 1500 seconds for cells grown under hexadecane for tg = 74 and 94 hours, respectively. The measured interfacial tensions are summarized in figure 3b. The large reduction of interfacial tension suggests that either A.borkumensis cells or likely a biosurfactant produced by cells and released into solution have moved to the oil droplet interface. These findings correlate with the up-regulation of biosurfactant genes when exposed to hydrocarbons.18

Figure 4. (a) Light microscopy images captured of droplets formed during energy input for BATH tests. (b) Fluorescence microscopy images of droplets formed during BATH tests, where live bacteria is stained green. (c) Light microscopy images captured after energy input for BATH tests after removing biosurfactant from cell membrane. Scale bars are 1mm.

The relative hydrophobicity of the bacteria grown under the two growth conditions was ascertained using BATH experiments. A.borkumensis was grown for tg = 94 hours in under spill conditions and for tg = 74 hours under clean environment conditions and BATH tests were conducted on the bacterial suspensions. OD600 values were 97% ± 3% of their values for the cells cultured under clean environment conditions suggesting only 3% of cells moved to the interface i.e. those were removed by hydrophobic interactions offered by BATH test. Conversely, OD600 10 ACS Paragon Plus Environment

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values were 41% ± 9% of their values for the cells grown under hexadecane suggesting ~60% removal of cells based on hydrophobicity. The vortexing of cells grown under hexadecane during the BATH test leads to discrete oil droplets, as seen in Figure 4a. As seen in figure 4b, there are cells, stained green, that stabilize and sit at the interface. When bacteria were grown using sodium pyruvate, no droplets were formed when vortexed with oil for the BATH test (Figure 4c). Clearly, the growth of A.borkumensis with a hydrocarbon oil layer leads to increased hydrophobicity of the cells, allowing them to adhere to oil/water interfaces. Conclusions from the performed experiments assume that A.borkumensis cells adhere

to the oil/water interface, based on physicochemical processes leading to hyrodphobic cells. We investigate further with interfacial tension measurements to ensure that is valid, and that it is not a synthesized biosurfactant released into the extra-cellular environment. Bacteria cells were grown for tg=94 hours in minimalistic broth under hexadecane, and cells were separated out of suspension through centrifugation. Bacterial cells resuspended in minimalistic broth and the separated growth solutions were exposed separately to single droplets of hexadecane. The results are shown in figure 5. Bacterial cells reduce the interfacial tension to 37% of its value at t0, similar to the original growth suspension as seen in figure 3. The separated supernatant has the same effect on interfacial tension as the minimalistic broth/hexadecane control. Thus it is hydrophobic bacterial cells that adhere to the oil/water interface, and not biosurfactant in solution.

Based on cited studies, we conclude that increased hydrophobicity is caused by

biosurfactant anchored to the surface of bacterial cells.


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Figure 5. Interfacial tension vs. time for separated bacteria and supernatant solutions.

Furthermore, we developed a binding-kinetics model to understand the effect of the hydrophobic bacteria on the oil/water interfacial tension. Based on the insight that biosurfactant bound to the bacterial cell membrane leads to cell adsorption to the oil/water interface, we develop a kinetics model based on Langmuir adsorption kinetics to describe how bacteria locates there and influences interfacial tension.33 Let us assume B t  (#/m2) is the bacteria concentration on the oil-water interface. The adsorption rate is proportional to the number of available binding spaces   B  , where Γ∞ is the total number of binding positions on the oilwater interface. The rate is also dependent on the bacteria concentration in the bulk suspension C B , where the detachment rate can be assumed to be negligible. Hence, the rate of accumulation

of bacteria can be written as dB   k B C B   A  dt

(1)

Here k B (m3/#-s) denotes the adsorption rate constant. At steady state, equation (1) results in   Bss . The solution of equation 1, B t   Bss 1  exp k B C B t  , can be used to describe the

observed adsorption kinetics. We use this result describing droplet surface coverage of bacteria to gain further insight into the reduction in IFT.

The reduction in IFT has been described as 12

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being linearly proportional to surface coverage,    0 ~ B , where γ is IFT, γ0 is the IFT at test initiation and ΓB is bacteria surface coverage.34 We find that

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where K and k2 are fitting coefficients. For this model we consider CB to be the concentration of bacteria in the stationary phase (CB = 6x108 cells/mL) and steady state bacterial coverage of the oil droplet is the considered to be ΓBss = 4πRdroplet2/πℓbacteria2, where ℓbacteria=1 µm. The data 3 resulted in a value of k B =1.5*10-18 m s.# , K = 8.5x10-11 N-m/# and k2 = 0.1 for A.borkumensis

bacteria samples grown for tg = 96 hours. For tg = 72 hours, kB = 9x10-19 m3/#-s, K = 6x10-11 Nm/# and k2 = 0.07. The model can be seen in figure 6 along with experimental data. kB is the binding coefficient in the Langmuir binding isotherm part of the model, and therefore describes the ability of the bacteria to adsorb to the oil/water interface. Therefore, kB likely correlates with the hydrophobicity of the cells. K and k2 are fitting coefficients, but can also be used to describe the system. The value of K describes the absolute IFT change of the system; therefore K may be related to the concentration of biosurfactant in the system, or again related to the hydrophobicity of the bacterial cells. k2 describes the rate of change in IFT at low surface concentrations of bacteria complex. These model values offer a new way to characterize bacteria/protein systems and their interaction with- and effect on the oil/water IFT. This model could be used to compare the ability of bacteria to adhere to oil/water interfaces.


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Figure 6. Interfacial tension, plotted as instantaneous interfacial tension minus interfacial tension measured at t0, vs time along with the kinetics model.

3. Onset of A.borkumensis biofilms Bacterial cells were fluorescently stained after reaching the stationary growth phase with the two culturing conditions and set next to an oil/water interface. Images of the interface were obtained immediately after forming the oil/water interface, and after 2.5 hours (Figure 7a-d). Grey scale, fluorescence intensity values were measured along the direction perpendicular to the interface and shown in Figure 6e. A uniform fluorescence signal is observed initially in the aqueous phase for both bacteria growth conditions, as bacteria are dispersed homogeneously in suspension. After 2.5 hours with bacteria cultured under oil spill conditions, a layer of strong fluorescence intensity is observed on the aqueous side of the oil/water interface, suggesting that cells cultured with oil are adhering to the oil/water interface and forming a biofilm. After 2.5 hours with bacteria grown under clean environment conditions, no increase in fluorescence intensity is observed at the oil/water interface. Thus A.borkumensis cells without prior exposure to hydrocarbons are not able to adhere to the interface.


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Figure 7. a) Schematic of experiment conducted to image bacteria adhering to oil/water interface. b) Fluorescence images at the oil/water interface after incubating stained bacteria, cultured in clean environment condition, for 0 and 90 minutes in vicinity of interface. c) Fluorescence images at the oil/water interface after incubating stained bacteria, cultured in oil spill condition, for 0 and 90 minutes in vicinity of interface. Scale bars are 50 μm. d) Grey scale values of pixels along red line in images (c, 90 minutes). e) Maximum grey scale intensity at different times in fluorescence images at oil/water interface with oil spill condition bacteria.

Measured gray scale along the direction perpendicular to the interface (as shown in Figure 7D) suggests bacteria population at the oil/water interface over 90 min of observation time. The maximum grey scale intensity (as shown in figure 7E) increases non-linearly ( I max ~ t ) with time. Assuming concentration of bacteria t  around the interface is proportional to measured maximum intensity I max , the observed time scale can be predicted by adsorption of bacterial cells via diffusion using t   2Cb Dt 

35

. Here, Cb is bulk concentration, and D is the diffusion

coefficient of the bacterial cell. Similarly, cells grown into the stationary phase and stained were incubated with an oil/water interface for 24 hours and the oil/water interface was imaged using a confocal microscope (figure 8), to gain insight into longer time scale biofilm formation. Bacteria cultured under oil spill 15 ACS Paragon Plus Environment

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conditions formed a complex-patterned biofilm at the oil/water interface (figures 8a-b). As seen in figure 8a, the film covers the entire interface, and has a thickness of approximately 25µm (figure 8b). Biofilms have a complex structure containing not only cells, but also proteins and extra-cellular polymeric substances synthesized by the bacteria. Here, we show the ability of bacteria cultured in oil spill conditions to adhere to the interface and form a complex structure, without thoroughly investigating the morphology of the entire biofilm.

Figure 8: Fluorescence confocal microscopy images of the oil/water interface with A.borkumensis in the aqueous phase, incubated for 24 hours. Bacteria were grown under oil spill conditions, a) top view and b) front view. Bacteria were grown under clean environment conditions, c) top view and d) front view. Scale bar is 100 µm.

Some bacteria cultured under clean environment conditions have adhered to the interface as individual cells, as seen in figure 8c,d. However, the cells have not formed a complex biofilm with significant thickness (see figure 7b) like those grown with hexadecane. These results further suggest that A.borkumensis needs previous exposure to hydrocarbons (i.e. oil spill) in order to form biofilms at the oil/water interface. In summary, the effects of the past exposure to hydrocarbons on A.borkumensis ability to adhere to oil/water interfaces through increased cell hydrophobicity are probed. Through interfacial tension, cell hydrophobicity and fluorescence imaging, it is clear that the exposure of 16 ACS Paragon Plus Environment

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A.borkumensis to hydrocarbons renders the bacterium hydrophobic and substantially increases its ability to adhere to oil/water interfaces. By culturing the bacteria with hydrocarbons as an energy source, cells adhere to the oil/water interface reducing interfacial tension and forming the early stages of a biofilm. Employing cell hydrophobicity and interfacial tension measurements we show that the A.borkumensis cells are hydrophobic, and that it is not a synthesized biosurfactant released into solution that affects interfacial tension. Considering other studies of A.borkumensis, which have focused on genetic factors and their effect on biosurfactant production and cell hydrophobicity17-18, 36, it is likely that the exposure to hydrocarbons allows for the synthesis of a biosurfactant that is bound to the bacterial cell which provides for cell hydrophobicity. The growth of A.borkumensis with sodium pyruvate as an energy source results in cells having no effect on interfacial tension and no reduction in cell hydrophobicity. These cells have limited ability to adhere to oil/water interfaces. This is the first study to provide insights into the influence of physiochemical effects of A.borkumensis cells on the bacterial cell ability to adhere to the oil/water interface. Results may help explain the physicochemical mechanisms by which A.borkumensis becomes the dominant strain in bacterial communities during oil spill situations. Acknowledgement This research was made possible by a grant from BP/The Gulf of Mexico Research Initiative. Disclosures The authors declare no competing financial interest. References


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Behavior of Marine Bacteria in Clean Environment and Oil-Spill

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