Manipulation of Microbial Extracellular Electron Transfer by Changing

Dec 17, 2012 - Here, the impacts of the substituent group on the reduction potentials of phenazine-type redox mediators in aqueous solution were inves...
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Manipulation of Microbial Extracellular Electron Transfer by Changing Molecular Structure of Phenazine-Type Redox Mediators Jie-Jie Chen,† Wei Chen,† Hui He, Dao-Bo Li, Wen-Wei Li, Lu Xiong, and Han-Qing Yu* Department of Chemistry, University of Science and Technology of China, Hefei 230026, China S Supporting Information *

ABSTRACT: Phenazines, as a type of electron shuttle, are involved in various biological processes to facilitate microbial energy metabolism and electron transfer. They constitute a large group of nitrogen-containing heterocyclic compounds, which can be produced by a diverse range of bacteria or by artificial synthesis. They vary significantly in their properties, depending mainly on the nature and position of substitutent group. Thus, it is of great interest to find out the most favorable substituent type and molecular structure of phenazines for electron transfer routes. Here, the impacts of the substituent group on the reduction potentials of phenazine-type redox mediators in aqueous solution were investigated by quantum chemical calculations, and the calculation results were further validated with experimental data. The results show that the reaction free energy was substantially affected by the location of substituent groups on the phenazine molecule and the protonated water clusters. For the main proton addition process, the phenazines substituted with electrondonating groups and those with electron-withdrawing groups interacted with different protonated water clusters, attributed to the proximity effect of water molecules on proton transfer. Thus, high energy conversion efficiency could be achieved by controlling electron flow route with appropriate substituted phenazines to reduce the biological energy acquisition. This study provides useful information for designing efficient redox mediators to promote electron transfer between microbes and terminal acceptors, which are essential to bioenergy recovery from wastes and environmental bioremediation.

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INTRODUCTION Electrochemically active bacteria have the ability to transfer electrons outside cells to extracellular electron acceptors in their respiratory pathway.1 Various mechanisms of extracellular electron transfer (EET), including direct contact through outer membrane proteins, diffusion of soluble redox mediators, electron transfer through cellular pili, and mediated energy taxis,2 have been proposed.3,4 The involvement of different redox mediators can effectively enhance the EET process.5−7 Phenazines, as one type of redox mediator, are well-known to play an important role in microbial redox processes,8,9 shuttling electrons between microbial cells and external oxidants. They can reduce molecular oxygen in cellular redox-cycling, resulting in accumulation of reactive oxygen species10,11 and promoting reduction of environmental minerals.12 The impacts of phenazine derivatives, both natural and synthesized, on bacterial interactions and metabolisms have been studied.13 In a microbial fuel cell (MFC), phenazines are found to be able to enable a high electrical conductivity of the multilayered biofilm on anode, leading to enhanced electricity generation from bacterial metabolism.14 The redox reaction of phenazines is a type of proton-coupled electron transfer (PCET) reaction,15,16 which is common in many chemical and biological processes, such as enzymatic and photosynthesis reactions.17 For example, both humic substances18,19 and their analogue anthraquinone-2,6-disulfonate (2,6-AQDS)20 possess redox-active moieties quinone groups. © 2012 American Chemical Society

These compounds can serve as exogenous electron shuttles (ESs) for Fe(III) reduction with PCET reactions. The electronaccepting capacity of an ES is determined by its principal reducible moieties, such as para-benzoquinone, ortho-benzoquinone, and nonquinone groups including nitrogen and sulfur functional groups in humic substances.21,22 Since humic substances contain a wide variety of reducible moieties, they can transfer electrons coupled with protons at a wide range of reduction potentials. Phenazines with nitrogen-containing heterocyclic ring structures have a similar function as humic substances and 2,6-AQDS and are also endogenous redoxactive molecules.23 Their electron-accepting capacity depends on the coupled electron−proton transfer, which is known to be associated with the tricyclic heteronuclear organic ring.11 However, information about how phenazines are involved in various electron transfer processes is very limited. Specifically, the characteristics of electron-withdrawing group (EWG) and electron-donating group (EDG) of phenazines, their chemical behavior, and the impact of the functional groups on electron transfer remain unclear so far. The substituent groups on the heterocyclic ring are the main reason for the different physical and chemical properties of the Received: Revised: Accepted: Published: 1033

October 13, 2012 December 13, 2012 December 17, 2012 December 17, 2012 dx.doi.org/10.1021/es304189t | Environ. Sci. Technol. 2013, 47, 1033−1039

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the calculation, an all-electron method within the Perdew− Wang 91 (PW91) form of generalized gradient approximation (GGA)33,34 for the exchange-correlation term was employed, as implemented in the DMol3 code.35,36 The double precision numerical basis sets including p polarization (DNP) were adopted. The energy in each geometry optimization cycle was converged to within 1 × 10−5 Hartree with a maximum displacement and force of 5 × 10−3 Å and 2 × 10−3 Hartree/Å, respectively. The solvent effect of the water medium was described using the conductor-like screening model (COSMO).37,38 COSMO is a continuum solvent model where the solute molecule forms a cavity within the dielectric continuum of permittivity that represents the solvent. Coupled Electron−Proton Transfer. Reduction of phenazine may involve an incomplete 1e−/1H+ process to yield semiphenazine radical or a complete 2e−/2H+ process to form stable dihydrophenazine (Scheme 1),23 such as the

individual phenazines. It has been reported that the nature and position of the substituents significantly affect their redox potential, polarity, and stability8,23 and can drastically influence the electron transfer and electricity generation in MFC.4 Thus, it is of great interests to elucidate the substituent effects of phenazines and to find out the most favorable substituent type and molecular structure of ES that enable efficient electron transfer for environmental bioremediation and energy conversion. To choose suitable ES, electrochemical tests16,23 or MFC operation experiments should be conducted, but they are usually complex, costly, and time-consuming. In contrast, quantum chemical calculations can offer an effective and rapid alternative to screen existing or creating novel ES structures.24 A combined quantum mechanical and molecular mechanical approach has been used to simulate the electron and proton addition reactions of the flavin bound quinone oxidoreductase 2.25 The energetics of electrochemical changes for several substituted flavins were investigated using M06-L density functional, and the functional groups were found to substantially affect the redox potential of flavin molecules.26 All of these studies suggest a high feasibility of applying quantum chemical calculations to explore the phenazine substituent effects. It is known that the versatility of flavin’s chemical behavior is largely ascribed to its unique ability to undergo both sequential (two 1e−/1H+) and simultaneous (one 2e−/2H+) reductions.25−27 Thus, the coupled electron−proton transfer process of phenazines, in which the electron transfer and proton reactions occur in sequence or concurrently, should be considered in the quantum chemical calculations. Another important factor that needs to be considered is the hydrated proton, the structure and properties of which are one of the most fundamental aspects of aqueous chemistry.28 In the water system, the protonated water clusters include hydronium ion (H3O+), Zundel cation (H5O2+),28 H7O3+,29 Eigen cation (H9O4+),28 and larger-sized cations with small clusters as the core species.30−32 The presence of such hydrated proton may also affect the PCET reactions, but this factor has been neglected in the existing calculations for the known PCET reactions.25−27 Therefore, the aim of this work was to explore the substituent effects on the energetics and electrochemical behaviors of several phenazine derivatives in aqueous solution by using a new method of quantum-chemical calculation based on density functional theory (DFT). The changes of free energy and redox potentials during the electron and proton addition processes of phenazine systems were computed. The types of protonated water clusters surrounding phenazines were studied in detail to gain insight into their roles in protoncoupled reductive reactions. Electrochemical experiments were also conducted to verify the calculation results. In this way, a molecular-level insight into the redox elementary reactions of nitrogen-containing heterocyclic compounds could be offered, and useful information for designing new-structure mediators with a low energy loss and high reaction efficiency could be provided.

Scheme 1. Possible Steps of Sequential Electron and Proton Addition Reactions for Phenazines in Aqueous Solution

reduction processes of flavins.25,26 Thus, phenazines have three oxidation states: fully oxidized (Phz), unstable anionic semiphenazine radical (Phz•−), and two electron reduced dianionic species (Phz2−). Each oxidation state has corresponding protonazed form (Scheme 1). Phz•− can quickly transform into a protonated neutral semiphenazine form, PhzH• (Step 2), or undergo another 1e− reduction to form the dianionic species, Phz2− (Step 5). The later process belongs to a concerted proton−electron transfer pathway.39 The anionic hydrophenazine, PhzH−, can be formed from the reduction of neutral semiquinone radical, PhzH• (Step 3), or from protonated Phz2− (Step 6). Because of their highly symmetric structure, the phenazine molecule only has two different monosubstituted positions (i.e., R1 and R2). Therefore, based on the theoretical calculations, the effects of the EWGs and EDGs on the redox potential of phenazines can be evaluated. In addition, the hydrated proton may take the place of isolated proton to undergo the protonated reactions; thus, the reaction and transformation of hydrated protons are also calculated. Electrochemical Experimental Procedures. To validate the calculation results, several substituted phenazines were selected and cyclic voltammetry (CV) was used to determine their redox potential. One selected phenazine was neutral red (NR),40 which has a similar reaction activity as cyanocobalamin for the reduction of hexachloro-1,3-butadiene, perchloroethene, and hexachlorobenzene by anaerobic microorganisms. Another was a planar electron-rich heterocyclic diamine, 2,3-diaminophenazine (DAP).41,42

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COMPUTATIONAL METHODS AND EXPERIMENTAL DETAILS Quantum Chemical Calculations. The structures of substituted phenazine molecules and protonated water clusters in aqueous solution are studied using DFT computations. In 1034

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Table 1. Charge Distribution and Geometry Structure Analysis of the Center Ring in the Minimum Energy Structures of Tricyclic Phenazine Ring phenazine

N13 (e)

N6 (e)

l (N13−C12) (Å)

l (N13−C14) (Å)

θ (C5−N6−C7) (deg)

θ (C12−N13−C14) (deg)

1-COOH 1-CONH 1-COH 1-OH 1-CH3 1-NH2 2-COOH 2-CONH 2-COH 2-OH 2-CH3 2-NH2 NR DAP

−0.328 −0.337 −0.311 −0.404 −0.384 −0.382 −0.363 −0.367 −0.370 −0.370 −0.382 −0.378 −0.377 −0.372

−0.368 −0.372 −0.374 −0.369 −0.381 −0.377 −0.364 −0.369 −0.370 −0.366 −0.378 −0.374 −0.370 −0.372

1.346 1.346 1.348 1.347 1.347 1.349 1.344 1.345 1.343 1.348 1.348 1.349 1.353 1.356

1.342 1.343 1.340 1.338 1.344 1.338 1.348 1.347 1.349 1.347 1.346 1.352 1.356 1.344

117.357 117.290 117.242 117.562 117.367 117.710 117.019 116.975 117.108 117.180 117.078 117.184 117.071 117.213

117.530 117.387 117.510 117.261 117.599 117.676 116.934 116.974 117.007 117.159 117.137 117.247 117.145 117.213

All solutions were prepared from reagent-grade chemicals without further purification. NR, K2HPO4, KH2PO4, and KCl were purchased from Sinopharm Chemical Reagent Co., China. Phenazine-1-hydroxy and DAP were purchased from J & K Tech Ltd., Beijing, China. CVs were recorded using a CHI852C electrochemical workstation (CHI Instruments Co., China). A 2.0-mm diameter Au plate, after being polished (alumina polishing paste), washed, and sonicated, was used as the working electrode. In addition, a Pt counter electrode and an Ag/AgCl reference electrode were employed. The CV tests were conducted in phosphate buffer solution (PBS)-buffered electrolyte (pH = 6.95, containing 0.05 M K2HPO4+KH2PO4, and 0.1 M KCl) at ambient temperature (about 25 °C). Before the tests, the electrolyte was deoxygenated by purging nitrogen. All the potential values were referenced to the standard hydrogen electrode (SHE) (E vs. SHE = E vs. Ag/AgCl + 0.197 V) to permit comparisons with other studies.

from the active center. For the NR molecule, the EDGs of −N(CH3)2, −NH2, and −CH3 attached on the three R2 positions have no significant influence on the charge distribution and geometry symmetry. These results show that the molecular properties of phenazines, including electron transfer thermodynamics and redox potential, are influenced by their molecular structure. Thermodynamic Properties of PCET Reactions. The phenazines reduction reaction may be described as a coupled electron−proton transfer process, in which the electron transfer reactions are rapidly followed or accompanied by proton transfer reactions. The redox potential of the two types of reactions is determined by the thermodynamic driving force, which substantially affects the electron transfer kinetics.15 According to the reaction steps in Scheme 1, the Gibb’s free energy change of semi 1e−/1H+ addition in aqueous solution can be obtained by summarizing Steps 1 and 2:

RESULTS AND DISCUSSION Charge Distribution and Geometry Structure Analysis. The nitrogen atoms (N13, N6) are active sites of the phenazine structure. Thus, the charge distribution and geometry change of the tricyclic ring can significantly affect the electron-accepting process. Table 1 lists the typical geometry parameters and charge distribution of the active sites in the phenazine systems calculated at PW91/DNP basis level. The charge distribution results indicate that the substituent group causes a charge difference in N13 and N6 atoms. The EWGs on the R1 position, such as −COOH, −CONH, and −COH, draw electrons away from the redox active center and lead to less electrons on the N13 atom. For example, the charge of N13 in Phz-1-COOH is −0.328, but that of N6 has a larger absolute value. Thus, the N13 in phenazine with the R1 position substituted EWGs (Phz-1-EWG) is less nucleophilic and much more easily receives electrons. In contrast, the EDGs including −OH, −CH3, and −NH2 push electrons toward the redox active sites, making it more nucleophilic and, therefore, more difficult to receive electrons. The geometry symmetry of the tricyclic nitrogen-containing rings is broken by the substituents. For the Phz-1-EWG systems, the C−N−C angle θ(C12−N13-C14) is larger than the θ(C5−N6-C7). However, the EWGs and EDGs on the R2 position pose less impact, attributed to a longer distance

The Gibb’s free energy change value of the complete 2e−/ 2H+ process can be obtained from the following equation:

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ΔG⊖(1e−/1H+) = ΔG1⊖ + ΔG2 ⊖

(1)

ΔG⊖(2e−/2H+) = ΔG1⊖ + ΔG2 ⊖ + ΔG3⊖ + ΔG4 ⊖ = ΔG⊖(1e−/1H+) + ΔG3⊖ + ΔG4 ⊖ (2)

The second proton addition depends on the solution pH and the pKa of the neutral hydrophenazine. The 2e−/1H+ process is for the deprotonated hydrophenazine. ΔG⊖(2e−/1H+) = ΔG1⊖ + ΔG2 ⊖ + ΔG3⊖ (or = ΔG1⊖ + ΔG5⊖ + ΔG6⊖)

(3)

The pKa values of the protonated species can be calculated from the ΔG⊖ of the proton additional steps: pK a = −ΔG⊖/2.303RT

(4)

For various substituted phenazines and hydrated protons, the aqueous-state free energy changes of the three coupled electron−proton transfer processes are calculated. The results indicate that the PCET reactions for the studied phenazines can spontaneously occur at room temperature and atmospheric pressure (Figure 1). The electrochemical experiments were conducted under neutral conditions (pH = 6.95) at ambient 1035

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instance, the ΔG⊖(1e−/1H+), ΔG⊖(2e−/1H+), and ΔG⊖(2e−/ 2H+) for Phz-1-OH are −83.116, −156.033, and −153.344 kcal/mol, respectively, while the values for Phz-1-COO− are −79.811, −149.982, and −149.877 kcal/mol, respectively. The −COOH group on the site R1 or R2 of phenazine has analogous structure to benzoic acid. The pKa value of benzoic acid is 4.20;44 thus the group of carboxylic acids on phenazine could be easily dissociated into a carboxylate anion at pH 7, for microbial systems usually preferred a neutral condition.45 The thermodynamic properties of PCET reactions are obviously influenced by the protonated water clusters. Figure S1 (Supporting Information) illustrates the energy-minimized structures. Under aqueous conditions in biological systems, hydrogen ions exist in hydrated form. The proximity effect of water molecules may play an important role in proton addition process, attributed to the weak interactions in proton transfer.46 For the same PCET reaction, the free energy of the process with isolated proton is higher than those of other protonated water cluster systems. Along with the increasing size of the water clusters, the free energy of the electron and proton transfer reactions also gradually increases, but the process remains thermodynamically feasible. Furthermore, there is a significant difference in free energy change between the systems of H3O+ and the other three proton forms caused by the proton addition (ΔG2⊖ and ΔG4⊖) (Figure 1a−c). However, such a difference becomes less when larger water clusters are involved in the reactions. Thus, it can be concluded that both the structure of the hydrated proton and substituents substantially influence the thermodynamic performance of phenazines for redox reaction in aqueous solution. Standard Redox Potential. The reduction potentials in aqueous solutions for both one- and two-electron reductions of these phenazines are calculated by DFT methods with COSMO to determine the solvent effect. The standard electrode potential (E⊖ vs. SHE) of the half-reaction is defined as

Figure 1. Standard Gibb’s free energy changes for three electron and proton transfer reactions of the substituted phenazines in aqueous solution with hydrated protons: (a) 1e−/1H+; (b) 2e−/1H+; and (c) 2e−/2H+.

E ⊖ = −ΔG⊖/nF − E H ⊖

(5)

where n is the number of electrons on the left side of the reaction, F is the Faraday constant, which equals 23.06 kcal/ mol V, and EH⊖ is the standard reduction potential of normal hydrogen electrode with a value of 4.28 V. The experimentally measured values of redox potentials for the selected phenazines are obtained from the CVs (Figure 2), and are used to verify the calculated results. The CVs of Phz-1OH in PBS at variable scan rates at pH 6.95 are also obtained (Figure S2, Supporting Information). The detailed analysis of redox potential is described in the Supporting Information. The results show that, as in the case of the free energy of the electron and proton transfer reactions, the redox potentials are also influenced by the functional groups and proton hydration. In addition, the free energy of the second proton addition step (ΔG4⊖) indicates that it is difficult for this step to proceed for most phenazines (Table S1, Supporting Information). For instance, the ΔG4⊖ values for Phz-1-OH, Phz-1-COO− and DAP are 2.689, 0.106, and 1.976 kcal/mol, respectively. The positive values suggest that the second proton addition is thermodynamically nonspontaneous. Although the redox potentials of the first proton addition (ΔG2⊖) are similar to those of the ΔG4⊖, the subsequent electron transfer step has a negative free energy and can spontaneously occur. Thus, the reaction equilibrium will be shifted to drive the forward elementary step. The type of the PCET reaction detected in the

temperature (about 25 °C), which is consistent with the calculation settings under standard conditions (298.15 K, 100 kPa). The detailed free energy changes for these elementary steps of isolated proton system are given in Table S1 (Supporting Information). More substantial changes of free energy can be observed after the electron addition than after the proton addition, suggesting that the free energy change of the overall reduction reaction is dominantly contributed by the electron addition reaction. Furthermore, it can also be seen that the energetics of the two sequential electron addition processes are of a similar level. The free energy is substantially affected by the location of substituent groups on the phenazine molecule (Figure 1). The EWGs, such as −COOH, −COH, and −CONH, make the phenazine less nucleophilic and tend to stabilize anions or electron-rich structures.43 Thus, the free energies of the phenazines with EWGs become more negative, making it easier to receive electrons when added with the same protonated water cluster. The EDGs such as −OH, −CH3 and −NH2 have the opposite effect and can make phenazine molecule more nucleophilic.43 As a result, the free energies of the phenazines with EDGs are higher than those with EWGs. However, it is still negative for the three PCET reactions. For 1036

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Table 2. Comparison between the Computed and Measured (Eexp⊖, at pH 7.0) Reduction Potentials (vs. SHE, V) of Phenazine Derivatives in Aqueous Solution phenazine Phz-1-OH

NR

DAP

Figure 2. CVs of the reduction of 0.2 mM phenazines in PBS (pH 6.95) recorded at a 2 mm planar Au electrode with a scan rate of 100 mV/s.

Phz-1-COO−

CV experiments can be identified by comparing the calculation results with the measured data. The incomplete 1e−/1H+ process of yielding the unstable radical semiphenazine (PhzH•) is illustrated in Scheme 1. Thus, it is difficult to experimentally detect the PhzH• formation process. Moreover, with the value of ΔG4⊖ (Table S1), the pKa of these dihydrophenazines can be calculated from eq 4. Here, pKa is less than 7, suggesting that all these dihydrophenazines would be substantially deprotonated at neutral pH. Therefore, the 2e−/1H+ reaction that produces the anionic hydrophenaiznes should be the type of PCET reaction detected in this case. The data in Table 2 suggest that the PCET reactions for the systems of phenazines substituted with EDGs may undergo the proton addition step with the protonated water cluster of H3O+. For instance, the calculated reduction potential of NR for the 2e−/1H+ process with H3O+ (−0.334 V) agrees well with the experimental values. However, the phenazines substituted with EWGs may mainly seize the proton from the protonated water cluster of H5O2+. For example, the calculated value of Phz-1-CONH2 is −0.154 V and is in good agreement with the experimental results reported by Wang and Newman.23 Therefore, our results imply that, for the 2e−/1H+ reactions, the phenazines substituted with EDGs and EWGs mainly undergo proton addition with H3O+ and H5O2+, respectively. The reduction potentials of phenazines with different ring substituents are shown in Figure 3. The presence of EWGs reduces the electron density of phenazine ring and makes it liable to stabilize the electron-rich structures. Thus, the EWGs in phenazine make it easier to receive electrons and increase the reduction potential for the 2e−/1H+ process. In contrast, EDGs show the opposite effect. The introduction of EDGs, such as −N(CH3)2, and −CH3, onto the R2 site of NR lowers the redox potential (Table 2). Energy Conversion Efficiency. The redox potential has important implications for redox activation and reactivity.15 The standard potential of the mediators (linking species), E⊖(link), should ideally be close to the redox potential of the primary substrate, E⊖(sub), as possible, or at least be significantly negative compared to that of the terminal

Phz-1CONH2

a

protons +

H H3O+ H5O2+ H7O3+ H9O4+ H+ H3O+ H5O2+ H7O3+ H9O4+ H+ H3O+ H5O2+ H7O3+ H9O4+ H+ H3O+ H5O2+ H7O3+ H9O4+ H+ H3O+ H5O2+ H7O3+ H9O4+

E⊖ (2e−/ 2H+)

E⊖ (2e−/ 1H+)

−0.955 1.046 0.387 0.244 0.039 −1.474 1.027 0.236 0.094 −0.111 −1.405 0.904 0.245 0.102 −0.103 −1.030 1.426 0.767 0.624 0.419 −0.863 1.310 0.651 0.508 0.303

−0.897 −0.108 −0.437 −0.509 −0.611 −1.497 −0.334 −0.730 −0.801 −0.904 −1.362 −0.265 −0.594 −0.665 −0.768 −1.028 0.186 −0.144 −0.215 −0.317 −0.750 0.176 −0.154 −0.225 −0.327

Eexp⊖ −0.17423 −0.152a

−0.32540 −0.345a

−0.281a

−0.11623 −0.17751

−0.14023 −0.11552

From CV experiments in this study according to Figure 2.

Figure 3. Redox potentials in aqueous solution for the two-electron (2e−/1H+) reductions in the electron and proton transfer reactions of substituted phenazines as shown in Scheme 1, E⊖(sub), E⊖(link), and E⊖(acc) are the redox potentials of primary substrates, electron shuttles, and terminal acceptors, respectively.

acceptors, E⊖(acc).4 In the electron transfer pathway, the biological energy acquisition could be defined as4 ΔΔG biol ⊖ = −nF[E ⊖(link) − E ⊖(sub)]

(5)

Depending on the different primary substrates for microorganisms, the value of E⊖(sub) may vary significantly. For example, the E⊖ values are −0.43 V for CO2/glucose, −0.28 V for CO2/acetate, and −0.19 V for pyrovate/lactate reactions.47 Thus, the appropriate linking species could be selected for specific systems to lower the energy acquisition for the microorganism. For example, for the oxidation of pyrovate, 1037

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phenazines substituted with 1-NH2, 1-CONH2, and 2-CONH2 could be appealing ESs to lower the energy loss. However, the Phz-2-NH2 and Phz-2-COO− are incapable of electronically linking microbial cells and terminal acceptors because their E⊖(link) are lower than E⊖(pyrovate/lactate). For a specific exogenous oxidant (i.e., external terminal electron acceptor), the energy output can be calculated from the following equation4 ΔΔGout ⊖ = −nF[E ⊖(acc) − E ⊖(link)]

AUTHOR INFORMATION

Corresponding Author

*Fax: +86 551 63601592. E-mail: [email protected]. Author Contributions †

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The numerical calculations were performed on the supercomputing system in the Supercomputing Center at the University of Science and Technology of China. The authors wish to thank the Fundamental Research Funds for Central Universities (WK2060190007) and Shanghai Tongji Gao Tingyao Environmental Science and Technology Development Foundation (STGEF) for the partial support of this work.

(6)

Under ambient environmental conditions, phenazines exhibit sufficiently low reduction potentials (E⊖(link)) to enable thermodynamically feasible redox reactions between reduced phenazines and terminal electron acceptors (Figure 3). For instance, E⊖(NO3−/NO2−) is 0.43 V and E⊖(Fe3+/Fe2+) is 0.77 V.47 Such a low E⊖(link) implies that, for a specific electron flow route, it is possible to lower the biological energy acquisition and increase the energy conversion efficiency by choosing or creating an appropriate ES. Significance of This Work. The quantum chemical calculation is demonstrated to be an effective method to explore the substituent effect as well as the energetics and electrochemical behaviors of phenazines. The redox active group of ESs can take the form of diketone as in the case of quinones such as 2-amino-3-carboxy-1,4-naphtho-quinone,48 and heteroatoms contained within the heterocyclic system such as nitrogen in the case of phenazines.23 Thus, the exogenous redox mediators could be designed by mimicking the structures of their active sites for tuning the reduction potentials of phenazine-type redox mediators. Furthermore, two or more substituents could be added simultaneously to phenazines to adapt to specific electron transfer pathways. The direct use of an artificial redox mediator is not a feasible approach,4 but many electrochemically active microorganisms can produce mediators via secondary metabolic pathways by themselves under environmental stress,49,50 and immobilization of redoxactive molecule on electrode is found to effectively promote the microbial EET by our group (data not shown). The results of this work imply that efficient electron transfer from microorganisms to terminal acceptors can be achieved by selecting or designing phenazines with suitable substituent groups, and that the protonated water cluster substantially affects the reduction potential of phenazines. These results also facilitate a better understanding about the electron shuttling processes by redox mediators, and provide useful guidance for designing efficient derivatives of linking species, which may govern the electron transfer routes and provide new possibilities for microbeassociated applications in the fields of environmental bioremediation and bioelectrochemical systems.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Discussion about redox potential analysis from CV experiments, standard Gibb’s free energy of the elementary protoncoupled electron transfer reactions of phenazines in aqueous solution, energy-minimized structures of protonated water clusters, and CVs of Phz-1-OH in PBS (pH = 6.95) at variable scan rates. This information is available free of charge via the Internet at http://pubs.acs.org/. 1038

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Environmental Science & Technology

Article

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