An electrical model able to decouple the electron pathway from microbial cell machinery impedance terms is introduced. In this context, capacitance characteristics of the biofilm are clearly resolved. In other words, the model allows separating, according to the advantage of frequency and spectroscopic response approach, the different terms controlling the performance of the microbial biofilm respiratory process and thus the directly related electricity production process. The model can be accurately fitted to voltammetry measurements obtained under steady-state conditions and also to biofilm discharge amperometric measurements. The implications of biological aspects of the electrochemical or redox capacitance are discussed theoretically in the context of current knowledge with regard to structure and physiological activity of microbial Geobacter biofilms.Fil: Bueno, Paulo R.. Universidade Estadual Paulista. Institute of Chemistry; BrasilFil: Schrott, Germán David. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigación En Ciencia y Tecnología de Materiales (i); Argentina. Universidad Nacional de Mar del Plata. Facultad de Ingeniería; ArgentinaFil: Bonanni, P. Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigación En Ciencia y Tecnología de Materiales (i); Argentina. Universidad Nacional de Mar del Plata. Facultad de Ingeniería; ArgentinaFil: Simison, Silvia Noemi. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigación En Ciencia y Tecnología de Materiales (i); Argentina. Universidad Nacional de Mar del Plata. Facultad de Ingeniería; ArgentinaFil: Busalmen, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigación En Ciencia y Tecnología de Materiales (i); Argentina. Universidad Nacional de Mar del Plata. Facultad de Ingeniería; Argentin

Bonanni, P. Sebastian

Bueno, Paulo R.

Busalmen, Juan Pablo

Schrott, Germán David

Simison, Silvia Noemi

ChemSusChem

An electrical model able to decouple the electron pathway from microbial cell machinery impedance terms is introduced. In this context, capacitance characteristics of the biofilm are clearly resolved. In other words, the model allows separating, according to the advantage of frequency and spectroscopic response approach, the different terms controlling the performance of the microbial biofilm respiratory process and thus the directly related electricity production process. The model can be accurately fitted to voltammetry measurements obtained under steady-state conditions and also to biofilm discharge amperometric measurements. The implications of biological aspects of the electrochemical or redox capacitance are discussed theoretically in the context of current knowledge with regard to structure and physiological activity of microbial Geobacter biofilms

Schrott, Germán D.

Bonanni, Pablo S.

Simison, Silvia N.

Busalmen, Juan P.

Acervo Digital da Unesp

Biochemical capacitance of geobacter sulfurreducens biofilms

Paulo R. Bueno

Germán D. Schrott

Pablo S. Bonanni

Silvia N. Simison

Juan P. Busalmen

Crossref

Biochemical Capacitance of<i>Geobacter Sulfurreducens</i>Biofilms

LAReferencia - Red Federada de Repositorios Institucionales de Publicaciones Científicas Latinoamericanas

English

CONICET Digital

Supporting InformationBiochemical Capacitance of Geobacter SulfurreducensBiofilmsPaulo R. Bueno,*[a] Germn D. Schrott,[b] Pablo S. Bonanni,[b] Silvia N. Simison,[b] andJuan P. Busalmen[b]cssc_201403443_sm_miscellaneous_information.pdf  S.1. Geobacter Biofilm electrical model Typically, electricity production by Geobacter biofilms is continuum if cells respiration is not disrupted by electrode depolarization, but it is to note indeed that biofilm cells are able of respiration at a certain level even after electrode fully depolarization, a fact attributed to electron storage in the haem groups of the vast amount of exocytoplasmic cytochromes found in this bacterial strain.[1] Since pioneer works, DC current electrochemical techniques have been particularly useful in exploring potential dependent properties of Geobacter biofilms.[2] The approach, identified as Electrochemical Capacitance Spectroscopy (ECS)[3], a method derived from Electrochemical Impedance Spectrocopy (EIS), is particularly useful in analyzing electron transfer kinetics and redox density of states in molecular scale and redox confined films, and we use it here for the first time to analyze the Geobacter biofilm/electrode interface. Between DC techniques, the cyclic voltammetry has been by far the most informative one by showing the gating effect of cytochromes in electricity production and the oxidative catalysis feature of the microbial colony over acetate consumption[4]. Complementary, chronoamperometry in combination with numerical modeling have provided insights into charge accumulation inside biofilms,[1b] while gate measurements and light spectroscopy techniques have been cooperative in confirming the existence of potential driven redox gradients across their thickness.[5] AC current techniques on the other hand, have the potential to inform about frequency dependent properties of the biofilm/electrode interface, but most of this potential remains unexploited, since only a few works[6] have explored the use of EIS, providing estimations of biofilm capacitive and/or conductive elements properties, without integrating the whole electrochemical response of biofilms in a single model. A step forward in the analysis of impedance spectroscopy data is through its phasorial conversion to complex capacitance data.[3a, 7] In doing this, problems related to potential dependence in the behavior of charge transfer (resistance) and charge accumulation (capacity) processes are readily resolved.[3a, 7] For biofilms grown under fuel saturation as in the case here, the porous model can be simplified as a transmission line of just one conductive channel (𝑍𝑐 in Figure 1b) and the general impedance of the system is then appropriately given by: 𝑍 =  𝜆𝑍𝑐coth (𝐿/𝜆),          (1) where 𝜆 = (𝑐/𝑍𝑐)1/2, with 𝑐 representing the impedance of each individual cell (Figure 1b) and where L is the active thickness of the biofilm (typically around 50 m).[8] 𝑐  can be divided into equivalent circuit terms named 𝑅𝑐𝑡, 𝐶𝑟 and 𝑅𝑡, as in the parallel and series configuration shown in Figure 1c. 𝑅𝑐𝑡 is a charge transfer resistance (𝑅𝑐𝑡 = 𝑟𝑐𝑡𝐿) while 𝐶𝑟 is an electrochemical or redox capacitance (𝐶𝑟 = 𝑐𝑟𝐿); both in series represent the balance of energy (loss and storage, respectively) during the microbial individual activity in metabolizing the electron source and are parallel to 𝑅𝑡 = 𝑟𝑡𝐿, a recombination resistance that acts as a shunt to the 𝑅𝑐𝑡 and 𝐶𝑟 serial process. It may be noted that differing from the classical picture, the electrochemical meaning and mechanical statistic description of these circuit elements are here as function of the electrochemical potential and in turn, of the biofilm energy state or respiratory level, thus appropriately representing potential gradients existing inside biofilms[5]). It is important to highlight that in spite of impedance parameters involved in Eqn. (1), in the present work we focus only on the physical/physiological meaning of 𝐶𝑟. This capacitive feature of the presented model can be obtained by fitting impedance spectral data to Eqn. (1), or either more easily by graphical analysis of capacitive Bode diagrams at lower frequency, as exemplified in Figs. S1b and S1c (see details below and in S2 section on how to construct such ECS diagrams from EIS data). In the limit of lower frequency (i.e. when the electron states at electrode are in equilibrium to those of redox exocytoplastic cytochromes) the respiration process is entirely controlled by the energy involved with 𝐶𝑟. Accordingly, 𝐶𝑟 is the truly relevant parameter to thermodynamic model discussed in the main text. In controllably moving electrode potential to different energetic levels, but keeping the equilibrium between them and those redox levels (or respiration level) associated with 𝐶𝑟, we observe that a Boltzmann statistics is obeyed. In summary, although impedance/capacitance spectroscopy analysis were key to identify the physical/physiological meaning and potential dependence of 𝐶𝑟 in the context of electrogenic Geobacter biofilms, once 𝐶𝑟 function is known (Eqn. 6 in the main text), it can be either and easily investigated and quantified performing voltammetric or chronoamperometric methods. In physical chemistry terms, 𝐶𝑟 is not the classical or electrostatic (geometric) capacitance, as frequently used in impedance modelling studies by following the general knowledge, but instead it is an electron chemical capacitance[9] that has a dependence with electrode potential (see Eqns. 5 and 6). 𝐶𝑟 energetic donor/acceptor states are associated with reduced and oxidized chemical states[9-10] in the biofilm, which arise specifically from the coupling of a supporting electrode density of states  to the redox sites molecular density of states. In Geobacter biofilms operating in their maximum current performance, capacitive contribution dominates the electron transfer process and negligible (or none) dc current is present (Figure S2a). It is resolved experimentally by EIS analysis as shown in Figure S1a, where typical Nyquist diagrams reveal the absence of an effective dc conductivity (when scanned or perturbed externally) at lower frequencies. This response is appropriately modelled in the equivalent circuit presented here by 𝑅𝑡 (as to be detailed elsewhere) that acts as an ideal isolator (i.e., 𝑅𝑡 → ∞) in the maximum respiratory level of the biofilm, thus hiding external access from the electrode to biofilm internal faradaic process. As clearly shown by results presented in Figure S1b, ECS has advantages over traditional EIS in specifically detecting 𝐶𝑟, revealing the coupling between the electrode and cytochrome redox sites in the biofilm.[3, 7] As external cytochromes are the natural electron sink for the respiratory process, ECS thus offers an unprecedented opportunity to explore for the first time and noninvasively the energetic internal path of the bacterial cell machinery, indeed revealing the cost of biological energy conservation (see main text). According to the model presented here (Figure 1c) the internal charge transfer resistance associated to 𝐶𝑟 is 𝑅𝑐𝑡 , both representing a typical redox process that, operating on the microbial colony in isolation from the external perturbation, may be energetically feed internally (in terms of electrochemical driving force) by the work performed by the colony on available (bio)chemical energy. In this context, 𝑅𝑐𝑡 is viewed as a measure of the average energy loss of the bioenergetic process, i.e. as the electric analogue of the biological energy spent during the respiration process exclusively related to transfer internalized electrons to exocytoplasmatic cytochromes (note the energy lost in the electron transport outside exocytoplasmatic membranes to electrode is though not contained on this term). As previously stated, 𝐶𝑟 is the energy storage element of 𝑐 and is of particular importance for the analysis of electrogenic biofilms, because of this energetic particular feature. The capacitive effects on kinetics is found at intermediate to lower frequencies (from 10 Hz to 5 mHz) in EIS/ECS diagrams (Figure S1), from which a time scale of  10 s can be obtained as 𝜏 = 𝑅𝑐𝑡𝐶𝑟 (see Figure S1d), for the occupancy process of redox (cytochrome) centres. 𝜏 thus can inform on the rate at which chemical energy is converted into electricity by the bacterial population.  S2. Experimental method Biofilms of Geobacter sulfurreducens were grown into continuous mode at 32 oC within a 100 cm3 stirred electrochemical cell. A graphite disk with a diameter of 0.4 cm was used as the working electrode. Before use, the electrode surface was polished with 1000 grade silicon carbide paper and sonicated three times in distilled water. The electrode potential steady state potential for EIS was set against Ag|AgCl – 3M NaCl reference electrode. Platinum wire was used as the counter electrode. A culture medium lacking electron acceptors and containing sodium bicarbonate (100 mM) and acetate (20 mM) as the carbon source was prepared as described in literature[1b, 11]. It was circulated by the electrochemical reactor using biocompatible Pharma pure tubings (Saint Gobain, OH, USA) and a low rate peristaltic pump (BT100 – 2J, Longerpump, USA). The pH of the medium was kept constant of 7.3 by bubbling all media reservoirs and the reactor with a mixture of N2/CO2 (80:20). The gas mixture was filtered through a Variant C553120 oxygen filter to eliminate oxygen traces. All the electrochemical assays were performed using an Autolab PGSTAT101 potentiostat controlled by Nova 1.6-dedicated software. All experiments were performed when the biofilms reached the stationary growth phase, which was approximately 8-9 days after inoculation. Final current densities were in all cases approximately 7 A m-2. AC frequencies ranged from 0.1 MHz to 10 mHz with an amplitude of 10 mV peak to peak were employed for EIS analysis. All the obtained impedance data were checked regarding compliance with the constraints of linear systems theory by Kramers-Kronig using the appropriate routine of the FRA AUTOLAB software. The complex 𝑍∗(𝜔) (impedance) function can be converted into 𝐶∗(𝜔) (capacitance) by 𝐶∗(𝜔) = 1/𝑖𝜔𝑍∗(𝜔), where 𝜔 is the angular frequency and 𝑖 = √−1 (i.e., complex number). Practically, this involves taking the data resolved in a standard impedance analysis [𝑍∗(𝜔)], sampled across a range of frequencies at any steady-state potential, and converting it phasorially into complex capacitance [𝐶∗(𝜔)] with its real and imaginary components. In processing 𝑍∗(𝜔) datasets in this way one obtains the imaginary part of the capacitance by noting that 𝐶′′ = 𝜑𝑍′ and 𝐶′ =  𝜑𝑍′′, where 𝜑 = (𝜔|𝑍|2)−1 and |𝑍| is the modulus of 𝑍∗.  S3. Cyclic Voltammetry (CV) and Electrochemical Capacitance Spectroscopy (ECS) curves obtained at different respiration levels  The ECS and EIS approaches are comparatively analysed in Figures S1 and S2. ECS has advantages over traditional electrochemical impedimetric approach in revealing faradaic processes when capacitive contribution dominates the electron transfer process and negligible (or none) dc current is present (see Figure S1), which is the case of maximum respiration level of the Geobacter biofilm. Accordingly, Figure S1a shows typical Nyquist impedance diagrams of biofilm where the absence of an effective dc conductivity (when scanned or perturbed externally) is evident at lower frequency, meanwhile ECS diagrams (Figure S1b) additionally reveal the internal charge transfer resistance associated with charging/discharging of 𝐶𝑟 term (effectively acting as the conductive internal path – where the redox current flows – of the cell machinery associated with the energy whose origin resides on the work done by the biofilm through its intrinsic biochemistry energy). The ECS and EIS are equally important to evaluate the variation of the equivalent circuit model during electrode polarization as shown in Figure S2. The experimental curves and spectra in Figures S1 are always compared with theoretical model (as fitted and visualized in blue line in Figure S1) developed and explained in the main text. The inverse dependence with potential of the shunt resistance with respect of redox capacitance is better visualized in comparing Figure S2b and S2c. The analysis of the resistance terms and kinetics involved will be published elsewhere.   Figure S1. a) The impedance diagram of microbial biofilm at 50 mV with respect to Ag|AgCl, i.e. at the maximum current delivered to the external circuit (maximum microbial respiratory activity). The inset depicts the high frequency region in which the slope dashed line comprising a 45 degree demonstrates the porous/transport behavior of the electron conduction into cytochrome chains (observe the minimum series conductance). The diagram shows a capacitive behavior that is resolved by ECS approach in b). From both b) and c) diagrams the value of 𝐶𝑟 is easily obtained as well as the time scale of the associated redox process as indicated in d), 𝜏 = 𝑅𝑐𝑡𝐶𝑟  10 s. Blue line is the fitting to theoretical model.  Figure S2. a) Current density versus potential curve for a MFC under equilibrium scan rate of 1 mV s-1 obtained for a Biofilm thickness larger than 60 m. The colouring lines are highlighting the potentials of +100 mV (red), -200 mV (green) and -300 mV (red). As lower the potential lower is the biofilm respiratory rate. b) The corresponding Nyquist impedimetric diagrams of highlighted potentials of Figure 3a demonstrate the effect in lowering the respiratory rate. Ultimately this is monitored by changes on 𝑅𝑡 within the exchange impedance term of the transmission line. The meaning of 𝑅𝑡 is stated in the main text and it can be noted that as higher the respiration rate higher is the magnitude of 𝑅𝑡. c) Nyquist capacitive diagrams of highlighted potentials of Figure 3a. Equivalently to b) as higher is the respiration rate higher is the capacitive behavior (higher the magnitude of 𝑅𝑡 term). References  [1] aG. D. Schrott, P. S. Bonanni, L. Robuschi, A. Esteve-Nuñez, J. P. Busalmen, Electrochimica Acta 2011, 56, 10791-10795; bP. Sebastian Bonanni, G. D. Schrott, L. Robuschi, J. P. Busalmen, Energy & Environmental Science 2012, 5, 6188-6195; cN. S. Malvankar, T. Mester, M. T. Tuominen, D. R. Lovley, Chemphyschem 2012, 13, 463-468; dA. Esteve-Nunez, J. Sosnik, P. Visconti, D. R. Lovley, Environ. Microbiol. 2008, 10, 497-505. [2] aK. Fricke, F. Harnisch, U. Schroder, Energy & Environmental Science 2008, 1, 144-147; bE. Marsili, J. B. Rollefson, D. B. Baron, R. M. Hozalski, D. R. Bond, Applied and Environmental Microbiology 2008, 74, 7329-7337. [3] aP. R. Bueno, J. J. Davis, G. Mizzon, J. Phys. Chem. C 2012, 116, 8822–8829; bP. R. Bueno, D. Gimenez-Romero, F. F. Ferreira, G. O. Setti, Electrochimica Acta 2010, 55, 6147-6155. [4] E. Marsili, J. Sun, D. R. Bond, Electroanalysis 2010, 22, 865-874. [5] aL. Robuschi, J. Pablo Tomba, G. D. Schrott, P. Sebastian Bonanni, P. Mariela Desimone, J. Pablo Busalmen, Angewandte Chemie-International Edition 2013, 52, 925-928; bR. M. Snider, S. M. Strycharz-Glaven, S. D. Tsoi, J. S. Erickson, L. M. Tender, Proceedings of the National Academy of Sciences 2012, 109, 15467-15472. [6] aS. Srikanth, E. Marsili, M. C. Flickinger, D. R. Bond, Biotechnology and Bioengineering 2008, 99, 1065-1073; bN. S. Malvankar, M. T. Tuominen, D. R. Lovley, Energy & Environmental Science 2012, 5, 5790-5797; cJ. T. Babauta, H. Beyenal, Biotechnology and Bioengineering 2014, 111, 285-294; dA. ter Heijne, H. V. M. Hamelers, M. Saakes, C. J. N. Buisman, Electrochimica Acta 2008, 53, 5697-5703. [7] P. R. Bueno, F. Fabregat-Santiago, J. J. Davis, Anal. Chem. 2012, 85, 411-417. [8] D. R. Bond, S. M. Strycharz, L. Tender, C. I. Torres, ChemSusChem 2012, 5, 1099-1105. [9] P. R. Bueno, G. T. Feliciano, J. J. Davis, Phys. Chem. Chem. Phys. 2015. [10] P. R. Bueno, J. J. Davis, Analytical Chemistry 2014, 86, 1337-1341. [11] G. D. Schrott, P. Sebastian Bonanni, L. Robuschi, A. Esteve-Nunez, J. Pablo Busalmen, Electrochimica Acta 2011, 56, 10791-10795. 

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Biochemical capacitance of geobacter sulfurreducens biofilms

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