Engineered Clays as Sustainable Oil Dispersants in the Presence of

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Engineered clays as sustainable oil dispersants in the presence of model hydrocarbon degrading bacteria: the role of bacterial sequestration and biofilm formation Marzhana Omarova, Lauren T Swientoniewski, Igor Kevin Mkam Tsengam, Abhishek Panchal, Tianyi Yu, Diane A Blake, Yuri M. Lvov, Donghui Zhang, and Vijay John ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b02744 • Publication Date (Web): 07 Sep 2018 Downloaded from http://pubs.acs.org on September 13, 2018

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ACS Sustainable Chemistry & Engineering

Engineered clays as sustainable oil dispersants in the presence of model hydrocarbon degrading bacteria: the role of bacterial sequestration and biofilm formation. Marzhana Omarova1, Lauren T. Swientoniewski2, Igor Kevin Mkam Tsengam1, Abhishek Panchal3, Tianyi Yu4, Diane A. Blake2, Yuri M. Lvov3, Donghui Zhang4, Vijay John1*.

1

Department of Chemical and Biomolecular Engineering, Tulane University, 300 Lindy Boggs

Building, New Orleans, LA 70118, United States 2

Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA 70118, United

States 3

Institute for Micromanufacturing, Louisiana Tech University, 911 Hergot Avenue, Ruston, LA

71272, United States 4

Department of Chemistry, Louisiana State University, 207 Choppin Hall, Baton Rouge, LA

70803, United States

* Corresponding author: Phone: 504-865-5883. E-mail: [email protected].

1 ACS Paragon Plus Environment

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Abstract

Particle stabilized emulsions provide an environmentally friendly alternative to the chemical dispersion of oil in the event of a spill over water. Mineral clay particles present in abundance in marine environments adsorb at oil-water interfaces forming stable emulsion droplets. We describe the carbonization of these clays using the sustainable biopolymer, chitosan, to optimize wetting characteristics and generate extremely stable 150 μm clay-armored droplets using a model crude (Anadarko). In addition to such droplet stabilization, the work is comprehensive in elucidating the microbial processes involved in oil biodegradation. Using a model alkane degrading organism Alcanivorax borkumensis acclimatized on n-hexadecane, the colonization of oil droplets and the growth of biofilm is clearly visualized through high resolution cryo-scanning electron microscopy. The results indicate ubiquitous colonization of the organism on the surface and between platelets of the armored droplet with extensive biofilm formation bridging these particle stabilized droplets. Such oil-mineral aggregates stay buoyant although excess clay particles embedded in biofilm sediment out carrying small amounts of entrapped oil. Biodegradation is monitored through the loss of hexadecane doped into the crude oil and it is found that 90% of the hexadecane is lost over a 6-day experiment. These findings provide a comprehensive description of oil dispersion by mineral clay particles in the presence of marine oil-degrading bacterium, with the potential of developing technology for the mitigation of the environmental impacts of oil spills.

Keywords: mineral clay, Pickering emulsion, exopolymer, armored droplets, oil spill


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Introduction The Deepwater Horizon incident has led to intense research efforts on improvements in the mitigation of oil spills and the understanding of the processes related to oil biodegradation.1 Such mitigation technologies traditionally involve oil recovery by skimming2-3, dispersion of oil 4 and in-situ burning,5-6 with dispersion being the preferred technology for large spills in open water sufficiently far from sensitive coastlines. The fate of oil left exposed in the marine environment involves weathering, photooxidation and biodegradation by marine organisms.7-8 Dispersed oil could in principle, become increasingly susceptible to biodegradation because the oil is in the water column allowing an increased accessibility to the degrading bacteria. 9-11 An example of a specific hydrocarbon degrading bacterial strain is Alcanivorax borkumensis, first isolated and described by Yakimov et al.12 and used as the model organism in this work. Alcanivorax borkumensis is a rod-shaped gammaproteobacterium that belongs to the genus Alcanivorax; it utilizes n-alkanes as the carbon source and is known to produce biosurfactants and biofilm.13-14 The growth of A.borkumensis is highly dependent on the nutrients available, specifically nitrogen and phosphorus.15 The Alcanivorax species of hydrocarbonoclastic bacteria has been reported to dominate in the microcosm of hydrocarbon-degrading bacteria during oil spills16, and is thus an attractive model organism for studying oil biodegradation and interactions between bacteria, oil, suspended minerals and dispersants. Much effort has been devoted to investigating the behavior of Alcanivorax borkumensis in environments where various parameters, such as the chain length of available alkanes17, availability of nutrients15, 18-19 and the presence of surfactants20-21, are controlled in order to study this microorganism. Recent work by Bookstaver et al. 18, 20 reports that surfactants inhibit A.borkumensis growth rates and indicates the use of nitrogen supplementation to enhance growth. Pioneering work by Abbasi et al.22 has shown



bacterial colonization at the hexadecane-water interface in the presence of particles of carbon black and the presence of biofilm. In the marine environment, suspended sediment particles are always present, particularly in estuarine environments.23-24 In the presence of oil, these particles attach to the oil-water interface and are referred to as oil-mineral aggregates.23-25 Such sediment particles stabilize oil-in-water emulsions in the marine environment formed from the agitation of waves on the surface of oil spills, and are a manifestation of the phenomenon of particle-stabilized emulsions or Pickering emulsions.25 The free energy of detachment of amphiphilic particles at the oil-water interface is large, leading to a strong attachment of the particles at the interface and the formation of emulsion droplets.26-28 Particle-stabilized emulsions also known as Pickering emulsions have been extensively studied for their relevance as a surfactant-free method of emulsion formation where droplet coalescence is impeded by steric constraints.29-32 A major method of oil spill remediation is the use of chemical dispersants to break surface oil to droplets that can be dispersed over a wide volume of the marine environment and subsequently be consumed through biodegrading organisms. Because of concerns with the use of surfactants and solvents in the formulation of dispersants33, it would therefore be beneficial to develop surfactant-free approaches to the dispersion of oil. The use of naturally occurring particles that are essentially benign to marine organisms and that can be engineered to break oil into stable droplets with wave action is therefore a viable approach to oil spill remediation. While there have been several examples of using particle stabilized emulsions34-35, there is little in the literature that describes aspects of bacterial adhesion to such particle-stabilized interfaces and to oilbiodegradation of such particle-covered droplets.


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Our approach in this paper is to modify a naturally occurring clay, kaolinite, to stabilize the oil water interface and generate stable oil-in-water emulsions. We address questions regarding the exposure of these stabilized oil droplets to a model marine organism and the generation of extracellular polymeric substances (EPS) that will additionally stabilize these droplets. We also seek to understand sequestration of the organisms on the particle stabilized interface, and the role of particle stabilization and EPS formation in the eventual biodegradation of the oil.34-37 Of specific focus is the characterization of interfacial aspects at the nano and microscale with a view to extrapolate these findings to the fate of dispersed oil. The system we have used is that of kaolinite to stabilize oil-in-water emulsions, and the behavior of a model organism, Alcanivorax borkumensis, in attaching to such interfaces, forming EPS as part of the biofilm and gradually degrading the oil. Kaolinite is a naturally occurring aluminosilicate composed of tetrahedra and octahedra layers packed at a ratio of 1:1. 38 The essentially flat sheet morphology of kaolinite allows formation of stable oil-in-water emulsions and earlier pioneering studies have described such emulsions as being made up of armored droplets39. Kaolinite is an abundant mineral in Gulf of Mexico sediments40 and is an appropriate model clay. The use of cryo-scanning electron microscopy leads to an understanding of bacterial EPS formation and bacterial sequestration in the presence of particle stabilized emulsions and allows nanoscale characterizations relevant to the understanding of biodegradation. In addition to the use of natural kaolinite, we also examine the use of engineered kaolinite with an environmentally benign carbon coating to optimize droplet characteristics through modifications of the particle wetting characteristics. Thus, we seek to provide a comprehensive view of droplet generation, bacterial colonization, biofilm formation and degradation in these systems with visualization at the nanoscale being integral to the understanding of the underlying phenomena.



Materials and Methods Materials. Kaolinite, medium molecular weight (190-310 kDa) chitosan, sodium pyruvate and resazurin sodium salt were obtained from Sigma-Aldrich and used as received. N-Hexadecane was obtained from Sigma Aldrich and was filtered twice through a 0.22 µm syringe filter (MillexGV, Millipore) before use. Deionized (DI) water generated by an ELGA reverse osmosis water purification system (MEDICA 15BP) with a resistance of 18.2 MΩ·cm was used in all experiments. Anadarko crude oil was obtained from the Bureau of Safety and Environmental Enforcement (BSEE) facilities at Ohmsett, New Jersey. Anadarko crude oil was filtered twice through an 0.22 µm syringe filter before use. ONR7a medium was used to simulate the composition of seawater41 and prepared according to the published protocol.42 Fluorescent assay plates (96-well, black, flat bottom) were a product of Costar. Alcanivorax borkumensis (ATTC-700651TM) was obtained from the American Type Culture Collection (Manassas, VA). Freeze-dried cultures were reconstituted according to ATCC instructions, using Difco Marine Broth and agar obtained from Thermo-Fisher (Fairlawn, NJ). For experiments, bacterial cultures were transferred to synthetic ONR7a medium supplemented with 1% pyruvate, hexadecane or Anadarko crude oil. Carbonization of kaolinite. Carbonized kaolinite was prepared as reported earlier43. Briefly, a 2% suspension of kaolinite in deionized water was mixed with a specific volume of 0.5% solution of chitosan in 0.2M acetic acid. The chitosan to kaolinite weight ratios were 0.005, 0.01, 0.025, 0.05. The pH of the mixture was then adjusted to 3.7 by 0.2M acetic acid and the mixture was stirred for 24 hours. NaOH (1M) was added to increase the pH to 7 after which the mixture was centrifuged and washed three times with deionized water. The solid was dried and pyrolyzed at 700oC for one hour under nitrogen.


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Carbonized kaolinite characterization. The obtained kaolinite powder was pressed into 13 mm in diameter pellets in a stainless steel evacuable die (International Crystal Laboratories) by a hydraulic press (Carver Laboratory Press, Model C) under 2 tons of load acting on the 13mm die plunger. Compressed disks were immersed into hexadecane phase in a rectangular quartz cell, water phase was injected from a 21 gauge needle onto the pelleted material. Three-phase contact angle was measured by a standard Rame-hart goniometer equipped with DROPimage Advanced Software at room temperature. Measurement of oil-water interfacial tension. Hexadecane-salt water interfacial tension was measured by the pendant drop method using a standard goniometer (Rame-Hart, model 250). 15 μl drop of saline water containing 0.05% of kaolinite particles was injected into 5 ml of hexadecane outside phase. Emulsion preparation and characterization. Emulsions were prepared by mixing crude oil with particle suspensions containing 0.25% w/v of kaolinite in ONR7a medium. The oil phase to aqueous suspension v/v ratio was 1:30. The aliquots of emulsion cream were used for emulsion droplet characterization by Micro Core Zeiss microscope in bright field. The average droplet diameter of the emulsions (at least 300 droplets per sample) was estimated by ImageJ software. Hitachi S-4800 field emission Scanning Electron Microscope with operating voltage of 3 kV was used to obtain cryogenic SEM images of emulsions. Samples were imaged at a distance of 9 mm. Samples were loaded into rivets mounted onto the cryo-SEM sample holder. The samples were then plunged into slushed liquid nitrogen to freeze the sample. This was followed by fracturing at −130 °C using a flat-edge cold knife and sublimation of the solvent at −95 °C for 15 min to etch the sample. The temperature was lowered back to -130°C and the sample was then sputtered with a gold–palladium composite at 10 mA for 132 s before imaging.



Bacterial growth experiments. A liquid culture of A. borkumensis was grown on hexadecane (1% v/v) in ONR7a synthetic medium. ONR7a synthetic medium is commonly used as a seawater substitute. The ionic composition of ONR7a mimics that of seawater,41 as it is composed of inorganic salts.42 At the same time, the nutritional requirements for the bacteria can be met with ONR7a by the presence of ammonium chloride, iron chloride and disodium phosphate. Nitrogen and phosphorus are known to be especially important nutrients for A.borkumensis. ONR7a is formulated without a carbon source and uses hydrocarbons from oil as a carbon source for bacterial growth.42 Thus, in all the experiments, the growth of bacteria occurs due to the metabolism of alkanes from crude oil, while the nutrients facilitating metabolism, especially nitrogen and phosphorus, are supplemented by the ONR7a medium. When the absorbance of the inoculum (measured at 600nm) reached 0.5, indicating that the culture has entered exponential growth phase, the culture was washed by centrifugation with ONR7a medium to get rid of traces of hexadecane and used for experiments. This suspension was used as an inoculum at a v/v ratio of 1:100 in all experiments. To monitor bacterial growth, A. borkumensis inoculum was introduced into 3 ml of ONR7a medium in 10 ml culture tubes (17 mm*100 mm), Anadarko crude oil was added at 1:100 v/v ratio and the tubes were kept on a shaker at 150 rpm at 30oC. The samples with carbonized kaolinite contained 0.75wt% of particles in ONR7a medium. Every sample was duplicated for every time point to be analyzed. Bacterial cell growth was monitored by resazurin assay.44 Aliquots (0.4 ml) of collected samples were reacted with 40 μl of resazurin stock solution (0.1 mg/mL in water). Resazurin conversion was allowed to proceed for 2 hours, after which samples were filtered through 0.22 nm sterile syringe filters. The filtered solutions (200 μl) were serially diluted with ONR7a into black 96-well plates and analyzed for fluorescence (excitation 540/35nm/emission, 600/40nm).


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Sedimentation monitoring. Glass vials (40 ml, 28*95 mm) with perforations in the cap were used. ONR7a medium (30 ml) amended with 0.3 ml of Anadarko crude oil was used for cell growth. For particle stabilized oil, the emulsion was prepared beforehand following the emulsion preparation procedure described earlier. The stir bars were placed in the vials before oil was added. Sterile 9-inch glass Pasteur pipettes were inserted through the perforations in the caps and were placed in the medium with 0.1 ml of inoculum before oil was added. Pasteur pipettes acted as tubes through which sediment was collected without disturbing the top oil layer with biofilm. All samples were prepared in triplicate and corresponding controls had no inoculum initially present. The vials were stirred for the duration of the experiment in an incubator at 30oC. To quantify the amount of oil in the precipitate the sediment was collected through the Pasteur pipettes introduced in the beginning of the experiment. The oil in the collected sediment was recovered by extracting it into dichloromethane. Absorbance measurements (Shimadzu UV-1700 PharmaSpec) in the range 340-400 nm were used to quantify the amount of oil present in the sediment. Average absorbance at wavelengths of 340, 370 and 400 nm was used for oil quantification. Oil content in the sediment was normalized against oil incubated for the same period of time in similar conditions with no bacterial culture and no particles. Biodegradation measurements. The experimental setup was analogous to the one used for growth curve monitoring. Anadarko crude oil, a light crude, was doped with anthracene (3 mg/mL) as the internal standard, and hexadecane (5 mg/ml) prior to addition to the bacterial culture. We note that hexadecane and anthracene are mutually insoluble but are both soluble in the crude oil. At specific time periods, aliquots of the samples were taken to assess biodegradation. The oil fraction was extracted using a dichloromethane/hexane mixture (1:1 v/v) and the extracts were used to quantify biodegradation of n-hexadecane in Anadarko crude oil by GC-FID (Agilent



7820A, HP-5 column). The program used was as follows: start at 50oC, hold for 2 min, temperature increase was 10oC/min until the temperature reached 280oC. The detector temperature was kept at 300oC.


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Results and Discussion Engineered kaolinite to modify emulsion stability. As a natural thin sheet clay, kaolinite is efficient at covering oil-water interfaces and has been studied in the literature as forming armored oil-in-water emulsions.39 Average size distributions for the kaolinite particles used in this work are given in Supporting Information (S1S2). Disc shaped particles are significantly more effective than sphere and rod shaped particles in stabilizing emulsions, and in recent years graphene oxide platelets have been shown to stabilize such droplets.45 Our emphasis on kaolinite here, is the relevance to the natural clay found in marine environments and the abundance of the mineral for oil spill remediation. The Supporting Information section S3 details measurements of the carbon content through thermogravimetric analysis. In all succeeding experiments, the engineered kaolinite with a contact angle of 890 was used. Figure 1 illustrates the size distributions and stability characteristics for droplets stabilized with unmodified and carbonized kaolinite. We clearly observe a significantly smaller particle size and a tighter distribution. Droplets created by unmodified kaolinite have a diameter of 300±150 μm while droplets created with carbonized kaolinite have a diameter of 150±50 μm. Droplets formed with carbonized kaolinite also show an enhanced stability over the four-week time period of size characterization. The armoring of crude oil droplets with kaolinite sheets can be observed clearly using cryoSEM. Thus, Figure 2a is a low-resolution image showing multiple droplets over the field of imaging. We focus on a single intact droplet, and with increasing magnification (Figures 2b, 2c and 2d) it is clear that much of the droplet is covered with kaolinite. Important and relevant work by Creighton and coworkers45 using thin sheets of graphene oxide showed full coverage of the interface and tight tiling inhibiting evaporation of a light hydrocarbon (hexane). While we find 11 ACS Paragon Plus Environment


almost full coverage with the thicker sheets of kaolinite, the natural clay is not as pliable as graphene oxide and the tiling is not perfect. As a result, we clearly observe crevices between the sheets on Figure 3. The fractured sample on Figure 3 illustrates face-on attachment of kaolinite sheets at the oil-water interface and their tiling with crevices in between sheets.

Figure 1. Emulsion droplet size distributions and optical images: (a, c) Anadarko crude oil stabilized by kaolinite, (b,d) Anadarko crude oil droplets stabilized by carbonized kaolinite. Emulsions were prepared with 2.5% w/v particle suspension in ONR7a medium. Oil to aqueous suspension volume ratio: 1:30.


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Figure 2. Cryo-SEM images of kaolinite sheets on Anadarko crude oil droplet show coverage of oil surface by particles. (a) Low magnification view on the frozen emulsion aliquot showing several oil droplets in the continuous water phase. (b) Focusing on one oil droplet from (a) shows a roughened oil surface. (c) Closer inspection of the droplets surface from (b) shows that the surface is covered with particles, but there are no protruding edges indicating side-on coating of the oil surface by kaolinite particles. (d) High magnification image of the surface from (c) shows outlines of kaolinite sheets.



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300 μm

20 μm

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kaolinite sheets

kaolinite sheets

crevices crevices 10 μm

1 μm

Figure 3. Cryo-SEM of a fractured Anadarko crude oil droplet showing sheets attached faceon to the oil-water interface. (a) Low magnification view on the frozen emulsion droplet. (b) Focusing on one oil droplet from (a) shows particles attached to the surface of oil. Smooth oil surface visible on the image is the result of removal of particles upon fracturing the sample to reveal droplets. (c) Kaolinite particles can be seen attached side-on onto the oil surface, revealing some crevices between the sheets. (d) Additional image of a sample showing tiling of kaolinite sheets with crevices allowing direct access to oil.


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Alcanivorax borkumensis colonization of kaolinite stabilized crude oil droplets. An important aspect of particle stabilized emulsions is the possibility that the particles will serve as anchoring sites for oil-degrading bacteria to proliferate and consume oil. Our work has therefore focused on the model organism A.borkumensis to try to understand the colonization on kaolinite stabilized emulsions. Figure 4a is a cryo-TEM of the gram negative rod-shaped aerobic A.borkumensis showing a characteristic outer membrane (OM) and an inner cytoplasmic membrane (CM)12. The two membranes constitute a 30 nm cell wall. The two cells shown in Figure 4a illustrate a variation in the aspect ratio from 3.8 for cell A to 2.8 for cell B, and indicate a roughness in the outer membrane. The dense patch observed in both cells is attributed to an electron dense polyphosphate inclusion body12 serving as an intracellular storage vesicle. We note that all cells imaged in our samples have a single such dense patch close to the end of the major axis. The inset shows further details of the cell wall morphology. Figure 4b shows the kinetics of resorufin production (the black curve is for growth in the presence of carbonized kaolinite, the red curve shows growth in the system without particles), a measure of the viable cell density in culture. In this assay the nonfluorescent resazurin salt is reduced to the highly fluorescent resorufin through cell respiration thus leading to an assessment of viable cell growth.44 We note that the creation of emulsion droplets through particle armor at the interface does not hinder bacterial growth. The growth of the cells in experimental conditions rapidly reaches a maximum concentration, beyond which the number of cells begins to decline possibly due to the built-up of metabolic by-products in the growth medium (Figure 4b). Upon closer inspection of the sample it was noticed that the pH of the culture dropped significantly after a high density of cells was reached. The acidification was thus determined to be the cause of the sudden



Figure 4. (a) Cryo-TEM of A.borkumensis shows rod shaped cells with a thick cell membrane and polyphosphate granule. Scale of the inset is 200 nm. (b) Growth of A.borkumensis in ONR7a medium as monitored by rezasurin assay, red trace shows growth in the system with no particles, black trace shows growth in the presence of carbonized kaolinite. decline in the bacterial population. In the confined volumes used in laboratory experiments, we found a decrease in pH from 7.6 to 6. Similar observations on A.borkumensis isolates46-48 using ONR7a media indicate that the pH decline occurs due to respiration and extracellular chemical compounds such as anionic glycolipid biosurfactants generated through enhanced metabolism. 16 ACS Paragon Plus Environment

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The remarkable aspect of the growth curves both without and with particles in the system is the possible adaptation of the species to a lowered pH and a reinvigoration of the growth. We note that in marine environments, such pH changes will not occur, and our main finding here is that viable cell growth is unimpeded by the presence of particles at the oil-water interface.

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150

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100 μm Figure 5. (a) Carbonized kaolinite stabilized emulsion droplets covered with A.borkumensis biofilm, (b) emulsion droplets connected due to bridging by the biofilm, (c) Anadarko crude oil droplets stabilized by carbonized kaolinite with A.borkumensis and their stability over four weeks. Emulsions were prepared with 2.5% w/v particle suspension in ONR7a medium. Oil to aqueous suspension volume ratio: 1:30. Figure 5a is an optical micrograph of an armored droplet showing biofilm tendrils emanating from the surface. Figure 5b indicates droplets adhered to each other by the bridging effect of bacterial biofilm as indicated by the arrows of Figure 5b. Marine microbial EPS is associated with a high degree of stickiness49 and the exopolymer framework grown on the surface



of particle covered oil droplets may serve to bridge droplets. However, as Figure 5c indicates, the armored droplets even with bridging biofilm are extremely stable against coalescence. Many of the droplets are aspherical as a consequence of interfacial rigidity brought about by the flat sheet morphology of the clays. In the complex system containing bacterial cells, biofilm and flat sheet clays, the visual observation of biofilm growth from the kaolinite covered oil-water droplet interface implies that cells may not simply be in the aqueous phase surrounding the droplets but may be attached to the surface of the clays. To understand such bacterial attachment to the clay surface, we conducted cryo-SEM imaging of the droplets. A.borkumensis has recently been reported to be able to adhere to other solid particle stabilized interfaces, such as halloysite stabilized oil droplets50 and carbon black stabilized oil droplets22. In recent pioneering work by Abbasi and coworkers22, it was found that A.borkumensis adheres to n-hexadecane droplets stabilized by carbon black nanoparticles which form fractal aggregates at the oil-water interface. Our work is complementary in the aspects that we use the natural and sustainable flat sheet kaolinite to armor the droplets and have extended the concept of particle and biofilm stabilized oil-water interfaces to realistic crude oil systems. Our work specifically involves the use of high resolution imaging to understand the characteristics of cell sequestration and exopolymer formation in this system (Figure 6) and we relate these characteristics to cell growth and biodegradation. Figure 6 provides a clear evidence of bacterial EPS formed on the surface of kaolinite covered oil droplets. The fracture of the sample droplets and subsequent sublimation of vitrified water, reveals the oil surface and gives an outline of the layer covering the oil surface (Figure 6a). The “crust” of one representative droplet (Figure 6b) is illustrated to contain kaolinite particles lying face-on along the interface (indicated by arrows) and bacterial cells attached to the particles by exopolymer (Figure 6c). We note that the crust is


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identified by the thin gap (arrows on Figure 6b) formed during rapid cooling where the contrasting differences in volumetric shrinkage between that of water and oil due to the difference in the coefficient of thermal expansion of the two fluids.35 The rod-shaped cells labeled in Figure 6d and 6e are attached to the oil surface by a dense weblike EPS fiber mat that also interconnects the cells. Thus, the microstructure of the biofilm resolved by cryo-SEM reveals that biofilm is made of bacterial cells immobilized by exopolymer that entirely covers that surface of cells and bridges individual cells together with the particles present at the oil-water interface. A.borkumensis thus appears to be able to colonize the surface of oil that has solid particles adsorbed onto it. In Figures 6d and 6e, we see cells that are adjacent to the edge of kaolinite sheets, and perhaps directly at the oil-water interface. In Figure 6f, we see cells that are clearly at the top of kaolinite sheets. CryoSEM additionally demonstrates that the exopolymer covers the entire cell surface (Figures 6d-e), forming “capsules” around cells, as is described to occur in biofilms forming in high salinity conditions51. Such capsules serve as protection against desiccation and osmotic stress.51 The estimate of the amount of biofilm attached onto armored droplets is performed by TGA, details of which can be found in Supporting Information S6. Analysis of a dried emulsion sample with biofilm and without biofilm shows 36.3 wt% and 33.3 wt% loss, respectively, which corresponds to 3 wt% of the biofilm attached to kaolinite particles. Thus, approximately 30 mg of biofilm is generated per 1 g of dried crude oil-kaolinite clay mixture. This suggests that although exopolymer fibers and cells appear to be present extensively, the additional weight due to solids being generated by microbial activity is relatively small.



Figure 6. Cryo-SEM of carbonized kaolinite droplets colonized by A.borkumensis. (a) Low magnification view of the emulsion aliquot shows frozen oil droplets in continuous water phase. (b) Focusing on one droplet from (a), arrows indicate gaps formed as a result of different volume shrinkage of oil and water during freezing. (c) The “crust” of the droplet shows bacterial cells and kaolinite particles. (d) Closer view of (c) shows individual cells and kaolinite sheets at the oil surface. (e) Bacterial cells produce exopolymer at the surface of oil droplets. (f) Additional cryo-SEM image of bacterial cells attached onto the particles that form an armor around oil droplets.


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Sedimentation in particle stabilized emulsions with bacterial growth The presence of extensive EPS produced in a short amount of time is an important characteristic of this microorganism. The possibility of its contribution to the transport of oil in the event of an oil spill in the form of oil-particle aggregates (OPA)52-53 has been considered through the experiments shown in Figure 7. Vials containing ONR7a medium and bacterial culture amended with crude oil stabilized by the carbonized kaolinite were kept gently stirred at 30 oC. A Pasteur pipette tip was introduced through the vial lid at the beginning of the experiment both to keep the system under aerobic conditions while minimizing oil evaporation, and to collect the sediment at the end of three weeks with minimal disruption of the oil layer. After three weeks of incubation a small amount of sediment was observed (Figure 7a). Control experiments without bacterial growth and biofilm formation do not show any evidence of such sedimentation. Optical micrographs of the sediment indicate gauze-like aggregates (Figures 7b and 7c) primarily containing biofilm strands with kaolinite. Figure 7c indicates small oil droplets (1-10 μm) trapped in the sediment. It is important to note that we do not see any of the emulsion droplets in the sediment. Based on the stacks observed through cryo-SEM (Figure 6), we can approximate the armored droplets to have a 500 nm thick shell of particles (specific gravity of 2.6). The armored oil droplets are, on average, of 150 μm in diameter (specific gravity of 0.8) with an effective specific gravity of 0.82, thus remaining buoyant. Cryo-SEM (Figure 7d) shows that the carbonized kaolinite particles are covered and interconnected by microbial exopolymer. Our explanation for such sedimentation is that bridging exopolymer fibers may compact loosely attached kaolinite sheets leading to the formation of negatively buoyant aggregates. On the other hand, emulsion droplets created by carbonized kaolinite remain buoyant and do not appear in the sediment. Extraction of the oil in the sediment shows that about 6% of oil initially introduced 21 ACS Paragon Plus Environment


Figure 7. Carbonized kaolinite and A.borkumensis biofilm induced precipitation: (a) photos of vials showing a precipitate formed of excess particles bridged by biofilm, (b) optical micrograph shows a large dense aggregate of particles and biofilm, (c) optical micrograph shows small droplets trapped in the biofilm-clay aggregate, (d) cryo-SEM images demonstrate exopolymer fibers attached to particles. is removed in the sediment either through adsorption to the carbonized kaolinite in the sediment or through entrapment in the biofilm. Bacterial cells are also trapped in the biofilm as observed in the cryo-SEMs of Figure 7d. Simple addition of clays therefore sink only a small amount of oil if the oil is dispersed and is in the form of particle stabilized emulsions. 22 ACS Paragon Plus Environment

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Biodegradation in the system containing kaolinite stabilized crude oil droplets. In analyzing characteristics of biodegradation, we used Anadarko crude spiked with both hexadecane (susceptible to biodegradation by A.borkumensis) and anthracene (not biodegraded by A.borkumensis and therefore used as an internal standard). The ratio of the hexadecane peak to anthracene is therefore used as a measure of biodegradation. We note that n-hexadecane and anthracene are mutually insoluble while both compounds are soluble in the crude oil thus facilitating analysis of two specific components in a single phase multicomponent mixture. As Figure 8 indicates, hexadecane in the emulsion system stabilized by carbonized kaolinite proceeds at approximately the same rate as in the non-emulsified system with over 50% degradation in the first two days of the experiment. Pseudo first order kinetic rate constants calculated from this data are in the range of 0.58 d-1 and the half-life is 1.2 days. Similar aliphatic degradation rates with A.borkumensis have been observed by Overholt and coworkers21 and by Nekouei and Nekouei.54 The result that biodegradation rates are comparable for oil in a flat layer and oil that is in the form of droplets and thus of higher interfacial area, is somewhat counter-intuitive to the concept that higher surface areas promote biodegradation rates. A rough calculation based on droplet sizes of 150 μm indicates that for the extremely small amount of oil (30 μl) used in the experiment, the ratio of the droplet interfacial area (12 cm2) to the flat surface interfacial area (2.3 cm2 for the culture tubes with the internal diameter of 17 mm) is about 5/1. In principle therefore, there is 5 times as much interfacial area available for bacteria to colonize. On the other hand, the growth curves of Figure 4b also support the observation that the growth curves do not depend on the oilwater interfacial area. This is possible if the organism consumes hydrocarbon directly dissolved in the bulk solution or within micelles formed by biosurfactant. In other words, the equilibrium partitioning between the bulk hydrocarbon phase and the hydrocarbon in water is quickly



established and the concentration of dissolved hydrocarbon available for bacterial consumption is the same regardless of whether the hydrocarbon bulk phase has a flat oil-water interface or is in the form of droplets with an increased oil-water interface. The rate limiting step in biodegradation is therefore the bacterial consumption of dissolved oil. We assume that there is a quick reestablishment of equilibrium between the bulk hydrocarbon and dissolved hydrocarbon to maintain the equilibrium and reach saturation in the water phase (0.0004 mg/L at 250C).55

100 90

Hexadecane remaining (%)

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1

2

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Time (days)

Figure 8. Degradation of hexadecane spiked into crude oil by A.borkumensis over 6 days. Crude oil was used with no particles added (black trace) and stabilized by carbonized kaolinite (red trace).


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Conclusions These observations provide insights into the fate of oil spills, the attachment of particles at the oil water interface, the colonization of hydrocarbon degrading bacteria at these interfaces and the characteristics of biodegradation. We have shown that surface modification of natural kaolinite clay leads to the formation of emulsions with high stability and uniform droplet size of 150 μm. These emulsions are formed as a result of sheet-like carbonized kaolinite particles strongly adsorbing to the crude oil-water interface. Crude oil-in-seawater emulsions made by carbonized kaolinite clay becomes quickly colonized by alkane-degrading Alcanivorax borkumensis. The colonization of droplets by bacteria results in extensive exopolymer formation around and between the cells and on the clay sheets. The high-resolution imaging provided by cryo SEM provides direct visual observation of such colonization. The biofilm formed as a result of microbial colonization of oil surface connects particle covered droplets. Sinking aggregates are the consequence of exopolymer bridging with loose clay particles, but the armored oil droplets remain buoyant. Biodegradation of oil is not hindered by the presence of clay particles attaching to droplets and the effective surface area has little effect on the rates of biodegradation. We propose that biodegradation occurs through the consumption of hydrocarbon solubilized in the bulk solution perhaps close to the oil-water interface, or within biosurfactant micelles. The conclusions drawn from this work indicate that the sinking of relatively fresh oil through particle attachment to the interface and the growth of biofilm is insignificant, and that degradation by A. borkumensis may be through the rate controlling step of consumption of the dissolved components of the oil with a more rapid partitioning of oil components from the bulk to the aqueous phase. We therefore propose that particle stabilized emulsion droplets are an alternate approach to chemical dispersion where the impacts of the use of dispersants on hydrocarbon degrading 25 ACS Paragon Plus Environment


marine bacteria20, 33 are minimized. The breakup of oil into droplets prevents the formation of migrating oil slicks. Although the particle stabilized emulsion droplets are buoyant and stay on the surface, they could be dispersed by ocean waves and currents and thus not reach sensitive coastal areas as a slick. The armoring of such droplets could mitigate direct adsorption of oil on plants in coastal marshlands if the oil reaches shorelines. Additionally, for small spills, it may be easier to skim solid stabilized oil droplets rather than highly thinned surface oil slicks. These are technologically relevant aspects that remain to be evaluated.


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Supporting Information SEM images of kaolinite and carbonized kaolinite particles (S1); characterization of the particle size distributions for kaolinite and carbonized kaolinite (S2); TGA analysis for determination of the carbon content (S3); contact angle measurements for the wettability characterization of kaolinite and carbonized kaolinite (S4); characterization of the emulsion formed by native kaolinite and biofilm (S5), TGA analysis of carbonized kaolinite and biofilm containing sample (S6); gas chromatograms for abiotic control and biodegraded samples (S7).



Acknowledgment Funding from the Gulf of Mexico Research Initiative is gratefully acknowledged. The dataset can be accessed through the Gulf of Mexico Research Initiative Information and Data Cooperative (GRIIDC): https://data.gulfresearchinitiative.org (doi: 10.7266/N7QC020J).


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Synopsis: The work shows that natural and engineered kaolinite clay minerals can be a sustainable and green method for oil dispersion and subsequent biodegradation



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Engineered Clays as Sustainable Oil Dispersants in the Presence of

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