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Sensors and Actuators A 195 (2013) 206– 212

Contents lists available at SciVerse ScienceDirect

Sensors and Actuators A: Physical

j ourna l ho me p age: www.elsev ier .com/ locate /sna

ptimal biofilm formation and power generation in a micro-sized microbialuel cell (MFC)

eokheun Choi ∗, Junseok Chaechool of Electrical, Computer, and Energy Engineering, Arizona State University, USA

r t i c l e i n f o

rticle history:eceived 2 April 2012eceived in revised form 11 July 2012ccepted 17 July 2012vailable online 9 August 2012

a b s t r a c t

Microbial fuel cells (MFCs) represent an emerging technology for generating electricity from renewablebiomass. Micro-sized MFCs show promising applications in certain niche applications. However, exist-ing micro-sized MFCs are generally limited by their low power density, rendering them insufficient forpractical applications. Here, we report a micro-sized MFC having optimal biofilm formation and minimal

eywords:icrobial fuel cellsEMS

eobacter sulfurreducensicro-sized

oxygen invasion into its anode chamber to generate high power density. The biofilm formed by exo-electrogen, Geobacter sulfurreducens, was studied by using four different thicknesses of photo-definablepolydimethylsiloxane (PDMS) spacer; 10, 20, 55, and 155 �m. Both current and power densities weresignificantly limited when the PDMS spacer was less than 55 �m thick. The maximum power densityof our MFC was 95 �W/cm2, the highest value among previously reported micro-sized MFCs and evencomparable to that of macro-scale counterparts.

© 2012 Elsevier B.V. All rights reserved.

. Introduction

Microbial fuel cells (MFCs) may become an alternative “green”nergy technology of the future as they generate sustainable elec-ricity from biodegradable organic compounds through microbial

etabolism. Miniaturizing MFCs is an interesting approach forotentially powering small portable electronics [2–6] with suc-essful validation of conceptual macro-sized MFCs as a low-costenewable energy technology [1]. However, existing micro-sizedFCs are generally limited by their extremely low power density,

endering them insufficient for practical applications [6,7]. Theirower density, ranging from 0.0023 to 0.4 �W/cm2, is up to fiverders of magnitude lower than that of macro-sized MFCs. We pre-iously reported that micro-sized MFCs suffer from O2 diffusionnto the anode chamber, which could compete with extracellularlectron transfer (EET) to the anode and, therefore, reduce powereneration [6,8,9]. Microorganism in the anode chamber mightonsume O2 immediately and mitigate abiotic O2 reduction onhe anode; this O2-scavenging reaction is independent of size of

FCs. The reaction occurs in macro-sized MFCs, yet these MFCs

ave high concentration of microorganism in the anode cham-er. That is why macro-sized MFCs may not be very sensitiveo the presence of O2 intrusion, while oxygen exposure to the

∗ Corresponding author.E-mail addresses: shchoi2@asu.edu, cshlkm@gmail.com (S. Choi).

924-4247/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.sna.2012.07.015

anode chamber in micro-sized MFCs can be very critical since themicro-sized MFCs have a very small population of O2-utilizingmicroorganism. We reported 4.7 �W/cm2 from a 4.5 �L-MFC byeffectively minimizing oxygen intrusion by adding oxygen scav-enger, l-cysteine [6]. The power density was relatively high yetstill an order of magnitude lower than that of macro-sized coun-terparts. This is primarily because the adherent cells forming abiofilm on the anode were not fully developed. Macro-sized MFCstypically have 50 �m-thick bacterial biofilm [10]. However, suchthicknesses for biofilms could not be developed in the micro-sized MFC since the anode chamber was only 20 �m tall, asdefined by the PDMS spacer (Fig. 1). Although the micro-sizedMFCs showed good reproducibility of the current generation [6],the 20 �m-thick space constraint might limit the accumulationof the bacteria, decreasing their power generation. Therefore, itis crucial to obtain a fully grown optimal biofilm to maximizethe power density of a micro-sized MFC. To study the optimalbiofilm formation in micro-sized MFCs, we designed four micro-sized MFCs having different PDMS spacer thicknesses (10, 20, 55,and 155 �m) and observed for their power density performance.To demonstrate the feasibility of increasing the MFC performancebased on our findings, we minimized oxygen intrusion and spaceconstraints by using 254 �m-thick PTEE (polytetrafluoroethylene)

spacers and PEEK (Polyetheretherketone) fluidic tubings. The thickPTEE spacer served to remove the space constraints in the anodecompartment. In Section 2, experimental methods and device fabri-cation are discussed. All measurements results and discussions are

S. Choi, J. Chae / Sensors and Actuators A 195 (2013) 206– 212 207

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ig. 1. Cross-sectional view of a micro-scale microbial fuel cell (MFC); space consnode and cathode chambers were formed between the glass chips and CEM (catiathode chambers to limit the bacterial biofilm formation.

resented in Section 3. Finally, concluding remarks follow in Sec-ion 4.

. Materials and methods

.1. Device fabrication for studying optimal biofilm formation

A schematic of the micro-sized MFC is shown in Fig. 2a. The MFCad a proton exchange membrane (PEM, nafion 117) sandwichedetween two glass chips pre-fabricated with gold electrodes. Fourcrews clamped the sandwitch to tightly hold them securely.he glass chips (VWR, 75 mm × 25 mm × 1 mm) were cut into5 mm × 25 mm and mechanically drilled six holes on each chip:ne inlet, one outlet, and four screws. The glass chips were thenoated with Cr/Au (20 nm/200 nm) by an electron-beam evapo-ator. The photo-definable PDMS (Dow corning WL-5351) waspin-coated on gold films to define an anode chamber (Fig. 2b). Thehickness of the PDMS layer was carefully controlled by the spinpeed (500, 1000, 2500, and 3000 rpm for 30 s) to define 155, 55, 20nd 10 �m anode chamber depth, respectively. The PDMS layer wasre-baked at 110 ◦C for 120 s prior to UV exposure (3000 mJ/cm2)

hrough a negative photo-mask. Following exposrue, the substrateas baked at 150 ◦C for 180 s and agitated in a developer (WL-9653,ow couring) for 30 sec. Finally, the PDMS-coated glass chips wereured at 180 ◦C for 1 h. and the final exposed anode electrode was

ig. 2. (a) Schematic of the MFC assembly to study the optimal biofilm formation in minode compartment has a spin-coated PDMS layer on top of sputtered Cr/Au (20/200 nmhe spin speed (500, 1000, 2500, and 3000 rpm for 30 s) to define 155, 55, 20 and 10 �m

54 �m-thick PTEE spacer. (c) Experiment setup for monitoring of the MFC power genera

(20 �m-tall anode chamber) by a photolithographically defined PDMS layer. Thechange membrane). The PDMS layer is a spacer to define the height of anode and

10 mm × 10 mm. Four MFCs were prepared having different PDMSthicknesses; 155 �m, 55 �m, 20 �m, and 10 �m, defining 15.5 �L,5.5 �L, 2 �L, and 1 �L of anode chamber volume, respectively. The254-�m-thick PTEE (polytetrafluoroethylene) spacer was used todefine a cathode chamber (Fig. 2c). The inlet and outlet of themicrofluidic channel were formed on the backside of the chipsvia nanoports (10-32 Coned assembly, IDEX Health & Science) andconnected with fluidic tubing (Fig. 2d) (outer diameter: 0.0625 in.,inter diameter: 0.002 in., 1548, IDEX Health & Science). We usedNafion 117 as a PEM to permit only cation transport for maintain-ing electroneutrality between anode and cathode chambers. Nafionmembrane was pretreated by sequentially boiling in H2O2 andwater, followed by soaking in 0.5 M sulfuric acid solution and thenwater, each for 1 h. The membrane was cut into 45 mm × 25 mmand the exposed surface area of PEM was 10 mm × 10 mm. Beforewe assembled the MFC, the anode/cathode chips were first steril-ized with 70% ethanol then blown dry with nitrogen. All the layerswere manually stacked in sequence while carefully aligning thetubing holes for the microfluidic channels.

2.2. Inoculum

We obtained the inoculum from an acetate-fed Microbial Elec-trolysis Cell (MEC) mother reactor that had Geobacter-enrichedmixed bacterial culture from anaerobic digester sludge. The anode

cro-sized MFCs, and photograph of (b) anode and (c) cathode compartments. The) on the glass substrate. The thickness of the PDMS layer is carefully controlled byanode chamber depth, respectively. The cathode chamber (25.4 �L) is defined by ation.

2 Actuators A 195 (2013) 206– 212

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hamber of our MFC was filled with 20 mM acetate in min-ral medium as the sole electron donor using a syringe pumpHarvard Apparatus, Inc.) for continuous-mode operation. Theomposition of the mineral medium was (per litter of deionizedater): Na2HPO4 (6.02 g), KH2PO4 (1.024 g), NH4Cl (0.41 g), min-

ral media (10 mL), 1 mL of 4 g/L Fe(II)Cl2 stock solution, 0.5 mLf 37.2 g/L Na2S·9H2O stock solution. The mineral media had theollowing composition in 1 L: EDTA (0.5 g), CoCl2·6H2O (0.082 g),aCl2·2H2O (0.114 g), H3BO3 (0.01 g), Na2MoO4·2H2O (0.02 g),a2SeO3 (0.001 g), Na2WO4·2H2O (0.01 g), NiCl2·6H2O (0.02 g),gCl2 (1.16 g), MnCl2·4H2O (0.59 g), ZnCl2 (0.05 g), CuSO4·5H2O

0.01 g), and AlK(SO4)2 (0.01 g). The catholyte was 50 mM ferri-yanide in a 100-mM phosphate buffer in which pH was adjustedt 7.5 ± 0.2 with 0.1 M NaOH.

.3. MFC operation

Acetate is used as an organic substrate. Protons, electrons,nd CO2 are generated via the metabolic products of the bacte-ia catabolism, as shown in Eq. (1). Electrons are transferred to thenode via the conductive matrix that the bacteria form and flow tohe cathode through the external electrical circuit.

H3COO− + 2H2O → 2CO2 + 8e− + 7H+ (1)

Protons released by bacteria catabolism travel through the PEMoward the cathode. At the cathode, ferricyanide, [Fe(CN)6]3−, cap-ures the electrons (Eq. (2)) and the cycle is completed.

Fe(CN)6]3− + e− → [Fe(CN)6]4− (2)

.4. Experiment setup

We operated the four MFCs with different anode chamberepths at 30 ◦C. Anolyte and catholyte were continuously suppliedsing a syringe pump at the rate of 1 �L/min. We measured theotential between the anode and cathode by a DAQ/68 data acqui-ition system (National Instrument) and recorded voltage every

min via customized LabVIEW (National Instrument) interface. Anxternal resistor (150 �) was connected between electrodes of theFCs to close the circuit. We calculated the current through the

esistor via Ohm’s law, I = V/R, and the output power via Joule’s law, = V × I.

.5. Bacterial fixation and imaging

The MFCs were disassembled, rinsed, and adherent bacteria onhe gold anode were immediately fixed in 2% glutaraldehyde solu-ion overnight at 4 ◦C. Samples were then dehydrated by serial,0 min transfers through 50, 70, 90, and 100% ethanol. Fixed sam-les were examined using a FESEM (Hitachi S-4700-II).

. Results and discussion

.1. Optimal biofilm formation

.1.1. Current generationFig. 3 shows current profiles of the four MFCs with different

node chamber depths after Geobacter-enriched mixed bacteria arenoculated. A lag period exists, followed by a rapid increase in cur-ent, which is associated with the time needed to establish initialacteria-anode electron transfer [4]. It has been suggested that bothuspended and attached cells can contribute to electron transfer,

et adherent cells on the anode is considered as the main contribu-or to current generation in most cases [11]. In particular, in the casef using Geobacter sp. as a biocatalyst, optimizing biofilm formations a critical element for high performance MFC because the main

Fig. 3. Currents produced from the four MFCs having 10, 20, 50, and 155 �m PDMSspacers with 150-� load. The potentials between the anode and cathode are mea-sured and the currents are calculated through the resistor via Ohm’s law.

electron transfers mechanism of Geobacter sp. is through a conduc-tive biofilm matrix [12]. Fig. 3 shows that the current generationincreases and the lag-time decreases as the anode chamber depthincreases. The MFC with 155 �m-thick anode chamber started togenerate current 6 h after the inoculation and rapidly increasedup to 26 �A in 40 h whereas the 10 �m-thick MFC needed 28 h toinitiate current generation and reached only one fifth of the cur-rent from 155 �m-thick counterpart at 40 h. This result stronglysuggests that the chamber depth, defined by PDMS spacers, limitsthe biofilm formation, controlling the lag time and current genera-tion. However, the current enhancement is rather saturated whenthe chamber depth becomes larger than 55 �m. This suggests thatthe optimal biofilm thickness may reach when the chamber depthis more than 55 �m which is in a good agreement with previ-ous literature [10]. Also, these current productions can be furtherexplained with electron microscopic examination of gold anodesremoved from the MFCs after the operation in 40 h (Fig. 4). Thebiofilm with 10 �m-thick MFC had sparsely distributed clustersin a single-layer structure. However, the 20 �m-thick MFC startedto form the bacterial colony in a multi-layer structure at severalplaces and finally 55 and 155 �m-thick MFCs contained denselypacked multi-layer biofilms. Picioreanu et al. reported a compu-tational model for the biofilm formation in MFCs, describing thatbacterial colony is initiated more intensely at some spots, formed ina multi layer, and then covered over the entire surface rather thanthe colony forms in a layer-by-layer step [13]. The SEM image of10 �m-thick MFC (Fig. 4d) supports their findings; the cells neithercover the entire surface area nor form multi layers, suggesting thatthe cell growth/attachment was limited by the space constraint.Multi-layer formation was observed for the spacer of 20, 55, and155 �m (Fig. 4).

3.1.2. Polarization curve and power outputFig. 5 shows polarization curves (Fig. 5a) and power outputs

(Fig. 5b) of the four MFCs with different anode chamber depths,which were derived and calculated based on the maximum cur-rent value at a given external resistance (910k, 482k, 270k, 150k,66k, 33k, 15k, 10k, 7k, 2.6k, 1k, 428, 333, and 150 �). Using thepolarization curve in Fig. 5a, we estimated their internal resis-tances (Rini) from the points where the polarization curve exhibiteda near-linear drop due to ohmic losses. Linear fitting of the curve

yielded Rini of ∼38 k� (155 �m), 45 k� (55 �m), 112 k� (20 �m)and 202 k� (10 �m), respectively. As the anode chamber depthdecreases, the total internal resistance increases. Given that (1) thetotal internal resistance consists of anodic, membrane, cathodic,

S. Choi, J. Chae / Sensors and Actuators A 195 (2013) 206– 212 209

Fig. 4. SEM images of the anode surface populated by Geobacter sp.; the MFCs having (a) 155 �m, (b) 55 �m, (c) 20 �m, and (d) 10 �m-thick PDMS spacers. Scale bar is 20 �m.Inset: individual bacterial cells of the (d) image (scale bar is 1 �m).

Fig. 5. (a) Polarization curve and (b) power output of the MEMS MFCs with four different thicknesses of the PDMS spacers, measured as a function of current. The values arederived and calculated based on the maximum current value at a given external resistance (910k, 482k, 270k, 150k, 66k, 33k, 15k, 10k, 7k, 2.6k, 1k, 428, 333, and 150 �).

Table 1Comparison of four MFCs with different thickness of spacers.

Condition PDMSthickness (�m)

Chambervolume (�L)

Surface-area-to-volumeratio (cm−1)

Lag-time (h) Internalresistance (k�)

Maximumcurrent density(�A/cm2)

Maximumpower density(�W/cm2)

500 rpm for 30 s 155 15.5 64.5 6 38 26 4.41000 rpm for 30 s 55 5.5 181.8 8 45 23 3.32500 rpm for 30 s 20 2 500 12 112 8 1.55000 rpm for 30 s 10 1 1000 28 202 5 1

210 S. Choi, J. Chae / Sensors and Actuators A 195 (2013) 206– 212

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nd electrolyte resistance [12] and (2) the anodic resistance is theain bottleneck in the micro-sized MFCs [6], these results suggest

hat the internal resistance increases probably due to high elec-rode energy losses from the small population of bacteria attachedo the surface. The 155 �m-thick MFC had the highest power den-ity among four MFCs, 4.4 �W/cm2, which is four times more thanhat of 10 �m-thick MFC. Table 1 summarizes performance of theour MFCs.

.2. High power micro-sized MFC

To demonstrate the feasibility of optimal biofilm formation onower density of MFC, we performed preliminary tests to construct

microsized MFC using 254 �m-thick PTEE (polytetrafluoroethy-ene) spacer. This spacer has five orders of oxygen permeabilityower than that of PDMS, which is expected to help the power den-ity of MFC substantianlly. In addition to the spacer we used PEEKpolyetheretherketone) fluidic tubings that show fifty times lowerxygen permeability than that of fluorinated ethylene propyleneubing that was used in our previous work [6]. The MFC was builty sandwiching five functional layers: an anode (Cr/Au), an anodehamber layer (PTEE), a PEM, a cathode chamber layer (PTEE) and

cathode (Cr/Au). All the dimensions and materials are unchangedrom the previous MFC for the experiment of optimal biofilm for-

ation.

.2.1. Current and power generationThe anolyte including bacteria and the catholyte were injected

ontinuously at a rate of 1 �L/min. As shown in Fig. 6a, currenteneration began 25 h after the injection. The population of bac-eria attached to the gold anode was small at the initial stages ofiofilm formation. The current continued to increase over time andtabilized to a value of 170 �A (170 �A/cm2) around 100 h afternitial injection. The population of bacteria was significantly aug-

ented, forming a densely packed film on the anode. Despite theact that biofilm formation was maximized, the lag time (35 h)ncreased compared to the previous micro-sized MFCs (6 h for55 �m-thick MFC), not consistent with the previous results (the

ag-time decreases as the anode chamber depth increases, as shownn Table 1). Normally, the short chamber height of the micro-sized

FCs should increase the probability of cell attachment and biofilm

ormation on the anode, resulting in short lag-time [4]. How-ver, as the chamber depth significantly decreases below 55 �m,he lag-time rather starts to increase. In this case, although the54 �m-thick MFC device has maximized the biofilm formation, it

operation for 40 h and 100 h, respectively. Scale bar is 5 �m. (b) Polarization curveent. (For interpretation of the references to color in this figure legend, the reader is

has rather increased the lag-time. One concern with the increasedchamber depth to maximize the biofilm formation is that its protontraveling distance from anode to cathode might be lengthened anddecrease the power density to some extent. The further researchneeds to be done to find out an optimal chamber thickness. How-ever, our experimental results show that the biofilm formation is amore dominant factor in determining the power density.

Polarization and power curves were derived from the volt-age recorded using a series of external resistors (Fig. 6b). Themaximum power was measured to be 95 �W at a current of170 �A and voltage of 0.56 V (Rext = 3 k�). This demonstrates themaximum power density of 95 �W/cm2, which is the highestvalue among previously reported micro-sized MFCs. The outputvoltage of a MFC is less than the predicted thermodynamic voltagedue to unavoidable losses. The three major losses are: activation,ohmic, and mass transport losses [15]. These losses are definedby the voltage increase required to compensate for the currentloss due to electrochemical reactions, charge transport, and masstransfer processes that take place in both anode and cathode com-partments. The polarization curve in Fig. 6b can be divided intothree zones: (i) activation loss region (0–20 �A) – starting fromthe OCV at zero current, there is an initial steep decrease of thevoltage, (ii) ohmic loss region (20–160 �A) – the voltage then fallsmore slowly and the voltage drop is linear with current, and (iii)mass transport region (>160 �A) – there is a rapid decline of thevoltage at higher currents. As shown in the polarization curve, theperformance of our MFC is limited by mass transport loss, whichprevents generation of higher current and power density [16].Furnishing a supply of sufficient substrate to the anodic biofilmat rates at least equivalent to the current generated is crucial. Inaddition, the accumulation products in the biofilm needs to beprevented because it might alter the redox conditions and hamperthe metabolic activity of the biofilm. In particular, a poor transferof protons can trigger a pH gradient to form between the anodeand cathode that severely degrades bioanode performance [17].Limited mass transfer results in concentration or mass transferlosses, causing the occurrence of an anode saturation potential.However, in our case, mass transport limitations may be governedby insufficient oxidant transport in the cathode compartment. Weused 50 mM ferricyanide, corresponding to 50 e− meq/L, which isless than a third of the number of electron equivalents available

at the anode (160 e− meq/L). This deficiency of electrons may beone factor attributing to mass transport losses limiting the overallpower density. However, since the gold electrode is vulnerable todecay through direct contact with the ferricyanide solution, a high

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concentration of ferricyanide certainly impairs the performance ofthe MFC over long-term use. Therefore, in order to more securelyincrease power and current density, the use of other types ofcathodes, carbon-based, air-, or bio- cathodes, may be considered.

Using the polarization curve in Fig. 6b, we obtained internalresistance of approximately 1 k� which is the smallest value amongall prior micro-sized MFCs [6]. We believe that the high powerdensity from this micro-sized MFC is mainly due to this low inter-nal resistance. However, the internal resistance of the micro-sizedMFC is still higher than that of macro-sized MFCs (a few �) [14].This large discrepancy in internal resistance might be due to thepoor interactions between bacteria and the gold electrode [6,8].Most micro-sized MFCs used gold as an electrode material as goldis one of the most popular materials used in microfabricationprocesses. If one can find alternative electrode materials meet-ing microfabrication process requirements and providing bettersurface characteristics for bacterial biofilm formation, the powerdensity of micro-sized MFC is expected to improve substantially.

4. Conclusion

Table 2 summarizes specifications of prior micro-sized MFCsand compares our MFC with them. Despite diverse experimentalconditions, the following conclusions can be drawn from Table 2.Our MFC has achieved highest current/power density among anypreviously reported micro-sized MFCs. We attribute the high cur-rent/power density to minimum oxygen intrusion and to optimalbiofilm formation. This work resulted in a leap forward for micro-sized MFCs through achievement of higher power density andcontributed to having more in-depth understandings of the inter-play between micro-sized device architecture and active microbes.There remains abundant room for improvement in their perfor-mance and the preliminary figure (95 �W/cm2 power density) forthe micro-sized MFCs may be considerably amplified. Once highpower micro-sized MFCs become available, they could be a powersource for implantable biomedical devices by directly consumingglucose in the bloodstream [3] and other organic substrates in thelarge intestines [19], fuel sources that are naturally available inthe body and replenished from food. Furthermore, the micro-sizedMFCs could be suitable for long-term powering of small wire-less telemetry systems as well as wireless sensors used at remotesites where frequent battery replacement is not practical [20]. Itis also expected that research of micro-sized MFCs enables crucialunderstanding of EET processes in a smaller population of microor-ganisms with excellent control in the microenvironment [21,22],thus serving as a versatile platform for fundamental MFC studies.

Acknowledgement

The authors thank Dr. Parameswaran for providingGeobacteraceae-enriched culture.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.sna.2012.07.015.

References

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Biographies

Seokheun Choi received the BS & MS degrees in Electrical engineering fromSungkyunkwan University, Korea, in 2003 and 2004, respectively. After graduat-ing, he was a research engineer at LG Chem, Ltd. from 2004 to 2006. He earnedhis PhD in Electrical Engineering at Arizona State University in 2011. From 2011 to2012, he was a research professor in the School of Electric & Computing Systemsat the University of Cincinnati. He is currently an assistant professor of electricalengineering at State University of New York, Binghamton. He has published over 40journal and conference articles, two book chapters, and one book and he holds oneU.S. patent. His areas of interest are BioMEMS/Biosensors/Biofuel Cells.

Junseok Chae received the BS degree in metallurgical engineering from KoreaUniversity, Seoul, Korea, in 1998, and the MS and PhD degrees in electrical engi-neering and computer science from the University of Michigan, Ann Arbor, in 2000and 2003, respectively. He joined Arizona State University, Tempe, in 2005 as anAssistant Professor, and he is currently an Associate Professor of electrical engi-neering. He has published more than 95 journal and conference articles, two bookchapters, and one book, and he holds three U.S. patents. His areas of interest are

MEMS sensors/actuators, integrating MEMS with readout/control electronics, andmicropackaging. Dr. Chae was the recipient of the First Place Prize and the Best PaperAward in the Design Automation Conference (DAC) Student Design Contest in 2001.He was the recipient of a National Science Foundation CAREER Award for a MEMSprotein sensor array.