Bioresource Technology 102 (2011) 263–271

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Bioresource Technology

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Hydrogen consumption in microbial electrochemical systems (MXCs): The roleof homo-acetogenic bacteria

Prathap Parameswaran *, César I. Torres, Hyung-Sool Lee, Bruce E. Rittmann, Rosa Krajmalnik-BrownCenter for Environmental Biotechnology, The Biodesign Institute at Arizona State University, P.O. Box 875701, Tempe, AZ 85287 – 5701, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 4 February 2010Received in revised form 23 March 2010Accepted 29 March 2010Available online 28 April 2010

Keywords:Microbial fuel cellsHomo-acetogensHydraulic retention time (HRT)Formyl tetrahydrofolate synthetase (FTHFS)Biofilm anode

0960-8524/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.biortech.2010.03.133

* Corresponding author. Tel.: +1 480 727 0432; faxE-mail address: prathap@asu.edu (P. Parameswara

Homo-acetogens in the anode of a microbial electrolysis cell (MEC) fed with H2 as sole electron donorallowed current densities similar to acetate-fed biofilm anodes (�10 A/m2). Evidence for homo-acetogensincluded accumulation of acetate at high concentrations (up to 18 mM) in the anode compartment; detec-tion of formate, a known intermediate during reductive acetogenesis by the acetyl-CoA pathway; and detec-tion of formyl tetrahydrofolate synthetase (FTHFS) genes by quantitative real-time PCR. Current productionand acetate accumulation increased in parallel in batch and continuous mode, while both values decreasedsimultaneously at short hydraulic retention times (1 h) in the anode compartment, which limited sus-pended homo-acetogens. Acetate produced by homo-acetogens accounted for about 88% of the current den-sity of 10 A/m2, but the current density was sustained at 4 A/m2 at short hydraulic retention time because ofa robust partnership of homo-acetogens and anode respiring bacteria (ARB) in the biofilm anode.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction lish syntrophic relationships with ARB (Parameswaran et al.,

Microbial electrochemical systems (MXCs) include two impor-tant approaches for bioenergy generation from organic streams:microbial fuel cells (MFCs) for producing electricity and microbialelectrolysis cells (MECs) for producing hydrogen (H2). Recent re-views on methodology, applications, and challenges in MXCs sum-marize the significance of this rapidly developing interdisciplinaryarea (Logan et al., 2006, 2008; Rozendal et al., 2008; Rittmann, 2008).

Several groups of bacteria present in MXCs are able to respireelectrons to the anode, and they are referred to as anode respiringbacteria (ARB). ARB remove electrons from an electron donor andtransfer them to the anode by extracellular electron transport(EET) (Marcus et al., 2007; Torres et al., 2010). A unique featureof either MXC producing high current densities is that its biofilmhas a conductive matrix, which makes it part of the anode; hence,this biofilm is called the biofilm anode (Marcus et al., 2007). Whenthe ARB act efficiently, the MXC has a high Coulombic efficiency(CE), which is the ratio of electron equivalents transferred intothe anode (and moving to the cathode) divided by the electronequivalents removed from the electron donor substrate in the an-ode compartment, and a high current density, such as �10 A/m2.

Bacteria other than ARB also inhabit biofilm anodes (Fung et al.,2006; Rabaey et al., 2004; Parameswaran et al., 2010), particularlyin MXCs fed with fermentable substrates. Although their role withrespect to EET is not well defined, several of the key non-ARB,including fermenters, methanogens, and homo-acetogens, estab-

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: +1 480 727 0889.n).

2009; Freguia et al., 2008) when fed with fermentable substrates.These syntrophies affect the performance of the MXCs in positiveor negative ways.

A negative impact of syntrophy is a reduction to the CE causedby diversion of electrons away from the anode, often to methanegas (CH4). A positive impact is channeling more electrons throughsubstrates that are readily oxidized by ARB. Acetate definitely is afavorable substrate for ARB (Torres et al., 2007; Lee et al., 2009);the role of H2 is less clear (Torres et al., 2007).

Whether positive or negative, the channeling of electronsthrough acetate and H2 seems to be a common feature of biofilmanodes (Lee et al., 2008; Parameswaran et al., 2009, 2010). The fol-lowing reactions indicate the stoichiometry for production of ace-tate and H2 from common fermentation products:

Propionate : CH3CH2COO� þ 2H2O! CH3COO� þ CO2 þ 3H2

DGo0 ¼ þ76:1 kJ mol�1

Butyrate : CHþ 3CH2CH2COO� þ 2H2O! 2CH3COO� þHþ þ 2H2

DGo0 ¼ þ48 kJ mol�1

Ethanol : CH3CH2OHþH2O! CH3COO� þHþ þ 2H2

DGo0 ¼ þ9:6 kJ mol�1

Lactate : CH3CHOHCOO� þH2O! CH3COO� þ CO2 þ 2H2

DGo0 ¼ �8:6 kJ mol�1

A significant fraction (up to 43% for propionate) of the elec-tron products ends up in H2. While acetate is readily consumedby ARB belonging to various genera, such as Geobacteraceae,

264 P. Parameswaran et al. / Bioresource Technology 102 (2011) 263–271

Rhodopseudomonas, and Shewanella (Nevin et al., 2008; Phamet al., 2007; Xing et al., 2008), H2-oxidizing methanogens canoutcompete H2 oxidation that generates current in the biofilmanode (Parameswaran et al., 2009; Call and Logan, 2008; Wanget al., 2009; Lee et al., 2009). Thus, CH4 production from H2

can be responsible for the decrease in CE when MXCs are fedwith fermentable substrates. H2 can be converted to electricalcurrent in cases in which methanogenesis is eliminated, leadingto higher CEs (Parameswaran et al., 2009), or in a well accli-mated biofilm anode previously fed only with acetate (Rozendalet al., 2008; Lee et al., 2009).

Selective inhibition of undesired H2 consumers should be anefficient strategy to evaluate if positive syntrophies can be estab-lished and lead to higher electron recoveries. 2-Bromoethane sul-fonic acid (BES) selectively inhibits (at 50 mM) the methylcoenzyme reductase A (mCrA) activity of both acetoclastic andhydrogenotrophic methanogens. Other H2 scavengers, such as den-itrifiers and sulfate-reducing bacteria, can be eliminated by notallowing the respective electron acceptors in the media (nitrateand sulfate, respectively). This should provide an opportunity foralternate H2 scavengers, such as homo-acetogens, to proliferatein the anode compartment.

Homo-acetogenic bacteria grow chemolithoautotrophically onH2 and CO2, producing acetate at higher H2 thresholds than meth-anogens or sulfate-reducers (Drake, 1993). They have highergrowth rates than other fermentative bacteria due to energy con-servation from a combination of substrate level phosphorylationand sodium-based chemiosmotic mechanisms (Muller, 2003).Though homo-acetogens are phylogenetically diverse and spreadover 16 genera, molecular microbial ecology tools targeting thehighly conserved formyl tetrahydrofolate synthetase (FTHFS) geneof homo-acetogens enables their identification and subsequentquantification (Leaphart and Lovell, 2001; Xu et al., 2009;Parameswaran et al., 2010).

We previously demonstrated that positive syntrophic interac-tions involving homo-acetogens produced higher coulombic effi-ciencies (CE) when ethanol was used as electron donor andwhen negative syntrophies, such as methanogenesis, were sup-pressed (Parameswaran et al., 2009, 2010). Homo-acetogenicbacteria also were observed in MFCs previously, although theirroles were not well understood then (Choo et al., 2006; Ishiiet al., 2008).

The goal of this research was to evaluate the hypothesis thatefficient H2 conversion to current is achieved in an anode suppliedwith H2 as the sole electron donor when a syntrophic relationshipbetween ARB and homo-acetogenic bacteria is established. Wedocument the positive syntrophy through a combination of chem-ical and genomic tools.

2. Methods

2.1. MEC batch and continuous experiments

We operated an H-type MEC with a volume of 325 mL each for theanode and the cathode compartments. An anion exchange mem-brane (AMI 7001, Membranes International, Glen Rock, NJ) sepa-rated the anode and cathode compartments. The two electrodes inthe anode compartment were graphite squared blocks with a totalsurface area of 21.14 cm2 each (www.graphitestore.com). The anodeelectrodes were poised at a potential of �350 mV with reference tothe Ag/AgCl electrode (BASI Electrochemistry, West Lafayette, IN)using a potentiostat (Princeton Applied Research, Model VMP3,Oak Ridge, TN); the potential was �80 mV versus the standardhydrogen electrode (SHE) (Torres et al., 2008). Each MEC compart-ment was agitated with a magnetic stirrer at 150 rpm and main-tained in a constant-temperature chamber at 30 �C.

The anode compartment had the following components in 1 L ofdeionized water: Na2HPO4 (12.04 g), KH2PO4 (2.06 g), NH4Cl(0.41 g), selective methanogenic inhibitor sodium 2-bromoethane-sulfonate (10.55 g to produce 50 mM), trace minerals (10 mL) ofthe same composition as in Parameswaran et al. (2009), 1 mL ofa 4 g/L Fe(II)Cl2 stock solution, and 0.5 mL of a 37.2 g/L Na2S�9H2Ostock solution. The liquid in the anode compartment was continu-ously sparged with 80% H2:20% CO2 gas mix at a flow rate 58 mL/min (Gen Pac, USA), and H2 served as the sole electron donorthroughout all experiments. The inoculum for the experimentswas 1 mL each of return activated sludge and anaerobic digestedsludge from Mesa Northwest Wastewater Reclamation Plant(Mesa, AZ). The cathode compartment contained NaOH solutionmaintained at a pH of 12 to enable the efficient operation of thereference electrode and the anion exchange membrane (AEM).

We performed the MEC experiments twice in successive timeperiods (referred to as Runs 1 and 2 throughout the article) fromthe same inoculum. After the current density stabilized at around3 A/m2 for Run 1, we switched to continuous operation with anHRT of 10 h (which corresponds to a media flow rate of 0.6 mL/min). We performed additional continuous-flow experiments dur-ing Run 1 by decreasing the HRT to 1 h (5 mL/min media flow rateand no change in gas supply) and then performed rapid kinetic-re-sponse tests explained in the next section.

After a constant current density was achieved during continu-ous-flow (10 h HRT) with Run 2, we successively varied the HRTin 5 steps – 17, 9, 4.5, 2.25, and 1 h to evaluate the washout of sus-pended homo-acetogens on MEC performance.

2.2. Rapid kinetic-response tests during continuous-flow in Run 1

We analyzed the kinetics of the consumption of electron donorsby ARB by performing rapid kinetic-response tests, which refer tothe sudden addition or removal of electron donor to the anodechamber at a time when the biofilm anode is producing a stablecurrent density. We could monitor the response to the additionor removal of electron donor by ARB instantly by recording electriccurrent. We previously demonstrated sharp differences among theARB’s utilization capabilities for acetate, H2, and ethanol by per-forming fast kinetic-response tests at the end of batch experiments(Parameswaran et al., 2009).

Here, we performed rapid kinetic-response tests during contin-uous-flow experiments in Run 1. The tests consisted of two parts:first to remove electron donor by changing gas supply from 80%H2:20% CO2 mix to 100% N2 gas, and second to restore the electrondonor supply when current density was at a stable minimum va-lue. The HRT of the continuous experiment was constant at 10 h,and only the gas supply was changed.

2.3. Chemical analyses

2.3.1. HPLCWe filtered �1 mL of sample from the anode compartment

through a 0.2 lm membrane filter for HPLC analyses for simpleacids and alcohols (acetate, formate, propionate, butyrate, iso-butyrate, valerate, iso-valerate) using high performance liquidchromatography (Model LC-20AT, Shimadzu). An Aminex HPX-87H (Bio-Rad) column maintained at 50 �C separated the acidsand alcohols. Sulfuric acid at 2.5 mM was the eluent at a flow rateof 0.6 mL/min.

2.3.2. Gas analysisWe monitored the gas percentages of H2, CO2, and CH4 (if any)

in the headspace of the MEC in samples taken with a gas-tight syr-inge (SGE 500 lL, Switzerland) using a gas chromatograph (GC2010, Shimadzu) equipped with a thermal conductivity detector

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and a packed column (ShinCarbon ST 100/120 mesh, Restek Corpo-ration, Bellefonte, PA) for separating sample gases. N2 was the car-rier gas fed at a constant flow rate of 10 mL/min, and thetemperature conditions for injection, column, and detector were110, 140, and 160 �C, respectively. Analytical grade H2, CH4, andCO2 were used for standard calibration curves. We carried outgas analyses in duplicate and averaged the data.

2.4. Genomic techniques

2.4.1. DNA extractionAt the end of all continuous-flow experiments in Runs 1 and 2,

we centrifuged the entire volume of liquid in the anode compart-ment to obtain a pellet for DNA extraction. We also separatelyscratched a known cross-section of the biofilm anode to collectbiofilm samples. We used 0.25 g of the pellet for extracting DNAfollowing the recommendations for the MoBio� Powersoil DNAextraction kit. We also extracted DNA from anodic and cathodicbiofilms from acetate-fed single-chamber upflow and modularMECs, as described in Lee et al. (2009) and Lee and Rittmann(2010), to evaluate the presence of FTHFS genes, and hence,homo-acetogenic bacteria.

2.4.2. Quantitative PCR analysisWe targeted 16S rRNA in General Bacteria and Geobacteraceae

(Parameswaran et al., 2009) and the formyl tetrahydrofolate syn-thetase (FTHFS) gene (Xu et al., 2009) in the DNA samples. We per-formed SYBR Green assays for the three targets with a Realplex 4Squantitative PCR unit for 10 lL total reaction volumes. Each reac-tion consisted of: 0.5 lL nuclease free H2O, 4.5 lL of SYBR Realmas-termix (5 Prime, CA) at 1X final concentration, forward and reverseprimers for a final concentration of 0.5 lM, and 4 lL of templatecDNA (130 nM final concentration). We performed each assay intriplicate with a six-point standard curve, along with the samples.

2.4.3. Clone-library analysisWe performed PCR amplification targeting the 16S rRNA gene of

General Bacteria with universal bacterial primers 8F and 1525R inthe DNA extracted from the biofilm anodes at the end of Runs 1and 2, as well as DNA obtained from the suspension sample atthe end of Run 2. We performed clone-library analysis using thepurified PCR products, as explained in Torres et al. (2009). A total

Fig. 1. Framework for all possible paths of H2 consumption under the experimental condour H2-fed anode. A represents direct H2 oxidation by ARB, B denotes acetate productioacetate production by homo-acetogens in the biofilm anode, and D denotes acetate-oxidgreen cells are homo-acetogens. AEM is anionic exchange membrane.

of 96 and 49 clones were obtained for biofilm anode clone librariesfor Runs 1 and 2, respectively. The clone-library for the suspensionsample from Run 2 was comprised of a total of 61 clones. Repre-sentative sequences were submitted to GenBank and can be ac-cessed at GU595066–GU595095.

3. Results and discussion

3.1. Framework for explaining H2 electron flow to electric current inanode

Fig. 1 presents a summary of various electron flow routes thatH2 could have taken in the anode chamber to produce electric cur-rent. Becasue addition of BES and the absence of sulfate and Fe(III)in the media ensured the elimination of methanogenesis, sulfatereduction, and Fe(III) reduction as H2 sinks in the anode, we donot include these paths in Fig. 1. Direct H2 oxidation by ARB isthe first possibile route, as indicated by route A in Fig. 1. H2 canbe converted to acetate when homo-acetogens are present eitherin the suspension (route B) or in the biofilm anode (route C). Ace-tate can then be consumed by acetate-oxidizing ARB to produceelectric current (route D). Acetate for D diffuses from the bulk li-quid into the biofilm anode when route B is present or is consumedin situ when route C is present.

3.2. Homo-acetogenesis leads to current generation – Runs 1 and 2

Current density and acetate concentrations throughout thebatch and initial continuous-flow stages of Runs 1 and 2 are pre-sented in Fig. 2a and b. The current increased in batch mode untilstable current densities were obtained in both runs. We thenswitched the MECs to continuous-flow, and current densities con-tinued to increase, which likely indicated that the biofilm anodescontinued to grow. Current densities eventually stabilized at 10–12 A/m2, although it took 20 days for Run 1 versus 7 days for Run2. Acetate levels increased in parallel with current in batch mode.Since the influent contained no organic substrate, the steady accu-mulation of acetate means that acetogenesis was faster than ace-tate consumption for current production during the batch mode.The high concentration of acetate (�18 mM) during batch opera-tion of Run 2 indicates a high rate of acetate production byhomo-acetogens. Eventually, acetate consumption by ARB

itions (which eliminated methanogenesis, sulfate reduction, and Fe(III) reduction) inn by homo-acetogens in the suspension of the anode chamber, C stands for in situizing ARB. HAc stands for acetate. Bacterial cells in orange denote ARB, while light

Fig. 2. Current density, acetate production, and formate formation in an MEC fed with 80% H2:20% CO2 started up from a diverse wastewater inoculum. (a) Run 1 and (b) Run2. Steady formate was quantifiable only for Run 2. Maximum current densities were around 10–12 A/m2 during continuous-flow, although the times to reach the values weredifferent between the two runs.

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matched homo-acetogenesis, and acetate concentration stabilizedat about 6 mM in continuous operation.

The current densities we observed when H2 was supplied as thesole electron donor were high (10–12 A/m2), comparable to ace-tate-fed biofilms (Torres et al., 2008; Fan et al., 2007). This indi-cates that the startup procedure of MEC anodes fed with H2 andinoculated with a mixed microbial community was crucial. Selec-tive inhibition of hydrogenotrophic methanogens enabled the pro-liferation of desirable H2 oxidizers, the homo-acetogens, asevidenced by the production of acetate in the anode.

3.3. Continuous-flow experiments in Run 1

We altered the HRT of the MECs to evaluate the limiting condi-tions for process performance. At the end of continuous steady-state operation (HRT = 10 h) in Run 1, we increased the flow rate

to operate at an HRT of 1 h. Current density decreased from 10 to4 A/m2, while the acetate concentrations dropped from 5.5 to0.2 mM (Fig. 3a). The parallel decreases in current density and ace-tate concentration show that acetate-oxidizing ARB were responsi-ble for a significant fraction of the current. However, the acetateconcentration declined more than the current density. The muchlower acetate concentrations with the 10 times shorter HRTs andthe relatively higher current density at very low HRTs can be ex-plained by one of two phenomena: (1) Most homo-acetogenic bac-teria were in the suspension (route B), and they were washed outwith the short HRT so that the few remaining homo-acetogenscould generate relatively little acetate. (2) The acetate was pro-duced by homo-acetogens mainly in the biofilm, but the acetatewas washed out with the short HRT. The effluent advection rateof acetate for the 10 h HRT was 0.18 mmol/h (5.5 mM � 0.325 L/10 h), while the acetate advection rate for the 1 h HRT was

Fig. 3. (a) Continuous-flow experiments followed by fast kinetic-response tests, at the end of Run 1, to understand the effect of low HRT and the significance of H2 as theultimate electron donor. Current densities were sustained even at low HRT of 1 h. (b) Change in current density and acetate concentration in the anode as a function ofdecreasing HRT at the end of Run 2.

P. Parameswaran et al. / Bioresource Technology 102 (2011) 263–271 267

0.065 mmol/hr (0.2 mM � 0.325 L/1 h). Thus, the washout rate ofacetate was smaller for the HRT of 1 h, which indicates that phe-nomenon 2 was not the main cause of low acetate.

Despite the low acetate concentrations in the medium, ARBwere able to produce significant current densities (�4 A/m2, asshown in Fig. 3a), which are still higher than the maximum currentdensities observed with H2-fed biofilm anodes in previous re-search, about 2.2–3.3 A/m2 (Rozendal et al., 2008; Lee et al.,2009). This indicates that a major portion of the 4 A/m2 shouldhave come from either route A or a combination of routes C andD (Fig. 1). To assess the contribution of direct H2 oxidation byARB (route A in Fig. 1), we performed rapid kinetic-response tests.

3.4. Rapid kinetic-response tests during continuous-flow in Run 1

When we removed electron donor by sparging 100% N2 gas inthe liquid space of the anode chamber, current density rapidly de-

creased from 10 to 8.8 A/m2 within the first 5 min (Fig. 4a). Currentdensity decreased gradually after that, reaching 8.3 A/m2 after thefirst 80 min and 8 A/m2 after 200 min. At the time of the gaschange, acetate had accumulated in the anode compartment at4 mM. The initial rapid drop in current density (by 1.2 A/m2) mostprobably was a response of ARB performing direct H2 oxidation(route A in Fig. 1), since the acetate concentration remained atclose to 4 mM in the first 5 min. After this initial drop in current,the gradual decrease in current probably resulted from continuousconsumption of acetate by the acetate-oxidizing ARB and from ace-tate washout in the effluent. This gradual decrease continued forabout 36 h until the current density was stable at 2.3 A/m2 forabout an hour, when the acetate concentration had declined toabout 0.3 mM.

After flushing the anode compartment with 100% N2 for 36 hand operating at a HRT of 10 h, we switched back to H2 as theelectron donor via gas of 80% H2:20% CO2. The current density

Time of continuous tests (minutes)

0 50 100 150 200 1250 1300

Cur

rent

Den

sity

(A

/m2 )

0

1

2

3

4

5

6

7

Ace

tate

(m

M)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

CurrentAcetate

Gas changed from 100% N2

to 80% H2 :20% CO2

a

b

Fig. 4. (a) Change in current density with time after stopping the electron donor (80% H2:20% CO2) supply by replacing the gas with 100% N2. An initial sharp decrease in5 min was followed by a gradual decrease in current density. (b) Current density change after the H2 supply was restored. Current density initially increased from 2.3 to 3.5 A/m2 in 20 min, which was followed by a gradual increase. Concurrent acetate concentrations are also shown for the two tests. Acetate measurements for the second-point in(b) could not be taken at the same time as the current density, and it was taken 1200 min after the fast kinetic-response test.

268 P. Parameswaran et al. / Bioresource Technology 102 (2011) 263–271

increased from 2.3 to 3.6 A/m2 (an increase of 1.3 A/m2) in the first20 min after the gas change, and, after that, it gradually increasedto 6 A/m2 over 4.5 h (Fig. 4b). The rapid increase in current densityby 1.2 A/m2 again reflects the activity of ARB that were directlyoxidizing H2. The gradual increase again indicates the productionof current by acetate-oxidizing ARB as the homo-acetogens replen-ished acetate (route B and/or route C in concert with D).

Evidence from the fast kinetic-response tests supports that H2-oxidizing ARB were producing around 1.2 A/m2. While significant,this current density was not the major source of current in the bio-film anode of Run 1 experiment. Although we did not perform ki-netic-response experiments in Run 2, the operating conditions andcurrent densities were quite similar for Runs 1 and 2. This suggeststhat the current generation from H2-oxidizing ARB may have beensimilar in Run 2.

Since the total current density was �10 A/m2 for both runs, ace-tate derived from homo-acetogenesis occurring by either routes Bor C and subsequently consumed by route D (Fig. 1) contributed tothe majority of the observed current density (8.8 A/m2 or 88% ofthe total current density = 10–1.2 A/m2). Our data do not enable

us to distinguish the contribution from routes B or C to electriccurrent.

3.5. Step HRT experiments in Run 2

We performed a new set of continuous-flow experiments at theend of Run 2 (Fig. 3b) to determine the effect of low HRT andhomo-acetogen washout on process performance. We decreasedthe HRT in a stepwise manner and allowed the current density toreach a stable value for each HRT. Acetate concentrations decreasedwith each successive decrease in the HRT from 17 to 4.5 h, while thedecrease in current density was less significant. However, for an HRTof 2.25 h, we observed a decrease in the current density by 50% of itsvalue at HRT of 4.5 h. This reinforces the impact of washing out sus-pended homo-acetogens on current generation.

3.6. Presence and impact of formate

Besides accumulation of acetate in the anode compartment, weconfirmed that homo-acetogens were producing acetate in the

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MECs by tracking formate in the effluent. Formate is an importantintermediate in the formyl tetrahydrofolate synthetase branch ofthe acetyl-CoA pathway (Drake, 1993). Moreover, previous studieshave shown that formate was not directly used by ARB such asGeobacter, while it was readily used by acetogens to produce ace-tate, which was then used by the ARB (Ha et al., 2008). We ob-served comparable levels of formate, which were consistentlylower than that of acetate in both runs, as shown in Fig. 2b forRun 2. This observation is in accord with Ha et al. (2008) and sup-ports that current densities from H2 were greater due to in situ pro-duction of formate and its subsequent transformation to acetate.

3.7. Presence of the FTHFS gene

We obtained direct evidence for the presence of homo-acetogensin the suspension and biofilm by detecting the FTHFS gene usingQPCR analysis. The results in Fig. 5 show that the FTHFS gene copieswere higher in the suspension than in the biofilm anode samples. Theratio of FTHFS/16S rRNA gene copies was 0.12% in the biofilm anodefor Run 1 (0.01% in biofilm anode for Run 2), as against 22% in the sus-pension fraction for Run 2. Since one FTHFS gene copy is present ineach cell (Xu et al., 2009), we can estimate the total number ofhomo-acetogenic cells for the entire volume of the suspension tobe 2.0 � 1011 cells (6.1 � 108 gene copies/mL of suspension in Run1 multiplied by the entire reactor volume of 325 mL) and for the en-tire biofilm anode surface area of the reactor to be 3.2 � 108 cells(1.5 � 107 gene copies/cm2 multiplied by the entire biofilm anodearea of 21.14 cm2). This analysis indicates that homo-acetogenswere predominantly in the suspension, compared to the biofilm an-ode when the HRT was 10 h. Fig. 5 also shows that the ratio of Geob-acteraceae/General Bacteria was 100 times greater in the biofilmanode, compared to the suspension, in Run 2. This low level of Geob-acteraceae levels in the suspension conforms to the fact that ARBmust be associated with the biofilm anode.

3.8. Microbial community structure at the end of Runs 1 and 2

Microbial community structures of the biofilm anodes, obtainedfrom clone libraries and summarized in Fig. 6a and b, were domi-nated by Geobacter in both runs. Most of the genus was repre-

Fig. 5. Summary of 16S rRNA for General Bacteria, 16S Geobacteraceae, and FTHFS genecalculated for each fraction as follows: suspension = (gene copies/mL) � total volume of(21.14 cm2). The suspension fraction had the highest amounts of FTHFS gene copies.

sented by Geobacter sulfurreducens PCA (shown in Table S2-1 ofSupporting Information). The community structure was signifi-cantly different from the structures of acetate-fed biofilms re-ported by Torres et al. (2009) and Xing et al. (2009), even thoughall MECs produced similar current densities and operated at a sim-ilar anode potential (�0.09 V versus SHE). The acetate-fed biofilmsshowed >95% predominance of G. sulfurreducens (Torres et al.,2009), but this fraction dropped to 71–77% for the H2-fed MEC.In the H2 case, 10–15% of the total clones were comprised of thephyla Actinobacteria and Bacteroidetes (mainly belonging to thegenus Dysgonomonas) during Runs 1 and 2, respectively. Previousresearch has observed that these phyla are associated with biofilmanodes, but with low fractions of total bacteria (Xing et al., 2009;Torres et al., 2009). The possibility that they are ARB remains spec-ulative and is not confirmed by these or other experiments.

A second major feature of the H2-fed biofilm anode communi-ties is the presence of 6–8% of the total clones as potentialhomo-acetogenic and other acetogenic bacteria (genera Acetobac-terium, Eubacterium, and Spirochaeta). This finding supports theexistence of route C (Fig. 1) and subsequent consumption of ace-tate by route D. While some of the acetate diffused out of the bio-film, most of it was utilized by the ARB, such as Geobacter, whichwere in close proximity inside the biofilm. Preferential presenceof homo-acetogens in the biofilm anode over the suspension wasalso observed in batch experiments that reported high electronrecoveries when fed with ethanol (Parameswaran et al., 2010).

In sharp contrast to the biofilm anode, the community structureof the suspension (which was characterized only at the end of Run2) had no Geobacter, Actinobacteria, or Bacteroidetes (Fig. 6c). 57% ofthe clones were from the genus Desulfovibrio, and the species Des-ulfovibrio desulfuricans alone accounted for 36% of the total clones(shown in Table S2-2 of Supporting Information), while Pseudomo-nas mendocina accounted for 14% of the total clones. With very lowsulfate concentration and in the presence of 80% H2:20% CO2 in theH2-fed MEC anode, members of the genus Desulfovibrio can per-form homo-acetogenesis and excrete acetate into the medium(Ljungdahl, 1986; Klemps et al., 1985). The presence of FTHFSgenes in Desulfovibrio has been confirmed recently (Xu et al.,2009). Both pieces of evidence reinforce the predominance ofhomo-acetogenesis in the suspension fraction.

copies in the various samples under study. Gene copies reported are total, and aresuspension fraction (325 mL); biofilm = (gene copies/cm2) � total area of electrodes

Fig. 6. Pie charts showing the results (at the genus level) from clone-library analysis targeting the 16S rRNA gene of bacteria. (a) Biofilm anode – Run 1 (total number ofclones = 96) (b) biofilm anode – Run 2 (total number of clones = 49), and (c) suspension – Run 2 (total number of clones = 61).

270 P. Parameswaran et al. / Bioresource Technology 102 (2011) 263–271

The role of P. mendocina is not clear in the suspension, while adiverse fraction of b- and c-proteobacteria also existed in the sus-pension. Members of the genus Pseudomonas have been implicatedas ARB that produce soluble electron shuttles for extracellular elec-tron transfer (Pham et al., 2007). However, the high current densi-ties that we observed could not be explained by the soluble shuttletransport from a less abundant microbial community, as shown byclone-library analysis of the suspension (Parameswaran et al.,2010; Torres et al., 2010). Obligate homo-acetogens such as Aceto-bacterium and Eubacterium, which were present in the biofilm an-ode, were not detected in the suspension. This might indicate thatthe biofilm anode might be a better niche for these specific homo-acetogens and their effect on the biofilm anode apart from acetateproduction (such as biofilm conductivity) has to be carefullyevaluated.

H2 scavenging by methanogens in MXCs results in significantelectron losses away from the anode, and methanogenesis isviewed as a problem for sustainable operation of these systems(Call and Logan, 2008; Parameswaran et al., 2009; Lee et al.,2009). As our results here show, altering H2 scavenging mecha-nisms in the anode of MXCs by favoring positive syntrophies be-tween homo-acetogens and acetate-oxidizing ARB holds promise.This could be achieved practically by either selectively enrichingthe preferred partner for the ARB such as homo-acetogenic bacteriain situ, or by adding enriched cultures of appropriate homo-aceto-gens to sustain their population in the anodes. Higher tolerance ofhomo-acetogens to atmospheric oxygen exposure and their abilityto sustain lower pH (Kusel et al., 2001; Phelps and Zeikus, 1984)present supplemental avenues to utilize practical ways for sustain-ing these populations in the anode compartment. Previous re-search has not focused on these alternate pathways in great

detail or has reported them to be absent at the end of short-termtests (Freguia et al., 2008).

While homo-acetogens hold promise for high electron recover-ies in a separate anode chamber, they could potentially account forH2 losses in a single-chamber MEC (presented in detail in Supple-mentary Information Section S1).

4. Conclusions

Homo-acetogens make effective positive syntrophic interactionin MXC anodes for efficient consumption of H2 to produce electri-cal current, leading to current densities of �10 A/m2, comparableto acetate-fed biofilms. We established the presence of homo-acet-ogens in the H2-fed MEC by detecting acetate, formate, and FTHFSgenes in the anode chamber. Continuous-flow experiments dem-onstrated that ARB- homo-acetogen partnership in the biofilm an-ode is robust, even at short HRTs that limit suspended homo-acetogens. This partnership can work in tandem with approachesto minimize the negative syntrophy between methanogens andARB in MXCs operating at low HRTs (Lee et al., 2009).

Acknowledgement

The research was supported by the Biohydrogen Initiative atArizona State University and OpenCEL, LLC.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.biortech.2010.03.133.

P. Parameswaran et al. / Bioresource Technology 102 (2011) 263–271 271

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