Research Carbon and Steel Surfaces Modified by Leptothrix discophora SP-6: Characterization and Implications T U A N A N H N G U Y E N , † Y U Z H U O L U , †,‡ XINGHONG YANG,§ AND X I A N M I N G S H I * ,† Corrosion, Electrochemistry & Analysis Laboratory (CEAL), Western Transportation Institute, PO Box 174250, College of Engineering, Montana State University, Bozeman, Montana 59717-4250, School of Materials Science and Engineering, Tianjin University, Tianjin 300072, P.R. China, and Veterinary Molecular Biology Department, College of Agriculture, 960 Technology Boulevard, Montana State University, Bozeman, Montana 59718-4000
Received May 19, 2007. Revised manuscript received August 20, 2007. Accepted September 19, 2007.
Leptothrix discophora SP-6, a type of manganese(Mn)oxidizing bacteria, has been known to accumulate Mn oxides from the aqueous environment and thus play a key role in microbiologically influenced corrosion by increasing the electrochemical potential of steel and other metals. Similarly, this bacterium was found to modify the surface of glassy carbon in aqueous solution and increase its potential (i.e., ennoblement). In the latter case, biomineralized Mn oxides can be used as cathodic reactants for a new generation of microbial fuel cells featuring a biocathode. In this preliminary study, factors affecting the biofilm formation and biomineralization processes were examined. The inflow of air into the culture medium was found essential to sustain the ennoblement of substrate electrodes. The OCP and FESEM/EDS data indicated that a smoother initial substrate surface generally led to better ennoblement. Polarizing the carbon electrode at +500 mVSCE for 15 min was found to facilitate the ennoblement on carbon electrodes, and so did coating it with a poly(L-lysine) layer. Independent of substrate material, initial surface roughness, and pretreatment, there were three parameters in the EIS equivalent circuit that correlated well with the OCP indicating the level of ennoblement by L. discophora SP-6, i.e., electrolyte resistance, double-layer capacitance, and low-frequencies capacitance. These fascinating findings merit further investigation as they may shed light on the fundamental bacteria/substrate interactions and help advance the knowledge base needed for the engineering applications.
Introduction Microbial fuel cells (MFCs) present an attractive pathway to directly convert chemical energy into electrical energy and have been extensively investigated in the past several decades. Numerous studies have focused on microorganisms * Corresponding author phone: 1-406-994-6486; fax: 1-406-9941697; e-mail:
[email protected]. † College of Engineering, Montana State University. ‡ Tianjin University. § College of Agriculture, Montana State University. 10.1021/es071178p CCC: $37.00
Published on Web 10/26/2007
2007 American Chemical Society
to work as a biocatalyst for the anodic reaction of the MFC, generating electricity via degradation of organic matter. Until recently, biocathode MFCs have been a relatively unchartered territory. The microbial colonization of passive metals has been reported to increase their open circuit potential (OCP) to final values between +200 mVSCE (1) and +450 mVSCE (2, 3) through a series of reactions collectively termed ennoblement. Initial studies have explained ennoblement by a wide variety of mechanisms (4): microbially generated protons near the surface (5); microbially generated hydrogen peroxide, possibly combined with low pH (6); microbially produced organometallic catalysts of oxygen reduction (1, 3); specific enzymes (7); and passivating siderophores (8). A number of studies have attempted to establish a direct link between biofilm formation and ennoblement. Ennobled potential has been correlated with cell density (9) and with biological activities in the biofilms by measuring the accumulation of adenosin triphosphate (ATP) (10) or electron transport activity and lipopolysaccharide content (11). Nonetheless, the structural heterogeneity of the biofilm may also affect its activities and mass transfer dynamics, and thus the OCP of its substrate (12). Recent studies have linked the ennoblement of passive metals with the activity of manganese-oxidizing bacteria (MOB), such as the freshwater Gram-negative Leptothrix discophora (4, 13). As an essential micronutrient for all living organisms, manganese (Mn) is Earth’s second most abundant transition metal, next to iron (14). In natural waters, soluble Mn(II) can reach up to millimolar concentrations, even in the presence of oxygen (14), and biological catalysis has been well established as the dominant mechanism of oxidizing Mn(II) to insoluble Mn(III, IV) oxides in circumneutral freshwater (15, 16). It was proposed that the microorganisms first oxidize Mn(II) to a soluble or enzyme-complexed Mn(III) intermediate, then subsequently to Mn(IV) oxides primarily a phyllomanganate most similar to δ-MnO2 or acid birnessite (14). A reasonable reaction concomitant with the bacterial Mn(II) oxidation reactions is the reduction of oxygen to water as shown in eq 1. O2 + 4H+ + 4e- f 2H2O
(1)
Biomineralized manganese oxides can be used as cathodic reactants significantly superior to oxygen. First, they are some of the strongest oxidizing agents (next to oxygen) found in nature. In many natural waters, microbial activities lead to the formation of particulate Mn oxides with average oxidation states exceeding 3.4 (14). Second, the reduction of manganese oxides deposited by L. discophora increased the potential of a MFC by approximately 300 mV and delivered a current density up to 2 orders of magnitude higher, compared with those reached with the reduction of oxygen (17). More importantly, manganese oxides are in direct contact with the electrode, where the cathodic reaction is thus not limited by the mass transfer process of cathodic reactants. Finally, manganese oxides are renewable. When used as a cathode for the MFC, manganese oxides are reduced to Mn(II), which in return is microbially reoxidized. This sequence of events produces renewable cathodic reactants, MnOOH and MnO2, in direct contact with the electrode (4). Mn(II)-oxidizing microorganisms, primarily bacteria and fungi, are ubiquitous in nature and play an integral role in the biogeochemical cycling of manganese, iron, nitrogen, carbon, sulfur, and trace metals (18). They accelerate the VOL. 41, NO. 23, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Biominerals on water-immersed metal surfaces also play a key role in microbiologically influenced corrosion (MIC) and the biomineralization by L. discophora and related metal/ microbe interactions have been mostly examined in this context (4, 19, 21, 22). Shi et al. studied the Mn biomineralization by L. discophora SP-6 (4) and argued that the following electrochemical half-reactions are responsible for the ennoblement of the electrochemical potential of 316L stainless steel and a low-iron titanium alloy (Ti–6Al–4V): MnO2 + H+ + e- f MnOOH E0 ) + 0.81 VSCE
(2)
MnOOH + 3H+ + e- f Mn2+ + 2H2O E0 ) + 1.26 VSCE (3) With the overall reaction shown in eq 4, the half-cell equilibrium potential of a noble cathode (such as graphite) covered with biomineralized Mn oxides could reach +0.36 VSCE: MnO2 + 4H+ + 2e- f Mn2+ + 2H2O E0 ) + 1.28VSCE, i.e., E′pH)7.2 ) + 0.36 VSCE(4) FIGURE 1. Schematic of the novel MFC, consisting of a sacrificial anode of aluminum alloy and a cathode of porous graphite covered by manganese dioxide and L. discophora biofilms. rate of Mn biomineralization several orders of magnitude faster than either abiotic catalysis on mineral surfaces or homogeneous oxygenation in aqueous solution (18). This was demonstrated by in situ immersion of 316L stainless steel into a freshwater creek, where the OCP of steel was increased by 350 mV in 30 days resulting from Mn biomineralization (19). MOB are likely to survive cold temperatures or other relatively harsh conditions in the field environment. For instance, the “Wild-type” L. discophora are mostly the sheathforming strain SP-6, which can be maintained indefinitely in slow growing conditions at temperatures between 20 and 25 °C. For culture management, it can be stored at 4 and -80 °C (20). Therefore, it is envisioned that the ennoblement phenomena can be utilized for a new generation of MFCs featuring a biocathode that involves L. discophora or other MOB. Such MFCs that harness the native population of bacteria abundant in natural waters could potentially be made self-sufficient with simply the pretreated electrodes and nutrients continuously available in the environment. As such, they would be ideal for a host of autonomous applications that demand a minimum amount of maintenance (e.g., monitoring water quality in lakes and streams), which is the focus of an ongoing research project funded by the National Academies. Figure 1 illustrates the conceptual design of the novel MFC, consisting of a sacrificial anode of aluminum alloy and a cathode of porous graphite covered by manganese dioxide and L. discophora biofilms. Unpublished results in our laboratory demonstrated the feasibility of this concept at the laboratory-bench scale. Within this context, it is crucial to investigate how various factors would influence the ennoblement of surfaces by L. discophora and thus the performance of novel MFCs. The ultimate goal is to maximize the energy output and reliability of a biocathode utilizing the L. discophora biofilm. For instance, initial surface roughness is a factor that can be controlled to optimize how well the bacteria initially attach to the substrate and form the biofilm and to minimize the biofilm detachment as well. Cell density and dissolved oxygen concentration are two factors that may evolve during the ennoblement and affect the adhesion, growth, and biomineralization processes of L. discophora. 7988
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The reversal of eq 4, i.e., the Mn(II) oxidation to Mn(IV) oxide, can be balanced with the oxygen reduction shown in eq 1 and lead to the following reaction: Mn2+ + 1/2O2 + H2O f MnO2 + 2H+
(5)
In addition to the practical applications such as MIC control and MFCs, the ennoblement by L. discophora is also a phenomenon of intellectual importance. The mechanistic details of biofilm formation and biomineralization by MOB are still not fully unraveled. A recent review of three MOB species suggested that in all of them proteins containing the highly conserved Cu-binding ligands characteristic of multicopper oxidases seemed to participate in the oxidation process (23). L. discophora has been known to oxidize Mn(II) and Fe(II) during its stationary growth phase (24, 25). Both c-type hemes and multicopper oxidase are believed to play a role in Mn(II) oxidation, whereas Mn(II)-oxidizing factors are speculated to be secreted as part of a complex originating from sheath remnants of L. discophora (23). In this work, we examined the influence of electrochemical polarization on the ennoblement of carbon and steel surfaces by L. discophora SP-6. Biomineralization by L. discophora has a physiological function, which may be supplying energy for growth, scavenging of harmful oxygen species, or serving as terminal electron acceptor that supports anaerobic growth. The vital processes that bacteria use to capture, store, and mobilize energy involve electron transport and catalysis of redox reactions and therefore, the hypotheses to be tested include the following. (1) Polarizing the substrate material may alter the ennoblement processes; and (2) Coating an electroactive polymer on the substrate material may alter the ennoblement processes. Such alternations are expected to occur by changing the metabolic pathways of L. discophora; affecting its growth kinetics; affecting the attachment and detachment behaviors of L. discophora; changing the pathways of Mn-oxidization by L. discophora; and/or changing the products of Mnoxidization by L. discophora. For instance, polarizing the inert substrate cathodically is expected to “pump” electrons to the electrode surface and thus promote the reactions shown in eq 3 or 4 while hindering the Mn(II) oxidation. It is also speculated to potentially inhibit the secretion of Mn(II)oxidizing factors, slowing down the bacterial growth by consuming available oxygen, accelerating the biofilm aging and detachment, or reducing the existing biogenic oxides. Polarizing the inert substrate anodically, on the other hand, may affect the ennoblement processes in an opposite manner.
FIGURE 2. OCP evolution during ennoblement by L. discophora SP-6, as a function of initial surface roughness: (a) on glassy carbon electrodes; and (b) on stainless steel electrodes. Modification of substrate surface by coating with an elecconnected to a computer via an 8-channel multiplexer to the troactive polymer is speculated to have combined effects of OCP monitoring and electrochemical impendance specreducing the initial surface roughness and electrochemical troscopy (EIS) measurements. The reactor and its accessories polarization of the substrate. were then placed in the plastic custom-made glovebox (60 In this preliminary study, we used the electrochemical, cm × 60 cm × 120 cm) that had been sterilized by UV light microbiological, and surface analytical tools to achieve a for 24 h. Prior to inoculating the reactor with L. discophora, better understanding of the influential factors in the enthe coupons were immersed in the culture medium for 24 h noblement of carbon and steel surfaces by L. discophora to make sure that the OCPs were stabilized. SP-6. Such knowledge is essential for us to better control the Biofilm Preparation Procedures. To grow the MOB we capabilities of the bacterium to form a healthy and reliable prepared 15 L of American type Culture Collection (ATCC) Mn-oxidizing biofilm. The factors investigated include disculture medium 1917 MSPV (similar to the one used in ref solved oxygen concentration, substrate material (glassy (4)). The nutrient solution was autoclaved on the liquid setting carbon vs carbon steel), initial surface roughness, electroat 123 °C and 1.2 atm for 25 min. After cooling the solution chemical polarization, and substrate pretreatment by an to room temperature, we aseptically added 15 mL of filterelectroactive polymer. In addition, the evolution of cell sterilized vitamin solution, as required by the MSPV medium density during the ennoblement was examined. (17), 60 mL of filter-sterilized 50 mM manganese sulfate solution, and 75 mL of filter-sterilized 20% sodium pyruvate Materials and Methods solution. All chemicals were from Fisher Scientific. Materials and Reactor. Glassy carbon (GC-20SS grade, The bacterium, L. discophora SP-6 (ATCC 51168) was thickness 3 mm, #800 finish) and 304 stainless steel were purchased from ATCC and stored at -80 °C before use. To purchased from Tokai Carbon USA Inc. (Hillsboro, OR) and inoculate the reactor, we poured 2 L of the MSPV medium Metal Supermarkets (Boise, ID), respectively. To make test with vitamins, sodium pyruvate, and manganese sulfate into coupons, both materials were cut from larger sheets into reactor. All the stock bacteria L. discophora SP-6 grown on small size of about 1 × 1 cm. The unexposed side of each one agar plate for over 12 h were then added in the aerated coupon was then electrically connected to a copper wire by reactor. using Ag-conductive glue, followed by sealing with epoxy Cell Density Evaluation. The cell density in culture resin (MAS epoxies-FLAG). After the curing of the epoxy medium was measured by using a spectrophotometer sealer, the coupons were polished to provide a specific level BioState 3 (Thermo Spetronic). The cell density was defined of uniform surface roughness. They were wet-sanded with as number of bacteria per milliliter and characterized in terms tap water on the metallographic grinding disk covered with of UV absorption at 600 nm, i.e., optical density (OD600). silicon carbide papers of decreasing grit sizes: 200, 400, 600, Electrochemical Measurements. Electrochemical mea800, and 1000. After sanding at each grit size, the holder and surements were conducted using a three-electrode system. coupon were rinsed with running tap water to remove any The OCP of 8 coupons (working electrodes) were monitored remaining grit. The last step in cleaning the surface was to simultaneously by using a Gamry Electrochemical Multiplexer sonicate the coupons in distilled water. The electrodes were ECM8, which was also used periodically to measure the EIS then rinsed with ethanol and mounted in a silicone stopper. of each coupon. The counter electrode used was a curved The reactor was of a glass bowl type, 10 cm tall, and 30 platinum wire placed in the middle of the reactor, whereas cm in top-diameter. Eight electrodes were mounted to their the reference electrode was a saturated calomel electrode holders, which were attached to the top of the reactor. Plastic (SCE) connected to the reactor through a salt bridge. For EIS tubes were mounted in the reactor to aerate the culture measurement, the working electrode was polarized at ( 10 medium using an air pump (Elite 799; with 900 mL/min rate). mV around its OCP. Frequency was ranging between 10 KHz Pall-Gelman sterile mini-capsule filters were attached to these and 10 mHz, with 10 points per decade. We used the Gamry tubes to prevent contamination of the reactor. A magnetic analysis software for plotting and fitting the EIS data. bar was placed at the bottom of the reactor to provide stirring. Surface Analyses. Before and after the ennoblement The reactor was autoclaved on the dry setting (via depresexperiment, surface analytical tools were used to examine surization method) at 123 °C and 1.2 atm for 30 min. Once the surface of glassy carbon and stainless steel coupons. In cooled to room temperature, the reactor was then sealed addition to visual inspection under a digital microscope, field with silicon gel. To study the effect of pumping on the cell emission scanning electron microscopy/energy-dispersive density in culture medium, two variable flow pumps (Fisher X-ray spectrometry (FESEM/EDS) investigation was conScientific) were used to pump the culture medium in and ducted. To analyze the surface of the ennobled coupons, the out the reactor, respectively. coupons were mechanically removed from the holders, airPrior to the experiment, all electrodes assembly and dried, and kept in a vacuumed desiccator for up to 48 h prior reference electrode were sterilized by 99% ethanol and then VOL. 41, NO. 23, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 3. SEM image of glassy carbon electrodes: (a), (b), (c) before ennoblement, with initial surface roughness of # 800 (as received), #600, and #200, respectively; and (a2), (b2), (c2) after ennoblement. to FESEM/EDS analysis to ensure dryness. The biofilms on these coupons were preserved by coating with a thin carbon layer (
