1 SUPPLEMENTARY MATERIAL 2 3 MICROBIAL ECOLOGY MEETS ELECTROCHEMISTRY: ELECTRICITY 4 DRIVEN AND DRIVING COMMUNITIES 5 6 Korneel Rabaey1,2*, Jorge Rodríguez1, Linda Blackall1, Jurg Keller1, Damien Batstone1, Willy 7 Verstraete2, Kenneth H Nealson4 8 9 Submitted to: The ISME Journal 10 11 1 12 Building, Brisbane, Queensland 4072, Australia 13 2 14 Links 653, 9000 Ghent, Belgium 15 3 16 USA The Advanced Wastewater Management Centre, University of Queensland, Gehrman Laboratory of Microbial Ecology and Technology (LabMET), University of Ghent, Coupure Department of Earth Sciences, University of Southern California, Los Angeles, CA 90089 17 18 *Corresponding author: Dr. Korneel Rabaey, The Advanced Wastewater Management Centre, 19 Gehrman Building (60), The University of Queensland, Brisbane, Queensland 4072, 20 Australia, Tel. +61 7 3365 7519, Fax. +61 7 3365 4726, k.rabaey@uq.edu.au 21 S1 22 S1. Nernst Planck interactions and the relationship to diffusivity 23 It can be assumed that in biofilms and diffusion limited systems, redox shuttles will 24 demonstrate differences in diffusivity through the biofilm based on their redox status. 25 Negatively charged shuttles will be attracted by the more positively charged electrode, while a 26 positively charged shuttle will be repulsed or at least less intensively attracted by the 27 electrode. The influence of these electrostatic interactions on overall diffusivity can be 28 evaluated using well established fundamental theory. 29 30 The Nernst Planck equation provides an adapted diffusivity of compounds, i.e. shuttles, 31 moving to and from differently charged objects (MacGillivray 1968): 32 nF E S J D SH S SH R T x x 33 With J the flux of the shuttle away from the electrode (molSH/m2·s), n the electric charge of 34 the shuttle, D the diffusivity constant (m2/s), F the Faraday’s number (C/mole), SSH the 35 concentration of the shuttle at position x (molSH/m3), E the electrostatic potential (V), R the 36 universal gas constant (C·V/mole·K), T the temperature (K), x distance to the electrode (m). 37 Here the first term applies to the diffusive driving force and the second to the electrostatic 38 driving force. Since the shuttle is the key electrochemically active compound in the potential 39 field it can be assumed that E = ESH. 40 The equation is discretized within the main biofilm model, for a static biofilm model with the 41 differential terms calculated as follows: 42 nF E S J D SH S SH SH R T x x 43 ESH, and SSH are calculated from the difference between the values at layer i and layer 44 i-1. Boundary conditions at the anode and biofilm surface are applied as normal. Taking a S2 45 shuttle similar to phenazine-1-carboxamide as an example, assuming the reduced form of the 46 shuttle as charged -1 and the oxidised as 0, with some estimations for parameters: Parameter Value Explanation D 6.61·10-6 cm2 s-1 Diffusion constant of phenazine-1-carboxamide* T 300 K Temperature pH 7.00 pH SSH 5·10-5 mol L-1 Total shuttle concentration nred -1 Electric charge of reduced shuttle nox 0 Electric charge of oxidized shuttle E01 -0.115 V Standard potential of shuttle at pH 7 47 *the diffusion was calculated according to La-Scalea and co-workers (La-Scalea et al. 2005), 48 using a McGowan Volume of 162.86 cm³/mol (Abraham & McGowan 1987) and of the 49 molecular volume-diffusion correlation of Othmer and Thakar (Othmer & Thakar 1953). 50 51 With these parameters provided, a simulation using a microbial fuel cell model (under 52 development) was conducted. The simulation consisted of a one hour experiment starting with 53 equal concentrations of reduced and oxidised electron shuttle through the biofilm. Once the 54 bioelectrochemical activity starts, the concentrations of reduced and oxidised electron shuttles 55 change with time and between biofilm layers (layers 1 to 10), where reduction of the shuttle 56 by the microorganisms occurs together with its transport. In particular, the transport of shuttle 57 between the last layer (layer 10) and the liquid bulk causes a loss of shuttle to the liquid bulk. 58 In this case no generation of shuttle by the microorganisms was considered to illustrate the 59 different rates of shuttle loss when both Nernst-Planck interactions and diffusion are 60 considered versus the conventional case, where only diffusion is considered. 61 S3 62 Figure S1 to Figure S4 show the results obtained by considering only diffusive driving forces 63 compare the relative retention of the redox shuttle in a 10-layer biofilm over time. The 64 quantitative analysis of the shuttle over time indicates that the difference in retention based on 65 Nernst-Planck interactions is very relevant in terms of the current production and the shuttle 66 distribution along the biofilm. Electrical current generation 0.0135 0.013 Current generation (A) 0.0125 0.012 0.0115 0.011 0.0105 0.01 Only diffusion 0.0095 Diffusion + Nernst-Planck 0.009 0 500 1000 1500 2000 2500 3000 3500 Time (s) 67 68 Figure S1. Modelled current generation by a microbial fuel cell over time, depending on 69 whether Nernst-Planck interactions were considered when calculating the diffusion of redox 70 shuttles. The current generation could be sustained for much longer time periods due to the 71 electrostatic attraction of the shuttle. 72 S4 Electrical Potential at the Biofilm Layer #1 -0.1 Only diffusion -0.11 Diffusion + Nernst-Planck Electrical potential (V) -0.12 -0.13 -0.14 -0.15 -0.16 -0.17 -0.18 0 500 1000 73 1500 2000 Time (s) 2500 3000 3500 74 Figure S2. Modelled evolution of the potential versus standard hydrogen electrode (V) in a 75 biofilm layer adjacent to the anodic electrode. The Nernst-Planck interactions cause a notable 76 increase in stability as redox shuttles are retained better. The initial potential drop follows 77 high bacterial activity and the subsequent generation of negatively charged shuttles, which are 78 electrostatically attracted to the anode. 79 S5 Concentration of Shuttle at the Biofilm Layer #1 2.0E-04 Total electron shuttles concentration (molSH/L) Only diffusion 1.8E-04 Diffusion + Nernst-Planck 1.6E-04 1.4E-04 1.2E-04 1.0E-04 8.0E-05 6.0E-05 4.0E-05 2.0E-05 0.0E+00 0 80 500 1000 1500 2000 2500 3000 3500 4000 Time (s) 81 Figure S3. Modelled evolution of the redox shuttle concentration over time in the biofilm 82 layer adjacent to an anode. Due to the increased attraction of redox shuttles when Nernst- 83 Planck interactions are included in the diffusion calculations, the concentration remains 84 notably higher over time, after an initial rise due to high bacterial activity 85 S6 Concentration of Shuttle at the Biofilm Layer #10 5.0E-05 Only diffusion Diffusion + Nernst-Planck Total electron shuttles concentration (molSH/L) 4.5E-05 4.0E-05 3.5E-05 3.0E-05 2.5E-05 2.0E-05 1.5E-05 1.0E-05 5.0E-06 0.0E+00 0 500 1000 1500 86 2000 Time (s) 2500 3000 3500 4000 87 Figure S4. Modelled evolution of the redox shuttle concentration over time in the top layer of 88 a biofilm. In the case Nernst-Planck interactions are incorporated in the diffusion calculations, 89 due to the high initial bacterial activity, the redox shuttles migrate to lower levels of the 90 biofilm. This causes a limited shuttle concentration in the top of the biofilm, which strongly 91 decreases the efflux of redox shuttles towards the bulk liquid. 92 93 Conclusion 94 The electric charge of the oxidized/reduced shuttles, causes profound differences in diffusion 95 rates and dynamic behaviour of bioelectrochemical systems. For this reason, it is essential to 96 include Nernst-Planck-calculated electrostatic interactions in the MFC model. Presented here 97 is only a limited approach that does not include the production of redox shuttles by bacteria 98 themselves. Experimental validation will have to confirm these modelled results, which do 99 corroborate the findings by several studies, in which the presence of shuttle producing 100 organisms was found, also in continuous systems (Aelterman et al. 2006; Bond & Lovley 101 2005; Rabaey et al. 2005; Rabaey et al. 2004) S7 102 References 103 1. Abraham MH, McGowan JC (1987) The Use of Characteristic Volumes to Measure Cavity 104 Terms in Reversed Phase Liquid-Chromatography. Chromatographia 23: 243-246. 105 2. Aelterman P, Rabaey K, The Pham H, Boon N, Verstraete W (2006) Continuous electricity 106 generation at high voltages and currents using stacked microbial fuel cells. Environ. 107 Sci. Technol. 40: 3388 -3394. 108 3. Bond DR, Lovley DR (2005) Evidence for Involvement of an Electron Shuttle in 109 Electricity Generation by Geothrix fermentans. Appl. Environ. Microbiol. 71: 2186- 110 2189. 111 4. La-Scalea MA, Souza Menezes CM, Ferreira EI (2005) Molecular volume calculation 112 using AM1 semi-empirical method toward diffusion coefficients and electrophoretic 113 mobility estimates in aqueous solution. Journal of molecular structure. Theochem 114 730: 111-120. 115 116 117 118 119 120 5. MacGillivray AD (1968) Nernst-Planck equations and the electroneutrality and Donnan equilibrium assumptions. Journal of Chemical Physics 48: 2903-2907. 6. Othmer DF, Thakar MS (1953) Correlating Diffusion Coefficients in Liquids. Industrial and Engineering Chemistry 45: 589-593. 7. Rabaey K, Boon N, Höfte M, Verstraete W (2005) Microbial phenazine production enhances electron transfer in biofuel cells. Environ. Sci. Technol. 39: 3401 -3408. 121 8. Rabaey K, Boon N, Siciliano SD, Verhaege M, Verstraete W (2004) Biofuel cells select for 122 microbial consortia that self-mediate electron transfer. Appl. Environ. Microbiol. 70: 123 5373-5382. S8