- Email: [email protected]
Electrochemical evidence for direct interspecies electron transfer between Geobacter sulfurreducens and Prosthecochloris aestuarii
Bioelectrochemistry 127 (2019) 21–25
Contents lists available at ScienceDirect
Bioelectrochemistry journal homepage: www.elsevier.com/locate/bioelechem
Electrochemical evidence for direct interspecies electron transfer between Geobacter sulfurreducens and Prosthecochloris aestuarii Lingyan Huang, Xing Liu, Jiahuang Tang, Linpeng Yu ⁎, Shungui Zhou Fujian Provincial Key Laboratory of Soil Environmental Health and Regulation, College of Resources and Environment, Fujian Agriculture and Forestry University, Fuzhou 350002, China
a r t i c l e
i n f o
Article history: Received 21 November 2018 Received in revised form 3 January 2019 Accepted 3 January 2019 Available online 5 January 2019 Keywords: Prosthecochloris aestuarii Geobacter sulfurreducens Direct interspecies electron transfer Syntrophic photosynthesis Microbial fuel cell
a b s t r a c t The syntrophic photosynthesis via direct interspecies electron transfer (DIET) between Geobacter sulfurreducens and Prosthecochloris aestuarii has opened a new paradigm of microbial phototrophy. However, it is still unknown whether this photosynthetic DIET can be mediated by an electrical conductor. Here we report first the photosynthetic DIET in a two-chamber microbial fuel cell (photo-MFC). The photo-MFC worked well and generated a maximum current of 0.6 mA/m2, which validated photosynthetic DIET via the titanium wire. Confocal laser scanning microscopy showed that G. sulfurreducens and P. aestuarii colonized the anode and cathode, respectively. P. aestuarii accepted extracellular electrons from G. sulfurreducens bioanodes under illumination at a current density of 0.6 mA/m2 (normalized to the cathode surface area), which could not be produced in the dark. Such a light-dependent electron uptake from solid electrodes by P. aestuarii was closely related with the presence of CO2, suggesting that P. aestuarii utilized extracellular electrons for photosynthetic CO2 reduction. Electrochemical in situ Fourier transform infrared (FTIR) spectroscopy revealed that certain outer membrane components of the two strains were involved in the DIET process. These results implied photosynthetic DIET can be mediated by electrically conductive materials in natural environments. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Direct interspecies electron transfer (DIET) is a recently discovered microbial syntrophy pathway involving metabolic cooperation of interdependent microbes for energy production [1,2]. Compared with conventional syntrophy that rely on the transport of metabolic products such as hydrogen and formic acid [3,4], DIET is independent of metabolic intermediates. It proceeds only via electron flow between microbes using their own conductive structures, such as pili and c-type cytochromes [5–7]. DIET between Geobacter and methanogenic archaea has been suggested to be a widespread phenomenon that accelerates methane production in anaerobic environments [8,9]. More recently, Ha et al. have demonstrated a new form of DIET between Geobacter sulfurreducens and phototrophic bacterium Prosthecochloris aestuarii [10]. In this co-culture, G. sulfurreducens oxidized acetate and donated electrons directly to P. aestuarii, which accepted the electrons for photosynthesis under illumination but not in the dark. Such photosynthetic DIET between G. sulfurreducens and P. aestuarii may have represented a new paradigm for syntrophic anaerobic metabolism. The discovery of photosynthetic DIET is of important ecological significance because it links anaerobic respiration closely to anoxygenic photosynthesis in the interface zone of sediments and water. However, to our ⁎ Corresponding author. E-mail address: [email protected] (L. Yu).
https://doi.org/10.1016/j.bioelechem.2019.01.002 1567-5394/© 2019 Elsevier B.V. All rights reserved.
best knowledge, photosynthetic DIET process has not been explored directly in microbial fuel cell. In this work, we visualized the intriguing photosynthetic DIET between G. sulfurreducens and P. aestuarii via the bio-current of a photosynthetic microbial fuel cell (photo-MFC) platform. The anodic and cathodic compartment of photo-MFCs were separated by a proton exchange membrane and inoculated with G. sulfurreducens and P. aestuarii, respectively. The anode and cathode were electrically connected by a metal wire with an external resistance. Cell voltages cross the external resistance were recorded to show the DIET currents between P. aestuarii and G. sulfurreducens. Light/dark cycle experiments were performed to investigate effects of cathodic illumination on the current production. Chronoamperometry, cyclic voltammetry (CV) and linear sweep voltammetry (LSV) were used to determine the electrochemical activity of photo-MFCs. The electron transfer mechanisms of P. aestuarii and G. sulfurreducens were analyzed using electrochemical in situ FTIR spectroscopy. 2. Material and methods 2.1. Microorganisms and cultivation conditions G. sulfurreducens PCA was cultured routinely in nutrient broth (NB) medium with acetate and fumarate (NBAF) at 30 °C as reported previously [11]. P. aestuarii strain (DSM 5030) was purchased from Deutsche
22
L. Huang et al. / Bioelectrochemistry 127 (2019) 21–25
Sammlung von Mikroorganismen und Zellkulturen (DSMZ). P. aestuarii was initially grown at 30 °C in a modified medium containing 3.5 mM sodium sulfide under illumination by a LED light (15 W, optical power density of 20 W·m−2) as previously described [10].
2.2. Construction and operation of photosynthetic microbial fuel cells Two-chamber photo-MFC with a 25-mL liquid volume and a 5-mL headspace for each chamber was constructed as previously reported [10,12,13]. Graphite plates (1.5 cm × 1 cm × 0.5 cm) were utilized as the anode and cathode electrode, respectively. Hg|Hg2Cl2 (sat. KCl) was used as the reference electrode. A proton exchange membrane (PEM, Nafion 117, DuPont Co., USA) was installed to separate the anodic and cathodic chamber. An external resistance (50 kΩ) was used to connect the anode and cathode electrode. The electrolytes in the anodic and cathodic chamber were modified media that were amended with 10 mM acetate and 0.5 mM thiosulfate, respectively [10]. Pure cultures of G. sulfurreducens and P. aestuarii cells (2 mL for each strain) at the logarithmic phase (OD600 = 0.3) were collected and washed three times with anaerobic anolyte and catholyte, and then transferred into the anodic and cathodic chamber, respectively. The anodic chamber was completely wrapped with aluminium foil to keep dark during the entire experiments. Illumination of cathodic chambers was conducted using a LED light (15 W) as mentioned above. All of the photo-MFCs were run in batch mode at 30 ± 1 °C.
2.3. Electrochemical measurements and analytical methods Cell voltages across the external resistance were recorded every 2 min by a data acquisition system (model 2700, Keithley Instruments). Open circuit potential (OCP) was measured with a VC890D digital multimeter. Electron uptake by P. aestuarii from the poised cathodes at −400 mV (vs. SCE) was determined in the absence of G. sulfurreducens using an electrochemical workstation (660E, CH instruments, USA). CV and LSV measurements were conducted in a potential range of −800 to +600 mV (vs. SCE) at a scan rate of 5 mV·s−1. Electrochemical in situ FTIR spectra were monitored with sample potentials set at −800 to 200 mV (vs. SCE) with an interval of 200 mV as described previously [13]. The anode and cathode biofilms were stained using a LIVE/DEAD Baclight Bacterial Viability kit (Life Technologies, USA) and observed under a confocal laser scanning microscope (CLSM, Carl Zeiss LSM 880, Germany) [13,14].
3. Results and discussion 3.1. Co-culture of P. aestuarii with G. sulfurreducens via metal-wire mediated DIET Two-chamber photosynthetic microbial fuel cells (photo-MFCs) were constructed to visualize the DIET process between P. aestuarii and G. sulfurreducens electrically (Fig. 1A). The proton exchange membrane (PEM) separated P. aestuarii and G. sulfurreducens, which prevented their physical contact [13,15] and the possible mediated electron transfer by endogenous electron shuttles such as flavin [16]. In this case, the anode served as the electron acceptor for G. sulfurreducens whereas the cathode served as the electron donor for P. aestuarii. G. sulfurreducens oxidized acetate in the anode compartment and electrons were transferred to an external metal wire. In the cathode compartment, P. aestuarii accepted electrons from the metal wire and performed photosynthesis. This was the sole possible survival pathways for G. sulfurreducens and P. aestuarii because no other potential electron acceptor (e.g. endogenous oxygen) was present in the cathode chamber. The main advantage of such photo-MFCs was that it could rule out all indirect interspecies electron transfer pathways mediated by intermediates (e.g. H2 and formate) which were often present in previous DIET studies [17–19]. Current generations by the photo-MFCs (G. sulfurreducens bioanode + P. aestuarii biocathode) were shown in Fig. 1B. After 12 h of cultivation, the current of photo-MFCs increased to a maximum value (0.6 mA/m2) and then stayed constant, revealing the DIET process between G. sulfurreducens and P. aestuarii. To further confirm that the current was derived from DIET via the metal wire connection, two control treatments with individual strains (abiotic anode + P. aestuarii biocathode or G. sulfurreducens bioanode + abiotic cathode) were performed as well (Fig. 1B). Negligible currents were produced in these control photo-MFCs, suggesting the absence of electron transfer. Therefore, the significant current production by the co-culture photo-MFCs could serve as a direct and visible phototrophic DIET indicator. 3.2. Light-dependent DIET between P. aestuarii and G. sulfurreducens To figure out whether the DIET between P. aestuarii and G. sulfurreducens was light-dependent, current generation by the photo-MFCs were monitored in light/dark cycles. As shown in Fig. 2A, when the LED light was turned off, the current generation decreased to near zero. However, once the photo-MFC regained illumination, the current returned to a maximum value of 0.64 mA/m2. This pattern of current generation under illumination and dark conditions was
Fig. 1. Schematic diagram of the co-culture photo-MFC (A) and current generation by the photo-MFCs under illumination with an external resistance of 50 kΩ (B). G. sulfurreducens bioanode + P. aestuarii biocathode; abiotic anode + P. aestuarii biocathode; G. sulfurreducens bioanode + abiotic cathode. The symbols of sun and moon represent the light and dark condition, respectively.
L. Huang et al. / Bioelectrochemistry 127 (2019) 21–25
23
Fig. 2A was mainly attributed to the biocathode compartment. In other words, the cathodic electron uptake by P. aestuarii depended on illumination. The phototrophic bacterium P. aestuarii is capable of reducing CO2 for photosynthesis under illumination [10,20]. To investigate whether the cathodic electron uptake process was related to photosynthetic CO2 reduction, current generation by the biocathode poised at −400 mV vs. SCE was monitored with a headspace of N2 or CO2. As shown in Fig. 2C, when the photo-MFCs were illuminated by light, the currents of photo-MFCs increased immediately for both CO2 and N2 headspace. However, the current (−0.5 mA/m2) of the photo-MFC with the CO2 headspace was obviously higher than that (−0.07 mA/m2) with the N2 headspace. This enhanced electron uptake by the CO2 headspace suggested that P. aestuarii could have reduced CO2 for photosynthesis under illumination. Theoretically, the cathode may substitute for the common photosynthetic electron donor (H2S) of P. aestuarii as follows: 34 e− + 8 CO2 + 7 H2O → 2(C4H7O3) [20]. The minor electron uptake under the N2 headspace was likely attributed to the residual CO2 released by bicarbonate in the medium. 3.3. Electrochemical characterization of electron uptake by P. aestuarii
Fig. 2. Current response of the abiotic ( ), individual strain ( P. aestuarii; G. sulfurreducens) and co-culture ( ) photo-MFCs to alternate light/dark circles after 48-hour operation under a CO2 headspace (A); the OCP of G. sulfurreducens bioanode (
) and P. aestuarii biocathode (
) after 120-hour operation (B); electron
uptake by P. aestuarii from the poised cathode (−0.4 V vs. SCE) without G. sulfurreducens after 120-h operation under a headspace of N2 ( ) or CO2 ( ) (C).
reproducible in three cycles of alternate operation. In addition, the OCP of photo-MFC (110 mV) produced under illumination was higher than that in the dark (79 mV) (Fig. 2B), revealing the light-dependent current production in the photo-MFCs. Moreover, the OCP of bioanode (−72 mV vs. SCE) under illumination of anodic chamber was almost identical with that in the dark (−70 mV vs. SCE), suggesting no effect of illumination on the bioanode. However, the OCP of biocathode decreased from +38 to +9 mV (vs. SCE) when the LED light was switched off. This indicated that the current response to light/dark cycles in
To figure out the biofilm activity and their spatial structure, the cathodic and anodic biofilms were stained with LIVE/DEAD staining and visualized by a CLSM. Large areas of green fluorescence were observed for both cathodic and anodic biofilms after one month of operation. Only small amounts of dead P. aestuarii and G. sulfurreducens (represented by red fluorescence) were detected on these electrodes. The thicknesses of cathodic and anodic biofilms were 25 and 63 μm, respectively (Fig. 3A, B). These indicated the survival and strong adaption of both P. aestuarii and G. sulfurreducens in the photo-MFCs. Thus, the results demonstrated the syntrophic growth of P. aestuarii and G. sulfurreducens via metal-wire connection. The electrochemical activities of P. aestuarii biocathode and G. sulfurreducens bioanode were analyzed by CV and LSV measurements. As shown in Fig. 3C, two pairs of oxidation and reduction peaks with midpoint potentials (E1/2) of around −0.56 and − 0.11 V vs. SCE were detected for P. aestuarii biocathode. The former E1/2 is close to the standard redox potential of NAD+ (to NADH, −0.56 V vs. SCE) [21,22]. Under a CO2 headspace, the reduction peak current at −0.57 V vs. SCE produced by the biocathode with illumination was obviously higher than that in the dark (Fig. 3C). However, under the N2 headspace, the reduction peak currents at −0.57 V vs. SCE were nearly identical for the illumination and dark conditions. In addition, the reduction peak currents under the N2 headspace were obviously lower than those under the CO2 headspace, implying the involvement of CO2 in the electrocatalysis. Similarly, LSV scans showed that the P. aestuarii biocathode exhibited a higher catalytic current at −0.57 V vs. SCE under the illumination and CO2 condition than those obtained in other treatments (Fig. 3D). These results suggested photosynthetic CO2 reduction by P. aestuarii and demonstrated the important role of illumination and CO2 in the electron uptake. Electrochemical in situ FTIR spectroscopy was performed to clarify the electron transfer mechanisms of P. aestuarii biocathode and G. sulfurreducens bioanode. There were seven signal bands at 1700, 1605, 1530, 1504, 1380, 1335, 1153 cm−1 for P. aestuarii biocathode (Fig. 3E) and six bands at 1650, 1552, 1458, 1440, 1248, 1153 cm−1 for G. sulfurreducens bioanode (Fig. 3F). The bands in the region of 1700–1600 cm−1 could be assigned to the vibration of amide I in the peptide backbone [13,23]. Bands at 1440, 1380, 1248, 1153 cm−1 were associated with amide II and amide III [23,24]. The FTIR bands at 1380, 1335, 1552, 1458 cm−1 have been reported to be related to c-type cytochromes [25]. The band intensity at 1380 and 1335 cm−1 for P. aestuarii increased significantly when the electrode potential increased gradually from −800 to +200 mV vs. SCE. This revealed some redox transformation reactions had occurred at the bacteria-electrode
24
L. Huang et al. / Bioelectrochemistry 127 (2019) 21–25
Fig. 3. 3D images of P. aestuarii biocathode (A) and G. sulfurreducens bioanode (B) at day 30. CV curves (C) and LSV curves (D) of P. aestuarii biocathode at day 6 under a headspace of N2 or CO2. P. aestuarii-CO2-light; P. aestuarii-CO2-dark; P. aestuarii-N2-light; P. aestuarii-N2-dark; Abiotic cathode-CO2; Abiotic cathode-N2. Electrochemical in situ FTIR spectra for P. aestuarii biocathode (E) and G. sulfurreducens bioanode (F). Green and red fluorescence indicates living cells and dead cells, respectively.
interface. The band signals for P. aestuarii and G. sulfurreducens suggested that certain outer membrane redox components had participated in their extracellular electron transfer processes.
Acknowledgements
4. Conclusions
References
Our results demonstrate that the current generation from photoMFCs can directly indicate DIET between G. sulfurreducens and P. aestuarii. The change of currents in the photo-MFCs under light and dark conditions revealed the light dependence of the DIET. Cathodic electron uptake by P. aestuarii was closely associated with the presence of CO2, suggesting the photosynthetic reduction of CO2. Overall, these results suggest photosynthetic DIET may be mediated by natural electrical conductors in anaerobic environments, and photo-MFCs can be developed as a simple and visible platform for evaluating microbial photosynthetic DIET.
This study was supported by the Natural Science Foundation of China (No. 91751109 and 41701270).
[1] Z.M. Summers, H.E. Fogarty, C. Leang, A.E. Franks, N.S. Malvankar, D.R. Lovley, Direct exchange of electrons within aggregates of an evolved syntrophic coculture of anaerobic bacteria, Science 330 (2010) 1413–1415. [2] P.M. Shrestha, A.E. Rotaru, Z.M. Summers, M. Shrestha, F.H. Liu, D.R. Lovley, Transcriptomic and genetic analysis of direct interspecies electron transfer, Appl. Environ. Microbiol. 79 (2013) 2397–2404. [3] F.A. De Bok, C.M. Plugge, A.J. Stams, Interspecies electron transfer in methanogenic propionate degrading consortia, Water Res. 38 (2004) 1368–1375. [4] M. Felchner-Zwirello, J. Winter, C. Gallert, Interspecies distances between propionic acid degraders and methanogens in syntrophic consortia for optimal hydrogen transfer, Appl. Microbiol. Biotechnol. 97 (2013) 9193–9205. [5] D.R. Lovley, S.M. Strycharz, R.H. Glaven, M.V. Coppi, S.M. Gannon, L.A. Perpetua, A. Liu, K.P. Nevin, Gene expression and deletion analysis of mechanisms for electron
L. Huang et al. / Bioelectrochemistry 127 (2019) 21–25
[6]
[7] [8]
[9]
[10]
[11] [12]
[13]
[14]
[15]
transfer from electrodes to Geobacter sulfurreducens, Bioelectrochemstry 80 (2010) 142–150. X. Liu, P.L. Tremblay, N.S. Malvankar, K.P. Nevin, D.R. Lovley, M. Vargas, A Geobacter sulfurreducens strain expressing Pseudomonas aeruginosa type IV pili localizes OmcS on pili but is deficient in Fe(III) oxide reduction and current production, Appl. Environ. Microbiol. 80 (2014) 1219–1224. P.M. Shrestha, A.E. Rotaru, Plugging in or going wireless: strategies for interspecies electron transfer, Front. Microbiol. 5 (2014) 237. V.C. Cruz, S. Rossetti, S. Fazi, P. Paiano, M. Majone, F. Aulenta, Magnetite particles triggering a faster and more robust syntrophic pathway of methanogenic propionate degradation, Environ. Sci. Technol. 48 (2014) 7536–7543. A.E. Rotaru, P.M. Shrestha, F.H. Liu, M. Shrestha, D. Shrestha, M. Embree, K. Zengler, C. Wardman, K.P. Nevin, D.R. Lovley, A new model for electron flow during anaerobic digestion: Direct interspecies electron transfer to Methanosaeta for the reduction of carbon dioxide to methane, Energy Environ. Sci. 7 (2014) 408–415. P.T. Ha, S.R. Lindemann, L. Shi, A.C. Dohnalkova, J.K. Fredrickson, M.T. Madigan, H. Beyenal, Syntrophic anaerobic photosynthesis via direct interspecies electron transfer, Nat. Commun. 8 (2017), 13924. . M.V. Coppi, C. Leang, S.J. Sandler, D.R. Lovley, Development of a genetic system for Geobacter sulfurreducens, Appl. Environ. Microbiol. 67 (2001) 3180–3187. J. Ye, A.D. Hu, G.P. Ren, T. Zhou, G.M. Zhang, S.G. Zhou, Red mud enhances methanogenesis with the simultaneous improvement of hydrolysis-acidification and electrical conductivity, Bioresour. Technol. 247 (2018) 131–137. G.Q. Yang, L.Y. Huang, L.X. You, L. Zhuang, S.G. Zhou, Electrochemical and spectroscopic insights into the mechanisms of bidirectional microbe-electrode electron transfer in Geobacter soli biofilms, Electrochem. Commun. 77 (2017) 93–97. D. Sun, S.A. Cheng, A.J. Wang, F.B. Li, B.E. Logan, K.F. Cen, Temporal-spatial changes in viabilities and electrochemical properties of anode biofilms, Environ. Sci. Technol. 49 (2015) 5227–5235. L. Zhuang, Y. Yuan, G.Q. Yang, S.G. Zhou, In situ formation of graphene/biofilm composites for enhanced oxygen reduction in biocathode microbial fuel cells, Electrochem. Commun. 21 (2012) 69–72.
25
[16] A. Okamoto, K. Saito, K. Inoue, K.H. Nealson, K. Hashimoto, R. Nakamura, Uptake of self-secreted flavins as bound cofactors for extracellular electron transfer in Geobacter species, Energy Environ. Sci. 7 (2014) 1357–1361. [17] M. Morita, N.S. Malvankar, A.E. Franks, Z.M. Summers, L. Giloteaux, A.E. Rotaru, C. Rotaru, D.R. Lovley, Potential for direct interspecies electron transfer in methanogenic wastewater digester aggregates, MBio 2 (2011) (e00159-11). [18] T. Storck, B. Virdis, D.J. Batstone, Modelling extracellular limitations for mediated versus direct interspecies electron transfer, ISME J. 10 (2016) 621–631. [19] Z.Q. Zhao, Y.B. Zhang, Y. Li, Y. Dang, T.T. Zhu, X. Quan, Potentially shifting from interspecies hydrogen transfer to direct interspecies electron transfer for syntrophic metabolism to resist acidic impact with conductive carbon cloth, Chem. Eng. J. 313 (2017) 10–18. [20] H. Biebl, N. Pfennig, Growth yields of green sulfur bacteria in mixed cultures with sulfur and sulfate reducing bacteria, Arch. Microbiol. 117 (1978) 9–16. [21] T. Matsui, Y. Kitagawa, M. Okumura, Y. Shigeta, Accurate standard hydrogen electrode potential and applications to the redox potentials of vitamin C and NAD/ NADH, J. Phys. Chem. A 119 (2015) 369–376. [22] D.F. Wilson, A.M. Erecińska, P.L. Dutton, Thermodynamic relationships in mitochondrial oxidative phosphorylation, Annu. Rev. Biophys. Bioeng. 3 (1974) 203–230. [23] K. Ataka, J. Heberle, Functional vibrational spectroscopy of a cytochrome c monolayer: SEIDAS probes the interaction with different surface-modified electrodes, J. Am. Chem. Soc. 126 (2004) 9445–9457. [24] L. Marboutin, A. Boussac, C. Berthomieu, Redox infrared markers of the heme and axial ligands in microperoxidase: Bases for the analysis of c-type cytochromes, J. Biol. Inorg. Chem. 11 (2006) 811–823. [25] J.P. Busalmen, A. Esteve-Núñez, A. Berná, J.M. Feliu, C-type cytochromes wire electricity-producing bacteria to electrodes, Angew. Chem. Int. Ed. Engl. 120 (2010) 4952–4955.
Contents lists available at ScienceDirect
Bioelectrochemistry journal homepage: www.elsevier.com/locate/bioelechem
Electrochemical evidence for direct interspecies electron transfer between Geobacter sulfurreducens and Prosthecochloris aestuarii Lingyan Huang, Xing Liu, Jiahuang Tang, Linpeng Yu ⁎, Shungui Zhou Fujian Provincial Key Laboratory of Soil Environmental Health and Regulation, College of Resources and Environment, Fujian Agriculture and Forestry University, Fuzhou 350002, China
a r t i c l e
i n f o
Article history: Received 21 November 2018 Received in revised form 3 January 2019 Accepted 3 January 2019 Available online 5 January 2019 Keywords: Prosthecochloris aestuarii Geobacter sulfurreducens Direct interspecies electron transfer Syntrophic photosynthesis Microbial fuel cell
a b s t r a c t The syntrophic photosynthesis via direct interspecies electron transfer (DIET) between Geobacter sulfurreducens and Prosthecochloris aestuarii has opened a new paradigm of microbial phototrophy. However, it is still unknown whether this photosynthetic DIET can be mediated by an electrical conductor. Here we report first the photosynthetic DIET in a two-chamber microbial fuel cell (photo-MFC). The photo-MFC worked well and generated a maximum current of 0.6 mA/m2, which validated photosynthetic DIET via the titanium wire. Confocal laser scanning microscopy showed that G. sulfurreducens and P. aestuarii colonized the anode and cathode, respectively. P. aestuarii accepted extracellular electrons from G. sulfurreducens bioanodes under illumination at a current density of 0.6 mA/m2 (normalized to the cathode surface area), which could not be produced in the dark. Such a light-dependent electron uptake from solid electrodes by P. aestuarii was closely related with the presence of CO2, suggesting that P. aestuarii utilized extracellular electrons for photosynthetic CO2 reduction. Electrochemical in situ Fourier transform infrared (FTIR) spectroscopy revealed that certain outer membrane components of the two strains were involved in the DIET process. These results implied photosynthetic DIET can be mediated by electrically conductive materials in natural environments. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Direct interspecies electron transfer (DIET) is a recently discovered microbial syntrophy pathway involving metabolic cooperation of interdependent microbes for energy production [1,2]. Compared with conventional syntrophy that rely on the transport of metabolic products such as hydrogen and formic acid [3,4], DIET is independent of metabolic intermediates. It proceeds only via electron flow between microbes using their own conductive structures, such as pili and c-type cytochromes [5–7]. DIET between Geobacter and methanogenic archaea has been suggested to be a widespread phenomenon that accelerates methane production in anaerobic environments [8,9]. More recently, Ha et al. have demonstrated a new form of DIET between Geobacter sulfurreducens and phototrophic bacterium Prosthecochloris aestuarii [10]. In this co-culture, G. sulfurreducens oxidized acetate and donated electrons directly to P. aestuarii, which accepted the electrons for photosynthesis under illumination but not in the dark. Such photosynthetic DIET between G. sulfurreducens and P. aestuarii may have represented a new paradigm for syntrophic anaerobic metabolism. The discovery of photosynthetic DIET is of important ecological significance because it links anaerobic respiration closely to anoxygenic photosynthesis in the interface zone of sediments and water. However, to our ⁎ Corresponding author. E-mail address: [email protected] (L. Yu).
https://doi.org/10.1016/j.bioelechem.2019.01.002 1567-5394/© 2019 Elsevier B.V. All rights reserved.
best knowledge, photosynthetic DIET process has not been explored directly in microbial fuel cell. In this work, we visualized the intriguing photosynthetic DIET between G. sulfurreducens and P. aestuarii via the bio-current of a photosynthetic microbial fuel cell (photo-MFC) platform. The anodic and cathodic compartment of photo-MFCs were separated by a proton exchange membrane and inoculated with G. sulfurreducens and P. aestuarii, respectively. The anode and cathode were electrically connected by a metal wire with an external resistance. Cell voltages cross the external resistance were recorded to show the DIET currents between P. aestuarii and G. sulfurreducens. Light/dark cycle experiments were performed to investigate effects of cathodic illumination on the current production. Chronoamperometry, cyclic voltammetry (CV) and linear sweep voltammetry (LSV) were used to determine the electrochemical activity of photo-MFCs. The electron transfer mechanisms of P. aestuarii and G. sulfurreducens were analyzed using electrochemical in situ FTIR spectroscopy. 2. Material and methods 2.1. Microorganisms and cultivation conditions G. sulfurreducens PCA was cultured routinely in nutrient broth (NB) medium with acetate and fumarate (NBAF) at 30 °C as reported previously [11]. P. aestuarii strain (DSM 5030) was purchased from Deutsche
22
L. Huang et al. / Bioelectrochemistry 127 (2019) 21–25
Sammlung von Mikroorganismen und Zellkulturen (DSMZ). P. aestuarii was initially grown at 30 °C in a modified medium containing 3.5 mM sodium sulfide under illumination by a LED light (15 W, optical power density of 20 W·m−2) as previously described [10].
2.2. Construction and operation of photosynthetic microbial fuel cells Two-chamber photo-MFC with a 25-mL liquid volume and a 5-mL headspace for each chamber was constructed as previously reported [10,12,13]. Graphite plates (1.5 cm × 1 cm × 0.5 cm) were utilized as the anode and cathode electrode, respectively. Hg|Hg2Cl2 (sat. KCl) was used as the reference electrode. A proton exchange membrane (PEM, Nafion 117, DuPont Co., USA) was installed to separate the anodic and cathodic chamber. An external resistance (50 kΩ) was used to connect the anode and cathode electrode. The electrolytes in the anodic and cathodic chamber were modified media that were amended with 10 mM acetate and 0.5 mM thiosulfate, respectively [10]. Pure cultures of G. sulfurreducens and P. aestuarii cells (2 mL for each strain) at the logarithmic phase (OD600 = 0.3) were collected and washed three times with anaerobic anolyte and catholyte, and then transferred into the anodic and cathodic chamber, respectively. The anodic chamber was completely wrapped with aluminium foil to keep dark during the entire experiments. Illumination of cathodic chambers was conducted using a LED light (15 W) as mentioned above. All of the photo-MFCs were run in batch mode at 30 ± 1 °C.
2.3. Electrochemical measurements and analytical methods Cell voltages across the external resistance were recorded every 2 min by a data acquisition system (model 2700, Keithley Instruments). Open circuit potential (OCP) was measured with a VC890D digital multimeter. Electron uptake by P. aestuarii from the poised cathodes at −400 mV (vs. SCE) was determined in the absence of G. sulfurreducens using an electrochemical workstation (660E, CH instruments, USA). CV and LSV measurements were conducted in a potential range of −800 to +600 mV (vs. SCE) at a scan rate of 5 mV·s−1. Electrochemical in situ FTIR spectra were monitored with sample potentials set at −800 to 200 mV (vs. SCE) with an interval of 200 mV as described previously [13]. The anode and cathode biofilms were stained using a LIVE/DEAD Baclight Bacterial Viability kit (Life Technologies, USA) and observed under a confocal laser scanning microscope (CLSM, Carl Zeiss LSM 880, Germany) [13,14].
3. Results and discussion 3.1. Co-culture of P. aestuarii with G. sulfurreducens via metal-wire mediated DIET Two-chamber photosynthetic microbial fuel cells (photo-MFCs) were constructed to visualize the DIET process between P. aestuarii and G. sulfurreducens electrically (Fig. 1A). The proton exchange membrane (PEM) separated P. aestuarii and G. sulfurreducens, which prevented their physical contact [13,15] and the possible mediated electron transfer by endogenous electron shuttles such as flavin [16]. In this case, the anode served as the electron acceptor for G. sulfurreducens whereas the cathode served as the electron donor for P. aestuarii. G. sulfurreducens oxidized acetate in the anode compartment and electrons were transferred to an external metal wire. In the cathode compartment, P. aestuarii accepted electrons from the metal wire and performed photosynthesis. This was the sole possible survival pathways for G. sulfurreducens and P. aestuarii because no other potential electron acceptor (e.g. endogenous oxygen) was present in the cathode chamber. The main advantage of such photo-MFCs was that it could rule out all indirect interspecies electron transfer pathways mediated by intermediates (e.g. H2 and formate) which were often present in previous DIET studies [17–19]. Current generations by the photo-MFCs (G. sulfurreducens bioanode + P. aestuarii biocathode) were shown in Fig. 1B. After 12 h of cultivation, the current of photo-MFCs increased to a maximum value (0.6 mA/m2) and then stayed constant, revealing the DIET process between G. sulfurreducens and P. aestuarii. To further confirm that the current was derived from DIET via the metal wire connection, two control treatments with individual strains (abiotic anode + P. aestuarii biocathode or G. sulfurreducens bioanode + abiotic cathode) were performed as well (Fig. 1B). Negligible currents were produced in these control photo-MFCs, suggesting the absence of electron transfer. Therefore, the significant current production by the co-culture photo-MFCs could serve as a direct and visible phototrophic DIET indicator. 3.2. Light-dependent DIET between P. aestuarii and G. sulfurreducens To figure out whether the DIET between P. aestuarii and G. sulfurreducens was light-dependent, current generation by the photo-MFCs were monitored in light/dark cycles. As shown in Fig. 2A, when the LED light was turned off, the current generation decreased to near zero. However, once the photo-MFC regained illumination, the current returned to a maximum value of 0.64 mA/m2. This pattern of current generation under illumination and dark conditions was
Fig. 1. Schematic diagram of the co-culture photo-MFC (A) and current generation by the photo-MFCs under illumination with an external resistance of 50 kΩ (B). G. sulfurreducens bioanode + P. aestuarii biocathode; abiotic anode + P. aestuarii biocathode; G. sulfurreducens bioanode + abiotic cathode. The symbols of sun and moon represent the light and dark condition, respectively.
L. Huang et al. / Bioelectrochemistry 127 (2019) 21–25
23
Fig. 2A was mainly attributed to the biocathode compartment. In other words, the cathodic electron uptake by P. aestuarii depended on illumination. The phototrophic bacterium P. aestuarii is capable of reducing CO2 for photosynthesis under illumination [10,20]. To investigate whether the cathodic electron uptake process was related to photosynthetic CO2 reduction, current generation by the biocathode poised at −400 mV vs. SCE was monitored with a headspace of N2 or CO2. As shown in Fig. 2C, when the photo-MFCs were illuminated by light, the currents of photo-MFCs increased immediately for both CO2 and N2 headspace. However, the current (−0.5 mA/m2) of the photo-MFC with the CO2 headspace was obviously higher than that (−0.07 mA/m2) with the N2 headspace. This enhanced electron uptake by the CO2 headspace suggested that P. aestuarii could have reduced CO2 for photosynthesis under illumination. Theoretically, the cathode may substitute for the common photosynthetic electron donor (H2S) of P. aestuarii as follows: 34 e− + 8 CO2 + 7 H2O → 2(C4H7O3) [20]. The minor electron uptake under the N2 headspace was likely attributed to the residual CO2 released by bicarbonate in the medium. 3.3. Electrochemical characterization of electron uptake by P. aestuarii
Fig. 2. Current response of the abiotic ( ), individual strain ( P. aestuarii; G. sulfurreducens) and co-culture ( ) photo-MFCs to alternate light/dark circles after 48-hour operation under a CO2 headspace (A); the OCP of G. sulfurreducens bioanode (
) and P. aestuarii biocathode (
) after 120-hour operation (B); electron
uptake by P. aestuarii from the poised cathode (−0.4 V vs. SCE) without G. sulfurreducens after 120-h operation under a headspace of N2 ( ) or CO2 ( ) (C).
reproducible in three cycles of alternate operation. In addition, the OCP of photo-MFC (110 mV) produced under illumination was higher than that in the dark (79 mV) (Fig. 2B), revealing the light-dependent current production in the photo-MFCs. Moreover, the OCP of bioanode (−72 mV vs. SCE) under illumination of anodic chamber was almost identical with that in the dark (−70 mV vs. SCE), suggesting no effect of illumination on the bioanode. However, the OCP of biocathode decreased from +38 to +9 mV (vs. SCE) when the LED light was switched off. This indicated that the current response to light/dark cycles in
To figure out the biofilm activity and their spatial structure, the cathodic and anodic biofilms were stained with LIVE/DEAD staining and visualized by a CLSM. Large areas of green fluorescence were observed for both cathodic and anodic biofilms after one month of operation. Only small amounts of dead P. aestuarii and G. sulfurreducens (represented by red fluorescence) were detected on these electrodes. The thicknesses of cathodic and anodic biofilms were 25 and 63 μm, respectively (Fig. 3A, B). These indicated the survival and strong adaption of both P. aestuarii and G. sulfurreducens in the photo-MFCs. Thus, the results demonstrated the syntrophic growth of P. aestuarii and G. sulfurreducens via metal-wire connection. The electrochemical activities of P. aestuarii biocathode and G. sulfurreducens bioanode were analyzed by CV and LSV measurements. As shown in Fig. 3C, two pairs of oxidation and reduction peaks with midpoint potentials (E1/2) of around −0.56 and − 0.11 V vs. SCE were detected for P. aestuarii biocathode. The former E1/2 is close to the standard redox potential of NAD+ (to NADH, −0.56 V vs. SCE) [21,22]. Under a CO2 headspace, the reduction peak current at −0.57 V vs. SCE produced by the biocathode with illumination was obviously higher than that in the dark (Fig. 3C). However, under the N2 headspace, the reduction peak currents at −0.57 V vs. SCE were nearly identical for the illumination and dark conditions. In addition, the reduction peak currents under the N2 headspace were obviously lower than those under the CO2 headspace, implying the involvement of CO2 in the electrocatalysis. Similarly, LSV scans showed that the P. aestuarii biocathode exhibited a higher catalytic current at −0.57 V vs. SCE under the illumination and CO2 condition than those obtained in other treatments (Fig. 3D). These results suggested photosynthetic CO2 reduction by P. aestuarii and demonstrated the important role of illumination and CO2 in the electron uptake. Electrochemical in situ FTIR spectroscopy was performed to clarify the electron transfer mechanisms of P. aestuarii biocathode and G. sulfurreducens bioanode. There were seven signal bands at 1700, 1605, 1530, 1504, 1380, 1335, 1153 cm−1 for P. aestuarii biocathode (Fig. 3E) and six bands at 1650, 1552, 1458, 1440, 1248, 1153 cm−1 for G. sulfurreducens bioanode (Fig. 3F). The bands in the region of 1700–1600 cm−1 could be assigned to the vibration of amide I in the peptide backbone [13,23]. Bands at 1440, 1380, 1248, 1153 cm−1 were associated with amide II and amide III [23,24]. The FTIR bands at 1380, 1335, 1552, 1458 cm−1 have been reported to be related to c-type cytochromes [25]. The band intensity at 1380 and 1335 cm−1 for P. aestuarii increased significantly when the electrode potential increased gradually from −800 to +200 mV vs. SCE. This revealed some redox transformation reactions had occurred at the bacteria-electrode
24
L. Huang et al. / Bioelectrochemistry 127 (2019) 21–25
Fig. 3. 3D images of P. aestuarii biocathode (A) and G. sulfurreducens bioanode (B) at day 30. CV curves (C) and LSV curves (D) of P. aestuarii biocathode at day 6 under a headspace of N2 or CO2. P. aestuarii-CO2-light; P. aestuarii-CO2-dark; P. aestuarii-N2-light; P. aestuarii-N2-dark; Abiotic cathode-CO2; Abiotic cathode-N2. Electrochemical in situ FTIR spectra for P. aestuarii biocathode (E) and G. sulfurreducens bioanode (F). Green and red fluorescence indicates living cells and dead cells, respectively.
interface. The band signals for P. aestuarii and G. sulfurreducens suggested that certain outer membrane redox components had participated in their extracellular electron transfer processes.
Acknowledgements
4. Conclusions
References
Our results demonstrate that the current generation from photoMFCs can directly indicate DIET between G. sulfurreducens and P. aestuarii. The change of currents in the photo-MFCs under light and dark conditions revealed the light dependence of the DIET. Cathodic electron uptake by P. aestuarii was closely associated with the presence of CO2, suggesting the photosynthetic reduction of CO2. Overall, these results suggest photosynthetic DIET may be mediated by natural electrical conductors in anaerobic environments, and photo-MFCs can be developed as a simple and visible platform for evaluating microbial photosynthetic DIET.
This study was supported by the Natural Science Foundation of China (No. 91751109 and 41701270).
[1] Z.M. Summers, H.E. Fogarty, C. Leang, A.E. Franks, N.S. Malvankar, D.R. Lovley, Direct exchange of electrons within aggregates of an evolved syntrophic coculture of anaerobic bacteria, Science 330 (2010) 1413–1415. [2] P.M. Shrestha, A.E. Rotaru, Z.M. Summers, M. Shrestha, F.H. Liu, D.R. Lovley, Transcriptomic and genetic analysis of direct interspecies electron transfer, Appl. Environ. Microbiol. 79 (2013) 2397–2404. [3] F.A. De Bok, C.M. Plugge, A.J. Stams, Interspecies electron transfer in methanogenic propionate degrading consortia, Water Res. 38 (2004) 1368–1375. [4] M. Felchner-Zwirello, J. Winter, C. Gallert, Interspecies distances between propionic acid degraders and methanogens in syntrophic consortia for optimal hydrogen transfer, Appl. Microbiol. Biotechnol. 97 (2013) 9193–9205. [5] D.R. Lovley, S.M. Strycharz, R.H. Glaven, M.V. Coppi, S.M. Gannon, L.A. Perpetua, A. Liu, K.P. Nevin, Gene expression and deletion analysis of mechanisms for electron
L. Huang et al. / Bioelectrochemistry 127 (2019) 21–25
[6]
[7] [8]
[9]
[10]
[11] [12]
[13]
[14]
[15]
transfer from electrodes to Geobacter sulfurreducens, Bioelectrochemstry 80 (2010) 142–150. X. Liu, P.L. Tremblay, N.S. Malvankar, K.P. Nevin, D.R. Lovley, M. Vargas, A Geobacter sulfurreducens strain expressing Pseudomonas aeruginosa type IV pili localizes OmcS on pili but is deficient in Fe(III) oxide reduction and current production, Appl. Environ. Microbiol. 80 (2014) 1219–1224. P.M. Shrestha, A.E. Rotaru, Plugging in or going wireless: strategies for interspecies electron transfer, Front. Microbiol. 5 (2014) 237. V.C. Cruz, S. Rossetti, S. Fazi, P. Paiano, M. Majone, F. Aulenta, Magnetite particles triggering a faster and more robust syntrophic pathway of methanogenic propionate degradation, Environ. Sci. Technol. 48 (2014) 7536–7543. A.E. Rotaru, P.M. Shrestha, F.H. Liu, M. Shrestha, D. Shrestha, M. Embree, K. Zengler, C. Wardman, K.P. Nevin, D.R. Lovley, A new model for electron flow during anaerobic digestion: Direct interspecies electron transfer to Methanosaeta for the reduction of carbon dioxide to methane, Energy Environ. Sci. 7 (2014) 408–415. P.T. Ha, S.R. Lindemann, L. Shi, A.C. Dohnalkova, J.K. Fredrickson, M.T. Madigan, H. Beyenal, Syntrophic anaerobic photosynthesis via direct interspecies electron transfer, Nat. Commun. 8 (2017), 13924. . M.V. Coppi, C. Leang, S.J. Sandler, D.R. Lovley, Development of a genetic system for Geobacter sulfurreducens, Appl. Environ. Microbiol. 67 (2001) 3180–3187. J. Ye, A.D. Hu, G.P. Ren, T. Zhou, G.M. Zhang, S.G. Zhou, Red mud enhances methanogenesis with the simultaneous improvement of hydrolysis-acidification and electrical conductivity, Bioresour. Technol. 247 (2018) 131–137. G.Q. Yang, L.Y. Huang, L.X. You, L. Zhuang, S.G. Zhou, Electrochemical and spectroscopic insights into the mechanisms of bidirectional microbe-electrode electron transfer in Geobacter soli biofilms, Electrochem. Commun. 77 (2017) 93–97. D. Sun, S.A. Cheng, A.J. Wang, F.B. Li, B.E. Logan, K.F. Cen, Temporal-spatial changes in viabilities and electrochemical properties of anode biofilms, Environ. Sci. Technol. 49 (2015) 5227–5235. L. Zhuang, Y. Yuan, G.Q. Yang, S.G. Zhou, In situ formation of graphene/biofilm composites for enhanced oxygen reduction in biocathode microbial fuel cells, Electrochem. Commun. 21 (2012) 69–72.
25
[16] A. Okamoto, K. Saito, K. Inoue, K.H. Nealson, K. Hashimoto, R. Nakamura, Uptake of self-secreted flavins as bound cofactors for extracellular electron transfer in Geobacter species, Energy Environ. Sci. 7 (2014) 1357–1361. [17] M. Morita, N.S. Malvankar, A.E. Franks, Z.M. Summers, L. Giloteaux, A.E. Rotaru, C. Rotaru, D.R. Lovley, Potential for direct interspecies electron transfer in methanogenic wastewater digester aggregates, MBio 2 (2011) (e00159-11). [18] T. Storck, B. Virdis, D.J. Batstone, Modelling extracellular limitations for mediated versus direct interspecies electron transfer, ISME J. 10 (2016) 621–631. [19] Z.Q. Zhao, Y.B. Zhang, Y. Li, Y. Dang, T.T. Zhu, X. Quan, Potentially shifting from interspecies hydrogen transfer to direct interspecies electron transfer for syntrophic metabolism to resist acidic impact with conductive carbon cloth, Chem. Eng. J. 313 (2017) 10–18. [20] H. Biebl, N. Pfennig, Growth yields of green sulfur bacteria in mixed cultures with sulfur and sulfate reducing bacteria, Arch. Microbiol. 117 (1978) 9–16. [21] T. Matsui, Y. Kitagawa, M. Okumura, Y. Shigeta, Accurate standard hydrogen electrode potential and applications to the redox potentials of vitamin C and NAD/ NADH, J. Phys. Chem. A 119 (2015) 369–376. [22] D.F. Wilson, A.M. Erecińska, P.L. Dutton, Thermodynamic relationships in mitochondrial oxidative phosphorylation, Annu. Rev. Biophys. Bioeng. 3 (1974) 203–230. [23] K. Ataka, J. Heberle, Functional vibrational spectroscopy of a cytochrome c monolayer: SEIDAS probes the interaction with different surface-modified electrodes, J. Am. Chem. Soc. 126 (2004) 9445–9457. [24] L. Marboutin, A. Boussac, C. Berthomieu, Redox infrared markers of the heme and axial ligands in microperoxidase: Bases for the analysis of c-type cytochromes, J. Biol. Inorg. Chem. 11 (2006) 811–823. [25] J.P. Busalmen, A. Esteve-Núñez, A. Berná, J.M. Feliu, C-type cytochromes wire electricity-producing bacteria to electrodes, Angew. Chem. Int. Ed. Engl. 120 (2010) 4952–4955.