Electromethanogenesis for Biogas Upgrading

Electromethanogenesis for biogas upgrading

People at SDU: Amelia-Elena Rotaru Mon Yee Oo & Bo Thamdrup Who is an electromethanogen?

How are they retrieving electrons?

How to use EET to upgrade biogas? Electron uptake by methanogens

Strategies of electron transfer:

H2 or formate Direct electron transfer

D. vulgaris S. fumarooxidans G. metallireducens

M. harundinacea M. hungateii M. hungateii M. concilii M. barkeri

Rotaru 2014a, Rotaru 2014b, Rotaru 2015

Methanosaeta Geobacter DIET in consortia

14 Ethanol C-bicarbonate Electrotroph Acetate Mseta. harundinacea Ace Electrogen DIET CH4 + CO2 - + - G. metallireducens 4e + 4H e 14 0.5 CH4 CO2

2 0.6 )

Methane -1 Ethanol 0.5

1.5 Acetate µ mol

× 0.4

1 (CPM 0.3 2 mmol CO

14 0.2 / Specific activitySpecific 0.5 4

CH 0.1 14

0 0 0 20 40 60 80 100 1 21 28 days days Rotaru 2014a Rotaru 2014a 6 30 28 26 24 22 20 18 16 14

Methane (%) 12 10 8 6 4 Pili k.o. Geobacter 2 Methanosaeta 0 0 20 40 60 80 100 120 140 160 180 200 Time (days) Rotaru 2014a Cytochrome-c containing Archaea

Kletzin 2015 What other methanogens are implicated in EET?

Methanogens associated with EET

DIET

M. harundinacea DIET M. concilii DIET Methanosaeta spp. WE Methanospirillum hungateii DIET Methanoplanus petrolearius DIET

DIET Methanobacterium sp. IM1 ZVI Methanobacterium palustre WE Methanobrevibacter spp. WE

M. maripaludis KA1 ZVI and Mic1c10 ZVI Methanococcus voltae DIET

Original tree by Ohtomo 2013 Environ. Sci. Technol. 2009, 43, 3953–3958 Environ. Sci. Technol. 2009, 43, 3953–3958

Direct Biological Conversion of a theoretical potential as high as 1.1 V under neutral pH a theoretical potential as high as 1.1 V under neutral pH Direct Biologicalconditions (1). TheConversion MEC is a type of modified MFC of that has Electrical Current into Methane by been used to efficiently store electrical energy as a biofuel conditions (1). The MEC is a type of modified MFC that has (hydrogen gas) (2). Hydrogen gas evolution from the cathode, been used to efficiently store electrical energy as a biofuel Electromethanogenesis Electricalhowever, Current is not spontaneous into (3- Methane5). The voltage produced by by electrogenic bacteria on the anode using a substrate such (hydrogen gas) (2). Hydrogen gas evolution from the cathode, as acetate (EAn =-0.2 V) is insufficient to evolve hydrogen however, is not spontaneous (3-5). The voltage produced SHAOAN CHENG, DEFENG XING,Electromethanogenesisgas at the cathode (E )-0.414 V, pH)7). By adding a DOUGLAS F. CALL, AND BRUCE E. LOGAN* cell small voltage, hydrogen gas can be produced using MECs at by electrogenic bacteria on the anode using a substrate such FIGUREEngineering 3. Environmental The cathode Institute and biofilm Department examined of Civil and by (A) SEM of cells on the carbon cloth and (B) fluorescence microscopy of extracted cells very high energy efficiencies evaluated in terms of just as acetate (EAn =-0.2 V) is insufficient to evolve hydrogen usingEnvironmental the Methanobacterium Engineering, 212 Sackett Building,-specificSHAOAN The probe CHENG,electrical MB1174-Alexa energy DEFENG alone (200 Fluor-400%) XING, 488or both (green). electrical energy Pennsylvania State University, University Park, Pennsylvania 16802 gas at the cathode (E )-0.414 V, pH)7). By adding a DOUGLAS F.and CALL, substrate heat AND of combustion BRUCE energy E. (82%) LOGAN* (3). One cell disadvantage of electrically assisted method of hydrogen small voltage, hydrogen gas can be produced using MECs at Received December 12, 2008. Revised manuscriptEngineering received Environmentalproduction (electrohydrogenesis) Institute and Department is that a precious of Civil metal and March 5, 2009. Accepted March 6, 2009. very high energy efficiencies evaluated in terms of just Environmental Engineering,catalyst such as 212 platinum Sackett is usually Building, used The on the cathode. Hydrogen compression is also an energy-intensive process, electrical energy alone (200-400%) or both electrical energy Pennsylvania Stateand University, hydrogen storage University can be problematic Park, Pennsylvania (6). 16802 Renewable biomethane is typically produced by metha- and substrate heat of combustion energy (82%) (3). One New sustainable methods are needed to produce renewable nogens from a few substrates such as acetate, formate, and disadvantage of electrically assisted method of hydrogen energy carriers that can be stored and usedReceived for transportation, Decemberbiohydrogen 12, 2008. gas in anaerobic Revised digesters manuscript (7). Based received on heating, or chemical production. Here we demonstrate that thermodynamic calculations, methane could also be pro- production (electrohydrogenesis) is that a precious metal methane can directly be produced using a biocathodeMarch containing 5, 2009.duced Accepted electrochemically March through 6, 2009. carbon dioxide reduction catalyst such as platinum is usually used on the cathode. methanogens in electrochemical systems (abiotic anode) or at a voltage of 0.169 V under standard conditions, or -0.244 Hydrogen compression is also an energy-intensive process, microbial electrolysis cells (MECs; biotic anode) by a process V under more biologically relevant conditions at a pH)7, by called electromethanogenesis. At a set potential of less the reaction and hydrogen storage can be problematic (6). than -0.7 V (vs Ag/AgCl), carbon dioxide was reduced to Renewable biomethane is typically produced by metha- CO + 8H+ + 8e- f CH + 2H O (1) methane using a two-chamber electrochemicalNew reactor containing sustainable methods are2 needed to produce4 2 renewable nogens from a few substrates such as acetate, formate, and an abiotic anode, a biocathode, and no precious metal catalysts. At -1.0 V, the current capture efficiencyenergy was carriers 96%. This that suggests can be that stored methane and could used be produced for transportation, without an biohydrogen gas in anaerobic digesters (7). Based on Electrochemical measurements made using linear sweep organic fuel, at about the same potential needed for hydrogen heating, or chemicalproduction production. with an organic Here fuel we (such demonstrate as acetate). Methane that thermodynamic calculations, methane could also be pro- voltammetry showed that the biocathode substantially increased methane can directlyproduction be produced by eq 1 has the using added a advantage biocathode of CO2 containingcapture duced electrochemically through carbon dioxide reduction current densities compared to a plain carbon cathode where into a fuel (but not sequestration). A purely electrochemical only small amounts of hydrogen gas could bemethanogens produced. Both inroute electrochemical of methane production systems in practice (abiotic is energy anode) intensive or at a voltage of 0.169 V under standard conditions, or -0.244 increased current densities and very small hydrogenmicrobial production electrolysisdue to high cells electrode (MECs; potentials biotic and anode) the lack by of a suitable process V under more biologically relevant conditions at a pH)7, by rates by a plain cathode therefore support a mechanism of catalysts able to efficiently reduce this overpotential (8). The methane production directly from current and notcalled from hydrogen electromethanogenesis.required voltage could At theoretically a set potential be eliminated of when less using the reaction gas. The biocathode was dominated by a single Archaeon, an organic substrate due to a positive electrochemical cell than -0.7 V (vspotential Ag/AgCl), for methane carbon production dioxide (E was) 0.071 reduced V with acetate), to Methanobacterium palustre.Whenacurrentwasgenerated cell CO + 8H+ + 8e- f CH + 2H O (1) by an exoelectrogenic biofilm on the anode growingmethane on using acompared two-chamber to a negative electrochemical potential for hydrogen reactor production. containing 2 4 2 acetate in a single-chamber MEC, methane was produced at This thermodynamically favorable reaction of acetate con- an abiotic anode,version a biocathode, to methane is theand reason no precious why acetoclastic metal metha- FIGUREan overall 4. energy Linear efficiency ofsweep 80% (electrical voltammograms energy and substrate of cathodes in the nogenesis occurs in nature and in engineered anaerobic This suggests that methane could be produced without an heat of combustion). These results show thatcatalysts. electrometha- At -1.0 V, the current capture efficiency was 96%. presence and absence of a biofilm (1.0digesters. mV/s using CO2 organic fuel, at about the same potential needed for hydrogen nogenesis can be used to convert electrical currentElectrochemical produced measurementsSubstantial methane made production using has linear been reported sweep in saturatedfrom renewable medium, energy sources 100 (suchmM as PBS). wind, solar, or recent MEC studies (5, 9-13), but the source of this methane production with an organic fuel (such as acetate). Methane biomass) into a biofuel (methane) as well as servingvoltammetry as a method showedappears that to be the acetate biocathode via acetoclastic substantially methanogenesis increased and production by eq 1 has the added advantage of CO capture for the capture of carbon dioxide. current densitieshydrogenotrophic compared to methanogensis a plain carbon using hydrogen cathode gas where pro- 2 did produce any current (29). Thereduced are in other the process. reports Methane generation in anaerobic into a fuel (but not sequestration). A purely electrochemical only small amountsdigesters of originates hydrogen mostly gas from could acetate be (70%) produced. with a smaller Both route of methane production in practice is energy intensive Introduction portion from hydrogen gas (14). In contrast, methane demonstratingIncreasing competition that for fossil pure fuels, and cultures theincreased need to avoidof currentShewanellageneration densities in MECs and oneidensis may very be small more likely hydrogen from hydrogen production gas due to high electrode potentials and the lack of suitable andtheGeobacter release of carbon sulfurreducens dioxide from combustionratesdo of these notby fuels, a produce plainthan cathode acetate. as much therefore Clauwaert power et support al. (10) measured a mechanism a methane of catalysts able to efficiently reduce this overpotential (8). The has increased the search for new and sustainable approaches production rate of 0.28-0.75 L/L-d in several MEC experi- for energy production. Two new methodsmethane of bioenergy productionments directly at an applied from voltage current of -0.8 and V but not found from only hydrogen 0.17 required voltage could theoretically be eliminated when using in MFCs as the mixed cultures in air-cathode MFCs (30-32∼). production from biomass include electricitygas. production The using biocathodeL/L-d with was no applieddominated potential, by suggesting a single that Archaeon, 23-61% of an organic substrate due to a positive electrochemical cell Isolatesmicrobial have fuel cells so (MFCs) far and not hydrogen been production obtained by the methane for biocathodes was derived from acetate. Liu et al. (15) found electrohydrogenesis using microbial electrolysisMethanobacterium cells (MECs) no methane palustre generation.Whenacurrentwasgenerated in a reactor consistently operated in potential for methane production (Ecell ) 0.071 V with acetate), demonstrating(1, 2). Electricity generation oxygen in an MFC and is spontaneous nitrate with reductionan open-circuit or mode hydrogen compared to otherwise identical MECs compared to a negative potential for hydrogen production. oxidation of organic matter such as acetateby by anelectrogenic exoelectrogenicoperated at biofilm-0.7 V, on suggesting the anode hydrogen growing was needed on for This thermodynamically favorable reaction of acetate con- evolutionbacteria on the(16, anode 33, (EAn 34=-).0.2 V We vs standardacetate are hydrogen in in the a single-chamber processmethane production. of MEC, isolating Eliminating methane bicarbonate was produced from the at electrode) and oxygen reduction at the cathode (ECat = 0.2 medium has helped to reduce methanogenesis is some version to methane is the reason why acetoclastic metha- microorganismsV), with a working cell potential from of this approximately methanogenican overall 0.4 V andenergystudies efficiency biocathode. (16) but of not 80% in others (electrical (10). Organic energy loading and is clearlysubstrate a factor in methane production, with longFIGURE reaction times 5. (A) Methanenogenesis gas occurs production in nature from and the in mixed engineered culture anaerobic Further* Corresponding evidence author phone: to (814)863-7908; supportheat e-mail: methane of blogan@ combustion).and production increased These substrate results without concentrations show that increasing electrometha- methane digesters. psu.edu. production (2, 5). Most evidence suggestsbiofilm that in these (CH4-mixed) is very large compared to hydrogen gas the need for hydrogen evolutionnogenesis was canobtained be used by to chemically convert electrical current produced Substantial methane production has been reported in 10.1021/es803531g CCC: $40.75 © 2009 American Chemicalfrom Society renewable VOL. energy 43, NO. 10, sources 2009 / ENVIRONMENTAL (such as SCIENCE wind, &production TECHNOLOGY solar, or9 3953 in the absencerecent MEC of studies a biofilm (5, 9 (H-13-abiotic).), but the sourceNo hydrogen of this methane removingPublished on Web hydrogen 03/26/2009 gas and examining current generation 2 biomass) into a biofuel (methane) as well as servinggas as was a method detectedappears in mixed to be biofilm acetate tests. via acetoclastic Inset: Methane methanogenesis gas and using LSV. When the cathodefor the was capture coated of carbon with a dioxide. hydrogen production is lowerhydrogenotrophic using a biocathode methanogensis with a pure using culture hydrogen of gas produced in the process. Methane generation in anaerobic scavenger (1,4-diphenyl-butadiyne) (35), current densities M. palustre (CH4-Mp), but this gas production is still larger were not increased compared to those obtained with an digesters originates mostly from acetate (70%) with a smaller Introduction than that of an abioticportion cathode from hydrogen (H2-abiotic). gas (14 A). trace In contrast, level of methane uncoated electrode. MoreIncreasing detailed competition information for fossil on fuels, the and thehydrogen need to avoid gas (<0.01%)generation was in detected MECs may in testsbe more with likelyM. from palustre hydrogen. gas hydrogen scavenging resultsthe and release additional of carbon control dioxide from experi- combustion(B) of Current these fuels, densitiesthan measured acetate. Clauwaert using the et mixed al. (10 culture) measured biofilm, a methane ments that were conductedhas to eliminate increased the the search possibility for new and that sustainable approaches production rate of 0.28-0.75 L/L-d in several MEC experi- for energy production. Two new methodsM. of palustre bioenergy, or inments the at absence an applied of voltage microorganisms of -0.8 V but (applied found only 0.17 ∼ hydrogen evolution supportedproduction methane from generation biomass include can electricity be productionpotential usingof -1.0 V).L/L-d with no applied potential, suggesting that 23-61% of found in the Supporting Information.microbial fuel cells (MFCs) and hydrogen production by the methane was derived from acetate. Liu et al. (15) found If direct current transferelectrohydrogenesis is indeed occurring using tomicrobial metha- electrolysisalso cells appears (MECs) to beno methane possible generation in both in directions a reactor consistently (i.e. to and operated in (1, 2). Electricity generation in an MFC is spontaneous with an open-circuit mode compared to otherwise identical MECs nogens, the mechanism(s) byoxidation which of this organic occurs matter will such need as to acetatefrom by electrogenic the cell). Theoperated iron at reducing-0.7 V, suggesting bacterium hydrogenGeobacter was needed for be discovered. Electron transferbacteria mechanisms on the anode to dissimilatory (EAn =-0.2 V vs standardsulfurreducens hydrogen canmethane transfer production. electrons Eliminating to an bicarbonate anode, for from the iron respiring bacteria (DIRB)electrode) are still and being oxygen debated, reduction with at the cathodeexample, (ECat = but0.2 it canmedium also accept has helped electrons to reduce from methanogenesis the cathode is some V), with a working cell potential of approximately 0.4 V and studies (16) but not in others (10). Organic loading is clearly most strains having been found to be capable of growth using for cell respirationa factor with in nitrate methane ( production,38). Whether with thelong samereaction times an electrode (1, 19, 20). However,* Corresponding some DIRB author cannot phone: (814)863-7908; grow pathway e-mail: blogan@ is used forand the increased cell to substrate donate concentrations and accept increasing electrons methane using an electrode, while otherspsu.edu. capable of growth with an is not known. Gramproduction positive (2, 5 bacteria). Most evidence capable suggests of glucose that in these electrode cannot use iron10.1021/es803531g (21-23). Moreover, CCC: $40.75 some© 2009 American iron Chemicalfermentation Society to hydrogen VOL. 43, NO. 10, have 2009 been / ENVIRONMENTAL found to SCIENCE respire & TECHNOLOGY in an 9 3953 reducing bacteria such as ShewanellaPublished on Web oneidensis03/26/2009 may have MFC using the anode (39), and recently it was found that a multiple mechanisms of electron transfer that include self- cathodic biofilm accepted electrons and released hydrogen produced flavins and nanowires (36, 37). Electron transfer gas (16). Bacterial biofilms on MFC cathodes have also been

3956 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 10, 2009 US. Patent May 14, 2013 Sheet 1 0f5 US 8,440,438 B2

US008440438B2

(12) United States Patent (10) Patent N0.: US 8,440,438 B2 Cheng et al. (45) Date of Patent: May 14, 2013

(54) ELECTROMETHANOGENICFIGURE 1 REACTOR AND rRNA sequence analyses. International Journal of Systematic Bacte PROCESSES FOR METHANE PRODUCTION riology. 1993. 43(2): 278-286.* Wolin, EA et al. Formation of methane by bacterial extracts. Journal of Biological Chemistry. 1963. 238(8): 2882-2886.* (75) Inventors: Shaoan Cheng, State College, PA (US); Bond, et al., Electrode-Reducing Microorganisms That Harvest Bruce Logan, State College, PA (U S) Energy from Marine Sediments, Science, 295: 483-485, 2002. Bond, et al. Electricity Production by Geobacter sulfurreducens (73) Assignee: The Penn State Research Foundation, Attached to Electrodes, Applied and Environmental Microbiology, University Park, PA (U S) 69(3):1548-155, 2003. Rabaey, et al., A microbial fuel cell capable of converting glucose to ( * ) Notice: Subject to any disclaimer, the term of this electricity at high rate and ef?ciency, Biotechnology Letters, 25:1531-1535, 2003. patent is extended or adjusted under 35 Kim, et al., A mediator-less microbial fuel cell using a metal reducing U.S.C. 154(b) by 767 days. bacterium, Shewanellaputrefaciens, Enzyme and Microbial Technol ogy, 30: 145-152, 2002. (21) Appl.No.: 12/488,951 Park, et al., A Novel Electrochemically Active and Fe(III)-reducing Bacterium Phylogenetically Related to Clostridium bulyricum Iso (22) Filed: Jun. 22, 2009 lated from a Microbial Fuel Cell, Anaerobe, 7: 297-306, 2001. Chauduri, et al., Electricity generation by direct oxidation of glucose (65) Prior Publication Data in mediatorless microbial fuel cells, Nature Biotechnology, 21(10): 1229-1232, 2003. US 2009/0317882 A1 Dec. 24, 2009 Park, et al., Impact of electrode composition on electricity generation in a single-compartment fuel cell using Shewanella putrefaciens, Applied Microbiology and Biotechnology, 59: 58-61, 2002. Related US. Application Data Kim, et al., Effect of initial carbon sources on the performance of microbial fuel cells containing Proteus vulgaris, Biotechnology and (60) Provisional application No. 61/074,296, ?led on Jun. Bioengineering, 70(1): 109-114, 2000. 20,2008. Park, et al., Electricity Generation in Microbial Fuel Cells Using Neutral Red as an Electronophore, Applied and Environmental (51) Int. C1. Microbiology, 66(4):1292-1297, 2000. 0121* 5/02 (2006.01) Logan, Electricity-producing bacterial communities in microbial fuel cells, Trends in Microbiology 14(12): 512-518, 2006. (52) U.S.Cl. Lovley, et al., Anaerobic Oxidation of Toluene, Phenol, and p-Cresol USPC ...... 435/167 by the Dissimilatory Iron-Reducing Organism, GS-15, Applied and (58) Field of Classi?cation Search ...... None Environmental Microbiology, 56(6): 1858-1864, 1990. See application ?le for complete search history. Lovley, et al. Novel Mode of Microbial Energy Metabolism: Organic Carbon Oxidation Coupled t0 Dissimilatory Reduction of Iron 0r (56) References Cited Manganese, Applied and Environmental Microbiology, 54(6): 1472 1480, 1988. Chernicharo, Anaerobic Reactors: Biological Wastewater Treatment, U.S. PATENT DOCUMENTS v01. 4, Biological Wastewater Treatment Series, IWA Publishing, 4,067,801 A 1/1978 Ishida et a1. 2007. 4,503,154 A 3/1985 Paton Cheng, S. et al., Direct Biological Conversion of Electrical Current 4,735,724 A 4/1988 Chynoweth et al. into Methane by Electromethanogenesis, Environmental Science & 5,185,079 A 2/1993 Dague Technology, 43(10): 3953-58, 2009. 5,443,706 A * 8/1995 Kuroda et a1...... 204/242 5,976,719 A 11/1999 Kim et a1. (Continued) 6,270,649 B1 8/2001 Zeikus et a1. 6,299,774 B1 10/2001 Ainsworth et a1. Primary Examiner * Allison Ford 6,664,101 B2 12/2003 Wild 7,250,288 B2 7/2007 Zeikus et a1. Assistant Examiner * Susan E Fernandez 7,491,453 B2 2/2009 Logan et al. (74) Attorney, Agent, or Firm * Gifford, Krass, Sprinkle, 2006/0011491 A1* 1/2006 Logan et al...... 205/637 Anderson & Citkowski, RC. 2007/0259217 A1 11/2007 Logan OTHER PUBLICATIONS (57) ABSTRACT W.M. Haynes, ed., CRC Handbook of Chemistry and Physics, 92nd Increasing competition for fossil fuels, and the need to avoid Edition (Internet Version 2012). 2012. CRC Press/Taylor and release carbon dioxide from combustion of these fuels Francis, Boca Raton, FL. pp. 3-1 through 3-3 and 3-344.* requires development of neW and sustainable approaches for Nelson, DL et al. Lehninger Principles of Biochemistry, 3rd Edition. energy production and carbon capture. Biological processes NeWYork, NY, Worth Publishers, 2000. p. 88* for producing methane gas and capturing carbon from carbon Wu, W et al. Microbial composition and characterization of prevalent dioxide are provided according to embodiments of the methanogens and acetogens isolated from syntrophic methanogenic present invention Which include providing an electrometha granules. Appl. Microbiol. Biotechnol. 1992. 38: 282-290.* nogenic reactor having an anode, a cathode and a plurality of DiaZ, E et al. Molecular ecology of anaerobic granular sludge grown methanogenic microorganisms disposed on the cathode. at different conditions. Water Science and Technology. 2003. 48(6): Electrons and carbon dioxide are provided to the plurality of 57-64.* Wu, W et al. Metabolic properties and kinetics of methanogenic methanogenic microorganisms disposed on the cathode. The granules. Appl. Microbiol. Biotechnol. 1993. 39: 804-811.* methanogenic microorganisms reduce the carbon dioxide to Kenealy, W et la. In?uence of corrinoid antagonists on methanogen produce methane gas, even in the absence of hydro gen and/ or metabolism. Journal ofBacteriology. 1981. 146(1): 133-150.* organic carbon sources. Zhao, H et al. Assignment of fatty acid-beta-oxidizing syntrophic bacteria to Syntrophomonadaceae fam. nov. 0n the basis of 16S 10 Claims, 5 Drawing Sheets Direct via Indirect using Indirect using H2 outermembrane electrochemically produced produced enzymacally at the cathode redox proteins H2/formate

H+ H+ gradient e- CO2 + - H e H+ - H -ase e H2 2 H2 CO2

CH4 CH4 CH4 Direct via Indirect using Indirect using acetate outermembrane electrochemically produced produced by acetogens redox proteins H2/formate

H+ e- CO2 H+ gradient CO2 + - H e H+

- e H2 Acetogen ACE CO2

CH4 CH4 CH4

a Methanosarcina

• which cannot use H2 or formate 1000 0 Disconnected cathode 900 Poised cathode -0.1 Potenal vs. ref. electrode (V) 800 -0.2

700 (Volt) -0.3 AgCl 600 -0.4 500 -0.5 400 µmol methane -0.6 300 CO -0.7 200 2 CO2 Poised potenal vs. Ag/

100 -0.8

0 -0.9 0 10 20 30 40 50 60 70 80 Days by Mon Yee Oo New potenostats

Geobacter sulfurreducens on a cathode polarized at -600mV by AMEL2550

1.15 2.31 3.47 25 -4.00E-03 days

-6.00E-03 15 mM mM 10 Current (A) -8.00E-03 5 Cells on non-poised cathode 0 -1.00E-02 0 1 2 3 4 5 me (days)

by Mon Yee Oo Cyclic voltametry – 3 redox couples

Full Paper E. Marsili et al.

redox proteins, or their redox proteins are not yet in optimal 2.50E-02 contact with electrodes, analogous to how many bacteria require retraction of type IV pili or flagellar motility to overcome local electrostatic interactions and firmly adhere to surfaces. -0.305 V vs. Ag/The fact that rates ofAgCl growth following this initial attachment phase rivaled those of planktonic cells utilizing soluble Fe(III)-0.105V vs. SHE showed that electron transfer across this cell- 1.50E-02 electrode connection was not limiting, in terms of its ability to carry electrons from cells to the electrode surface. This rate could be sustained for days when the lowest levels of inoculum were used, (Fig 1B), and was never affected by removal of planktonic bacteria. Individual G. sulfurreducens cells can clearly respire, generate ATP, and double as 5.00E-03 Marsili 2010 fast using an-0.225 V vs. Ag/ insoluble acceptor (inAgCl this case an electrode) as with a soluble-0.025 V vs. SHE acceptor, when the surface area and attachment is not a limiting factor. While the first layers of cells experience near-optimal conditions, the slowing rate of anodic current increase as colonization progressed revealed a developing limitation to electron transfer as multiple cell layers were added. A -5.00E-03 simple hypothesis for this could be the transition from daughter cells being able to expand in two (sideways or current (A) upwards), to only one (upwards) dimension, limiting the area available for new colonization. The final plateau in current density suggests the existence of a barrier to accumulation of a biofilm beyond this thickness. Multiple researchers have proposed proton escape from the thick- -1.50E-02 -0.435 V vs. Ag/ening biofilmAgCl as a key factor in this limit [44 – 46]. This Fig. 7. (A) Representative cyclic voltammetry (1 mV/s) of calculation explains forces that oppose charge transfer, and biofilm starved of electron donors for 24 h, showing baseline--0.235 V vs. SHE agrees well with many experimental observations. It does subtracted data. (B) Comparison of baseline-subtracted peak Media heights for two independent films subjected to increasing scan not explain why these high rates of respiration (e.g., the high rates, showing linear dependence on the square root of scan rate. current/unit protein) are not resulting in progressively thicker biofilms. To explain this phenomenon,Electrode + Media cells may be -2.50E-02 responding to the lack of available expansion (within the biofilm development, we were able to quantify apparent biofilm) or attachment (on top of the biofilm) sites, or may -1 growth rates, yield,-0.8 and electron transfer-0.6 rates by this model-0.4 be expending significant-0.2 metabolic energy0 to compensate0.2 0.4 metal-reducing bacterium at different growth stages and for local pH differences. across a range of thermodynamic conditions. In general, We observed no evidence for a dramatic change in the these experiments reveal an early phase where this pathway redox interface, or model for electron transfer between cells must be established, and a subsequentPotenal versus Ag/ network of redox and electrodesAgCl at any phase (Volts) of growth. At all time points, the proteins connecting cells to surfaces that is remarkably rate-controlling electron transfer process was centered atby Mon Yee a Oo consistent, and able to communicate electrons across similar potential, and resistive behavior or new electron relatively long distances, with little change in the overall transfer reactions were not detected. No unusual waveforms mechanism. appeared at later stages of growth, which would have G. sulfurreducens shows rapid self-assembly on electro- suggested developing heterogeneity in protein orientation des, and even cells grown with soluble fumarate as an or binding. This has significant implications for simplifying electron acceptor are apparently immediately capable of the model of electron transfer from cells to electrodes, as some degree of extracellular electron transfer. For an was described by [26]. organism selected in the environment (and the laboratory) for interactions with charged Fe(III) and Mn(IV) surfaces, the increase in anodic (oxidation) current reflects a fortu- 3.8. Insights from Lower Potential Experiments itous affinity between cells and polycrystalline graphitic electrodes, as well as favorable orientation of at least one Another surprising finding was that, even when grown at redox protein close enough to the surface to allow direct 0.24 V, G. sulfurreducens reached its maximum rate of electron transfer. However, the low electron transfer rates electronþ transfer at a driving force of about 0.05 V, per unit of initially attached protein (Fig. 2) also shows that essentially ignoring over 50% of the energy in the availableÀ these first colonizers may not possess a full complement of electron acceptor. Even when grown at lower potential

872 www.electroanalysis.wiley-vch.de 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2010, 22, No. 7-8, 865 – 874

Time line

Oct-Nov 2015 Mon tests A. ordered potenostats Methanosarcina and June 2015 sludge in MES using a Jan 2015 Sept 2015 Mon tests AMEL Mon started powersource potenostats with Geobacter NEXT

Feb 2016 Feb 2016 The 8-channel Mon tests the Nov 2015 Jan 2016 mulstat arrived PalmSense mulstat A. started ordered a 8- with Geobacter channel mulstat Special Feb 2016 glassware End of dec 2015 A. tests Clostridium in MES using ordered Potenostats arrived the AMEL potenostats

What next? Who? How?

Digester community on cathodes Opmize consora on cathodes H sensor test 2

Methanosarcina Gene-deleon studies Methanosaeta Comparave Methanosarcina on cathodes Methanosarcina Methanosaeta Transcript-/prote-ome

PEOPLE: Mon, Karen, Amelia, Bo, Lars, Lars-Peter, Niels-Peter, Cornelia