Enhanced Microbial Electrosynthesis by Using Defined Co-Cultures

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The ISME Journal (2017) 11, 704–714 © 2017 International Society for Microbial Ecology All rights reserved 1751-7362/17 www.nature.com/ismej ORIGINAL ARTICLE Enhanced microbial electrosynthesis by using defined co-cultures

Jörg S Deutzmann1 and Alfred M Spormann1,2 1Department of Civil and Environmental Engineering, Stanford University, Stanford, CA, USA and 2Department of Chemical Engineering, Stanford University, Stanford, CA, USA

Microbial uptake of free cathodic electrons presents a poorly understood aspect of microbial physiology. Uptake of cathodic electrons is particularly important in microbial electrosynthesis of

sustainable fuel and chemical precursors using only CO2 and electricity as carbon, electron and energy source. Typically, large overpotentials (200 to 400 mV) were reported to be required for cathodic electron uptake during electrosynthesis of, for example, methane and acetate, or low electrosynthesis rates were observed. To address these limitations and to explore conceptual alternatives, we studied defined co-cultures metabolizing cathodic electrons. The Fe(0)-corroding strain IS4 was used to catalyze the electron uptake reaction from the cathode forming molecular hydrogen as intermediate, and Methanococcus maripaludis and Acetobacterium woodii were used as model microorganisms for hydrogenotrophic synthesis of methane and acetate, respectively. The IS4-M. maripaludis co-cultures achieved electromethanogenesis rates of 0.1–0.14 μmol cm− 2 h − 1 at − 400 mV vs standard hydrogen electrode and 0.6–0.9 μmol cm − 2 h − 1 at − 500 mV. Co-cultures of strain IS4 and A. woodii formed acetate at rates of 0.21–0.23 μmol cm − 2 h − 1 at − 400 mV and 0.57–0.74 μmol cm − 2 h − 1 at − 500 mV. These data show that defined co-cultures coupling cathodic electron uptake with synthesis reactions via interspecies hydrogen transfer may lay the foundation for an engineering strategy for microbial electrosynthesis. The ISME Journal (2017) 11, 704–714; doi:10.1038/ismej.2016.149; published online 1 November 2016

Introduction been emerging as promising platforms for a sustainable energy landscape. In particular, biocathodes in Microbial metabolism of electrons that are not ‘ ’ microbial electrosynthesis reactors are of interest, associated with a chemical element, that is, free where microorganisms at the cathode convert elec- electrons, is an intriguing metabolic capacity that has tricity plus CO2 into useful chemical products. From been primarily investigated in insoluble metal- a fundamental perspective, the mechanisms reducing microorganisms, such as Shewanella or involved in cathodic electron uptake are most Geobacter species (Bond and Lovley, 2003; intriguing, however, largely unknown. The most Hartshorne et al., 2009; Coursolle et al., 2010; basic biocathode is the hydrogen-evolving bio- Clarke et al., 2011; Lovley, 2012; Liu et al., 2014; cathode. Most studies on hydrogen-evolving bio- Malvankar and Lovley, 2014; Pirbadian et al., 2014; cathodes in a microbial electrosynthesis reactor have TerAvest et al., 2014). In addition to these anodic been carried out using mixed microbial cultures microbial processes, recent studies have revealed ‘ ’ (Jafary et al., 2015), and the ecology of the different that some microorganisms can take up free cathodic microorganisms in a community is poorly under- electrons from conductive minerals during interspe- stood. A few studies showed hydrogen formation by cies electron transfer (Kato et al., 2012) or from pure cultures of Geobacter sulfurreducens (Geelhoed abiotically reduced surfaces such as deep sea et al., 2010) or Desulfovibrio species (Croese et al., hydrothermal vent chimneys (Nakamura et al., ‘ ’ 2011; Aulenta et al., 2012), and the molecular 2010). This use of free electrons as electron donor mechanism of the electron uptake reaction in most in microbial catabolism finds also relevance in hydrogen-evolving, biocathodic microorganisms is engineered bioelectrochemical systems, which have unknown (Jafary et al., 2015). A more specialized type of biocathode is found in microbial electro- Correspondence: AM Spormann or JS Deutzmann, Department of synthesis. In this process, microorganisms convert Civil and Environmental Engineering, Stanford University, 318 cathode-derived electrons plus CO2 into organic Clark Center, Campus Drive, Mail Code 5429, Stanford, CA 94305, compounds rather than free molecular hydrogen. USA. E-mail: [email protected] or [email protected] Electrosynthetic methane formation has been Received 25 May 2016; revised 2 September 2016; accepted achieved using mixed cultures (Cheng et al., 2009, 18 September 2016; published online 1 November 2016 Siegert et al., 2015) or pure strains such as Enhancing electrosynthesis using co-cultures JS Deutzmann and AM Spormann 705 Methanococcus maripaludis and Methanobacterium Materials and methods sp. (Lohner et al., 2014; Beese-Vasbender et al., 2015a). Multi-carbon compounds such as acetate Microbial strains and cultivation have been synthesized on biocathodes using a Strain IS4 (DSM 15630) and A. woodii (DSM 1030) diversity of homoacetogenic strains (Nevin et al., were obtained from the German Collection of 2010, 2011; Deutzmann et al., 2015). Microorganisms and Cell Cultures. M. maripaludis Low electron transfer rates from the cathode strain MM901 was obtained from the laboratory of Dr during microbially catalyzed electrosynthesis were John Leigh, UW (Costa et al., 2013). generally considered to be limiting the feasibility of Strain IS4 was routinely cultivated in artificial this process on a commercial scale (Blanchet et al., seawater medium containing 28 mM sodium sulfate 2015). Moreover, overpotentials of 4200 mV had to as electron acceptor (Dinh et al., 2004). A. woodii be applied repeatedly to achieve significant electron was initially cultivated in a modified DSMZ medium transfer rates (Villano et al., 2010; Aulenta et al., 311. M. maripaludis was routinely cultured in a 2012). At these low potentials, the electrochemical modified DSMZ medium 141. A detailed description formation of small reduced molecules as potential of the media can be found in the Supplementary Information. electron carriers such as H2, CO or formate at the cathode cannot be excluded (Villano et al., 2010; For co-cultivation, A. woodii and M. maripaludis Yates et al., 2014; Deutzmann et al., 2015). To our were transferred to artificial seawater medium knowledge, all methanogens and homoacetogens medium (amended with 0.1% yeast extract in case studied for their electrosynthetic properties are able of A. woodii) and transferred at least twice in this to metabolize at least some of these small reduced medium before the co-culture experiment. molecules. All cultures were maintained in butyl-rubber Besides methanogenic archaea and homoaceto- stopper sealed 120 ml or 160 ml serum vials contain- genic bacteria, Fe(0)-corroding microorganisms ing 50 ml of medium with a headspace of 80% H2 have been intensively investigated for their out- and 20% CO2 as electron donor and sole carbon standing electron transfer capabilities (Dinh et al., source, respectively. 2004; Uchiyama et al., 2010; Enning et al., 2012; Venzlaff et al., 2013; Enning and Garrelfs, 2014; Kato et al., 2015; Beese-Vasbender et al., 2015b). Electrochemical experiments Although direct electron uptake has been proposed Electrochemical reactors were setup as described in these microorganisms, no mechanism has been previously (Lohner et al., 2014). The electrochemical identified to date (Enning et al., 2012; Venzlaff cell consisted of a two chambered, gas-tight borosi- et al., 2013; Enning and Garrelfs, 2014; Beese- licate H-cell, in which anode and cathode chambers Vasbender et al., 2015b). Recently, Fe(0)-corroding (150 ml each) were separated by a Nafion 117 proton- microorganisms, in particular strain IS4 (‘Desulfo- exchange membrane as described previously pila corrodens’, previously named Desulfobacter- (Lohner et al., 2014). The medium exposed area of ium corrodens (Dinh et al., 2004), but no name has the graphite electrodes (conductive graphite bars ¼ been validly published to date) have been investi- × ¼ inch, McMaster-Carr) was 8 cm2, and this area gated in electrode systems, and a direct electron was used to calculate specific electron transfer and transfer mechanism has been repeatedly postu- product formation rates. The reference electrodes lated (Venzlaff et al., 2013; Beese-Vasbender used were Ag/AgCl electrodes (model RE-5B, BASi, et al., 2015b). West Lafayette, IN, USA) in 3 M NaCl. Autoclaved In the present study, we investigated the Fe(0)- reactors were flushed with a sterile mix of 80% N2 corroding sulfate-reducing strain IS4 as a biocata- and 20% CO2 until oxygen was completely flushed lyst to enhance the electron uptake rate in a out, the reference electrode was inserted and 90 ml bioelectrosynthesis system. Strain IS4 was pre- sulfate-free artificial seawater medium was anoxi- viously shown to form molecular hydrogen tran- cally and aseptically transferred into the anode and siently during cultivation on Fe(0) (Dinh et al., cathode compartments with a N2/CO2 flushed plastic 2004). To provide the proof of concept that the syringe while continuously flushing the reactor. overall rates of electrosynthesis can be increased by Reactors were disconnected from the gas stream co-cultivation of a specialized electron-uptaking and maintained at 30 °C. A volume of 3–6 ml of early strain with another microbe capable of producing a stationary phase culture of the indicated organisms desired product, we constructed and examined was added to the cathode compartment, resulting in electron flow in defined co-cultures of strain IS4 an initial gas phase volume of 43–46 ml. The change with the methanogen M. maripaludis and the in gas to liquid ratio due to sampling was considered homoacetogen Acetobacterium woodii. Our data when calculating absolute amounts of different demonstrate the importance of interspecies hydro- substances in the reactor. In all, 250 μM sulfate was gen transfer in cathode-associated microbial com- added to reactors containing strain IS4. The cathode munities and highlights a primary role of electron was poised at the indicated potentials after inocula- uptake strains as ecosystem engineers in these tion. Electric current and product formation were communities. followed over time. Cyclic voltammograms were

The ISME Journal Enhancing electrosynthesis using co-cultures JS Deutzmann and AM Spormann 706 recorded after a steady current or product formation in the same setup to obtain hydrogen evolution rate had established, that is, after the establishment rates for a model inorganic catalyst under identical of the IS4 biocathode shown in Figure 1 and the conditions. subsequent cultivation time needed to establish a stable co-culture with M. maripaludis in a subset of the reactors and after rate measurements shown in Analytical procedures Figure 3 (total of 312 h cultivation time). The gas Methane and high concentrations of hydrogen were phase of all reactors was flushed with N2/CO2 at determined using a gas chromatograph with nitrogen the time of introducing a second organism. Platinum as the carrier gas (equipped with both a thermal foil (17.5 cm2 immersed surface) was used as cathode conductivity detector and a flame ionization detection detector). Low concentrations of H2 were determined using a reductive trace gas analyzer, and soluble compounds such as formate and acetate were determined using high-performance liquid chromatography as described previously (Lohner et al., 2014). To allow for a better comparison of formation rates of different products and electron flux, concentrations were converted to concentrations in electron equivalents (for example, μmol eeq) where appropriate. Concentrations in μmol eeq were calculated by multiplying the concentration of a given compound by the number of electrons required for its formation from CO2.

Electron microscopy Samples were fixed in 4% paraformaldehyde with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 24–48 h at 4 °C, rinsed in the same buffer and post-fixed in 1% aqueous OsO4 for 2 h before dehydration in an increasing ethanol series (50–70–90–100%, 15 min each). Samples were then critical point dried (Tousimis 815B, Rockville, MD, USA) with liquid CO2, mounted on aluminum pin stubs and sputter-coated with Au/Pd using a Denton Desk II sputter-coater (Denton Vacuum, Moorestown, NJ, USA) before visualizing with a Zeiss Sigma FESEM (Carl Zeiss Microscopy, Thorn- wood, NY, USA) using InLens secondary electron detection at 2–3 kV. At least three different positions encompassing at least 5000 μm2 electrode surface have been analyzed to estimate cell counts on the electrode surface.

Results Establishment of an active strain IS4 biocathode After inoculation of the biocathode in the presence of 250 μM sulfate, electron uptake in form of electrical Figure 1 Startup of a bioreactor with strain IS4 as biocathode. current consumption by strain IS4 cells increased Strain IS4 was inoculated into a bioelectrochemical reactor with a over time when the cathode was poised at − 400 mV cathode potential of − 400 mV vs standard hydrogen electrode and (vs standard hydrogen electrode) (see Figure 1 for one compared with an un-inoculated control. (a) Current consumption representative experiment, Supplementary Figure S1 of strain IS4 (black) and uninoculated control (gray). (b) Hydrogen accumulation by strain IS4 (black squares) and uninoculated for coulombic efficiencies, and Supplementary control (open squares). (c) Sulfide accumulation by strain IS4 (gray Figure S2 for all other replicates). No current con- circles; red in online version) and control (open circles) and sumption was observed using cell-free spent culture sulfate consumption by strain IS4 (black squares) and control medium or without initial sulfate addition (data not (open squares). For clarity, one representative reactor out of ten replicates (five for controls) is shown. The trend is identical in all shown), indicating that strain IS4 cells initially replicates, but the onset of current consumption and activity is required sulfate as electron acceptor to establish a shifted among the replicates (see Supplementary Figure S2 for all working biocathode. Current consumption increased replicates). for several days in the presence of sulfate before

The ISME Journal Enhancing electrosynthesis using co-cultures JS Deutzmann and AM Spormann 707 current consumption decreased sharply. Within this time period, sulfate concentration decreased and sulfide formed in equimolar concentrations as expected based on an active metabolism of this sulfate-reducing bacterium (Figure 1c). The sharp decrease in current consumption coincided with sulfate depletion and hydrogen accumulation in the cathode compartment, indicating that strain IS4 cells utilized cathode-derived electrons for sulfate reduction. We also observed that electrochemical reactors containing cells of strain IS4 transiently accumulated molecular hydrogen even in the presence of sulfate (Figure 1b). Notably, after sulfate depletion, rates of molecular hydrogen accumulation increased to ca. 0.2 μmol h − 1 cm − 2 relative to the initial rates of ca. 0.08 μmol h − 1 cm − 2 in the presence of sulfate. This indicates that upon depletion of sulfate electron uptake continued and electrons were used to reduce H+ to Figure 2 Cyclic voltammograms of graphite electrodes colonized by strain IS4 (black), strain IS4+M. maripaludis (medium gray; red form H2, while sulfate reduction as H2 (or electron) in online version) and pure M. maripaludis (light gray) after sink was blocked. Hydrogen accumulated to partial incubation at − 400 mV vs standard hydrogen electrode. Scanning pressures of up to 0.08 bar at − 400 mV vs standard speed was 1 mV s − 1. For clarity one out of three replicates is hydrogen electrode. These partial pressures approached shown. See Supplementary Figure S4 for CVs of all replicates. in our experimental setup the thermodynamic equilibrium concentration of the reaction 2 H++2 − → e H2, which prevented further consumption of reactors containing previously established strain IS4 cathodic electrons and explains the sharp decline of biocathodes were thoroughly flushed with N2/CO2 current consumption at − 400 mV once sulfate was for 30 min to eliminate any residual hydrogen. consumed (see Supplementary Figure S3 and Thereafter, hydrogen was formed at rates of Supplementary Information for detailed calculation). 0.4–0.5 μmol cm− 2 h − 1 at a cathode potential of The transient accumulation of H2 raised the − 400 mV (Figure 3a). Hydrogen formation rates interesting possibility that H2 formation at the increased by one order of magnitude to cathode may be reversible in this bioelectrochemical 4–7 μmol cm − 2 h − 1 when the potential was lowered system. If this were the case, we expected the to − 500 mV, but a further decrease in potential did formation of electric current from molecular hydro- not result in a significant change of hydrogen gen at a suitable electrode potential. Indeed, cyclic formation rate (Figures 3b and c). Under these voltammetry revealed the typical wave pattern for conditions, strain IS4 formed hydrogen from catho- reversible hydrogen evolution (Figure 2, Supple- dic electrons at a coulombic efficiency of 90–110% mentary Figure S4). This cyclic voltammetry (CV) in all experiments. These hydrogen formation rates profile also showed the absence of a noteworthy were more than one order of magnitude higher overpotential (o5 mV) for the hydrogen evolution than hydrogen evolution rates obtained with reaction: the equilibrium potential of reversible platinum foil (0.002 μmol cm − 2 h − 1 at − 400 mV hydrogen formation and hydrogen oxidation in our and 0.14 μmol cm − 2 h − 1 at − 500 mV, respectively system was at about − 425 mV under our experi- (Supplementary Figure S6)) instead of the IS4 mental conditions, which is close to the calculated biocathode in an identical setup. thermodynamic equilibrium potential of − 421 mV at pH = 7 and 30 °C. At more positive potentials, current was produced indicating hydrogen oxidation, Electrosynthetic formation of methane by a co-culture whereas at more negative potentials hydrogen was of strain IS4 and M. maripaludis formed. Sulfide produced by strain IS4 during the To investigate whether the observed highly efficient incubation (o250 μM after flushing) did not result in hydrogen formation by strain IS4 cells can be coupled a pronounced signal in the CVs and did not interfere to the hydrogenotrophic production of carbon com- with the catalytic wave of hydrogen production or pounds from CO2, we constructed a defined co-culture oxidation (see Supplementary Figure S5). of strain IS4 with the methanogenic archaeon M. maripaludis.WhenM. maripaludis was added to a thoroughly N2/CO2-flushed, sulfate-depleted cathode Product formation rates at different electrode potentials chamber with an established strain IS4 biocathode, To investigate hydrogen formation by strain IS4 cells no accumulation of hydrogen but formation of in greater detail, we performed experiments under methane was observed. Presumably, hydrogen was sulfate-free conditions at different cathode potentials consumed rapidly to steady state concentrations of and followed hydrogen formation and current con- o500 p.p.m. in the gas phase due to methanogenesis. sumption over time. Before each experiment, all At − 400 mV cathode potential this led to a small

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Figure 3 Hydrogen and methane formation as well as current consumption by strain IS4 (black) and strain IS4 in co-culture with M. maripaludis (gray; red in online version) at different potentials. Methane (circles) and hydrogen (squares) are shown in μmol electron equivalents (μmol eeq) per reactor; current consumption (in μmol electrons consumed) by strain IS4 (black) or the co-culture (gray; red in online version) is shown as dotted line. Note the different scales between (a) and (b)or(c).

increase in current consumption (0.7–1.1 μmol hydrogen partial pressure of more than two orders electrons cm − 2 h − 1) compared with the reactors with- of magnitude. CVs of un-inoculated reactors out methanogens (0.5–0.7 μmol electrons cm − 2 h − 1), (Supplementary Figure 2) or reactors inoculated most likely because the end product of the electron with axenic M. maripaludis (Figure 2) only showed uptake step, hydrogen, was effectively scavenged by marginal catalytic currents. the methanogen (Figure 3a). Lowering the cathode potential from − 400 to − 500 mV increased the rates of − − methane formation from 0.10–0.14 μmol cm 2 h 1 to Colonization of the graphite surface by strain IS4 0.6–0.9 μmol cm − 2 h − 1 (Figure 3b). This change in When the cathode chamber of an electrochemical rate, however, was slightly lower than the change in H-cell was inoculated with strain IS4, electron the rate of hydrogen formation in the axenic IS4 microscopy revealed after 1 week of incubation culture (Figure 3b). In agreement with those findings, a (corresponding to current consumption rate of further decrease in potential to − 600 mV did only 0.1 A m − 2 at this time) that cells on the electrode result in a small increase in methanogenesis rate in the surface formed a patchy monolayer (Figure 4). Cells methanogenic co-culture (Figure 3c). Notably, at were found clustered in ‘cracks’ and ‘crevices’ in the cathode potentials of − 400 mV and − 600 mV, the rate graphite surface, suggesting that cells might only be of methane formation by the co-culture corresponded irregularly attached to the graphite surface. No to the hydrogen formation rate by axenic strain IS4. No pronounced production of visible extracellular poly- product formation was detected in un-inoculated meric substance was observed. A rough estimation of controls or with an axenic M. maripaludis culture, cell number on the electrode surface yielded cell and only marginal background current of densities of 107–108 cells cm − 2 electrode surface. 0.005 − 0.007 μmol electrons cm − 2 h − 1 was observed However, these cell counts and extrapolated values (Supplementary Figure S7). Collectively, these data have to be treated with caution, because the graphite show that a co-culture of strain IS4 and M. maripalu- surface is very rough and cells can be buried below dis efficiently converted cathodic electrons plus the visible surface of the electrode. CO2 into methane without the accumulation of intermediates. Cyclic voltammetry measurements of the metha- Formation of acetate by a co-culture of strain IS4 and nogenic co-culture indicated hydrogen formation A. woodii was irreversible (Figure 2). No current production To generalize the finding that a co-culture of strain was recorded in this co-culture at potentials more IS4 and a hydrogenotrophic ‘production strain’ positive than the thermodynamic equilibrium poten- results in enhanced microbial electrosynthesis, we tial for H2 formation and oxidation. This confirmed co-cultured the homoacetogenic bacterium A. woodii that the methanogenic archaeon efficiently sca- and strain IS4 in the electrochemical setup. Due to venged the molecular hydrogen formed at negative higher concentrations required for experimental potentials during the sweep and prevented hydrogen detection of acetate (41 μM), no short-term product from reaching the electrode surface during formation rates were determined, but we estimated a hydrogen-oxidizing conditions. In addition, the formation rate from long term incubations. When onset potential of current consumption and presum- A. woodii cells were added to reactors preincubated ably hydrogen formation shifted by 460 mV to more with strain IS4, acetate was produced after a positive potentials, indicating a decrease in lag-phase at rates of 0.21–0.23 μmol cm − 2 h − 1

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Figure 4 Electron micrograph of plain graphite electrodes (a and c) and graphite electrodes colonized with strain IS4 after incubation in electrochemical reactors (b and d). Graphite electrodes were fixed after current consumption rates had reached a plateau that was stable for several days. Scale bars represent 10 μm(a and b) and 1 μm(c and d), respectively.

(Figure 5a). In the lag-phase, hydrogen and formate practically any overpotential at a biocathode con- accumulated transiently and were subsequently taining strain IS4 cells (Figures 2 and 3a). Electron consumed correlating with acetate synthesis uptake and hydrogen evolution rates of the estab- (Figures 5c and e). Upon changing the potential lished biocathode were an order of magnitude higher to − 500 mV, current consumption as well as rate than rates obtained with an abiotic platinum cath- of acetate formation increased significantly to ode. Comparisons to other inorganic catalysts are 0.57–0.74 μmol cm − 2 h − 1 (Figure 5b). Also here, a difficult, because most catalysts are not evaluated in transient accumulation of formate and hydrogen was the potential range suitable for microbial growth. In observed (Figures 5d and f). Coulombic efficiencies addition, biofouling of inorganic catalysts and the of acetate formation were low during the initial influence of medium components is not well studied phase of the co-cultivation, but approached 90% at for most inorganic catalysts. As cells attached the end of the incubation. Presumably, the popula- directly to the electrode surface, and cell-free culture tion size of A. woodii was insufficient to consume all supernatant or cell-free extracts did not catalyze H2 electron donor generated by strain IS4 at the start of formation (data not shown), cathodic electron uptake the co-culture and after the decrease of the potential is likely not facilitated by cathode-sorbed enzymes as to − 500 mV. We surmise that the population size of demonstrated in M. maripaludis (Deutzmann et al., A. woodii adapted to the supply of electron donor via 2015). The findings on electron uptake by Fe(0)- strain IS4 after a few days of incubation. Formate corroding microorganisms such as strain IS4 suggest presumably accumulated due to a formate hydrogen that these microbes are obvious candidates for lyase activity catalyzed by A. woodii (Kantzow and mediating the initial biological uptake of cathodic Weuster-Botz, 2016). Acetate synthesis by the co- electrons with several fundamental and technologi- culture of strain IS4 and A. woodii was stable for cal implications. + more than 2 weeks (Figure 5). Under the conditions The high rate of H2 formation from H and used in this study, a pure culture of A. woodii was electrons by strain IS4 at − 400 mV corresponds to a unable to consume current and produce acetate at high cellular electron uptake rate of 106– the potential range investigated. 107 e − s− 1 cell − 1 based on estimated cell counts obtained by electron microscopy in this study. These rates agree well with electron uptake rates of 6·106– 7 − − 1 − 1 Discussion 1.5·10 e s cell estimated from H2 formation from elemental Fe measured with washed strain

H2 evolution by microorganisms that take up cathodic IS4 cells (Enning, 2012). Another Fe(0)-corroding electrons microorganism, the methanogenic archaeon strain Our study showed that molecular hydrogen was IM1 was also shown to take up electrons at a high effectively formed at high rates and in the absence of rate of 6·106 and 6·107 e − s− 1 cell − 1 at − 400 and

The ISME Journal Enhancing electrosynthesis using co-cultures JS Deutzmann and AM Spormann 710 production of strain IS4, and the coulombic efficiency of hydrogen formation can achieve values of close to 100%. Thus, during long-term operation a continuous or batch fed system with additions of small amounts of sulfate could sustain a stable population of strain IS4 thriving on sulfate reduction while slowly re-supplying a reduced sulfur source for anaerobic microorganisms unable to assimilate sulfate. During continuous operation at neutral pH, the gas stream will slowly strip sulfide out of solution and, thus, deplete the sulfur source. Due to the differences in the reporting of product formation rates in the literature (for example,, as specific rate per electrode surface area, reactor volume or total amount per reactor), associated projected areal current densities are used in this study for normalization to allow direct comparisons. However, faradaic efficiencies are not always reported and, thus, projected areal current densities might slightly overestimate actual product formation rates. Current densities of 1.1–3.3 A m − 2 at − 700 mV were reported during hydrogen formation using mixed cultures in microbial electrolysis cells (Rozendal et al., 2008; Jeremiasse et al., 2010; Croese et al., 2011; Jeremiasse et al., 2012), and current densities of 0.76 A m − 2 were measured using a pure culture of the non-corroding sulfate reducer Desulfovibrio G11 (Croese et al., 2011). In our study, strain IS4 achieved similar current densities of − Figure 5 Co-cultures of strain IS4 and A. woodii in the cathode 1.5 A m 2 already at a cathode potential of compartment of electrochemical reactors. Acetate formation (filled − 500 mV and, thus, at a potential 200 mV more symbols and solid lines) and cumulative charge (dotted lines) positive than in previously published studies. In consumed at − 400 mV (a) and − 500 mV (b)inμmol electron equivalents (μmol eeq) per reactor. Hydrogen accumulation at addition, we used plain graphite electrodes which − 400 mV (c) and − 500 mV (d) and formate formation at − 400 mV likely provide a lower effective surface area than (e) and − 500 mV (f) are shown in μmol eeq for comparison. Two graphite felt electrodes used in other studies replicate reactors are shown in different shades of gray (red in the (Rozendal et al., 2008; Jeremiasse et al., 2010; online version) and using different symbols to allow identification of each replicate across graphs. The continuous experiment is Croese et al., 2011; Jeremiasse et al., 2012). Besides displayed in two separate parts for reasons of clarity due to the electrode surface area, the cell density on the different scales required to display the product formation at electrode surface impacts overall electron transfer different potentials. Diamonds: co-cultures of strain IS4 and rates. However, all isolates described to perform A. woodii; triangles: A. woodii pure cultures; circles: un- electrical microbially influenced corrosion, includ- inoculated controls. ing strain IS4, do not form thick biofilms but form dispersed patchy monolayers of cells (Beese- Vasbender et al., 2015a, b). Thus, improving attach- − 600 mV, respectively (Beese-Vasbender et al., ment and biofilm growth of these strains might 2015a). Interestingly, these rates are roughly in greatly increase areal current densities and should be the same order of magnitude as the single cell addressed in future research. anodic reduction reaction in Shewanella oneidensis (105–106 e − s− 1 cell − 1) (Gross and El-Naggar, 2015). Notably, only some Fe(0) corroding strains form Efficient interspecies hydrogen transfer in mixed hydrogen from cathodic electrons while other strains electrosynthetic cultures such as strain IS5 do not accumulate hydrogen (Dinh The rates of electromethanogenesis by a co-culture et al., 2004). Therefore, only H2 producing strains, of strain IS4 and M. maripaludis presented here including strain IS4, represent a promising biocata- are the highest reported to date. Compared with a lyst for the generation of H2 at a cathodic surface. recent study using Methanobacterium strain IM1 Another benefit of using sulfate reducers lies in the (Beese-Vasbender et al., 2015a), methanogenesis fact that growth of the microorganism can be rates reported here were about one order of magni- controlled by limiting the sulfate concentration in tude higher at − 400 mV (100–140 nmol h − 1 cm − 2 the reactor solution. Thus, during the electrosynth- compared with 12–14 nmol h − 1 cm − 2 in (Beese- esis phase, only a negligible fraction of the electrical Vasbender et al., 2015a)). However, electron uptake energy and electrons are directed towards biomass rates were comparable between both studies, and

The ISME Journal Enhancing electrosynthesis using co-cultures JS Deutzmann and AM Spormann 711 molecular hydrogen was likely a side product proceed in the bulk liquid, which allows better use of during electromethanogenesis by strain IM1, the volume of the cathode compartment; (3) two which became obvious at more negative poten- microbial organisms adapt to each other’s metabolic tials (Beese-Vasbender et al., 2015a). The rates capabilities via a thermodynamic-kinetic feedback obtained here with the co-culture are about 20 loop resulting in an optimized metabolic flux with- times higher compared with electromethanogenesis out accumulation of intermediates. In addition, using axenic M. maripaludis at − 600 mV (Lohner mixed communities are often reported to show et al., 2014) (0.6–1.2 μmol h − 1 cm − 2 compared with higher electron transfer rates or higher current 0.05 μmol h − 1 cm − 2). In addition, the overpotential efficiencies than pure cultures (Dinh et al., 2004; for electromethanogenesis by the co-culture was Nevin et al., 2008; Call et al., 2009; Fast and 4200 mV lower, thus resulting in less energy loss Papoutsakis, 2012; Ganigue et al., 2015). Thus, in this cathodic reaction. Few studies have shown especially with the possibility to engineer and microbial electrosynthesis of acetate, butanol or improve each specialist in the co-culture, a stable other multi-carbon compounds by pure cultures of and probably faster electrosynthesis system can be homoacetogenic microorganisms. Electron transfer achieved compared with engineering existing iron rates at a potential of − 400 mV in these systems corroding methanogens or homoacetogens varied between strains and ranged from no electron (Uchiyama et al., 2010; Kato et al., 2015). uptake using A. woodii to 0.01 A m − 2 using Moorella thermoacetica and to 0.2 A m − 2 using Sporomusa ovata (Nevin et al., 2010, 2011). Thus, the electron Electron uptake strains as ecosystem engineers in uptake rates achieved by our co-culture of strain IS4 cathodic communities with A. woodii (0.2–0.5 A m − 2 at − 400 mV) are Our finding that electrosynthesis rates can be slightly higher than the rates published with pure improved substantially by co-cultivating an electron cultures of homoacetogens (Nevin et al., 2011). Other uptake strain that produces hydrogen with hydro- studies using mixed cultures achieved high electro- genotrophic strains prompts the question about the synthesis rates when applying large overpotentials ecological relevance of such strains and their role in (3–20 A m − 2 at − 800 mV, (Ganigue et al., 2015)) at more natural environments. Successful co- which the apparent ‘electro’synthetic activity is most cultivation of strain IS4 with a homoacetogen and a likely due to the abiotic formation of hydrogen. At methanogen shows that interspecies hydrogen trans- the cost of high overpotentials and higher energy fer can effectively distribute cathodic electrons to losses, hydrogen is formed under these conditions microorganisms that by themselves do not have the even at electrodes with low catalytic activity for H2 capacity for direct electron uptake. According to evolution. This overpotential can be reduced when Jones, organisms directly or indirectly modulating cell-derived enzymes or cofactors adsorb to the the availability of resources (other than themselves) surface (Yates et al., 2014; Deutzmann et al., 2015). to other species by causing state changes in biotic or In our defined co-culture, hydrogen was formed abiotic materials are called ‘ecosystem engineers’ in already at − 400 mV, which enabled electrosynthesis ecology (Jones et al., 1994). Microbial strains that with A. woodii that has been shown previously to facilitate electron uptake from a cathodic surface and be unable to carry out electrosynthesis (Nevin supply electrons to the surrounding community can, et al., 2011) due to efficient interspecies hydrogen therefore, be considered ecosystem engineers. transfer. Although this possibility has already been men- tioned in one of the first studies on electrosynthesis by mixed species biofilms (Marshall et al., 2012), it General benefits of a decoupled microbial electron lacked evidence for single strains catalyzing hydro- uptake—microbial synthesis system gen formation at high rate. Based on the high cellular In a microbial electrosynthesis system, the combina- electron uptake rates (4106 e − s − 1 cell − 1), even a tion of an efficient electron uptake step with an relatively small number of electroactive cells could efficient synthesis step of a compound of interest has result in substantial current consumption rates and several distinct advantages over a single microorgan- thereby provide an effective electron donor for a ism process: (1) It becomes possible to engineer and diverse microbial ecosystem. Accordingly, the operate two fundamentally different metabolic pro- hydrogen evolution rate of a single strain IS4 cell cesses independently. Each microorganism can be (up to 30 fmol h − 1) would be sufficient to supply all optimized for its purpose, for example, through thehydrogenconsumedbyoneM. maripaludis cell genetic engineering, without the need to consider under optimum growth conditions (2 h doubling additional tradeoffs imposed by the other process, time), and provides hydrogen at a rate an order of and there are numerous efforts to engineer homo- magnitude higher than hydrogen uptake rates of acetogenic microorganisms to produce, for example, other more slowly growing organisms (Goyal et al., ethanol, butanol and isobutanol (Köpke et al., 2010; 2015). Besides intact cells that transform cathodic Leang et al., 2013; Banerjee et al., 2014; Cho et al., electrons into a widely accessible substrate, such 2015); (2) Only the electron uptaking strain has to as hydrogen, strains that release electroactive contact the cathode while end product formation can enzymes such as hydrogenases have to be

The ISME Journal Enhancing electrosynthesis using co-cultures JS Deutzmann and AM Spormann 712 considered in cathodic communities. Even small Acknowledgements amounts of electrocatalytically active enzymes could be sufficient to sustain biofilms on a cathodic We thank Lydia-Marie Joubert and the Cells Science Imaging Facility at Stanford University for providing surface due to the high turnover rates of support in preparing the samples and acquisition of the hydrogenases. ‘ SEM images. We thank David Kleiman for his help in Examples for candidate strains ( ecosystem engi- conducting experiments. This research was supported by neers’) mediating the key electron uptake to support the Global Climate and Energy Project (GCEP) and the complex cathodic biofilms can be found in the Office of Naval Research (ONR) through grants to AMS. literature. The genus Methanobacterium became enriched in several studies investigating electromethanogenesis (Pozo et al., 2015; Siegert et al., 2015) and it has been shown in pure culture that Methanobacterium strain IM1 not only catalyzed References electrosynthetic methanogenesis but also accumulated hydrogen during electromethanogenesis Aulenta F, Catapano L, Snip L, Villano M, Majone M. (2012). Linking bacterial metabolism to graphite (Beese-Vasbender et al., 2015a), which can create a cathodes: electrochemical insights into the H2- niche for other microorganisms. In homoacetogenic producing capability of Desulfovibrio sp. Chem- communities the role of distinct electron uptake suschem 5: 1080–1085. strains seems to be more elusive. A variety of Banerjee A, Leang C, Ueki T, Nevin KP, Lovley DR. (2014). homoacetogenic bacteria were shown to use cathodic Lactose-inducible system for metabolic engineering of electrons in pure culture (Nevin et al., 2010, 2011). Clostridium ljungdahlii. Appl Environ Microbiol 80: However, in acetate-producing electrosynthesis reac- 2410–2416. tors inoculated with undefined mixed cultures the Beese-Vasbender PF, Grote J-P, Garrelfs J, Stratmann M, cathode-associated community was often dominated Mayrhofer KJJ. (2015a). Selective microbial electro- by Acetobacterium spp., which had been reported to synthesis of methane by a pure culture of a marine lithoautotrophic archaeon. Bioelectrochemistry 102: lack the ability to utilize cathodic electrons (Nevin 50–55. et al., 2011). Often, sulfate reducers such as Beese-Vasbender PF, Nayak S, Erbe A, Stratmann M, Desulfovibrio sp. are also present in these enrich- Mayrhofer KJJ. (2015b). Electrochemical characteriza- ments (Marshall et al., 2013; LaBelle et al., 2014; tion of direct electron uptake in electrical microbially Patil et al., 2015), suggesting that hydrogen produced influenced corrosion of iron by the lithoautotrophic by the sulfate reducers might have a role in these SRB Desulfopila corrodens strain IS4. Electrochimica biofilms. However, the role of individual strains in Acta 167: 321–329. mixed communities and the molecular mechanism Blanchet E, Duquenne F, Rafrafi Y, Etcheverry L, Erable B, of microbial electron uptake from the cathode is Bergel A. (2015). Importance of the hydrogen route in unclear in most cases. Thus, further research is up-scaling electrosynthesis for microbial CO2 reduction. Energ Environ Sci 8: 3731–3744. needed on the electron uptake mechanism(s) acting Bond DR, Lovley DR. (2003). Electricity production by in different electron uptake strains and identification ‘ ’ Geobacter sulfurreducens attached to electrodes. 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