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Mediating Electron Transfer from Bacteria to a Gold Electrode via a Self-Assembled Monolayer Scott R. Crittenden,† Christian J. Sund,† and James J. Sumner* US Army Research Laboratory, Sensors and Electron DeVices Directorate, Adelphi, Maryland 20783 ReceiVed June 28, 2006. In Final Form: September 12, 2006 Numerous bacterial genera are known to respire anaerobically using macroscopic electrodes as electron acceptors. Typically, inexpensive graphite electrodes, which are readily colonized, are used to monitor electrogenic bacterial metabolism for microbial fuel cell and bioelectronics studies. We compare current production by electrogenic bacteria on gold electrodes coated with various alkanethiol self-assembled monolayers to current production on glassy carbon electrodes. Current production is correlated to chain length and headgroup of the monolayer molecules as expected. Relative to graphite, the coated gold electrodes achieve more reproducible experimental conditions and certain headgroups enhance electronic coupling to the bacteria.
Introduction Bacteria capable of anaerobic respiration utilize an electron acceptor other than oxygen. It has recently become clear that there are a number of genera capable of using macroscopic electrodes for this purpose (e.g., Geobacter,1 Shewanella,2 Rhodoferax,3 Pseudomonas,4 Clostridium,5 Geothrix,6 and Desulfitobacterium,7 as well as many bacteria that occur naturally in wastewater8,9). The natural soil environment of many of these electron donating bacteria contains significant concentrations of humates,10 which consist of hydrophobic compounds (long alkylchain alkanes, alkenes, fatty acids, sterols, terpenoids, and phenylalkyl residues of lignin degradation) that self-associate into complex polymeric colloids, leading to long residence times in the environment. Humates frequently have polar/acidic functional groups, mainly carboxylic acids but also peptides, alcohols, saccharides, amines, and quinones, that allow multivalent chelation of metal ions. It is reasonable to suppose that soil dwelling anaerobic or facultative bacteria may have the facility to utilize, in addition to commonly occurring metals such as manganese or iron,11,12,13 these humates as electron acceptors. Many researchers studying electrogenic bacteria, both in the laboratory and in the field, commonly utilize graphite electrodes because they are inexpensive and these bacteria will readily colonize them. This is consistent with the bacterial use of humates * To whom correspondence should be addressed. Phone: (301) 3940252. Fax: (301) 394-0310. E-mail:
[email protected]. † Oak Ridge Affiliated Universities Postdoctoral Research Fellows at the U.S. Army Research Laboratory. (1) Bond, D. R.; Lovley, D. R. Appl. EnViron. Microbiol. 2003, 69, 15481555. (2) Kim, H. J.; Hyun, M. S.; Chang; I. S.; Kim, B. H. J. Microbiol. Biotechnol. 1999, 9, 365-367. (3) Chaudhuri, S. K.; Lovley, D. R. Nat. Biotechnol. 2003, 21, 1229-1232. (4) Rabaey, K.; Boon, N.; Siciliano, S. D.; Verhaege, M.; Verstraete, W. Appl. EnViron. Microbiol. 2004, 70, 5373-5382. (5) Dobbin, P. S.; Carter, J. P.; Garcia-Salamanca, C.; von Hobe, M.; Powell, A. K.; Richardson, D. J. FEMS Microbiol. Lett. 1999, 176, 131-138. (6) Bond, D. R.; Lovley, D. R. Appl. EnViron. Microbiol. 2005, 71, 21862189. (7) Finneran, K. T.; Forbush, H. M.; Gaw VanPraagh, C. V.; Lovley, D. R. Int. J. Syst. EVol. Microbiol. 2002, 52, 1929-1935. (8) Cheng, S.; Liu H.; Logan B. E. Electrochem. Commun. 2006, 8, 489-494. (9) Min, B.; Logan, B. E. EnViron. Sci. Technol. 2004, 38, 5809-5814. (10) Lovley, D. R. Microbial Fuel Cells Powered by Electricigens; Abstract, 209th Electrochemical Society Meeting, Denver, CO, May 10, 2006. (11) Lovley, D. R.; Coates, J. D.; Blunt-Harris, E. L.; Phillips, E. J. P.; Woodward, J. C. Nature (London) 1996, 382, 445-448. (12) Lovley, D. R.; Fraga, J. L.; Coates, J. D.; Blunt-Harris, E. L. EnViron. Microbiol. 1999, 1, 89-98. (13) Coates, J. D.; Cole, K. A.; Chakraborty, R.; O’Connor, S. M.; Achenbach, L. A Appl. EnViron. Microbiol. 2002, 68, 2445-2452.
as electron acceptors, as the conductive end planes of graphite can possess many of the same functional groups that are present in humates, including carboxylic acids, alcohols, and quinones. The surface of the graphite electrodes is therefore very similar to the natural habitat for the bacteria, except that graphite is more conductive allowing the bacteria to more easily dispose of their waste electrons. In many cases, it is thought that bacterial cytochromes are responsible for the final electron transfer from the bacteria and that the carboxylic acid termini of self-assembled monolayer (SAM)-modified electrodes can accommodate cytochromes on an electrode surface through strong hydrogen bonding with the peptide bonds in the protein backbone.14,15 In trying to clarify the mechanism of bacterial interaction with conductive carbon electron acceptors, we can leverage the large body of work that has characterized electron transfer between cytochrome c and acid-terminated SAMs of alkanethiols on gold.14,16-18 In addition, there is extensive work by Whitesides that concerns the attachment, and long term viability, of cells on SAMs.19-23 In particular, it has been observed that bare gold is a poor choice of electron acceptor for both cytochromes and electrogenic bacteria, possibly due to a band gap mismatch. We conjectured that we could enhance the electronic coupling between gold electrodes and electrogenic bacteria by forming a carboxylic acid terminated alkanethiol SAM on the electrode surface. In this report, we describe marked increases in current production for carboxylic acid terminated alkanethiol SAMs over both bare gold and methyl terminated SAMs. Electrogenic metabolism has been observed by cells in a microbial fuel cell (MFC) assembly. A MFC is a power generating device that utilizes electrogenic bacteria to catalyze the oxidation (14) Lowy, D. A.; Tender, L. M.; Zeikus, J. G.; Park, D. H.; Lovley, D. R. Biosens. Bioelectron. 2006, 21, 2058-2063. (15) Stams, A. J. M.; de Bok, F. A. M.; Plugge, C. M.; van Eekert, M. H. A.; Dolfing, J.; Schraa, G. EnViron. Microbiol. 2006, 8, 371-382. (16) Tarlov, M. J.; Bowden E. F. J. Am. Chem. Soc. 1991, 113, 1847-1849. (17) Wang, W. Y.; Lee, T. H.; Reed, M. A. Phys. ReV. B 2003, 68, 035416. (18) Chen, X.; Ferrigno, R.; Yang, J.; Whitesides, G. M. Langmuir 2002, 18, 7009-7015. (19) Lopez, G. P.; Albers, M. W.; Schreiber, S. L.; Carroll, R.; Peralta, E.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 5877-5878. (20) Mrksich, M.; Whitesides, G. M. Annu. ReV. Biophys. Biomol. Struct. 1996, 25, 55-78. (21) Roberts, C.; Chen, C. S.; Mrksich, M.; Martichonok, V.; Ingber, D. E.; Whitesides, G. M. J. Am. Chem. Soc. 1998, 120, 6548-6555. (22) Ostuni, E.; Chapman, R. G.; Liang, M. N.; Meluleni, G.; Pier, G.; Ingber, D. E.; Whitesides, G. M. Langmuir 2001, 17 (20), 6336-6343. (23) Jiang, X.; Ferrigno, R.; Mrksich, M.; Whitesides, G. M. J. Am. Chem. Soc. 2003, 125, 2366-2367.
10.1021/la061869j CCC: $33.50 © 2006 American Chemical Society Published on Web 10/06/2006
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of organic fuels and deliver free electrons to an anode.24 MFCs are similar in function to the better understood enzymatic fuel cells but differ in that they utilize live microorganisms instead of their isolated and purified enzymes. Conventional MFCs have the disadvantage that typical experiments take on the order of weeks to months for the bacteria to colonize the electrode, produce power, and exhaust the fuel. To reduce the time required for these experiments, we developed a microliter-scale MFC and injected a sufficient number of bacteria at the start of the experiment to enable relatively instantaneous colonization of the electrode. This allows us to “turn-on” the fuel cell in seconds to minutes instead of days and complete a full run in hours instead of weeks. Our membraneless MFC, with a total volume of approximately 10 µL, is constructed from closely spaced standard electrochemical electrodes. Current production is seen almost immediately upon the addition of a suspension of cells in minimal medium. Typical MFCs utilize two chambers with the anodic and cathodic compartments separated by a proton exchange membrane. However singlechamber systems have been demonstrated and are simpler to construct at the 10 µL scale.8,25 We describe our use of these microliter-scale MFC assemblies to rapidly explore electronic coupling between electrogenic bacteria and various electrode materials including glassy carbon, gold, and SAM modified gold. Materials and Methods Materials and Reagents. Growth medium was either BBL Trypticase Soy Broth (Fisher Scientific, Swannee, GA) or a minimal medium consisting of 1 mL/L of Trace Mineral Supplement and Vitamin Supplement (ATCC, Manassas, VA), 1.5 g/L ammonium chloride, 0.6 g/L sodium phosphate monobasic monohydrate, and 0.1 g/L potassium chloride (Sigma). The electron donor in the minimal medium was 10 mM D,L-lactate obtained as 60 w/w% syrup from Sigma. All water was purified in a Barnstead EasyPure RF to 18 MΩ cm. Thiol-terminated alkanes and carboxylic acids for selfassembled monolayer formation on the gold electrodes were obtained from Aldrich. Bacteria. Shewanella putrefaciens (ATCC 49138) was obtained from Microbiologics, Inc. as a Lyfo-Disk. For the experiments, S. putrefaciens was grown on trypticase soy agar plates in a controlled gas incubator (0.5% oxygen, 10% carbon dioxide, and 89.5% nitrogen) at 30 C for approximately 20 h. The cells were harvested with the minimal medium with 10 mM lactate, and the resulting suspension was reduced to a pellet in a centrifuge and resuspended in 2 mL of the minimal medium with lactate three times to remove any residual soy broth. The resulting cell suspension was normalized to 0.5 A600 by dilution in the minimal medium with lactate. MFC Assembly and Instrumentation. As illustrated in Figure 1, single-chamber, microliter-scale microbial fuel cells were assembled by placing a Gortex gasket between two disk-shaped working electrodes (CH Instruments, Inc., Austin, TX). The working electrodes were 1.6 mm diameter platinum for the cathode and 1.6 mm diameter gold or 2.0 mm glassy carbon for the anode. Evaporated gold interdigitated arrays with 15 micron widths and 15 micron spacing were purchased from ABTECH Scientific (Richmond, Va.). Modification of the gold electrodes was achieved by soaking freshly polished electrodes in a 1 mM thiol ethanolic solution for at least 12 h. The Gortex gasket (∼1 mm thick) was cut to fit the outer diameter of the working electrodes and the inner diameter (∼1.6 mm) to completely expose the electrode surface. The electrodes have planar faces leaving the distance between the electrodes the same as the gasket thickness, yielding a total volume of approximately 10 µL. Cell suspensions were added in 20 µL aliquots to the electrode assembly thus allowing some mixing with the previous suspension (24) Bullen, R. A.; Arnot, T. C.; Lakeman, J. B.; Walsh, F. C. Biosens. Bioelectron. 2006, 21, 2015-2045. (25) Park, D. H.; Zeikus, J. G. Appl. Microbiol. Biotechnol. 2002, 59, 58-61.
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Figure 1. Current response of µL-scale MFC with a glassy carbon anode (3.8 nA peak current). Killed Shewanella putrefaciens was injected twice before the live organism injection at 0 min. The inset is a diagram of the assembly of the single chamber µL-scale MFC. and an overflow of the excess. All data was collected on either a CH Instruments electrochemical workstation model 760b or a Keithley Electrometer model 6514, both giving comparable results.
Results and Discussion Characteristic operation of the single-chamber, microliterscale microbial fuel cell with glassy carbon anode and platinum cathode is shown in Figure 1. In addition, during the initial assembly and filling of the fuel cells, there is a short-time-constant capacitive discharge from changing the ionic concentration with the minimal medium and from handling the electrode bodies (data not shown) which does not interfere with the much longer time scale of bacterial current production. This effect was observed whether lactate was included in the medium as the only oxidizable organic fuel or not (data not shown). The fuel cell was allowed to settle to the baseline of approximately 0.1 nA and then a dense cell suspension in minimal medium with lactate was injected. Figure 1 shows two injections of killed Shewanella putrefaciens as a negative control followed by one injection of live bacteria. The killed cells cause negligible change in the current, whereas the live cells produce multiple nanoamperes for several hours. Similar current response was seen for cases without addition of killed cells. The current generation lasts for 2-3 h, and even though there is theoretically enough lactate to allow operation for days at the observed level of current, premature current termination is most likely due to small leaks in the electrode assembly which causes a loss of electrical connection between the cathode and the electrolyte. Whenever the electrode assembly was opened after an experiment, it was noted that some number of bubbles had formed inside the cell. The anode material plays a crucial role in current collection in a microbial fuel cell. In Figure 2, the inability of a bare gold anode to rapidly couple with the metabolizing organisms is shown. These data include two injections of live Shewanella putrefaciens approximately 2 h apart as indicated. A relatively large and rapid discharge occurs immediately after the first injection but no sustained current production follows. Even though gold is highly conductive, the organisms are unable to immediately couple to the electrode. The goal of mediating bacterium-gold coupling was achieved by using 11-mercaptoundecanoic acid to form a self-assembled monolayer on gold electrodes. Figure 3 compares four different anode surfaces including glassy carbon and three self-assembled monolayers on gold, exploring chain length and headgroup modifications. We conjectured that if the surface of the gold were modified to have chemical characteristics more like humates
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Figure 2. Current response with a bare gold anode showing a capacitive discharge and minimal current generation from the bacteria. Injections of live cells are at 0 and 120 min.
found in soil, then microorganisms would be able to more readily donate electrons to it. This hypothesis is supported by the 11mercaptoundecanoic acid modified gold which had the highest current collection (Figure 3A). The carboxylic acid terminus of the SAM has very strong hydrogen bonding with peptide bonds in proteins, such as the cytochromes, which are suspected to play a major role in transferring electrons from the interiors of the cells. The exact mechanism of this enhancement is not known. However, the SAM is covalently attached to the gold so it cannot intercalate into the cells. Therefore, this phenomenon must be the result of interactions with the surface of the cells, presumably with cytochromes, or an electron shuttle that Shewanella produces. When the chain length was extended on the carboxylic acid terminated SAM by using 16-mercaptohexadecanoic acid, significantly less current was collected than with the 11mercaptoundecanoic acid monolayer, as would be expected from electron-transfer kinetics literature17,26,27 (Figure 3B). As the chain length is increased, the electron-transfer rate decreases logarithmically. If we assume that when a molecule in the SAM accepts an electron it becomes charged and cannot accept another until the first electron is donated to the electrode, we would expect the current to decrease with decreasing electron-transfer rate, which is what was observed. Figure 3C also shows that we were able to effectively insulate the gold electrode with 11mercaptoundecane, which lacks a polar headgroup. Current generation, although small (well less than 1 nA), was measurable and not dominated by capacitive discharge, in contrast with the bare gold. It is reasonable to assume that the number of bacteria binding to the electrode surface could play a significant role in the current collected. Therefore, an experiment was designed where three evaporated gold electrodes on glass were cleaned in piranha (3:1 concentrated sulfuric acid:30% hydrogen peroxide) and rinsed thoroughly. One electrode was left bare, one was modified with 11-mercaptoundecanoic acid, and one with 11-mercaptoundecane. The electrodes were placed in a 0.5 A600 cell suspension for 120 min, gently dipped in minimal medium, and stained with crystal violet. The electrodes were then observed with an optical microscope and cells counted. The two monolayer coated electrodes had comparable number density of cells on the surface even though they produced dramatically different signals. The bare gold had 4 times the number of cells attached than the monolayer coated electrodes and did not exhibit electronic coupling. (26) Adams, D. M.; et al. J. Phys. Chem. B 2003, 107, 6668-6697. (27) Sumner, J. J.; Weber, K. S.; Hockett, L. A.; Creager, S. E. J. Phys. Chem. B 2000, 104, 7449-7454.
Figure 3. Comparisons of current response between a 1-mercaptoundecanoic acid (7.6 nA peak current) modified gold anode with various anode materials including: (A) glassy carbon (3.8 nA), (B) 1-mercaptohexadecanoic acid (0.8 nA), and (C) 1-mecaptoundecane (0.3 nA) modified gold. Note: the glassy carbon electrodes have 56% more surface area than the gold electrodes.
In summary, a microliter-scale MFC was assembled to provide a platform for the investigation of the electronic coupling of Shewanella putrefaciens to different anode materials. The small size and high cell density of the MFC provided rapid current signal onset and shortened experimental run times. Several anode materials were tested including glassy carbon as an example of a completely organic electrode, bare gold and alkane thiol SAM modified gold. Glassy carbon produced no current with killed bacteria but live bacteria showed significant signal. Bare gold electrodes only exhibited a capacitive discharge, while gold modified with a carboxylic acid headgroup produced significantly more current than the glassy carbon anodes. Current collection was decreased significantly when the acid-terminated SAM was replaced with a chain extended by five methylene units and almost
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completely suppressed when the SAM was replaced with one with an identical chain length but terminated with a methyl headgroup. Acknowledgment. The authors thank Dr. Doran Smith and Prof. Lee Harrell for helpful discussions in this research. This research was supported in part by appointments of S.R.C. and
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C.J.S. to the U.S. Army Research Laboratory Postdoctoral Fellowship Program administered by Oak Ridge Associated Universities through a contract with the U.S. Army Research Laboratory. LA061869J
