Article pubs.acs.org/Langmuir
Fabrication of Photoreactive Biocomposite Coatings via Electric Field-Assisted Assembly of Cyanobacteria Oscar I. Bernal,† Bhuvnesh Bharti,‡ Michael C. Flickinger,*,†,§ and Orlin D. Velev*,† †
Department of Chemical and Biomolecular Engineering and §Golden LEAF Biomanufacturing Training and Education Center (BTEC), North Carolina State University, Raleigh, North Carolina 27695, United States ‡ Cain Department of Chemical Engineering, Louisiana State University, Baton Rouge, Louisiana 70803, United States S Supporting Information *
ABSTRACT: We report how dielectrophoresis (DEP) can be used as a tool for the fabrication of biocomposite coatings of photoreactive cyanobacteria (Synechococcus PCC7002) on flexible polyester sheets (PEs). The PE substrates were precoated by a layer-by-layer assembled film of charged polyelectrolytes. In excellent agreement between experimental data and numerical simulations, the directed assembly process driven by external electric field results in the formation of 1D chains and 2D sheets by the cells. The preassembled cyanobacteria chains and arrays became deposited on the substrate and remained in place after the electric field was turned off due to the electrostatic attraction between the negatively charged cell surfaces and the positively charged polyelectrolyte-coated PE. The DEP-assisted packing of cyanobacteria is close to the maximal surface coverage of ∼70% estimated from convectively assembled monolayers. Confocal laser scanning microscopy and spectrophotometry confirm that the photosynthetic pigment integrity of the Synechococcus cells is preserved after DEP immobilization. The significant decrease of the light scattering and the enhanced transmittance of these field-assembled cyanobacteria coatings demonstrate reduced self-shading compared to suspension cultures. Thus, we achieved the assembly of structured cyanobacteria coatings that optimize cell surface coverage and preserve cell viability after immobilization. This is a step toward the development of flexible multilayered cell-based photoabsorbing biomaterials that can serve as components of “biomimetic leaves” for utilizing solar energy to recycle CO2 into fuels or chemicals.
1. INTRODUCTION Composite materials that mimic the function of natural plant leaves have been a subject of extensive research over the last several decades.1−4 The next step toward the development of highly photoreactive and robust biocomposite materials is the incorporation of live cyanobacteria as an integrated part of their microscopic structure. The potential for high commercial impact of these biocomposites stems from the recent advances in genetic engineering of cyanobacteria and algae, making it possible to harvest solar light and convert carbon dioxide into useful chemical intermediates and fuels.5−8 One major factor governing the efficiency of such biocomposites is the spatial distribution of the photoactive bacteria within the material.9 The current photobioreactors operate using large volumes of suspension cultures, where the majority of the cells self-shade, limiting the overall light-harvesting efficiency. Because of this, predicting how to optimize the interaction of photosynthetic cells with solar illumination in photobioreactors is a complex problem.10 Recently, photosynthetic bacteria immobilization onto flat surfaces has been proposed as a more efficient alternative than bulk photobioreactors for recycling carbon dioxide and producing biofuels.11 Biophotocatalysis by planarimmobilized cells in biocoatings is a promising approach for enhancing the efficiency of the light-harvesting devices.12 Our © XXXX American Chemical Society
goal is to develop inexpensive methods to rapidly assemble biocomposites from reactive unicellular photosynthetic microorganisms11,13 into ordered structures with reduced self-shading and optimal surface coverage.14,15 Current methods for immobilizing cyanobacteria onto substrates include physical absorption,16−18 chemical crosslinking,19−21 and entrapment within a gel matrix.22−25 However, none of these methods is capable of organizing cells into closed-packed arrays similar to the structure of cell layers in natural leaves. Hence, the lack of optimal light trapping due to partial self-shading in these materials restricts their operational efficiency. The performance of biocomposite coatings as photobiocatalysts can be maximized by mimicking the structure of naturally occurring leaves, where the lightharvesting cells are arranged into closely packed arrays. Several literature studies report the assembly of cellular aggregates either in bulk dispersion26−28 or on surfaces.29−32 However, to the best of our knowledge no previous work has been reported on the assembly of viable, reactive cyanobacteria in bulk using Received: February 11, 2017 Revised: May 1, 2017 Published: May 8, 2017 A
DOI: 10.1021/acs.langmuir.7b00335 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir
Figure 1. (a) Schematic of the experimental setup used for AC-electric-field-driven assembly of cyanobacteria onto a polyester film surface. (b) Schematic of the electric field-initiated cellular coating deposition technique. The cells are first aligned in suspension and then deposited onto the surface of the activated substrate by a combination of DEP and gravitational forces. methods described previously.35 Cell suspensions for dielectrophoretic deposition were prepared as follows: 1.0 mL of an optical density (absorbance) of approximately 1.0 au at wavelength 540 nm, 3-day old culture, was centrifuged in 1.5 mL Eppendorf tubes in a benchtop centrifuge for 15 min at 3000g and room temperature. The BG11 supernatant was removed by pipetting, and the wet cell pellet was resuspended in 1.0 mL of deionized water for a final cell density of ∼8.0 × 107 cells mL−1 (viability experiments in suspension). For high cell density coating experiments, the wet cell pellet was resuspended in 300 μL of deionized water for a final cell density of ∼3−5 × 109 cells mL−1. The final pH of all cell suspensions was ∼7.0 with no added electrolyte. No cell lysis was observed after DI water resuspension of the pellet. 2.3. Dielectrophoretic Coating Deposition Procedure. The dielectrophoretic alignment of the suspended cyanobacteria was carried out in a 1 mm thick cylindrical perfusion chamber of diameter 20 mm (PCI-1.0, Grace Bio-Laboratories, OR, see Figure 1a). The assembly setup consisted of two coplanar gold electrodes separated by a 3 mm gap. The activated polyester substrate was cut to 10 × 5 mm shape to fit within the perfusion chamber that was used to house the concentrated cell suspension. A square wave AC signal was applied across the gold electrodes using a function generator (Agilent Technologies, Santa Clara, CA) connected to a voltage amplifier (Burleigh Instruments, Fishers, NY). In a typical experiment, the perfusion chamber was loaded with 300 μL of cell suspension, and an AC-electric field of 2−10 V mm−1 at frequency 50−250 Hz was applied to the dispersion for 1 h. During the course of the assembly, the cells align in bulk solution and selfimmobilize in densely packed arrays onto the activated surface. The cell-coated substrate was rinsed in deionized water prior to imaging in order to remove any unbound cells. 2.4. Microscopic Characterization of Surface Coatings. The alignment of the cyanobacteria induced by the AC-electric field was monitored using an Olympus BX-61 optical microscope (Olympus America, Center Valley, PA) in bright-field and fluorescence modes. The effect of the applied field intensity and frequency on cell chaining was evaluated by analyzing a set of five microscope images acquired after 2 min of initial field application. The values reported for the chain length correspond to an average number of cells per chain based on five microscope images. The microstructure of the coating and cell viability were determined from deconvoluted z-plane images obtained by confocal laser scanning microscopy (CLSM) using 515 nm argon ion laser (CVI MellesGriot, Albuquerque, NM). Cyanobacterial natural pigment florescence was detected in the red region of the spectra at wavelengths >650 nm (excitation at 488 nm). A stack of 50 CLSM images in the direction perpendicular to the coating plane were recorded to a total depth of ∼10 μm. ImageJ software package was then used to reconstruct the topographic profile of the samples.36 The
DEP-assisted assembly, followed by their immobilization as cell monolayers onto flexible substrates. We report here a robust method to assemble and immobilize photoreactive cells on flexible planar substrates, resulting in a biocomposite material capable of capturing and harvesting sunlight over a wide range of the visible spectrum. We direct the assembly of cyanobacteria into linear chains and 2D hexagonally packed arrays by using an external alternating current (AC)-electric field.33 The assembly was carried out over a planar surface of tunable surface-charge and charge-density. Normally cellular arrays formed in an AC-electric field are not permanent and disassemble upon turning off the field.26 To achieve permanent linking of the cells and to restrict their disassembly upon switching off the field, we introduce an additional electrostatic attraction component between the cell surface and the substrate. We characterize the packing efficiency of the cells and investigate the assembly mechanism using COMSOL simulations. The microstructure and in situ viability of these cellular coatings was investigated using optical, scanning electron, and confocal microscopy. We expect that each cell within such densely packed cyanobacteria monolayers will be illuminated uniformly, significantly increasing the lightabsorbing efficiency of the sunlight-harvesting biocomposites.11,12,14,15
2. EXPERIMENTAL SECTION 2.1. Materials and Substrate Activation Procedure. Aqueous solutions of poly(sodium 4-styrenesulfonate) (PSS) (Mw = 70 kDa) and poly(allylamine hydrochloride) (PAH) (Mw = 15 kDa) (Aldrich Chemical, MO) were prepared with deionized water (Milli-Q system, 18.2 MΩ cm) at a final concentration of 1 mg/mL. A 125 μm thick polyester substrate (DuPont Melinex 454, Tekra Corp, NJ) was precut into 75 × 25 mm rectangular sheets. A net negative charge on the surface was created by partial hydrolysis of the PE sheet with a boiling solution of 0.37 M NaOH for 30 min. Polyelectrolyte multilayers were deposited onto the charged polyester substrates with a layer-by-layer deposition technique.34 The PE substrate was sequentially dipped into PAH and PSS solutions. The final activated substrate contains three PAH/PSS bilayers and a final positively charged PAH layer on its surface (Figure 2). 2.2. Cyanobacterium Strain, Media, Growth, and DEP Preparation Conditions. Synechococcus PCC7002 was grown aerobically in 250-mL flasks containing 50 mL of BG11 medium at 100 oscillations/min in an orbital shaker with 70 μmol photons m−2 s−1 cool fluorescent light (light intensity measured using a LI-COR, LI190SA Quantum Sensor, NE) at 25 °C. BG11 was prepared using B
DOI: 10.1021/acs.langmuir.7b00335 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir
deposition technique to form a ∼100 nm thick polyelectrolyte coating on the substrate from the two oppositely charged polyelectrolytes, PSS and PAH (Figure 2a). The positively
Tikhonov−Miller algorithm was used for image deconvolution, noise reduction, and improving the image quality. The fractional surface area coverage by cells was estimated by image analysis performed using ImageJ software package. The micrographs were converted to binary images and processed using the "analyze particles" capability of the program. The background subtraction, particle size threshold, and circularity were adjusted manually to detect all cells in the field and estimate the void space. Surface coverage was calculated as the product of the total number of cells detected times the average area per cell divided by the total field area. Polyelectrolyte coating microstructure was observed by scanning electron microscopy (SEM) using a Hitachi 3200-N variable pressure scanning electron microscope equipped with a 4Pi Isis EDS system for digital image acquisition. Samples were sputter-coated prior to imaging with a thin layer of gold in a mild vacuum (∼100 mTorr of Ar gas pressure; 600 V accelerating voltage) for 5 min and immediately placed in the SEM vacuum chamber for analysis. The SEM imaging was performed at 5 kV accelerating voltage at magnification factors between 1000× and 10 000×. 2.5. Optical Properties. The optical characteristics of planktonic Synechococcus PCC7002, the 125 μm thick transparent polyester substrate, and DEP-generated cellular coatings of Synechococcus were measured using a 1 mL cuvette filled with BG11 media (for coatings or substrates) or cell suspensions in a Lambda 35 PerkinElmer UV−vis spectrophotometer equipped with an integrating sphere (RSA-PE-20, Labsphere, North Sutton, NH) at ambient temperature. A 4 × 25 mm cyanobacteria DEP coating was laid flat (vertical) on the back wall of the BG11-filled cuvette. Values for transmittance were measured between 400 and 900 nm at 480 nm min−1. No evidence of cell release or outgrowth to the liquid phase was observed during the experiments as indicated by hemocytometer chamber light microscope visualization of the medium in contact with the coatings.
Figure 2. Polyelectrolyte multilayer deposition onto polyester substrate. (a) Schematic representation of the polyelectrolyte layerby-layer coating of the polyester substrate. (b) SEM image of the alkali-treated negatively charged polyester sheet showing the microscale smooth surface. (c) Final multilayer coating (PAH/PSS)3PAH. (d) Synechococcus PCC7002 immobilized onto the polyelectrolyte coating. Scale bars = 2 μm.
3. RESULTS AND DISCUSSION Application of an external AC-electric field is an efficient means to assemble cells into closely packed chains.26,37 Our aim here is to permanently immobilize these oriented cellular arrays onto flexible polyester substrates following their assembly. The electric field assisted in the formation and immobilization of an ordered cellular layer onto the substrate in a dual-step process (Figure 1b). Step 1: Cells are assembled into linear chains in bulk suspension by the AC-electric-field-induced dipole−dipole interactions. Step 2: The assembled chains settle at the bottom of the activated polyester substrate by the combination of the vertical component of the DEP force (FDEP, eq 1)38 and the sedimentation force (FS, eq 2).39 The corresponding scaling relationships for the forces involved are 2 FDEP ∝ Vcell∇Erms
(1)
FS ∝ Vcell(ρcell − ρm )
(2)
charged PAH was used to form the first layer on the negative polyester substrate. This PAH coating serves as a base layer for the sequential deposition of alternatively charged layers of PSS and PAH. SEM imaging of the native partially hydrated charged substrate shows a clean surface (Figure 2b) primed to be covered by the polyelectrolyte coating. As the coating grows in thickness with subsequent polyelectrolyte layers, topographic features and surface roughness increase (Figure 2c). In our experiments, PAH was used for the final layer, such that the net charge of the multilayered surface is positive. This positive charge facilitates the binding and permanent immobilization of negative cyanobacteria (Figure 2d).43 No evidence of cell wall fusion to the polyelectrolyte layer can be observed in these electron micrographs. 3.2. Effect of Voltage and Frequency on Cell Alignment. Before immobilization on the surface, the cyanobacteria cells assemble in chains and 2-D closely packed lattices in suspension above the polyelectrolyte PE substrate. We investigated the effect of applied electric field voltage and frequency on cell chaining and their viability. The applied voltage and frequency were varied in the range 10−50 V and 50−250 Hz, respectively. These electric field parameters minimized electrohydrodynamic flows near the electrode edges that may disrupt the assembly process. All experiments were carried out at a cell density of 8.0 × 107 cells mL−1 at pH 7.0. The chaining outcome was expressed in terms of average number of cells per chain after 2 min of AC-electric field exposure (Figure 3). For low frequencies, the average number of cells in the chains increases with increasing applied voltage. Lower frequencies promote longer chain assembly, but the process is significantly
where Vcell is the volume of individual cell, ∇Erms is the gradient of electric field orthogonal to the plane containing the electrodes, ρcell and ρm, respectively, are the mass density of the cells and the dispersion medium. Here we specifically focus on the cellular assembly in low frequency (
