Direct or H2-mediated microbial electrosynthesis of acetate
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This document discusses direct microbial electrosynthesis or hydrogen-mediated microbial synthesis of acetate. It presents two potential electron transfer mechanisms for homoacetogenic bacteria to produce acetate from carbon dioxide at the cathode of a bioelectrochemical system: direct electron transfer, or hydrogen-mediated electron transfer via hydrogen gas. The document outlines experiments conducted with six homoacetogenic bacteria strains to determine which mechanism they use, including cyclic voltammetry to examine electron transfer and current production measurements. The results are inconclusive as to the dominant mechanism, suggesting both direct and mediated pathways may be involved in microbial electrosynthesis of acetate.
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Direct or H2-mediated microbial electrosynthesis of acetate
- 1. Direct microbial electrosynthesis or H2 mediated microbial synthesis of acetate? Alessandro Carmona*, Eric Trably and Nicolas Bernet *http://alessandrocarmona.blogspot.fr/
- 2. -Context- NADH NAD+ NAD+ Fossils fuels e- H2 + CO2 Acetate Classical fermentation e- NADH NAD+ NAD+ e- e- +CO2 Acetate e- Residual CO2 Electrode Cathode Microbial electrosynthesis
- 3. Electron transfer in Bioelectrochemical systems: bridge between natural environments and applied technologies NADH NAD+ NAD+ Potentiostat or Power source e- e- Organic matter CO2 e- Microbial anode Electricity production from waste! NADH NAD+ NAD+ e- + CO2 Potentiostat or Power source e- e- Reduced product e- Microbial cathode Molecules from renewable electricity and CO2! NADH NAD+ NAD+ e- Organic matter CO2 Fe(III) oxide
- 5. H2 evolution (H2 mediated acetate production) Direct MES Marshall (2012) Marshall (2013) Labelle (2014) Lovley‘s group: S ovata S. silvacetica S. Sphaeroides A. woodii C. ljunggahli C. aceticum M. thermoacetica Batlle (2015) Jiang (2013)Jourdin (2014) Xafenias (2014) Sun (2013) of acetate
- 6. Experimental approach Cronoamperometry + Chromatograpy Scanning electron microscopy Cyclic voltammetry current consumption electron transfer biofilm formation What type of electron transfer mechanism do these homoacetogens perform?
- 7. Cathode NADH NAD+ NAD+ e- e- e- + CO2 Potentiostat e- CH3COOH Medred Medox ? ? ? ? Moorella thermoacetica Clostridium aceticum Sporomusa sphaeroides Clostridium ljungdahlii Sporomusa silvacetica Acetobacterium woodii Sporomusa ovata carbon source T° nutrients pH biofilm Microbial electrosynthesis (MES) and e- transfer mechanisms in monocultures of homoacetogens What electron transfer mechanisms are used? Common growth conditions for all homoacetogens
- 8. “Classic microbiology” Electrochemical reactor CEM RE Cathode Anode Pt/IrFelt Gas Liquid DSMZ strains Liquid culture Grown strains 48 h 3000 rpm “washed“ pellet -Electron source: Electrode at -900 mV (vs. SCE) and other potentials -Carbon source: NaHCO3 -Electron source: H2 in gas phase -Carbon source: Pyruvate/ Fructose *DSMZ media: 135, 311, 642, 777 and 879 Average medium* 1. Sporomusa sphaeroides 2. Sporomusa silvacetica 3. Acetobacterium woodii 4. Moorella thermoacetica 5. Clostridium aceticum 6. Clostridium ljungdahlii 1. Sporomusa sphaeroides 2. Sporomusa silvacetica 3. Acetobacterium woodii 4. Moorella thermoacetica 5. Clostridium aceticum 6. Clostridium ljungdahlii “Electro-microbiology”
- 9. Electrochemical reactor: -Cathode: carbon felt 73 cm2 -Anode: Pt/Ir grid 2.5cm x 2.5cm -Reference electrode: SCE -Membrane: CEM Fumasep -Temperature: 35°C CEM RE SCE Anode: Pt/Ir 2.5 x 2.5 cm Gas samples Liquid samples Magnetic stirrer Magnetic stirrer Cathode: felt 1.0 x 3.5 x 7.0 cm
- 10. -Results-
- 12. Detected traces of: Propionate, Butyrate and Valerate only when yeast extract, vitamins and minerals are added Acetobacterium woodii Sporomusa sphaeroides ~ Sporomusa silvacetica< • Previously grown biofilms at -900 mV for 20 days in rich medium • Wasted medium was replaced by fresh „basal medium“: Vit, Min or Yeast • Multiple potential tests were carried out with only „basal medium“ Production rate per electrode surface area Pesa was calculated according to Patil, S. et al, Biotech. Adv. 2015: Additional experimental details: -Graphite felt as cathode material ~ 73 cm2 (projected surface cathode area) -Electron acceptor: excess of NaHCO3 (10 g/L) 10 mL of cells [OD620 nm: 1.041)] 10 mL of cells [OD620 nm: 0.741)] 10 mL of cells [OD620 nm: 0.044)] MET DET
- 13. Comparison of data on microbial electrosynthesis of acetate N.B.: taken from Patil S. et al. Environ. Sci. Technol. 2015, 49 (14): Sporomusa sphaeroides Carbon felt -556 mV vs. SHE (applied in CA) ~30 g/m2 d 0.987 g/L-0.08 A/m2 (observed) Carmona, Trably and Bernet, 2015
- 14. Scanning electron microscopy exemplary pictures biofilm formation
- 15. rod-shaped cell slightly curved spherical spores Kuhner et al., Int. J. Syst. Evol. Microbiol. 1997 Sporomusa silvacetica spores rodes flagella
- 20. Cyclic Voltammetry: Voltammograms showing the development of a reduction peak Example: S. silvacetica
- 21. -0,8 -0,6 -0,4 -0,2 0,0 -0,6 -0,5 -0,4 -0,3 -0,2 -0,1 0,0 0,1i/mA E/ V vs. SCE Eapp./ mV vs. SCE during CA: Control -200 -400 -600 -800 (-) Eapp./ mV vs. SCE during CA (+) Peakarea Acetate
- 22. -Conclusion- Then… what electron transfer mechanisms are used in MES of acetate? Direct? Mediated? Both?
- 23. Cathode Nanowire? 0.0 -250 -500 -750 -1000 ←Potentialvs.SCE(mV)→ e- e- e- e- Cytochrome? NADH NAD+ NAD+ CO2 e- CH3COOH Ef,1 -400 mV H+/H2 Ef,1 -900 mV Potentiostat e- NADH NAD+ NAD+ CO2 e- CH3COOH e- e- e- e- e- XY-Direct e- tranfer H2-Mediated e- tranfer
- 24. E.Trably, PhD We thank you for your attention… A. Carmona, PhD
Notes de l'éditeur
- Classical fermentation versus the concept of microbial electrosynthesis (MES): The main advantage that MES brings is the opportunity to use renewable sources of electrons to biologically convert residual (industrial) carbon dioxide into added value molecules.
- How can we go from the concept of Metal reducing bacteria to the so called Electrochemically active bacteria? -In the environment electrochemically active bacteria (EAB) can transfer electrons to solid terminal electron acceptors such as Fe(III), Mn(III) or Cr(VI). -In a Bioelectrochemical system such as microbial fuel cells (MFCs) these bacteria transfer the electrons to carbon materials for example. These bacteria not only play a key role in nature’s oxidation-reduction cycles but also are the key component of microbial bioelectrochemical systems (BES). Thus, the elucidation of the different microbial electron transfer pathways is of fundamental interest as well as technological relevance.
- Opportunities in microbial electrosynthesis: Most of the works reported so far use experimental conditions that electrochemically favor the production of hydrogen as electron carries for the production of acetate. Thus, at INRA Narbonne we have wondered whether it is feasible to conduct microbial electrosynthesis via a direct electron uptake of electrons between the electrode material and the bacterial culture.
- To investigate which type of electron transfer mechanisms is performed during microbial electrosynthesis of acetate we used different techniques: -Chronoampeometry to apply a fixed potential on the working electrode (or cathode) surface that electrochemically drives the uptake of electrons -Chromatography to quantify the amount of acetate (an other trace molecules) synthesized during this process -Scanning electron microscopy to inspect as to what extent the surface of the electrode was colonized by the pure cultures and whether a biofilm has been formed -Cyclic voltammetry to analyze possible electron transfer mechanisms occurring between the electrode material and the bacterial culture
- Now our question was… „What type of bacterial culture would allow us to unambiguously study such electron transfer mechanisms?“ For this reason we decided to test different homoacetogenic monocultures under strictly similar experimental conditions.
- Microbial electrosynthesis: classic and electro-microbiology -Six bacterial strains were ordered from the German collection of microorganisms and tested for growth in a homogenized medium. -During bacterial growth in a hommogenized medium the most favorable conditions were provided such as pyruvate/ fructose as carbon source, hydrogen in the gas phase as electron carrier, 35°C as incubation temperature and continuous stirring. -Only those strains able to grow in such homogenized medium were centrifuge-harvested, „washed“ and inoculated into the electrochemical reactor.
- Electrochemical reactor used: To test each monoculture, we employed a simplified lab scale electrochemical reactor. -A easily accessible commercial working electrode or cathode with a high surface area such as carbon felt, -A non catalyrically limiting commercial counter electrode or anode such as a Pt/Ir grid, -A reliable commercial saturated calomel reference electrode and -A cationic membrane to separate both chamber and idially avoid product transfer.
- Chronoampeometric biofilm growth: -After baterial cultures were inoculated* the electrodes were polarized at -900 mV vs SCE for 20 days to assure the formation of a biofilm. -Once this first growth phase concluded we carefully removed the wasted medium and subtituted it for fresh medium without Vitamins, Minerals, Yeast extract or any other carbon source such as piruvate or fructose. -Immediatly after, reactors were sealed and a gently flush of CO2 was used to maintain anaerobic conditions. -For each applied potential we performed the described proccedure and kept the fixed potential during at least 3-4 days. *Note: the concentration in OD at 620 nm for each culture is the typical concentration measured for each culture vial of 50 mL after 48 h of growth. Then, the vial was opened, centrifuged, the pellet resuspended in 10 mL of electrochemical-reactor-medium (thus, no pyruvate or fructose but yeast extract, minerals and vitamins) and injected into the electrochemical reactor.
- When we compare the data on microbial electrosynthesis of acetate previously compiled by the Patil and colleagues we noticed the following: -The maximum projected surface based rate obtained here is quite well in agreement with the current literature (around 30 via MET and 15 via DET (in g/m2 d)).
- Scanning electron microscopy to inspect the electrode surface for possible biofilm formation. -When compared to the first report microbiologically characterizing S. silvacetica we can see that in our micrograpghs we have found the three typical shapes for this bacterium: spores, rodes and flagella. -Something that we noticed is the clear difference between the typical bacterial deposition on the electrode of a well formed anodic bacterial biofilm and the deposition by this homoacetogenic monoculture. This means that we did not observe a uniform biofilm as in the case of electroactive Geobacter sp. dominated biofilms for example.
- Something that we found interesting and surprising while closely inspecting the electrode surface colonized by S. silvacetica is these two cells „sticked“ to the surface. When we inspect closer, we see that they seem to be attached to the surface… but by which means? Right now it is only a supossition but between the bacterial cell and the electrode fiber there seems to be a wire connecting both will they be conductive?
- While using cyclic voltammetry we found two types of voltammograms. CV type 1: Voltammograms showing an onset potential signal. CV type 2: Voltammograms showing the development of a reduction peak. Example: S. silvacetica.
- The second ty of CV showed the progressive development of a reduction peak. N.B.: only one voltammogram per applied potential is shown due to the similarity among them which indicates that the signal is due to the electrode-bacteria interaction. For example, when we subject the bacterial biofilm to a single applied potential and we perform CV afterwards we see a clear development of a reduction peak due to the interaction of the bacterial biofilm and the electrode material. Something to be notice is the absence of peak at -200 mV very likely due to the lack of activity/synthesized acetate at that potential. Additionally, there seems to be a correlation among the applied potential, the synthesized acetate and the area under the peak. Finally, and most importantly, the reduction peaks detected, independently of the applied potential, seem to be centered around -500 mV vs SCE which might indicate the potential of the molecule/protein use in the electron transfer mechanism.
- Due to the combination of electrochemistry, chromatography and microscopy we conclude from this study that during the MES of acetate two electron transfer mechanisms seem plausible depending on the applied potential: For applied potentials equal or more positive than the electrolysis of water (-650 mV vs SCE (-400 mV vs SHE)) certain homoacetogens will be able to perform MES via direc acceptance of electrons from the electrode. On the other side, for applied potentials equal or more negative than the electrolysis of water (-650 mV vs SCE (-400 mV vs SHE)) certain homoacetogens will be able to perform MES via direct and mediated acceptance of electrons from the electrode (likely both mechanisms).