Subscriber access provided by YORK UNIV
Article
Understanding Biophotocurrent Generation in Photosynthetic Purple Bacteria Matteo Grattieri, Zayn Rhodes, David P. Hickey, Kevin Beaver, and Shelley D. Minteer ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b04464 • Publication Date (Web): 18 Dec 2018 Downloaded from http://pubs.acs.org on December 20, 2018
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Catalysis
Understanding Biophotocurrent Generation in Photosynthetic Purple Bacteria Matteo Grattieri,† Zayn K. Rhodes,† David P. Hickey,† Kevin Beaver, †§ Shelley D. Minteer †* † Departments of Chemistry and Materials Science & Engineering, University of Utah 315 S 1400 E Room 2020, Salt Lake City, 84112 Utah (USA) § Departments of Biology and Chemistry, Lebanon Valley College 101 N College Ave, Annville, 17003 Pennsylvania (USA)
ACS Paragon Plus Environment
1
ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 2 of 23
ABSTRACT Establishing an efficient extracellular electron transfer (EET) process between photo-electroactive microorganisms and an electrode surface is critical for the development of photo-bioelectrocatalysis. Soluble and immobilized redox mediators have been applied with purple bacteria Rhodobacter capsulatus for this purpose. However, detailed information on its EET with an electrode surface is not available and, therefore, choice of mediators has been by trial and error. Herein, we experimentally evaluated the capability of different soluble, quinonebased redox mediators to support EET and compared the experimental data with a computational model based on density functional theory calculations. We show that computed electrochemical redox properties of redox mediators in a lipophilic environment correlate to EET processes of Rhodobacter capsulatus, suggesting that intermembrane mediator characteristics are more diagnostic than redox properties of the mediators in an aqueous solution, and that the limiting electron transfer step takes place in the lipophilic membrane of the bacterial cells. This knowledge provides critical insight into designing future mediated bioelectrocatalysis systems.
KEYWORDS photo-bioelectrocatalysis; extracellular electron transfer; Rhodobacter capsulatus; quinones; lipophilic membrane; rate determining step.
ACS Paragon Plus Environment
2
Page 3 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Catalysis
INTRODUCTION The continuous increase in global population together with improving life quality worldwide has resulted in a substantial increase in energy consumption. Accordingly, new approaches to non-renewable energy sources are necessary, and renewable power sources are expected to consistently improve to meet global energy demands.1 In this context, solar power is one of the most attractive energy sources with technological developments resulting in the achievement of record efficiencies.2 In addition, photosynthetic biomaterials have been explored for the development of biological photovoltaic devices,3 where such systems are capable of converting light energy into electrical current.4 Photosynthetic biomaterials comprise intact photosynthetic organisms and parts of cellular or subcellular machinery, such as the photosystem.5 Efficient generation of bioelectrocatalytic photocurrent using these biomaterials requires a coherent electrochemical interface between the biophotocatalyst and an electrode surface.6 It has been reported that some photosynthetic bacteria can secrete endogenous redox mediators, thus facilitating electron transfer with an electrode.7 Additionally, different approaches have been investigated to improve the electron transfer process, such as immobilization of intact organisms or a subcellular apparatus in redox polymers,8 as well as the preparation of mutants deficient in natural electron sinks.3 An isolated apparatus often yields enhanced electron transfer (ET) due to improved orientation on the electrode surface and decreased ET distance.9 This system also exploits the fact that energy is not required for organism growth and maintenance. However, intact organisms have repair systems that can replace photo-damaged photosynthetic proteins, thus giving the possibility to extended device lifetime.10 Nonetheless, establishing extracellular electron transfer (EET) with complete photosynthetic organisms is challenging due to the
ACS Paragon Plus Environment
3
ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 4 of 23
necessity of extracting electrons from their cellular membrane. Complete organisms are not designed to transfer electrons with an electrode surface and, with the exception of few organisms, EET is one of the major issues in bioelectrocatalysis.11 Detailed mechanisms of protein-protein interactions, contributions of microbial nanowires in the electron transport, and bacterium-electrode interactions remains strongly debated and poorly understood in the field.12 For photosynthetic microorganisms extremely little information is available on detailed EET mechanisms. The majority of these microorganisms require an exogenous redox mediator;13 however, specific interaction between redox mediators and different bacterial cells remains unclear, resulting in the absence of an efficient mediating system for several microorganisms.14 Rhodobacter capsulatus (R. capsulatus) is a purple photosynthetic bacterium, which is defined as non-sulfur, because it does not utilize sulfur compounds as substrates, but rather organic compounds such as malic or succinic acids. It is characterized by outstanding metabolic versatility, as it can rapidly grow under either anaerobic photosynthetic conditions, or aerobic dark conditions, adapting to the availability of light, oxygen or carbon sources.15 Its high versatility makes it interesting for several applications due to its possible growth even in saline and hypersaline environments. For example, it is extremely effective in anaerobic photoheterotrophic conditions, where it utilizes sunlight as an energy source and organic compounds present in solution as a substrate, opening pathways for its application in the removal of organic carbon while converting sunlight into electrical energy. This peculiarity can be of great interest for the development of self-sustained, sunlight-powered decontamination systems with the possibility of on-line monitoring thanks to the produced current.16 However, R. capsulatus has an outer and inner membrane, with a peptidoglycan cell wall in between. Further, it is encapsulated by an outer layer of slimy lipopolysaccharides, thus hindering the electron
ACS Paragon Plus Environment
4
Page 5 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Catalysis
transfer between the active redox center buried in the bacterial cells and an electrode. The research group of Lo Gorton pioneered bioelectrochemical studies on R. capsulatus, where osmium-based redox polymers (ORPs) were utilized to successfully wire bacterial cells to an electrode surface,17 specifically with gold and graphite electrodes.17a Furthermore, it was shown that changing the structure of the ORP mediator could strongly influence the production of electricity, and the addition of soluble p-benzoquinone in solution resulted in an increase of the biophotocurrent generated. However, the fundamental aspects of the microorganism-redox mediator-electrode interactions remain to be fully understood, calling for detailed studies on the specific physico-chemical and electrochemical properties of an effective mediator.18 With this issue in mind, we aimed to investigate how the properties of quinone-based soluble redox mediators influenced the electron transfer between R. capsulatus cells and carbon-based electrodes during photoheterotrophic metabolism. We used malic acid as the substrate for bacterial cells, and thus the photoinduced reduction of quinones is coupled to the photoinduced oxidation of the organic acid.19 Experimental data for biophotocurrent generation were obtained for different mediators. Specifically, we utilized p-benzoquinone and six other monomeric quinone-derivatives,
which
were
previously
shown
to
differentially
mediate
photobioelectrocatalysis between thylakoid membranes and gold electrodes modified with gold nanoparticles.20 Subsequent computational analysis of geometry optimized mediators was performed using density functional theory (DFT). Determination of electronic and structural parameters allowed investigating the redox process of different quinone forms, and computationally-derived electrochemical mediator properties were identified that correlate to experimental EET rates.
ACS Paragon Plus Environment
5
ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 6 of 23
EXPERIMENTAL SECTION Chemicals All chemicals were obtained from Sigma-Aldrich, except MgCl2 (Fisher Scientific), K2HPO4 (VWR Anaytical), KH2PO4 (Fisher Scientific), and KOH (Macron Chemicals), and used with no further purification.
Bacteria growth Rhodobacter capsulatus strain ATCC 33303 was obtained from American Type Culture Collection (ATCC). R. capsulatus cells were grown in a liquid broth medium with the following composition (for 1L of MilliQ water): 20 mg EDTA, 1.0 g (NH4)2SO4, 4.0 g malic acid, 200 mg MgSO4*7H2O, 75 mg CaCl2*2H2O, 12 mg FeSO4*7H20, 1 mg thiamine, 15 mg biotin, 0.9 g K2HPO4, 0.6 g KH2PO4, 1 ml of trace elements solution. The composition of the trace element solution was (for 250 ml of milliQ water): 700 mg H3BO3, 398 mg MnSO4, 188 mg Na2MoO4*2H2O, 60 mg ZnSO4 *7H2O, 10 mg Cu(NO3)2*3H2O, 50 mg CoCl2. The pH of the medium was adjusted to 6.8 using 10M KOH prior sterilization at 125˚C for 25 min. Trace elements were added after sterilization, by filtration through a 0.20 μm filter (VWR International). Bacteria cells were grown in sterile 20 ml scintillation vials, sealed with airtight rubber stoppers. An incandescent light bulb was used to maintain a light intensity of 3000 LUX during the growth. After 72 hours the cells were collected by centrifugation at 5,000g for 20 min (Allegra X-15R benchtop centrifuge, Beckman Coulter). The cells were resuspended in 1 ml of 20 mM MOPS buffer (pH 7) + 10 mM MgCl2 + 50 mM malic acid, and further concentrated by
ACS Paragon Plus Environment
6
Page 7 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Catalysis
centrifugation at 15,000 rpm for 10 min (Eppendorf Centrifuge 5424R). Finally, a solution with a bacterial cell concentration of 1 g ml-1 was prepared using 20 mM MOPS buffer (pH 7) + 10 mM MgCl2 + 50 mM malic acid.
Electrode preparation The electrodes for this study were obtained by cutting a Toray carbon paper electrode (TGP-H060 non-wet proof, Fuel Cell Earth) with an area of 1 cm2. The electrodes were sterilized by exposure under UV-light. 30 μL of the 1 g ml-1 solution of bacterial cells were deposited on every electrode. The solution was allowed to dry for 1 hour under N2 gas atmosphere. The dry electrodes were stored under N2 gas until use in the electrochemical experiments.
Electrochemical setup All of the electrochemical measurements were performed at 20±1 ˚C. The biophotocurrent generation of R. capsulatus with different soluble mediators was investigated in a three-electrode electrochemical cell by cyclic voltammetry (CV) and amperometric i-t tests (CH660 potentiostat, CH instruments). The working electrode was Toray carbon paper with bacterial cells immobilized on the surface, or in the absence of bacterial cells (sterile electrode) for the control experiments. The counter electrode was a Pt mesh, and the reference electrode was a saturated calomel electrode (SCE, CHI 150, CH Instruments Inc.). All of the potentials in this work refer to this reference electrode. The electrolyte used for the study was 20 mM MOPS buffer (pH 7) + 10 mM MgCl2 + 50 mM malic acid with differing concentrations of redox mediators. A Dolan-
ACS Paragon Plus Environment
7
ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 8 of 23
Jenner Fiber-Lite lamp (Model 190-1 quartz−halogen illumination system with an optical light guide providing a light intensity of 76 mW cm−2) was used to excite the photocurrent generation of R. capsulatus on working electrodes. CVs were performed at 1 mV s-1, to determine the redox potential for the oxidation of the mediators (EAN). Amperometric i-t tests were performed applying an anodic overpotential of 200 mV (η) for each redox mediator. After a dark step of 900 s, an illumination step of 500 s was performed, and the current was valuated at the end of the illumination step.
Mediator preparation Quinone based redox mediators were utilized in this study in concentration ranging from 30 to 200 μM. Specifically, the biophotocurrent generation was investigated using p-benzoquinone, 2 chloro-1,4 benzoquinone, 2,6 dichloro-1,4 benzoquinone, 2,3,5,6 tetrachloro-1,4 benzoquinone, 2,3,5,6 tetrafluoro-1,4 benzoquinone, 2,3,5,6 tetrabromo-1,4 benzoquinone, and menadione. Stock solutions for the mediators were dissolved in the MOPS buffer. Only for 2,3,5,6 tetrachloro-1,4 benzoquinone, 2,3,5,6 tetrafluoro-1,4 benzoquinone, 2,3,5,6 tetrabromo-1,4 benzoquinone, and menadione 20% dimethyl sulfoxide was added to the stock solution to solubilize the compounds (final concentration of dimethyl sulfoxide in the electrolyte below 0.1%).
Biophotocurrent calculation
ACS Paragon Plus Environment
8
Page 9 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Catalysis
The biophotocurrent was calculated by subtracting the current obtained with bacterial cells immobilized on the Toray paper electrode from the current obtained with bare Toray paper electrode (control experiment), at the end of the first illumination step (500 s) (Figure S1). All of the experiments (with and without bacterial cells) were performed in triplicate. The average value was calculated and standard deviation was used to calculate the error.
Density function theory calculations For each quinone, density functional theory (DFT) calculations were accomplished using Gaussian 09 21 with an ultrafine integration grid in three computations: geometrical optimization, frequency check, and finally a single-point energy calculation and NBO analysis 22 with a better basis set. Gaussian defaults were used in all computations, unless otherwise specified. In order to calculate the reduction potentials, inclusion of a more diffuse M06-2X/6-31+G(d,p)23 basis set for H, O, and C while M06-2X/LANL2DZ for Br atoms in the tetrabromobenzoquinone for the geometry optimization and frequency single-point, and a M06-2X/ jun-cc-pVTZ basis set for H, O, and C atoms with M06-2X/SDD for Br atoms in the single-point energy calculation. These computations all included water in an SMD solvent model and reduction potentials were calculated directly in condensed phase. Ground-state conformations of each quinone were discovered given the absence of imaginary frequencies in the final structures of each.
ACS Paragon Plus Environment
9
ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 10 of 23
RESULTS AND DISCUSSION Direct Bioelectrocatalytic Analysis Possibility of biophotocurrent generation under direct electron transfer (DET) conditions was first investigated performing cyclic voltammetry and amperometric i-t tests in the absence of any exogeneous mediator in solution. As indicated in Figure 1, a very limited biophotocurrent was obtained when no soluble mediator was present in solution and R. capsulatus cells were immobilized on the electrode (
