Bacteria Interface Pickering Emulsions Stabilized by Self-assembled

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Bacteria Interface Pickering Emulsions Stabilized by Self-assembled Bacteria−Chitosan Network Pravit Wongkongkatep,† Khajohnpong Manopwisedjaroen,†,§ Perapon Tiposoth,‡ Somwit Archakunakorn,‡ Thunyarat Pongtharangkul,† Manop Suphantharika,† Kohsuke Honda,∥ Itaru Hamachi,⊥ and Jirarut Wongkongkatep*,†,§ †

Department of Biotechnology and ‡MDL, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400 Thailand, § Center of Excellence on Agricultural Biotechnology: (AG-BIO/PERDO−CHE), Bangkok, Thailand, ∥ Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan, ⊥ Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura Kyoto 615-8510 Japan S Supporting Information *

ABSTRACT: An oil-in-water Pickering emulsion stabilized by biobased material based on a bacteria−chitosan network (BCN) was developed for the first time in this study. The formation of self-assembled BCN was possible due to the electrostatic interaction between negatively charged bacterial cells and polycationic chitosan. The BCN was proven to stabilize the tetradecane/water interface, promoting formation of highly stable oil-in-water emulsion (o/w emulsion). We characterized and visualized the BCN stabilized o/w emulsions by scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM). Due to the sustainability and low environmental impact of chitosan, the BCN-based emulsions open up opportunities for the development of an environmental friendly new interface material as well as the novel type of microreactor utilizing bacterial cells network.

1. INTRODUCTION An emulsion is a system consisting of dispersed droplets of one immiscible liquid in another immiscible liquid. The system is stabilized by surfactants or emulsifiers.1 Since the original work of Ramsden2 and Pickering,3 solid colloidal particles have been proven to adsorb at the interfaces to form “Pickering emulsions”. Interest in Pickering emulsions has increased over the past 10 years, especially in health-related and cosmetics applications where the use of surfactants can cause adverse effects. Indeed, Pickering emulsions not only present good mechanical properties, but also produce good stability, thereby leading to a reduction in the use of nonenvironmentally friendly surfactants.4 The mechanism of emulsion stabilization by colloidal particles relies on their accumulation at the oil−water interface in a form of a densely packed layer, consequently preventing both emulsion flocculation and coalescence by a steric barrier.5,6 The extent of the steric barrier depends on how difficult it is to remove those particles from the interface, for example, the steric barrier is higher when most of the particles’ surfaces locate on the outer side of the oil emulsions.6 Therefore, the contact angle θ made by stabilizing colloidal particles at the water−oil contact line determines the particle location at the interface and the nature of the emulsion. Contact angles less than 90° indicate the hydrophilic property of the colloidal particles contributing to the formation of o/w emulsions, whereas the contact angles greater than 90° imply © 2012 American Chemical Society

the hydrophobic nature of the particles that favors formation of w/o emulsion.7,8 In addition, the energy of desorption per particle, predicted to be of the order of several thousand kT, is strictly related to the contact angle as long as contact angle is not close to 0° or 180°.6,9 This implies that once the particles are at the interface, they are adsorbed effectively and irreversibly. Therefore, one of the most important features of the particle-stabilized emulsions is that they are extremely stable to coalescence even when the emulsions are quite large.6,10 Most of the literatures dealing with Pickering emulsion concern various types of inorganic particles such as silica,11,12 graphene oxide sheet,13 clay,14 carbon nanotubes,15 latexes,11,16 and nanocrystals.17,18 In addition to various rigid particulate systems, Pickering emulsions can also be stabilized by soft material such as microfibrillated cellulose,19,20 microgels21,22 and botanical spore.23 However, the most natural materials are usually not surface active due to their lack of hydrophilicity/ hydrophobicity, as well as their great tendencies to aggregate. Methods involving hydrophobicity modifications or an addition of cosurfacting compounds are required to produce particles/ material suitable for Pickering stabilization.24 Such methods not only increase the use of hazardous chemicals but also make the Received: August 31, 2011 Revised: March 6, 2012 Published: March 23, 2012 5729

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(0.5 mL) of the preculture was transferred to a 500-mL Erlenmeyer flask containing 50 mL of fresh medium. This culture was grown with orbital shaking (200 rpm). Cells were harvested by centrifugation at 6953 × g for 5 min at room temperature, washed twice with a saline solution (0.85% sodium chloride). Finally, the washed cells were resuspended in 0.1 M sodium phosphate buffer solution (pH 6.8) and adjusted the optical density at 660 nm OD660 = 2, which is equivalent to 4.73 ± 0.55 × 108 cfu/mL. 2.3. BCN Stabilized Emulsion Preparation. Emulsions were prepared by combining of 0.03% w/v chitosan, dissolved in 0.03% acetic acid (0.3 mL) with the bacterial cell suspension (0.9 mL) prior to a vigorously shaking by hand with n-tetradecane (1.2 mL). Although simple, this method produced emulsions in a reproducible pattern, i.e., with the same final average diameter and size distribution. Stability of BCN stabilized emulsion was obtained by measuring the volume of the emulsion layer compared to total volume of the mixture over a period of 2 months as expressed in term of Emulsification Index (EI). 2.4. Zeta Potential Measurements. The ζ potentials were recorded on Nano Zetasizer ZS, Malvern Instrument at 25 °C. The bacterial cell suspensions were prepared in 0.1 M sodium phosphate buffer solution pH 6.8. BCN solutions were prepared by mixing 3 mL of bacterial cell suspension with 1 mL of 0.03% chitosan dissolved in 0.03% acetic acid aqueous solution. In the control experiment, 3 mL of bacterial cell suspension were analyzed without modification with chitosan. The ζ potential values were measured using Dynamic Light Scattering technique. Each value represents the mean of at least three independent experiments. 2.5. Contact Angle Measurements. A 50 mL of suspension of E. coli cells or BCN was filtered on a nylon membrane with mean pore size of 0.45 μm, 47 mm in diameter (Millipore, U.S.). The lawns were kept on a 1% agar plate at room temperature to prevent drying. The lawn was then immersed in n-tetradecane bath, and a 2-μL droplet of distilled water was placed gently on the cell lawn. Then the threephase contact angle (θ) between the water drop, bacterial lawn, and ntetradecane was recorded using a contact angle meter (Kyowa DMCE1, Japan). 2.6. Fluorescence Microscopy Analysis. Fluorescent imaging was carried out using epifluorescent light microscope (Olympus BX 51, Japan) and laser scanning confocal microscope (LSCM 510 Meta, Zeiss, Jena, Germany). A blue fluorescent 4,6-diamidino-2-phenylindole (DAPI) (Invitrogen, CA) at the final concentration of 2 μg/mL was added to the cell suspension and vigorously mixed. After incubation in a dark at room temperature for 15 min, cell pellets were collected, washed, and resuspended with 0.1 M sodium phosphate buffer (pH 6.8). Preparation of nile-red-stained ntetradecane was performed by mixing n-tetradecane with the nile red (Invitrogen, CA) at the final concentration of 0.5 μg/mL. The DAPIstained cell suspension (0.3 mL) was then mixed with 0.03% (w/v) chitosan dissolved in 0.03% acetic acid (0.1 mL). Then, n-tetradecane (0.4 mL) was then vigorously shaken with the DAPI-stained E. coli based BCN. The reaction mixture was allowed to stand in dark at room temperature for 24 h before fluorescent analysis. 2.7. Scanning Electron Microscopy. An observation was recorded using a scanning electron microscope (SEM, S-2500, Hitachi, Japan). A suspension of E. coli DH5α grown 15 h in NB was harvested, washed twice and then centrifuged at 6953 × g for 5 min. The pellet was first fixed with 2.5% (w/w) glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) at 4 °C for 2 h, washed three times, and second fixed with 1% Osmium tetroxide in 0.1 M cacodylate buffer (pH 7.2) at 4 °C for 1 h. The samples were dehydrated in a graded ethanol series and then coated with a platinum/palladium alloy. The BCN was prepared as mentioned above and imaged using the similar procedure. 2.8. Droplet Size Measurements. Because the BCN-stabilized emulsions provided a polydisperse emulsion with sizes ranged from 0.01 mm and sufficiently disperse to be observed, the size distribution was estimated using Microsoft Office Excel 2003. Images were recorded and the dimensions of about 500 droplets were measured by Olympus Digital Imaging Solution CellA cell Family imaging software (Olympus Soft Imaging Solution GMBH, Munster, Germany) so that the averaged particle diameter D(1,0) and the surface-averaged

process complicated and not suitable for large-scale production. Research efforts are being focused on the development of an environmentally friendly, biobased Pickering emulsion with simple preparation. Only a few studies have described stabilization by particles derived from renewable resources9,17−20,23 while bacteria-based Pickering emulsion has never been reported before. Bacteria are prokaryotic microorganisms, typically a few micrometers in length, ubiquitous in every habitat on earth. The bacterial cell is surrounded by negatively charged lipid membrane which encloses the contents of the cell and acts as a barrier to hold nutrients, protein, enzyme, and other essential components of the cytoplasm within the cell.25 An electrostatic interaction between the negatively charged bacterial cell surface and polycationic chitosan has been well studied.26,27 Chitosan [poly (β-(1→4)-2-amino 2-deoxy-D-glucose)] is the Ndeacetylated derivative of chitin, found naturally in the exoskeletons of insects shells of crustaceans and in fungal cell walls. Since chitosan is a positively charged linear polysaccharide at pH < 6.5, it is applied as a polyelectrolyte in numerous applications.28 Chitosan by itself was found to display only a weak surface activity due to its lack of large hydrophobic segments. However, at pH > 6 the free amino groups of chitosan become less protonated and the hydrophobic characteristic along the chitosan chain become stronger.28 Chemical modifications of chitosan could improve such surface activity by introducing hydrophobic substituents into its glucosidic groups,29 but the process requires tremendous effort. We envisioned that the strong binding affinity of polycationic chitosan toward the negatively charged cell surface would neutralize the surface charges of both bacterial cells and chitosan. Consequently, the hydrophobic properties will be promoted, resulting in a self-assembled bacteria-chitosan network (BCN) capable of stabilizing the novel type of bacteria-based o/w emulsion. In this work, Escherichia coli (E. coli) DH5α and other four bacterial strains were selected as model bacteria in order to demonstrate our unique bacteria-based Pickering emulsion regarding to their industrial importance as a genetic engineering host.25 By mixing with chitosan, a self-assembled BCN was formed through an electrostatic interaction between negatively charged bacterial cells and positively charged chitosan. An emulsion was formed by vigorously shaking an equal volume of n-tetradecane which was employed as a model of oil phase due to its low toxicity. The stability of the emulsion, droplet size, and location of BCN were investigated. In addition, effect of pH, buffer types, organic solvents, and species of bacteria as well as the bacterial cell viability were evaluated.

2. MATERIALS AND METHODS 2.1. Materials. Chitosan was purchased from Sigma-Aldrich (Product Number C3646) with deacetylation degree of 96% (LOT 068K00851). All other chemicals including n-tetradecane (ρ = 0.767 g/mL) were commercially available from Sigma-Aldrich at 99% or greater purity and used without further purification. 2.2. Microorganisms and Culture Conditions. Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 14579 were cultivated on nutrient broth (NB; Difco Laboratories, Detroit, MI, U.S.) at 37 °C for 24 h. Escherichia coli DH5α, were grown on Lysogenic Broth (LB) medium for 15 h. Lactobacillus sakei ATCC 15521 was cultured on MRS broth (MRS; Difco Laboratories, Detroit, MI, U.S.) at 37 °C for 24 h. Cells were inoculated to a test tube (16 × 150 mm) containing 5 mL of the medium and cultivated on a reciprocal shaker (200 rpm). An aliquot 5730

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Table 1. Characteristics of BCN Compared to Hydrophobic/Hydrophilic Bacteriab hydrophobic bacteria ζ potential (mV) contact angle (deg) a

hydrophilic bacteria

R. opacus B-4a

R. erythropolis PR4a

E. coli. JM109a

E. coli DH5α

BCN consisted of E. coli DH5α and chitosan

−1.67 ± 0.8 118 ± 2.1

−18.0 ± 0.6 132 ± 8.7

−15.9 ± 1.9 21.4 ± 0.1

−19.4 ± 0.9 15.8 ± 1.0

5.1 ± 0.1 54.5 ± 2.0

Data referred from Hamada et al., 2008.31 bData are presented as the average ±SD, which was derived from three individual experiments.

diameter D(3,2) defined by the following equations, could be estimated.

D(1, 0) =

D(3, 2) =

moderate contact angle of BCN demonstrated in our study is not limited to some specific species of hydrophobic bacteria which is quite rare in nature but can be applied to any species of hydrophilic bacteria which is ubiquitous in every habitat on earth. Scanning electron micrographs also provided a clear image of BCN self-assembled network compared to the nontreated E. coli (Figure 1). The nontreated E. coli cells

∑i nidi ∑i ni

(1)

∑i nidi3 ∑i nidi2

(2)

where ni is the total number of droplets with diameter di.

3. RESULTS AND DISCUSSION 3.1. Charactistics of BCN. In this study, the physiology of E. coli-based BCN was assessed in comparison with unmodified E. coli through ζ potential measurement.30 Because ζ potential is an indirect determination of the bacterial cell surface charge, which represents the level of ionized carboxylate and phosphoryl components on the bacterial cell surface, therefore bacterial cells always exhibit net negative ζ potential. In 0.1 M sodium phosphate buffer (pH 6.8), the surface charge of E. coli (−19.4 ± 0.9 mV) increased dramatically to 5.1 ± 0.1 mV in BCN system as summarized in Table 1. The ζ potential of E. coli DH5α measured in our study is in good correspondence with −15.9 ± 1.9 mV of E. coli JM109 or −18.0 ± 0.6 mV of Rhodococcus erythropolis PR4 reported previously.31 However, some specific strains of hydrophobic bacteria have been reported to show a smaller negative electrostatic charge including −1.67 ± 0.8 mV of Rhodococcus obacus B-4.31 The reduction of a net negative electrostatic charge on the outer cell surface after association with chitosan was considered as a dominant factor leading to a formation of bacterial interface o/ w emulsion. Contact angle measurement (CAM) was another useful technique for evaluating bacterial cell surface hydrophobicity.31 A lawn of bacterial cells was prepared on a nylon membrane by filtering 30 mL of cell suspension with or without 75 mg/L chitosan. The bacterial lawn was then immersed in ntetradecane, and a 2-μL droplet of distilled water was placed onto the lawn. The three-phase contact angle between the aqueous drop, bacterial lawn, and n-tetradecane were measured and presented in Table 1. The contact angle of E. coli without BCN formation was 15.8 ± 1.0°, which is in good agreement with 21.4 ± 0.1° of E. coli JM10931 and 15.5 ± 2.1° of Brevibacillus agri strain 1332 reported recently. However, the contact angle of the associated E. coli significantly increased to 54.5 ± 2.0° after being self-assembled with chitosan. The increase in contact angle of the BCN system indicates a successful surface coating of the bacterial cells with chitosan. The association between chitosan and bacterial cells causes a reduction in the free amino groups along the chitosan chain, thereby promoting the hydrophobic characteristic of the polymer. Although the contact angle of chitosan-modified E. coli was not as high as those of hydrophobic bacteria, for example, 118 ± 2.1° and 132 ± 8.7° reported for Rhodococcus obacus B-4 and Rhodococcus erythropolis PR4, respectively,31 the

Figure 1. SEM images of E. coli DH5α (A) and BCN obtained by mixing E. coli DH5α and chitosan at the final concentration of 75 mg/ L after heated at 80 °C for 15 min (B). Scale bars represent 3 μm.

were observed as rod-shaped cells with the length of about 2−3 μm distributed independently (Figure 1A), while cells in BCN formed a dense network with chitosan (Figure 1B). The increase in surface hydrophobicity and the reduction of cell surface charge after BCN formation were considered to be key mechanisms contributing to the formation of a bacterial interface Pickering emulsions. 3.2. BCN Stabilized Emulsion Formation. The aboveconfirmed prerequisite encouraged us to introduce an organic solvent to BCN suspension. First, 0.03% chitosan dissolved in 0.03% acetic acid (0.3 mL) was combined with the E. coli cell suspension (OD660 = 2 in 0.1 M sodium phosphate buffer pH 6.8, 0.9 mL). Then an equal volume of n-tetradecane (1.2 mL) was added prior to a vigorous shaking, consequently a stable layer of emulsion was obtained (Figure 2A, bottle d). The formation of emulsion was not observed in a mixture of chitosan aqueous solution and n-tetradecane (Figure 2A, bottle b) or a mixture of nontreated E. coli and n-tetradecane (bottle c), indicating that self-assembled BCN was essential for stabilization of the emulsion as illustrated in Figure 2D. It could be explained that when the shaking stopped, the partially unprotected droplets coalesce, thus reducing the total area of oil/water interface. Since the BCNs are irreversibly adsorbed, the coalescence process is prohibited as long as the oil/water interface is sufficiently covered. The resulting emulsions exhibit a drop diameter that is controlled by degree of coverage of BCNs. Although the bacterial cells is fragile and soft and have a limited lifetime compared to the nanocrystal or other inorganic 5731

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Figure 2. (A) phase separation observed when n-tetradecane was shaken with 0.1 M sodium phosphate buffer (pH 6.8) without (bottle a) and with 75 mg/L chitosan (bottle b), macroscopic view of n-tetradecane shaken with E. coli suspended in 0.1 M sodium phosphate buffer (pH 6.8) without (bottle c) and with 75 mg/L chitosan (bottle d). Microscopic view of the bacteria interface Pickering emulsion observed in bright field (B) and red fluorescent field (C) after staining n-tetradecane with nile red. Scale bars represent 500 μm. (D) Schematic illustration of the bacteria interface Pickering emulsion stabilized by self-assembled BCN. Dimensions of the chitosan and bacteria are for clarification purpose. The emulsion can be produced by either partial or full coverage of BCN.

Figure 3. Confocal images of emulsion stabilized by self-assembled BCN consisting of chitosan and E. coli DH5α stained with fluorescent DAPI. The bacterial cells were observed mainly at the interface of n-tetradecane droplet in bright field (A), blue fluorescent field at z = 10 (B), 20 (C), 30 (D), and 40 μm (E). Scale bars represent 100 μm.

particle, the lifetime of our bacterial interface emulsion ranges from days to months, comparable to the lifetime of the o/w emulsions stabilized by rigid nanocrystal particles17,18 and emulsion stabilized by soft microgel.21,22 The results emphasized the high efficiency of the BCN in stabilizing an emulsion. 3.3. BCN Stabilized Emulsion Characterization. In order to classify the structure of the emulsion system, ntetradecane was stained with nile red32 (1.5 μg/mL, red fluorescence) before mixing with BCN prepared as mentioned previously. Red fluorescence from nile red was imaged inside the spherical droplets (Figure 2C), indicating that an o/w emulsion was formed in the presence of moderate hydrophobic BCN (contact angle θ = 54°, Table 1). This result is in good agreement with previous findings that particles with θ < 90° yield the o/w emulsions while those with θ > 90° can stabilized w/o emulsions.7,8 Digital microscopy, particularly laser scanning confocal microscopy (LSCM), offers an opportunity to observe microbial distribution in 3D system. LSCM is generally used to produce a more accurate 2D image compared with epifluorescent microscopy since the blur originating from unfocused light is eliminated.34 Staining E. coli with DAPI32 (2 μg/mL, blue fluorescence) prior to a modification with chitosan allowed us to localize the position of bacteria in the emulsion. The cross sections of the droplet at z = 10, 20, 30, and 40 μm

were observed under blue fluorescent field. Because the emulsion was mounted on the concave slide glass in semidried state, the weight of the droplet and the soft property of bacteria-chitosan shell turned the spherical shape of the emulsion into a sessile drop in which the horizontal diameter is 114 μm while the vertical diameter was recorded at about 40 μm (Figure 3E). The round-shaped bright fluorescence of DAPI was observed only at the perimeter of the emulsion while the inside remained dark (Figure 3B−E). It has been clarified that the self-assembled BCN was located at the interface of the o/w emulsion resulting in a bacterial interface emulsion. BCN, imaged clearly using SEM (Figure 1B), showed almost no surface/interfacial activity (data not shown) similar to chitosan29,35 and E. coli bacterial cells,31 therefore it may not be able to emulsify the oil emulsion by lowering the surface/ interfacial tension. One possible explanation is that the BCN would interrupt the coalescence of oil droplets and stabilize the emulsion by covering a larger surface of the oil droplet as a mechanical barrier as observed in the case of Pickering emulsion.5,6 In a Pickering emulsion system, it has been proposed that the steric particle-based barrier is not a simple bilayer or monolayer which is densely packed, but a network of particles, adsorbed at the oil−water interface, with the entire aggregated structure held together by attractive interparticle 5732

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Figure 4. (A) EI and (B) droplet size of the BCN stabilized emulsion during storage at 4, 25, and 45 °C. (C) Initial size distribution calculated based on an average particle diameter D(1,0) and cumulative distribution stored at 25 °C for 5 d.

Figure 5. Effect of pH in 0.1 M sodium phosphate buffer (A) and type of buffer (B) at pH 6.8 on EI5d (bar) and particle size (●). NaPi: sodium phosphate; KPi: potassium phosphate; C/P: citrate-phosphate; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; MES: 2-(Nmorpholino)ethanesulfonic acid; MOPS: 3-(N-morpholino)propanesulfonic acid. Values obtained from 3 individual replicates.

forces.6,16,36,37 This phenomenon was also observed in our BCN-based emulsion, in which a steric BCN layer is selfassembled through the electrostatic interactions between bacterial cells and chitosan, and a dense network of BCN is adsorbed at the oil−water interface as clearly imaged in Figure 3. Experimentally, it was also observed by fluorescent microscopy that BCN-stabilized emulsion systems can be produced without a full monolayer coverage of particles network around the emulsions, which is in good agreement with the case of Pickering emulsion reported previously.6 3.4. Emulsion Stability. Bacteria interface Pickering emulsions were prepared at a ratio of 1: 1 (water:oil) with a BCN formulated by 75 mg/L chitosan self-assembled with around 473 millions bacterial cells per mL (4.73 ± 0.55 × 108 cfu/mL) in the water phase (E. coli DH5α, OD660 = 2). These emulsions were subjected to a stability test under three temperature conditions. High stability was obtained without significant change in EI and droplet size D(3,2) after a storage of 2 months at 25 and 45 °C (Figure 4A and B). However, some coalescence occurred at the beginning of the storage because the D(3,2) increased from 0.11 mm at day 1 to 0.18 mm on day 2 of observation at room temperature. After day 5, the size was 0.22 mm and almost no variation in droplet size was observed until 2 months. These results implied that the rearrangement of the BCN to the interface of oil droplet is a time-consuming process. The stabilization kinetic of BCN on the oil droplets was accelerated when keeping the samples at 45 °C. The stability in droplet size consolidates the fact that although the bacterial cells are fragile and soft, the irreversible absorption occurred at the interface of oil droplets resulting in a highly stable bacteria interface Pickering emulsion that can last for months. Even with different storage temperatures, normal

distribution of droplet size was observed throughout the storage in most cases as shown in Figure 4C. The freeze−thaw stability of the emulsion was tested when the samples were stored at 4 °C. The oil droplets were solidified at a temperature below its freezing point at 5.8 °C. After 2 cycles of freeze−thaw, destabilization and phaseseparation of the emulsion were observed, therefore D(3,2) data could not be collected since day 2 (Figure 4B). After day 2, the emulsions separated into three layers: an upper free oily layer, an intermediate bubbly layer and a lower clear aqueous layer. In Figure 4A, the EI values represent the thickness ratio of an intermediate bubbly layer which decreased gradually during the first 20 days of storage at 4 °C. The intermediate layer finally shrank into a flat white sheet located in between the upper oil phase and the lower aqueous phase. The Microscopy technique provided further evidence of a bacteriachitosan continuous network formed during freezing by a mechanism related to a coalescence which later on collapsed during lipid melting to allow phase separation. 3.5. Effect of pH and Type of Buffer. Since both chitosan and bacteria are pH-dependent ionized materials, effects of pH and type of buffer on the bacterial interface emulsion formation were investigated. The washed cells of E. coli were suspended in 0.1 M sodium phosphate buffer at different pH ranging from 5 to 8. Then the BCN was formed by addition of 0.03% w/v chitosan dissolved in 0.03% acetic acid (pH 4, 0.3 mL) to the bacteria suspension (OD660 = 2, 0.9 mL). A bacterial cell surface was evaluated by a ζ potential measurement. The ζ potential of E. coli DH5α from pH 5−8 was around −20 mV without large variation (see Supporting Information). This is in good agreement with the previous report where the ζ potential of P. multocida ATCC 11039 decreased drastically when pH was changed from 2 to 4, but remained stable during pH 5−8.30 5733

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However, after associated with chitosan, the ζ potential of the BCN increased almost to a similar level to that of the chitosan itself (see Supporting Information). These results confirmed the full coverage of chitosan onto the E. coli cell’s surface and also indicated the pH-dependent polyelectrolyte property of chitosan. After shaking vigorously with an equal volume of organic solvent, the EI5d at pH 5 was only 32% compared to that of the pH 6.0−8.0 which showed the stable emulsification ability of 70−80% (Figure 5A). At pH 5, the ζ potential of the BCN showed the highest positive value of 20.80 mV due to the large amount of free amino groups along the chitosan chain, however, the smallest positive ζ potential (1.2 mV) was recorded at pH 7.5, indicating the most effective hydrophobic BCN. At pH 7.5 the smallest D(3,2) was obtained. Our pHdependent formation of bacterial interface emulsion indicated the effectiveness of electrostatic force between bacterial cells and chitosan. In addition, six types of generally used buffer were employed to suspend the bacterial cells before formation of BCN. Figure 5B showed that EI5d of BCN stabilized emulsion in 6 selected buffers were about 70−80% without an influence from the buffer type. However, the D(3,2) was slightly higher in small molecular buffer such as sodium phosphate (NaPi), potassium phosphate (KPi) and citrate-phosphate (C/P) buffer, compared to that of the high molecular weight buffer such as HEPES, MES, and MOPS (Figure 5B). The slightly smaller size of the emulsions when prepared in the high molecular weight buffer suggests lateral repulsive forces among adsorbed chitosan-modified bacterial cells due to the lower screening effect of the charged chitosan chains. 3.6. Bacterial Cell Viability. It was previously reported that chitosan with Mw of 50 kDa showed the highest inhibitory activity toward E. coli compared to the longer chain of chitosan with Mw of 1000 kDa.27 In our study, chitosan with Mw of 1000 kDa was selected because it provided an adequate length for BCN formation and exhibited low cytotoxicity. We here confirmed the toxicity of chitosan toward E. coli DH5α by evaluating the number of cell viability in the aqueous phase upon an exposure to chitosan with and without emulsion formation via a conventional serial dilution technique. As presented in Figure 5, the chitosan-modified E. coli in aqueous solution (75 mg/L w/v final concentration of chitosan) showed a large reduction in viable population of approximately 5-order on the first 2 days. In contrast, the bacterial cells in BCN stabilized emulsion system showed higher viability, only 2-order reduction in the viable population at the same period, and even a positive sign of proliferation since the viable population of E. coli was surprisingly increased about 1-order from approximately 106 to 107 cfu/mL during 2 to 5 days, which is comparable to the population of an unmodified E. coli. These results demonstrated that chitosan showed some toxicity toward E. coli in an aqueous solution, on the other hand the toxicity of chitosan was significantly reduced when BCN stabilized emulsion was introduced. Observation under light microscope also offered a movie of viable population of E. coli attached on the surface of the emulsion. Approximately 65% of viable E. coli cells was disappeared from aqueous phase which may attribute to the BCN attached at the interface of the emulsion, the rest of 35% viable cell population remained in the aqueous phase. They were capable of moving freely in the emulsion system. Although an emulsion provided from a small molecular surfactant such as Tween 80, a nonionic surfactant has been reported, but the high toxicity of small molecular surfactant hinders its practical

Figure 6. Cell viability of E. coli DH5α when exposed to 0.0075% w/v acetic acid (○) and 75 mg/L chitosan dissolved in 0.0075% w/v acetic acid in 0.1 M sodium phosphate buffer (pH 6.8) without (▲) and with (■) emulsion formation with n-tetradecane. Values obtained from 3 individual replicates.

applications due to the strong detergent action which denaturing the surface proteins and decreases the membrane integrity.38 Using a cationic surfactant such as cetyltrimethylammonium bromide (CTAB) in the similar system caused worse effect since the bacterial cells could not withstand its toxicity.38 Using a small molecular surfactant does not usually increase the microbial growth on a hydrophobic substrate that is dispersed in small globules because the emulsion diameter is more important to favor a contact. An increased microbial growth was reported when fatty acid globules were significantly bigger or smaller than cells and a decreased growth was reported with comparable emulsion sizes, since in that case the contact was more difficult.38 In our BCN stabilized emulsion system, the D(3,2) of the oil emulsion was 0.22 mm, about 100 fold larger than the size of the bacterial cell, therefore it may be favorable for the bacterial growth. However, the quantitative analysis of bacterial cell viability in the BCN stabilized emulsion 3D system is under investigation in our laboratory. 3.7. Effect of Organic Solvent and Strain of Bacteria. To prove that our newly developed BCN stabilized emulsion system can generally be applied to a system other than aqueous/n-tetradecane, five industrially important organic solvent were also investigated. Figure 7A shows that acetate derivatives solvent demonstrated the EI5d of less than 20% while the other types of solvent showed the EI5d higher than 70%. Since chitosan has been known to have good complexing ability with metals, surfactants28 or acetic acid,39 therefore complexation between chitosan and acetate derivatives solvent is highly possible. In the experiment, addition of acetic acid or ethyl acetate to the emulsions induced an immediate phase separation, which implied that the affinity between chitosan and bacterial cells was lower than that of chitosan/acetic-acetate derivatives. As a result, the self-assembled BCN was destroyed easily in the presence of acetic acid or ethyl acetate, but only partially destructed in case of steric butyl acetate. Our results implied that the self-assembled BCN has a high capability to induce the emulsion formation in various types of organic solvent except ethyl acetate. It has been reported that some specific strains of hydrophobic bacteria such as Rhodococcus obacus B-4 was capable of stabilizing the emulsions,33 but those specific bacteria is quite rare in nature, compared to the hydrophilic bacteria which are ubiquitous in every habitat on earth. To check the generality of our original BCN-based emulsion toward other species of hydrophilic bacteria, BCN of four hydrophilic bacteria with 5734

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Figure 7. (A) EI5d (bar) and D(3,2) (●) of o/w emulsion stabilized by E. coli DH5α associated with chitosan in various organic solvent. EA: ethyl acetate; BA: butyl acetate; CH: cyclohexane; HX: n-hexane; PB: n-propyl benzene; TD: n-tetradecane. (B) Emulsions formed in n-tetradecane using different species of bacteria. LS: Lactobacillus sakei ATCC 15521; SA: Staphylococcus aureus ATCC 25923; EC: Escherichia coli DH5α; BS: Bacillus subtilis ATCC 6633; and BC: Bacillus cereus ATCC 14579. Values obtained from 3 individual replicates.

industrial importance, namely Lactobacillus sakei ATCC 15521, Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633, and Bacillus cereus ATCC 14579 were prepared and compared with those obtained from E. coli DH5α. The EI5d and D(3,2) provided by those selected five species of bacteria ranged between 70 and 80% and 0.25 mm, respectively with no significant difference between each species (Figure 7B). The results confirmed that our newly developed BCN stabilized emulsion can be generally used with any type of hydrophilic bacteria as all bacteria normally possess the net negative charge on the surface, and therefore easily interacts with the polycationic chitosan through electrostatic forces.

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

S Supporting Information *

Tabulation of ζ potential of E. coli DH5α before and after BCN formation at different pH. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author

*Tel: 66 (2) 201-5317; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research project is supported by the Faculty of Science, Mahidol University, the National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand, and the Japan Society for the Promotion of Science (JSPS). This research is partially supported by the Center of Excellence on Agricultural Biotechnology, Science, and Technology Postgraduate Education and Research Development Office (PERDO), Commission on Higher Education, Ministry of Education. We use the laser scanning confocal microscope (LSM 510 Meta, Zeiss, Jena, Germany) at the Division of Medical Molecular Biology, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University. W.V. is grateful for his technical help on CAM measurement.

4. CONCLUSIONS In summary, we reported a completely new and unique BCN stabilized emulsion system consisting of bacteria, chitosan, and organic solvent. By introducing the high molecular weight chitosan to bacteria, the self-assembled BCN was obtained through the electrostatic interactions between polycationic chitosan and the negative charge of the bacterial cell surface. After mixing vigorously with organic solvent, the stable o/w emulsion containing bacterial cell at the interface was obtained. The stable BCN stabilized emulsions can be created using any kind of organic solvent except ethyl acetate or any type of bacteria. The bacteria interface emulsion is not restricted to the hydrophobic bacteria which is extremely rare in nature, but can be created by using any species of hydrophilic bacteria, which are ubiquitous in every habitat on earth. Due to the sustainability and low environmental impact of chitosan and bacteria, our original BCN-based emulsion will open up opportunities for the development of environmentally friendly new interfacial materials as well as the utilization of our BCNbased emulsion in organic syntheses assisted by microorganisms. These developments will promote the cleaner technology useful for industrial synthesis of bulk chemicals, pharmaceutical and agrochemical intermediates, active pharmaceuticals, and food ingredients. The roles of bacteria and chitosan in our BCN-based emulsion system, factors influencing BCN-stabilized emulsion, as well as the integration of our novel BCN-based emulsion system into chemical manufacturing are being investigated in our laboratory.

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REFERENCES

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