Curr Microbiol DOI 10.1007/s00284-014-0539-2
Metabolic Efficiency of Geobacter sulfurreducens Growing on Anodes with Different Redox Potentials Julian Bosch • Keun-Young Lee • Siang-Fu Hong • Falk Harnisch • Uwe Schro¨der • Rainer U. Meckenstock
Received: 20 September 2013 / Accepted: 24 December 2013 Ó Springer Science+Business Media New York 2014
Abstract Microorganisms respiring Fe(III) in the environment face a range of redox potentials of the prospective terminal ferric electron acceptors, because Fe(III) can be present in different minerals or organic complexes. We investigated the adaptation of Geobacter sulfurreducens to this range by exposing the bacteria to different redox potentials between the electron donor acetate and solid, extracellular anodes in a microbial fuel-cell set-up. Over a range of anode potentials from -0.105 to ?0.645 V versus standard hydrogen electrode, G. sulfurreducens produced identical amounts of biomass per electron respired. This indicated that the organism cannot utilize higher available energies for energy conservation to ATP, and confirmed recent studies. Either the high potentials cannot be used Electronic supplementary material The online version of this article (doi:10.1007/s00284-014-0539-2) contains supplementary material, which is available to authorized users. J. Bosch (&) R. U. Meckenstock Institute of Groundwater Ecology, Helmholtz Zentrum Mu¨nchen, Ingolsta¨dter Landstr. 1, 85764 Neuherberg, Germany e-mail: [email protected] R. U. Meckenstock e-mail: [email protected] K.-Y. Lee Korea Atomic Energy Research Institute, Daejeon, Republic of Korea S.-F. Hong F. Harnisch U. Schro¨der Institute of Environmental and Sustainable Chemistry, TU Braunschweig, Hagenring 30, 38106 Brunswick, Germany Present Address: F. Harnisch Department of Environmental Microbiology, UFZ-Helmholtz Centre for Environmental Research, Permoserstrasse 15, 04318 Leipzig, Germany
due to physiological limitations, or G. sulfurreducens decreased its metabolic efficiency, and less biomass per unit of energy was produced. In this case, G. sulfurreducens ‘‘wasted’’ energy at high-potential differences, most likely as heat to fuel growth kinetics.
Introduction Microbial extracellular electron transfer, i.e. the transfer of respiratory electrons to insoluble terminal electron acceptors (TEAs) like ferric iron [21] or anodes [15], is highly relevant for biogeochemical oxidation–reduction cycles [12] and a key element of microbial bioelectrochemical systems [5]. In nature, redox potentials of extracellular TEA metal oxide species are ranging from ?1.1 to -0.4 V versus standard hydrogen electrode (SHE) at neutral pH [13]. Abundant iron oxides from natural environments such as goethite, magnetite, or ferrihydrite exhibit potentials ranging from -310 to ?10 mV versus SHE at neutral pH [17]. In addition, iron oxides often do not occur in pure form in the environment, but as complex mixtures, often composed of different types of iron oxides, iron sulphides, and iron carbonates, coated with humic substances, or bound to clay minerals. In consequence, iron-reducing microorganisms face a wide range of redox potentials of their TEAs. The maximum energy gain microorganism can harvest is directly determined by the redox potential difference of h0 h0 electron donor Edon and acceptor Eacc according to Eq. 1. 0
0
0
h h Edon Þ DGh ¼ n F ðEacc
ð1Þ
Here, n represents the number of transferred electrons; F Faraday’s constant; Eh0 represent the formal potentials, derived from the standard potentials, Eh of a redox reaction
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J. Bosch et al.: Metabolic Efficiency of G. sulfurreducens Growing on Anodes
by taking into account the actual reaction conditions, including e.g. pH, ionic strength and temperature. Consequently, an iron-reducing microorganism like Geobacter sulfurreducens utilizing a specific susbtrate will have access to a higher energy gain per transferred electron when reducing an electron acceptor with a more positive redox potential. However, to conserve this higher DGh0 a flexible adjustment of the metabolism would be necessary. This means, microbes would have to be able to transfer different amounts of protons across their cytoplasmic membrane upon different redox potentials of the TEAs. In contrast, in present models for bacterial respiratory chains, the stoichiometry of transported protons per transferred electron, and thus the energy gain per electron, is fixed. For the majority of all electron acceptors other than metal oxides, this makes perfect sense as the redox potentials that can be achieved by changing concentrations within the physiological range are close to the redox potentials at standard conditions. Thus, the question arises if iron-reducing bacteria can adjust their energy gain to different TEAs with different redox potentials. Iron-reducing bacteria have a basic respiratory chain, where only two protons per two electrons are pumped across the membrane via the quinone pool. Apart from this proton transferring step, the electron transport chain is extremely versatile. In iron-reducing bacteria, up to 111 multiheme cytochrome c-type proteins might be involved in electron transport from the quinole oxidase towards the extracellular or periplasmic electron acceptor [4, 10, 11, 16]. Furthermore, deletions of outer membrane cytochromes showed a strong impact on the efficiency of iron reduction [20]. An adaptation of the energy conservation by the respiratory chain seems possible with respect to this respiratory toolbox [1]. Several scenarios are conceivable: (i) additional energy at higher redox potentials of the TEA is not conserved at all, and is either released as heat or left unused in the first place, (ii) an adjustment within the respiratory chain takes place, where the amount of transported protons per transferred electron is changed, and higher redox potential differences lead to higher biomass, (iii) the respiratory chain does not change, but additional available energy at higher redox potential differences is exploited for faster growth. Consequently, the aim of this study was to investigate the adaptation of the iron-reducing bacterium G. sulfurreducens to different redox potentials of its TEAs. Geobacter species are key model organisms for direct electron transfer [8] and thus for ‘‘electroactive’’ or ‘‘exoelectrogenic’’ microorganisms in general. Within this study, G. sulfurreducens was cultivated at anodes using a potentiostatic set-up to secure an accurate control of the applied TEA potentials. During the microbial bioelectrochemical oxidation of acetate, the amount of biomass formation and electron transfer to the anode were monitored
123
in order to allow the determination of the metabolic efficiency as a function of the anode, i.e. TEA, potential.
Materials and Methods General Conditions All chemicals were of analytical, biochemical grade and were purchased from Sigma-Aldrich and Merck. If not stated otherwise, all experimental potentials provided in this article were derived from a Ag/AgCl reference electrode (sat. KCl, 0.195 V vs. SHE). These data were recalculated versus SHE for all thermodynamic calculations. All microbial experiments were performed under strictly sterile and anoxic conditions. Micro-organism and Growth Medium Geobacter sulfurreducens DSMZ 12127 [2] was obtained from the German Collection of Micro-organisms and Cell Cultures (Braunschweig, Germany). For pre-cultivation and experiments, Geobacter was grown at 30 °C in anoxic medium as described before [3], and 50 mM fumarate was used as electron acceptor. Cells were cultivated until late exponential phase, when the OD[570] reached *0.7, and then centrifuged. The resulting cell pellets were collected in fresh, fumarate-free medium. Bioelectrochemical Experiments All electrochemical experiments were carried out under potentiostatic control (Potentiostat/Galvanostat VMP3, BioLogic Science Instruments, France), utilizing a three electrode arrangement. This consisted of a carbon rodworking electrode (4 cm height 91.0 cm diameter, CPGraphite GmbH, Germany), a Ag/AgCl reference electrode (sat. KCl, Sensortechnik Meinsberg, Germany) and a carbon rod counter electrode (5 cm high 91.0 cm diameter). Sealed and thermostated vessels (250 mL) served as electrochemical cells which hosted the anoxic media solution (200 mL) and the electrodes. Microbial growth was performed in potentiostatic halfcell experiments at 35 °C at different potentials, respectively. After start of the exponential growth of the biofilms (about 170 h), nitrogen gas was utilized to flush the head space of the experimental vessels at a pressure of 0.1 bar to remove excess hydrogen. In total, 18 individual bioelectrochemical experiments were conducted; three independent biological replicates for each of the 6 anode potentials. Two out of the 18 experiments had to be excluded from further calculation due to unspecified contamination (see supporting information).
J. Bosch et al.: Metabolic Efficiency of G. sulfurreducens Growing on Anodes
Metabolite Determination Substrate consumption and non-gaseous fermentation product formation were monitored by HPLC analysis. The HPLC (Spectrasystem P400, FINNIGAN Surveyor RI Plus detector, Fisher Scientific, Germany) was equipped with a Rezex HyperREZ XP Carbohydrate H? lm column. The chromatograms were recorded at room temperature with 0.005 N sulphuric acid as eluent. The acetate consumption data was used to determine the coulombic efficiency (CE). Biomass Determination After depletion of acetate and a decline of the oxidative current flow, biomass formation was assessed by three different methods: measuring the protein content, measuring the total organic carbon content in 0.2 lm-filtrates and direct cell counts. Cell counts (derived from flow cytometry) were used as an additional confirmation, but not included in further calculations due to higher deviations. Biological samples were taken from the medium inside the fuel-cell vessels and from the biofilm on the anode surface. Samples from the medium were fixed in paraformaldehyde and used for the determination of planktonic cells. Biofilms on the electrode were mechanically removed with a brush and fixed using a similar procedure. All samples were measured in triplicate for protein content, total organic carbon content (TOC) and cell number. Cell numbers were determined by flow-cytometry. A LSRII FACS flow cytometer (Becton Dickson Bioscience, Franklin Lakes, NJ, USA) was applied to count the DNA-stained cells: two ml of 2.4 % paraformaldehydefixed cells from the cell suspensions was stained by 1 9 SYBRÒ Green I nucleic acid stain (Molecular Probes, Eugene, OR, USA), diluted in 0.22 lm-filtered PBS and counted at a wavelength of 510 nm in TrucountTM bead (Becton Dickson) calibrated measurements. For measuring cell organic carbon, samples were filtered on glass fibre filters with a cut-off at 0.2 lm. The filters were dried at 60 °C for a minimum of 24 h. Afterwards, these samples were analysed with a Shimadzu 5050 TOC-analyser (Shimadzu, Japan). Protein content was measured via TCA precipitation. Samples were photometrically analysed at 595 nm after addition of Bradford dye. Thermodynamic Calculations The chronoamperometrically measured charge data (and calculated number of electrons) of transferred electrons were used for calculating the coulombic efficiency (CE). In combination with the redox potentials of the electron donor h0 acetate (Edon ¼ 0:290 mV versus SHE, [18]), charge data
were used according to Eq. 1 to calculate the maximum available energy. The experimentally assessed amount of formed biomass was then related to the available energy in order to calculate the metabolic efficiency of the microbial reaction.
Results and Discussion Bioelectrochemical Growth and Characterization Geobacter sulfurreducens readily transferred electrons from acetate oxidation to the anode, and biomass was formed during the chronoamperometric experiments at all applied potentials (Fig. 1). The mean coulombic efficiency was 45.5 ± 21.0 %. More than 99 % of the acetate was used up by the bacteria in all experiments. The gained maximum current densities of about 400 lA cm-2 are well in line with previous studies using similar electrode materials and pure cultures of G. sulfurreducens [6, 9]. The total mean charge transfer, derived from integration of the chronoamperometric curves and reflecting the number of transferred electrons, showed a slight increase from 523 C at an anode potential of -0.105 V to 864 C at ?0.645 V versus SHE, yet significant deviations among the individual experiments were observed (data shown in the supporting information). In general, this observation confirms the general metabolic versatility of G. sulfurreducens. It is able to grow at a large range of redox potentials of the available TEAs, down to redox potential differences of as little as ?0.185 V versus SHE between the electron donor acetate and an anode at -0.105 V versus SHE. To assess the adaptations of the energy gain of G. sulfurreducens to the redox potential of the TEA, we determined the amount of produced biomass per respired electron. As biomass production is directly dependent on the ATP generated during this process, an increase in biomass formation at higher redox potentials would indicate a change in the respiratory chain. However, on the basis of the measured electron turnover, i.e. the total charge, a more or less constant biomass formation was observed (Fig. 1). At low (-0.105 and ?0.045 V, all vs. SHE) and high (?0.345 and ?0.495 V) applied anode potentials, biomass formation per electron seemed to be enhanced as compared to the mean (?0.195 V) and highest applied anode potentials (0.645 V). However, the changes in the ratio of biomass per electron were not significant within the experimental error range. The strong deviation was ascribed to differences among the independent experimental runs e.g. putative, slight oxygen contaminations of the inoculum or differences in biofilm mechanics. Thus, biomass formation had
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Fig. 1 Biomass production (protein, cells, TOC) per transferred Coulomb in microbial fuel-cell experiments. G. sulfurreducens was grown with 10 mM acetate on anodes with different redox potentials
Fig. 2 Average available Gibb’s free energy harvested by G. sulfurreducens growing with 10 mM acetate on anodes with different potentials. The values were determined by the measured charge turnover and Eq. 1 Grey line: linear regression. Number of replicates per anode potential =3 (except for the two highest potentials, where only two replicates were performed)
to be considered as constant at all applied electrode potentials. We concluded that G. sulfurreducens cannot change the number of transferred protons per electron respired under the applied conditions, even if more energy was available with electrons respired at higher redox potential differences according to Eq. 1. Biomass Formation Per Energy as a Function of the Available Redox Potential Difference Geobacter sulfurreducens showed a constant biomass formation per electron at all applied potentials. However, the available Gibbs free energy, which is determined by the
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as TEA. n per potential =3, except the two highest potentials, where n = 2 (see ‘‘Materials and Methods’’ section). Grey line linear regression
Fig. 3 Relative biomass formation per kJ of G. sulfurreducens grown with 10 mM acetate on electrodes in an anoxic bioelectrochemical system as a function of the applied electrode potential. Data represent the means of triplicate protein and TOC production measurements of 16 independent experimental replicates, with all data normalized to the highest observed biomass formation data (at -0.105 V vs. SHE). Error bars show the averaged relative standard deviation of the protein and TOC measurements. Black line: modelled exponential decay function
applied anode potentials for the used experimental conditions (Eq. 1), implicitly increased with increasing potential (Fig. 2). Plainly, correlating the constant biomass formation of all experiments (Fig. 1) to the available Gibb’s free energy (Fig. 2), and relating the resulting absolute biomass production per unit of energy to the highest observed biomass production, revealed an inverted exponential correlation between available energy and metabolic efficiency (Fig. 3). Thus, Fig. 3 indicated that an increase in the differences of the redox potentials of electron donor and acceptor (an
J. Bosch et al.: Metabolic Efficiency of G. sulfurreducens Growing on Anodes
increase of the available energy) resulted in a decrease of the metabolic efficiency in terms of biomass formation per unit of available energy. This clearly implied that G. sulfurreducens used the available energy for biomass formation at lower potential differences more efficiently, or in turn indicated that the bacteria either ‘‘wasted’’ energy at higher potential differences or cannot use high-anodic potentials at all [9]. Clearly, a waste of energy has to result in some kind of energy dissipation. Heat dissipation is very likely, which might also have fuelled growth kinetics. In fact a recent study showed faster growth rates of G. sulfurreducens in fuel-cell experiments with anodes set to ?0.400 and 0 V versus SHE in comparison to -0.160 V, but not ?500 V [22]. Using different solid iron oxides, anthraquinone-2,6disulfonate (AQDS) or U(VI) [7, 14], it had already been suggested that reduction rates for G. sulfurreducens are dependent on the redox potential of the electron acceptor. If this holds true for our experiments, our data would support theoretical considerations on the driving force of microbial growth [19]. According to this model, microorganism can thrive close to thermodynamic equilibrium, with a high efficiency, small heat losses and a high-biomass yield, but slow growth rates. In contrast, microorganisms facing an energy-abundant environment would grow fast, with strong heat dissipation and a low-biomass yield. Considering the redox potential range of the different forms of ferric iron in the natural environment, an enhancement of the driving force (faster but less efficient growth) appears to be a beneficial metabolic adaptation. In contrast, this adaptation would ensure that iron-reducing microorganisms always exploit the maximum of the available redox energy under energy limiting conditions. However, we did not observe consistent patterns of current production as a function of anode potential, from which such growth behaviour could be deduced (see supporting information). The non-systematic current production rates rather imply experimental limitations by e.g. electrode surface area. Even more, faster growth can be achieved either by a higher turnover per cell (higher respiration rate) or by a higher yield coefficient (higher biomass per respired electron). As the electron donor consumption in all experiments was the same, and the absolute biomass remained more or less constant in all experiments, a change of the biomass yield is not likely based on our data. Thus, the latter conclusion remains speculative. If no redox potential-dependent changes of growth kinetics occurred in our experiments, this would imply that G. sulfurreducens could only effectively utilize energy which was fed into the metabolism at a discrete potential range, putatively close to -0.1 V versus SHE. If more energy at higher redox potentials was available, this surplus was either left completely unused or was probably just
dissipated as heat. Growth rates and biomass yield would then be identical across all redox potential; only the efficiency of biomass formation per unit of energy would decline at higher redox potentials. Still, our findings showed that G. sulfurreducens was able to grow at a large potential range. A constant biomass formation is maintained even at low available energies. This versatility suits to an ecology which implies the dissimilatory reduction of soluble, poorly soluble or insoluble electron acceptors with a large range of redox potentials, and it enables Geobacter to maintain at least a minimum of metabolic activity on almost all kinds of ferric iron and other metal oxide electron acceptors. Acknowledgments Financial support was provided by the DFG research unit FOR 580, the EU-project AQUAREHAB (FP7—Grant Agreement #226565) and the EU-project NANOREM (FP7—Grant Agreement #309517). F.H. acknowledges support by the BMBF (Research Award ‘‘Next generation biotechnological processes— Biotechnology 2020?’’) and the Helmholtz-Association (Young Investigators Group). US acknowledges the Energy Research by the Volkswagen AG, the Verband der Deutschen Biokraftstoffindustrie e.V. and the foundation of the professorship Sustainable Chemistry.
References 1. Bonanni PS, Schrott GD, Busalmen JP (2012) A long way to the electrode: how do Geobacter cells transport their electrons? Biochem Soc Trans 40:1274–1279 2. Caccavo F, Lonergan DJ, Lovley DR, Davis M, Stolz JF, McInerney MJ (1994) Geobacter sulfurreducens sp. nov., a hydrogen- and acetate oxidizing dissimilatory metal-reducing microorganism. Appl Environ Microbiol 60:3752–3759 3. Fritzsche A, Bosch J, Rennert T, Heister K, Braunschweig J, Meckenstock RU, Totsche KU (2012) Fast microbial reduction of ferrihydrite colloids from a soil effluent. Geochim Cosmochim Acta 77:444–456 4. Harada E, Kumagai J, Ozawa K, Imabayashi S, Tsapin AS, Nealson KH, Meyer TE, Cusanovich MA, Akutsu H (2002) A directional electron transfer regulator based on heme-chain architecture in the small tetraheme cytochrome c from Shewanella oneidensis. FEBS Lett 532:333–337 5. Harnisch F, Schro¨der U (2010) From MFC to MXC: chemical and biological cathodes and their potential for microbial bioelectrochemical systems. Chem Soc Rev 39:4433–4448 6. Katuri KP, Kavanagh P, Rengaraj S, Leech D (2010) Geobacter sulfurreducens biofilms developed under different growth conditions on glassy carbon electrodes: insights using cyclic voltammetry. Chem Comm 46:4758–4760 7. Lloyd JR, Leang C, Myerson ALH, Coppi MV, Cuifo S, Methe B, Sandler SJ, Lovley DR (2003) Biochemical and genetic characterization of PpcA, a periplasmic c-type cytochrome in Geobacter sulfurreducens. Biochem J 369:153–161 8. Lovley DR (2008) The microbe electric: conversion of organic matter to electricity. Curr Opin Biotechnol 19:564–571 9. Marsili E, Sun J, Bond DR (2010) Voltammetry and growth physiology of Geobacter sulfurreducens biofilms as a function of growth stage and imposed electrode potential. Electroanalysis 22:865–874
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J. Bosch et al.: Metabolic Efficiency of G. sulfurreducens Growing on Anodes 10. Methe BA (2003) Genome of Geobacter sulfurreducens: metal reduction in subsurface environments. Science 302:1967–1969 11. Meyer TE, Tsapin AI, Vandenberghe I, De Smet L, Frishman D, Nealson KH, Cusanovich MA, Van Beeumen JJ (2004) Identification of 42 possible cytochrome c genes in the Shewanella oneidensis genome and characterization of six soluble cytochromes. Omics-a J Integr Biol 8:57–77 12. Nielsen LP, Risgaard-Petersen N, Fossing H, Christensen PB, Sayama M (2010) Electric currents couple spatially separated biogeochemical processes in marine sediment. Nature 463:1071–1074 13. Pierre JL, Fontecave M, Chrichton RR (2002) Chemistry for an essential biological process: the reduction of ferric iron. Biometals 15:341–346 14. Roden EE, Zachara JM (1996) Microbial reduction of crystalline iron(III) oxides: influence of oxide surface area and potential for cell growth. Environ Sci Technol 30:1618–1628 15. Schro¨der U (2007) Anodic electron transfer mechanisms in microbial fuel cells and their energy efficiency. Phys Chem Chem Phys 9:2619–2629 16. Shi L, Squier TC, Zachara JM, Fredrickson JK (2007) Respiration of metal (hydr)oxides by Shewanella and Geobacter: a key role for multihaem c-type cytochromes. Mol Microbiol 65:12–20
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17. Stumm W, Morgan JJ (1996) Aquatic chemistry—chemical equilibria and rates in natural waters, 3rd edn. Wiley, New York 18. Thauer RK, Junkermann K, Decker K (1977) Energy conservation in chemotrophic anaerobic bacteria. Bacteriol Rev 41: 100–180 19. von Stockar U (2006) Thermodynamics of microbial growth and metabolism: an analysis of the current situation. J Biotechnol 121:517–533 20. Voordeckers JW, Kim BC, Izallalen M, Lovley DR (2010) Role of Geobacter sulfurreducens outer surface c-type cytochromes in reduction of soil humic acid and anthraquinone-2,6-disulfonate. Appl Environ Microbiol 76:2371–2375 21. Weber KA, Achenbach LA, Coates JD (2006) Microorganisms pumping iron: anaerobic microbial iron oxidation and reduction. Nat Rev Microbiol 4:752–764 22. Wei JC, Liang P, Cao XX, Huang X (2010) A new insight into potential regulation on growth and power generation of Geobacter sulfurreducens in microbial fuel cells based on energy viewpoint. Environ Sci Technol 44:3187–3191
Metabolic Efficiency of Geobacter sulfurreducens Growing on Anodes with Different Redox Potentials Julian Bosch • Keun-Young Lee • Siang-Fu Hong • Falk Harnisch • Uwe Schro¨der • Rainer U. Meckenstock
Received: 20 September 2013 / Accepted: 24 December 2013 Ó Springer Science+Business Media New York 2014
Abstract Microorganisms respiring Fe(III) in the environment face a range of redox potentials of the prospective terminal ferric electron acceptors, because Fe(III) can be present in different minerals or organic complexes. We investigated the adaptation of Geobacter sulfurreducens to this range by exposing the bacteria to different redox potentials between the electron donor acetate and solid, extracellular anodes in a microbial fuel-cell set-up. Over a range of anode potentials from -0.105 to ?0.645 V versus standard hydrogen electrode, G. sulfurreducens produced identical amounts of biomass per electron respired. This indicated that the organism cannot utilize higher available energies for energy conservation to ATP, and confirmed recent studies. Either the high potentials cannot be used Electronic supplementary material The online version of this article (doi:10.1007/s00284-014-0539-2) contains supplementary material, which is available to authorized users. J. Bosch (&) R. U. Meckenstock Institute of Groundwater Ecology, Helmholtz Zentrum Mu¨nchen, Ingolsta¨dter Landstr. 1, 85764 Neuherberg, Germany e-mail: [email protected] R. U. Meckenstock e-mail: [email protected] K.-Y. Lee Korea Atomic Energy Research Institute, Daejeon, Republic of Korea S.-F. Hong F. Harnisch U. Schro¨der Institute of Environmental and Sustainable Chemistry, TU Braunschweig, Hagenring 30, 38106 Brunswick, Germany Present Address: F. Harnisch Department of Environmental Microbiology, UFZ-Helmholtz Centre for Environmental Research, Permoserstrasse 15, 04318 Leipzig, Germany
due to physiological limitations, or G. sulfurreducens decreased its metabolic efficiency, and less biomass per unit of energy was produced. In this case, G. sulfurreducens ‘‘wasted’’ energy at high-potential differences, most likely as heat to fuel growth kinetics.
Introduction Microbial extracellular electron transfer, i.e. the transfer of respiratory electrons to insoluble terminal electron acceptors (TEAs) like ferric iron [21] or anodes [15], is highly relevant for biogeochemical oxidation–reduction cycles [12] and a key element of microbial bioelectrochemical systems [5]. In nature, redox potentials of extracellular TEA metal oxide species are ranging from ?1.1 to -0.4 V versus standard hydrogen electrode (SHE) at neutral pH [13]. Abundant iron oxides from natural environments such as goethite, magnetite, or ferrihydrite exhibit potentials ranging from -310 to ?10 mV versus SHE at neutral pH [17]. In addition, iron oxides often do not occur in pure form in the environment, but as complex mixtures, often composed of different types of iron oxides, iron sulphides, and iron carbonates, coated with humic substances, or bound to clay minerals. In consequence, iron-reducing microorganisms face a wide range of redox potentials of their TEAs. The maximum energy gain microorganism can harvest is directly determined by the redox potential difference of h0 h0 electron donor Edon and acceptor Eacc according to Eq. 1. 0
0
0
h h Edon Þ DGh ¼ n F ðEacc
ð1Þ
Here, n represents the number of transferred electrons; F Faraday’s constant; Eh0 represent the formal potentials, derived from the standard potentials, Eh of a redox reaction
123
J. Bosch et al.: Metabolic Efficiency of G. sulfurreducens Growing on Anodes
by taking into account the actual reaction conditions, including e.g. pH, ionic strength and temperature. Consequently, an iron-reducing microorganism like Geobacter sulfurreducens utilizing a specific susbtrate will have access to a higher energy gain per transferred electron when reducing an electron acceptor with a more positive redox potential. However, to conserve this higher DGh0 a flexible adjustment of the metabolism would be necessary. This means, microbes would have to be able to transfer different amounts of protons across their cytoplasmic membrane upon different redox potentials of the TEAs. In contrast, in present models for bacterial respiratory chains, the stoichiometry of transported protons per transferred electron, and thus the energy gain per electron, is fixed. For the majority of all electron acceptors other than metal oxides, this makes perfect sense as the redox potentials that can be achieved by changing concentrations within the physiological range are close to the redox potentials at standard conditions. Thus, the question arises if iron-reducing bacteria can adjust their energy gain to different TEAs with different redox potentials. Iron-reducing bacteria have a basic respiratory chain, where only two protons per two electrons are pumped across the membrane via the quinone pool. Apart from this proton transferring step, the electron transport chain is extremely versatile. In iron-reducing bacteria, up to 111 multiheme cytochrome c-type proteins might be involved in electron transport from the quinole oxidase towards the extracellular or periplasmic electron acceptor [4, 10, 11, 16]. Furthermore, deletions of outer membrane cytochromes showed a strong impact on the efficiency of iron reduction [20]. An adaptation of the energy conservation by the respiratory chain seems possible with respect to this respiratory toolbox [1]. Several scenarios are conceivable: (i) additional energy at higher redox potentials of the TEA is not conserved at all, and is either released as heat or left unused in the first place, (ii) an adjustment within the respiratory chain takes place, where the amount of transported protons per transferred electron is changed, and higher redox potential differences lead to higher biomass, (iii) the respiratory chain does not change, but additional available energy at higher redox potential differences is exploited for faster growth. Consequently, the aim of this study was to investigate the adaptation of the iron-reducing bacterium G. sulfurreducens to different redox potentials of its TEAs. Geobacter species are key model organisms for direct electron transfer [8] and thus for ‘‘electroactive’’ or ‘‘exoelectrogenic’’ microorganisms in general. Within this study, G. sulfurreducens was cultivated at anodes using a potentiostatic set-up to secure an accurate control of the applied TEA potentials. During the microbial bioelectrochemical oxidation of acetate, the amount of biomass formation and electron transfer to the anode were monitored
123
in order to allow the determination of the metabolic efficiency as a function of the anode, i.e. TEA, potential.
Materials and Methods General Conditions All chemicals were of analytical, biochemical grade and were purchased from Sigma-Aldrich and Merck. If not stated otherwise, all experimental potentials provided in this article were derived from a Ag/AgCl reference electrode (sat. KCl, 0.195 V vs. SHE). These data were recalculated versus SHE for all thermodynamic calculations. All microbial experiments were performed under strictly sterile and anoxic conditions. Micro-organism and Growth Medium Geobacter sulfurreducens DSMZ 12127 [2] was obtained from the German Collection of Micro-organisms and Cell Cultures (Braunschweig, Germany). For pre-cultivation and experiments, Geobacter was grown at 30 °C in anoxic medium as described before [3], and 50 mM fumarate was used as electron acceptor. Cells were cultivated until late exponential phase, when the OD[570] reached *0.7, and then centrifuged. The resulting cell pellets were collected in fresh, fumarate-free medium. Bioelectrochemical Experiments All electrochemical experiments were carried out under potentiostatic control (Potentiostat/Galvanostat VMP3, BioLogic Science Instruments, France), utilizing a three electrode arrangement. This consisted of a carbon rodworking electrode (4 cm height 91.0 cm diameter, CPGraphite GmbH, Germany), a Ag/AgCl reference electrode (sat. KCl, Sensortechnik Meinsberg, Germany) and a carbon rod counter electrode (5 cm high 91.0 cm diameter). Sealed and thermostated vessels (250 mL) served as electrochemical cells which hosted the anoxic media solution (200 mL) and the electrodes. Microbial growth was performed in potentiostatic halfcell experiments at 35 °C at different potentials, respectively. After start of the exponential growth of the biofilms (about 170 h), nitrogen gas was utilized to flush the head space of the experimental vessels at a pressure of 0.1 bar to remove excess hydrogen. In total, 18 individual bioelectrochemical experiments were conducted; three independent biological replicates for each of the 6 anode potentials. Two out of the 18 experiments had to be excluded from further calculation due to unspecified contamination (see supporting information).
J. Bosch et al.: Metabolic Efficiency of G. sulfurreducens Growing on Anodes
Metabolite Determination Substrate consumption and non-gaseous fermentation product formation were monitored by HPLC analysis. The HPLC (Spectrasystem P400, FINNIGAN Surveyor RI Plus detector, Fisher Scientific, Germany) was equipped with a Rezex HyperREZ XP Carbohydrate H? lm column. The chromatograms were recorded at room temperature with 0.005 N sulphuric acid as eluent. The acetate consumption data was used to determine the coulombic efficiency (CE). Biomass Determination After depletion of acetate and a decline of the oxidative current flow, biomass formation was assessed by three different methods: measuring the protein content, measuring the total organic carbon content in 0.2 lm-filtrates and direct cell counts. Cell counts (derived from flow cytometry) were used as an additional confirmation, but not included in further calculations due to higher deviations. Biological samples were taken from the medium inside the fuel-cell vessels and from the biofilm on the anode surface. Samples from the medium were fixed in paraformaldehyde and used for the determination of planktonic cells. Biofilms on the electrode were mechanically removed with a brush and fixed using a similar procedure. All samples were measured in triplicate for protein content, total organic carbon content (TOC) and cell number. Cell numbers were determined by flow-cytometry. A LSRII FACS flow cytometer (Becton Dickson Bioscience, Franklin Lakes, NJ, USA) was applied to count the DNA-stained cells: two ml of 2.4 % paraformaldehydefixed cells from the cell suspensions was stained by 1 9 SYBRÒ Green I nucleic acid stain (Molecular Probes, Eugene, OR, USA), diluted in 0.22 lm-filtered PBS and counted at a wavelength of 510 nm in TrucountTM bead (Becton Dickson) calibrated measurements. For measuring cell organic carbon, samples were filtered on glass fibre filters with a cut-off at 0.2 lm. The filters were dried at 60 °C for a minimum of 24 h. Afterwards, these samples were analysed with a Shimadzu 5050 TOC-analyser (Shimadzu, Japan). Protein content was measured via TCA precipitation. Samples were photometrically analysed at 595 nm after addition of Bradford dye. Thermodynamic Calculations The chronoamperometrically measured charge data (and calculated number of electrons) of transferred electrons were used for calculating the coulombic efficiency (CE). In combination with the redox potentials of the electron donor h0 acetate (Edon ¼ 0:290 mV versus SHE, [18]), charge data
were used according to Eq. 1 to calculate the maximum available energy. The experimentally assessed amount of formed biomass was then related to the available energy in order to calculate the metabolic efficiency of the microbial reaction.
Results and Discussion Bioelectrochemical Growth and Characterization Geobacter sulfurreducens readily transferred electrons from acetate oxidation to the anode, and biomass was formed during the chronoamperometric experiments at all applied potentials (Fig. 1). The mean coulombic efficiency was 45.5 ± 21.0 %. More than 99 % of the acetate was used up by the bacteria in all experiments. The gained maximum current densities of about 400 lA cm-2 are well in line with previous studies using similar electrode materials and pure cultures of G. sulfurreducens [6, 9]. The total mean charge transfer, derived from integration of the chronoamperometric curves and reflecting the number of transferred electrons, showed a slight increase from 523 C at an anode potential of -0.105 V to 864 C at ?0.645 V versus SHE, yet significant deviations among the individual experiments were observed (data shown in the supporting information). In general, this observation confirms the general metabolic versatility of G. sulfurreducens. It is able to grow at a large range of redox potentials of the available TEAs, down to redox potential differences of as little as ?0.185 V versus SHE between the electron donor acetate and an anode at -0.105 V versus SHE. To assess the adaptations of the energy gain of G. sulfurreducens to the redox potential of the TEA, we determined the amount of produced biomass per respired electron. As biomass production is directly dependent on the ATP generated during this process, an increase in biomass formation at higher redox potentials would indicate a change in the respiratory chain. However, on the basis of the measured electron turnover, i.e. the total charge, a more or less constant biomass formation was observed (Fig. 1). At low (-0.105 and ?0.045 V, all vs. SHE) and high (?0.345 and ?0.495 V) applied anode potentials, biomass formation per electron seemed to be enhanced as compared to the mean (?0.195 V) and highest applied anode potentials (0.645 V). However, the changes in the ratio of biomass per electron were not significant within the experimental error range. The strong deviation was ascribed to differences among the independent experimental runs e.g. putative, slight oxygen contaminations of the inoculum or differences in biofilm mechanics. Thus, biomass formation had
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Fig. 1 Biomass production (protein, cells, TOC) per transferred Coulomb in microbial fuel-cell experiments. G. sulfurreducens was grown with 10 mM acetate on anodes with different redox potentials
Fig. 2 Average available Gibb’s free energy harvested by G. sulfurreducens growing with 10 mM acetate on anodes with different potentials. The values were determined by the measured charge turnover and Eq. 1 Grey line: linear regression. Number of replicates per anode potential =3 (except for the two highest potentials, where only two replicates were performed)
to be considered as constant at all applied electrode potentials. We concluded that G. sulfurreducens cannot change the number of transferred protons per electron respired under the applied conditions, even if more energy was available with electrons respired at higher redox potential differences according to Eq. 1. Biomass Formation Per Energy as a Function of the Available Redox Potential Difference Geobacter sulfurreducens showed a constant biomass formation per electron at all applied potentials. However, the available Gibbs free energy, which is determined by the
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as TEA. n per potential =3, except the two highest potentials, where n = 2 (see ‘‘Materials and Methods’’ section). Grey line linear regression
Fig. 3 Relative biomass formation per kJ of G. sulfurreducens grown with 10 mM acetate on electrodes in an anoxic bioelectrochemical system as a function of the applied electrode potential. Data represent the means of triplicate protein and TOC production measurements of 16 independent experimental replicates, with all data normalized to the highest observed biomass formation data (at -0.105 V vs. SHE). Error bars show the averaged relative standard deviation of the protein and TOC measurements. Black line: modelled exponential decay function
applied anode potentials for the used experimental conditions (Eq. 1), implicitly increased with increasing potential (Fig. 2). Plainly, correlating the constant biomass formation of all experiments (Fig. 1) to the available Gibb’s free energy (Fig. 2), and relating the resulting absolute biomass production per unit of energy to the highest observed biomass production, revealed an inverted exponential correlation between available energy and metabolic efficiency (Fig. 3). Thus, Fig. 3 indicated that an increase in the differences of the redox potentials of electron donor and acceptor (an
J. Bosch et al.: Metabolic Efficiency of G. sulfurreducens Growing on Anodes
increase of the available energy) resulted in a decrease of the metabolic efficiency in terms of biomass formation per unit of available energy. This clearly implied that G. sulfurreducens used the available energy for biomass formation at lower potential differences more efficiently, or in turn indicated that the bacteria either ‘‘wasted’’ energy at higher potential differences or cannot use high-anodic potentials at all [9]. Clearly, a waste of energy has to result in some kind of energy dissipation. Heat dissipation is very likely, which might also have fuelled growth kinetics. In fact a recent study showed faster growth rates of G. sulfurreducens in fuel-cell experiments with anodes set to ?0.400 and 0 V versus SHE in comparison to -0.160 V, but not ?500 V [22]. Using different solid iron oxides, anthraquinone-2,6disulfonate (AQDS) or U(VI) [7, 14], it had already been suggested that reduction rates for G. sulfurreducens are dependent on the redox potential of the electron acceptor. If this holds true for our experiments, our data would support theoretical considerations on the driving force of microbial growth [19]. According to this model, microorganism can thrive close to thermodynamic equilibrium, with a high efficiency, small heat losses and a high-biomass yield, but slow growth rates. In contrast, microorganisms facing an energy-abundant environment would grow fast, with strong heat dissipation and a low-biomass yield. Considering the redox potential range of the different forms of ferric iron in the natural environment, an enhancement of the driving force (faster but less efficient growth) appears to be a beneficial metabolic adaptation. In contrast, this adaptation would ensure that iron-reducing microorganisms always exploit the maximum of the available redox energy under energy limiting conditions. However, we did not observe consistent patterns of current production as a function of anode potential, from which such growth behaviour could be deduced (see supporting information). The non-systematic current production rates rather imply experimental limitations by e.g. electrode surface area. Even more, faster growth can be achieved either by a higher turnover per cell (higher respiration rate) or by a higher yield coefficient (higher biomass per respired electron). As the electron donor consumption in all experiments was the same, and the absolute biomass remained more or less constant in all experiments, a change of the biomass yield is not likely based on our data. Thus, the latter conclusion remains speculative. If no redox potential-dependent changes of growth kinetics occurred in our experiments, this would imply that G. sulfurreducens could only effectively utilize energy which was fed into the metabolism at a discrete potential range, putatively close to -0.1 V versus SHE. If more energy at higher redox potentials was available, this surplus was either left completely unused or was probably just
dissipated as heat. Growth rates and biomass yield would then be identical across all redox potential; only the efficiency of biomass formation per unit of energy would decline at higher redox potentials. Still, our findings showed that G. sulfurreducens was able to grow at a large potential range. A constant biomass formation is maintained even at low available energies. This versatility suits to an ecology which implies the dissimilatory reduction of soluble, poorly soluble or insoluble electron acceptors with a large range of redox potentials, and it enables Geobacter to maintain at least a minimum of metabolic activity on almost all kinds of ferric iron and other metal oxide electron acceptors. Acknowledgments Financial support was provided by the DFG research unit FOR 580, the EU-project AQUAREHAB (FP7—Grant Agreement #226565) and the EU-project NANOREM (FP7—Grant Agreement #309517). F.H. acknowledges support by the BMBF (Research Award ‘‘Next generation biotechnological processes— Biotechnology 2020?’’) and the Helmholtz-Association (Young Investigators Group). US acknowledges the Energy Research by the Volkswagen AG, the Verband der Deutschen Biokraftstoffindustrie e.V. and the foundation of the professorship Sustainable Chemistry.
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