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Biosensors and Bioelectronics 26 (2011) 4169–4176

Contents lists available at ScienceDirect

Biosensors and Bioelectronics

journa l homepage: www.e lsev ier .com/ locate /b ios

ffect of conductive polymers coated anode on the performance of microbial fuelells (MFCs) and its biodiversity analysis

hao Li, Libin Zhang, Lili Ding, Hongqiang Ren ∗, Hao Cuitate Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210093, Jiangsu, PR China

r t i c l e i n f o

rticle history:eceived 28 January 2011eceived in revised form 7 April 2011ccepted 11 April 2011vailable online 16 April 2011

eywords:FC

onductive polymer

a b s t r a c t

Conductive polymer, one of the most attractive electrode materials, has been applied to coat anode of MFCto improve its performance recently. In this paper, two conductive polymer materials, polyaniline (PANI)and poly(aniline-co-o-aminophenol) (PAOA) were used to modify carbon felt anode and physical andchemical properties of the modified anodes were studied. The power output and biodiversity of modifiedanodes, along with unmodified carbon anode were compared in two-chamber MFCs. Results showedthat the maximum power density of PANI and PAOA MFC could reach 27.4 mW/m2 and 23.8 mW/m2,comparing with unmodified MFC, increased by 35% and 18% separately. Low temperature caused greatlydecrease of the maximum voltage by 70% and reduced the sorts of bacteria on anodes in the three MFCs.

node biofilmicrobial diversity

Anode biofilm analysis showed different bacteria enrichment: a larger mount of bacteria and higherbiodiversity were found on the two modified anodes than on the unmodified one. For PANI anode, thetwo predominant bacteria were phylogenetically closely related to Hippea maritima and an unculturedclone MEC Bicarb Ac-008; for PAOA, Clostridiales showed more enrichment. Compare PAOA with PANI,the former introduced phenolic hydroxyl group by copolymerization o-aminophenol with aniline, which

al com

led to a different microbi

. Introduction

In anode chamber of microbial fuel cells (MFCs), electrochem-cally active microorganisms adhere to anode, generate electronsy substrate oxidation, and then transfer the electrons to anode.t is believed that the process of electron transfer from bacteria tonode is the limiting step of power output of MFC. Several electronsransfer mechanisms are proposed as: rely on bacteria membranective proteins (Bond and Lovley, 2003), by electron conductiveanowires (Gorby et al., 2006), or via metabolites mediator andrtificial mediator (Park and Zeikus, 2000).

Anode for MFC, as the bacteria carrier, can influence not onlyhe bacteria adhesion but also the electrons transfer process fromhe microorganisms to the anode. So an excellent anode, shouldrompt the electrons transfer process described above and suit-ble for microbial growth. Various strategies have been developedo improve MFC anode, such as metal and nonmetal materials mod-fied (Rosenbaum et al., 2007; Cheng and Logan, 2007).

Conductive polymer materials, which have both strong conduc-

ivity and the properties of polymers, are widely used in electronicsnd biosensors (Chen et al., 2005). Recently, they have earned morettention to improve MFC performance by modifying its anode.

∗ Corresponding author. Tel.: +86 25 89680512; fax: +86 25 89680569.E-mail address: hqren@nju.edu.cn (H. Ren).

956-5663/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.bios.2011.04.018

munity and the mechanism of group effect was proposed.© 2011 Elsevier B.V. All rights reserved.

Among all the conductive polymers, which had been studied,polyaniline (PANI), polypyrrole (PPy) and the composite materialsbased on them can effectively improve MFC performance.

Qiao et al. (2007) employed carbon nanotube/PANI compos-ite as anode material and obtained maximum power density42 mW/m2, with Escherichia coli as the microbial catalyst. Scottet al. (2007) investigated a range of materials base on PANImodified carbon anodes, which all achieved better performancethan the unmodified. Feng et al. (2010) used a copolymers,polypyrrole/anthraquinone-2,6-disulphonic (PPy/AQDS)-modifiedanode to gain a maximum power density 13 times larger thanunmodified one.

Polyaniline is the most widely used conductive polymers forits environmentally stability and low cost. It can also be furtherimproved by introducing groups to its original backbone by copoly-merization of aniline with its derivatives, such as copolymerizationof aniline with aminophenol (Mu, 2004). The further decoratedPANI, can intensively differ from the primary properties of PANI(Lee et al., 2009) and may bring new influent to MFC anodes.

The anode biofilm play the key role in electron transfer to anodeand the potential of anodic half-cell (Cheng et al., 2008). However,conductive polymers, due to the special characteristics, are likely

to affect the bacteria attachment on anode and the process of elec-trons transfer from bacteria to anode, which can change the biofilmand further affect the MFC power generation. Even the power den-sity can also affect microbial diversity of anode biofilm, in return.

4170 C. Li et al. / Biosensors and Bioelect

Table 1Experiment of three repeated runs.

Run 1 Run 2 Run 3

Purpose Pre-experiment Pre-experiment ExperimentTemperature 6–10 ◦C 33–35 ◦C 33–35 ◦COperation date December 6,

2009January 7, 2010

January 11, 2010February 20,2010

March 2, 2010May 3, 2010

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pPice

2

2

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pHaa

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1× TAE buffer at 90 V for 14.5 h at 60 ◦C. Gels were photographedusing Kodak 1D Image Analysis Software after stained by ethidiumbromide.

For further identification of DGGE bands in each sample, sev-

Operation time 32 days 39 days 62 daysAnodes Each anode was made based on new carbon felt at each run

aturi et al. (2011) reported the anode biodiversity fell down ashe current density increased by applying different external resis-ance, which is similar result as Rismani-Yazdi et al. (2010). So, theelationship between conductive polymers and the anode biofilms very complex and important.

Most studies which applied conductive polymer in MFC, usedole kinds of bacteria (pure culture) for inoculation such as: E. coliZou et al., 2008) or Shewanella decolorationis (Feng et al., 2010) andeported the increasing number of bacteria on conductive polymersodified anodes. However, only investigating the quantity change

f bacteria in these researches is not enough to fully reflect theunction of conductive polymers in MFC anode. Therefore, in thistudy, we use mixed culture for inoculation, which was not onlydvantage in practical wastewater treatment but can also reflectacterial diversity change of conductive polymer modified anode.

To study electrochemical and microorganism effect that con-uctive polymers bring to MFC, we use PANI, and poly(aniline-co--aminophenol) (PAOA), with phenolic hydroxyl further decoratedANI, these two materials to modify our MFC anodes.

The aims in this study are to (i) investigate the influence on MFCerformance by different conductive polymer materials based onANI, (ii) compare the bacterial diversity of modified and unmod-fied anode in order to study the biofilm biodiversity effect whichonductive polymers bring to anode and (iii) explore the groupffect on microbial community compare with parent PANI.

. Material and methods

.1. Anode material polymerzation

According to previous protocols (Zhang et al., 2007), “proton-oped” PANI and PAOA that contain aniline and aminophenol0:1 (molar ratio) anodes were prepared. The relevant solution

nvolved:Aniline monomer was distilled under reduced pressure before

olymerization. Solution I (SI): aniline (4 mL) was mixed in 1 mol/LCl 100 mL. Solution II (SII): aniline (4 mL) was mixed with O-minophenol (0.5 g) in 1 mol/L HCl 100 mL. Solution III (SIII):mmonium persulfate (APS, 5 g) was mixed in 1 mol/L HCl 100 mL.

The PANI anode was modified by submersing a carbon felt intohe mixture of SI and SIII, while the PAOA anode was submersednto the mixture of SII and SIII. After stirring constantly for 8 h ofolymerization, the PANI anode showed a green coat and a blueoat for PAOA. The two anodes were dried in ovens at 80 ◦C for 24 hnd then were rinsed successively with HCl (1 M), deionized water,cetone, deionized water before used in MFCs.

.2. Experimental device

The H-type MFC devices consisted of two cylinder transpar-nt polyacrylic plastic bottles (9 cm × �8 cm), which separated by

proton exchange membrane (PEM) (Nafion 117, Dupond, USA),hose inner diameter was 2 cm. Each chamber has a carbon felt

lectrode (7 cm × 2 cm × 0.2 cm, US Morgan). The electrode spaces 10 cm and connected with an external resistance of 1000 �.

ronics 26 (2011) 4169–4176

2.3. Experimental methods

Experiments were conducted in batch mode with three paralleltwo chambers MFCs containing three different material anodes:bare carbon felt, PANI modified carbon felt and PAOA modifiedcarbon felt.

The two chambers, both filled with electrode medium (pH 7.0),contained:NH4C1 0.31 g/L, KC1 0.13 g/L, NaH2PO4·H2O 4.97 g/L,Na2 HPO4·H2O 2.75 g/L (Liu and Logan, 2004), and 1 ml/L oftrace elements solution (Logan et al., 2005). Sodium acetatewas controlled at COD of 1000 mg/L in the anodic solution. Theanaerobic digester sludge from a pharmaceutical was inocu-lated in the anode compartment (15%, v/v) as biological catalyst,whereas the cathode compartment was continuous aerated withair.

The whole experiment consists of three stages (Table 1): firstrun in low temperature (6–10 ◦C) and another two runs in middletemperature (33–35 ◦C). For each run, three anodes were made bynew carbon felt, and the other operation condition of the three runswas same as above.

2.4. Calculations and analyses

The power destiny versus anode surface area is calculated aspreviously described (Oh et al., 2004). Polarization curves for theMFCs were generated by changing the external resistance in therange of 100–10,000 �. Soluble COD was estimated according tothe Standard Methods (APHA, 1998).

2.5. Characterization of modified anode

The morphology of three anodes, before used in MFCs wassubjected to scanning electron microscopy (SEM) by cutting off0.5 cm × 0.5 cm × 0.2 cm pieces. Simultaneously, infrared spectrawere obtained by Nexus 870 FT-IR with an attenuated total reflec-tion (ATR) method.

After operation of 62 d, the MFCs were dismantled. A squarepiece (0.5 cm × 0.5 cm × 0.2 cm) of each anode carbon felt, withbiofilm adhesion, was cut and subjected to SEM after freeze dryingat 4 ◦C

2.6. Bacterial community analysis from anode electrode

Biofilm was scratched from the anodes and the total genomicDNA of each sample was extracted as the protocol developed byZhou et al. (1996).

PCR was performed with the primers 518r and 338f (with GC-clamp) under the following conditions: 94 ◦C/5 min denaturationstep; 30 cycles of 94 ◦C/30 s, 58 ◦C/30 s, 72 ◦C/45 s; and a final exten-sion step at 72 ◦C/10 min.

DGGE was carried out in a denaturing gradient gel electrophore-sis system for the PCR products. Polyacrylamide gels (10%, w/v)were 18 cm × 18 cm, thickness of 0.75 mm and made with dena-turing gradients of 35–65% gel. The electrophoresis was conduct in

eral representative DGGE bands were cut off from the DGGEgel and amplified with the primes 518r and 338f (withoutGC-clamp), and then were sent for sequencing by Genscript,Nanjing.

C. Li et al. / Biosensors and Bioelectronics 26 (2011) 4169–4176 4171

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Fig. 1. The infrared spectra of three anodes by attenuated total

. Results

.1. Anode modification

The surface of PANI modified anode was green, which sug-ested that the protonic acid doping “Green imine” was obtained.t means that the PANI obtained, is the most important form of con-uctive of all the states of PANI, which possesses stable structurend highest conductivity (Chiang and MacDiarmid, 1986). How-ver, the color of PAOA is blue, as some other reports (Huang et al.,001).

The original infrared spectra obtained by attenuated total reflec-ion were shown in Fig. 1a. For better comparison, we removed theackground of unmodified curve, and obtained Fig. 1b.

For the curve of PANI modified (Fig. 1a): the peaks at137 cm−1 and 1578 cm−1 are assigned to the stretching vibra-ion of the quinoid ring. The feature which appears at 1499 cm−1

nd 1304 cm−1 is the evidence of presence of benzenoid ring.he obvious peak at 1235 cm−1 can be explained by the C–Ntretching vibration, which indicated the PANI is at proton-dopedtate.

For the curve of PAOA modified (Fig. 1b): it is same as PANIxcept for the peak at 3555 cm−1 which indicated –OH group suc-

essfully introduced to PANI.

From Fig. 1, we can conclude that the PANI and PAOA wereuccessfully polymerized on the two anodes and earned the goodonductivity by proton-doped.

tion (ATR). (a) Original image; (b) background removal image.

3.2. Performance of MFCs

3.2.1. Operation in low temperature (run 1)Three MFCs first run in room temperature in winter (6–10 ◦C).

There was no increasing in voltage after the 28th day, whichindicated the three MFCs had completed the start-up period.The maximum voltage (extra resistance 1000 �) of threeMFCs was 61.9 mV, 89 mV and 75 mV for unmodified, PANIand PAOA anode separately. The comparison of the three is:PANI > PAOA > unmodified.

3.2.2. Operation in middle temperature (run 2 and run 3)These two runs both had finished the start-up period at the day

35–38, and no significant increase after the day 14–16, as Fig. 2showed. For run 2, the maximum voltages (extra resistance 1000 �)of the three were: 214 mV, 267 mV, 231 mV for unmodified, PANIand PAOA anode separately. While for run 3, after six batches ofoperation, the voltage were: 222 mV, 276 mV, 246 mV, separately.The power output in middle temperature was much higher thanlow temperature above, and the comparison of the three MFCs is:PANI > PAOA > unmodified, which is same as low temperature.

Fig. 2 also showed that, during the start-up period, the modi-fied MFCs had a faster speed of increasing than the unmodified. For

example in run 3, after a week’s operation, the voltage was: 85 mV,160 mV, 143 mV for bare, PANI and PAOA modified MFC, respec-tively and reached the 38.3%, 58.0%, 58.1% of their each maximumvoltage.

4172 C. Li et al. / Biosensors and Bioelectronics 26 (2011) 4169–4176

an external resistance of 1000 � for the three runs.

3

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Fig. 2. Cell voltage as a function of time with

.2.3. Polarization curvesPolarization curves were obtained after six batches’ operation

n run 3. Fig. 3a and b showed that voltage versus current densitynd power density versus current density of the three MFCs.

It can be clearly seen that the conductive polymers material hadnfluence on the performance of MFCs. The lowest performancef power density 20.2 mW/m2 was observed in unmodified MFChereas the highest power density 27.3 mW/m2 appeared in PANIodified MFC (at a cell voltage of 276 mV and current density of

8.6 mA/m2).PAOA, as a new conductive polymer material, first used in

FC anode modified, had a relatively good performance for itsaximum power density 23.8 mW/m2, but lower than the PANIodified one.These two materials (PANI and PAOA modified) enhanced the

ower output of MFC by 35% and 18% separately comparing withnmodified MFC.

.3. SEM image of anodes

Before operation, from Fig. 4a–c, the unmodified carbon felt pos-essed smooth surface and had an average diameter of about 20 �m.he PANI modified (Fig. 4b) and PAOA modified (Fig. 4c) carbon feltnodes had irregular amorphous material coats so that they hadore rough surface than the unmodified one (Fig. 4a).Therefore, conductive polymers can change the morphology of

nodes surface and bring much larger area to anodes, so it wasxpected that the changed anode surface could affect the powerutput and influence the colonization of anode biofilm.

After 62 days’ operation of MFC, the morphology of each elec-rode was showed by SEM (Fig. 4d–f). The biofilm morphologyf the three had a little difference, and the two modified anodesFig. 4e and f) had increased number of bacteria compare withhe unmodified one (Fig. 4d). It may due to the rough surface ofhe conductive polymers coats and is more suitable for bacteriadhesion.

.4. Microbial community

To understand the influence of conductive polymers to bacterialiversity on anodes after a period of operating in this study, PCR-GGE was used to analyze bacterial compositions at each end of

he 3 runs.Fig. 3. Polarization curves of the three MFCs. (a) Voltage versus current density; (b)power density versus current density.

C. Li et al. / Biosensors and Bioelectronics 26 (2011) 4169–4176 4173

Fig. 4. SEM images of anode surface of three MFCs. (a) Unmodified anode (×2000); (b) PANI anode (×2000); (c) PAOA anode (×2000) and after operation of 62 days, themicroorganism on the three anodes; (d) unmodified anode (×4000); (e) PANI anode (×6000); (f) PAOA anode (×6000).

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.4.1. Comparison of the three runs from DGGEFrom Fig. 5, the two runs in middle temperature showed much

imilarity in DGGE fingerprint and had much more detectable bandshan the run in low temperature and run 3 was taken to the objectf biodiversity analysis.

.4.2. Analysis of microbial communityFig. 5 shows the significant shift in microbial community struc-

ures between the sludge “before inoculation” and the bacterian anodes “after inoculation” in middle temperature. At least 19etectable bands were observed in the sludge before inoculation,hile bacteria from the three anodes showed much fewer bands. It

uggested that the anode of MFC provide a selective environmentor bacteria and only electrochemically active bacteria or theacteria which benefit to electron transfer can be enrichment onnodes of MFCs.

There were significant differences of DGGE fingerprints (includ-ng both band number and band intense) on the three anodes,

hich were represented by the seven marked bands in middle tem-erature (Fig. 5). Obviously, the two modified anodes had moreetectable bands than the unmodified (15 for the two modified, 12or the unmodified), which mean the modified had enriched moreacterial species. B1, B2, B3, B4, B6, B7 were more intense compareith the unmodified, especially B4 earned the largest enrichment in

ANI lane. While for the unmodified, B1, B2, B4 even not appeared.owever, B5 is a more remarkable band in unmodified than the

wo modified.Though PAOA hold polyaniline-like structure and similar prop-

rties to the parent PANI, DGGE result showed that PANI andAOA modified anodes tended to have different fingerprints, for

1, B4 are brighter in PANI while B6, B7 were more intense inAOA.

These seven representive bands were sequenced, and blastedith NCBI (Table S1).

B1 only appear in PANI and PAOA lanes whose correspond-ing microorganism became more advantage species in conductivemodified MFCs. According to sequencing and blast, B1 was closelyrelated to Hippea maritima, and it is phylogenetic close to Desul-furella, which contains several electrochemically active bacteria(Ieropoulos et al., 2005). Both of them are included in family Desul-furellaceae which belong to ð-proteobacteria.

The two slight band B2, B3 were closely related to a kind ofuncultured bacteria and a prevotella sp. oral clone AO096 (Table S1).The enrichment of those two bacteria is similar on PANI and PAOAmodified anode.

B4, a remarkably intense band in the two modified anodes,especially in PANI lane, was closely related to an uncultured bac-terium clone MEC Bicarb Ac-008, which was reported previously(Call et al., 2009) in a microbial electrolysis cells (MECs). This indi-cated that the corresponding microorganism of B4 has potentialelectrochemically activity. So, the great increase in richness of B4is probably the reason of enhanced MFC performance.

B5 was representative of the bands which was more intenseon unmodified anode than modified anode, who is closelyrelated to Azotobacter chroococcum, in the family of Pseudomon-adaceae, belong to the �-proteobacteria. The fading of B5 fromunmodified to modified anodes indicated that it was likely thetraditional electricity generation bacteria. Furthermore, this strainof bacteria had the ability of nitrogen assimilate, since livedfor long period in anaerobic with nitrogen aeration in anodechamber.

The sequences B6 and B7 emerged more in the modified, espe-cially in PAOA lane, the corresponding bacteria of them were bothbelonged to Firmicutes, Clostridiales (Table S1), which contain many

kinds of traditional electrochemically active bacteria (Niessen et al.,2004). Most of them had much flagella on their surface, which mayassist themselves easily adsorbed and grown on the anode withrougher surface conductive polymers bring.

4174 C. Li et al. / Biosensors and Bioelectronics 26 (2011) 4169–4176

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Fig. 5. DGGE analysis of bacterial community

. Discussion

.1. Influence factors of MFC performance

Many factors influence MFC performance, such as temperature,H, different bacteria inoculation, substrates, MFC configuration,he deviation of MFC device, and electrode material (metal, non-

etal, carbon nanotubes or catalyst modified electrodes). Recently,any researches applied conductive polymers in MFC, such as

ANI/CNT (42 mW/m2, Qiao et al., 2007), PPy/CNT (228 mW/m2,ou et al., 2008) and PPy/AQDS (1303 mW/m2, Feng et al., 2010).reat difference in power density existed among these studies, notnly because they applied diversified material, but also for the rea-on of different culture inoculation, electron acceptor in cathodend other operated condition. Pure culture inoculation was used inll these studied above which can effectively increase anode per-ormance. However, pure culture is not suitable for waste waterreatment and cannot fully reflect the effect of conductive polymersring to anode biofilm. Beside, though single-chamber MFC canreatly improve MFC performance, but it fail to rapidly enrich elec-rochemically active bacteria on anode for much aerobic microbialxidation of substrate (Kim et al., 2007).

Therefore, two-chamber MFC and mixed culture (commonnaerobic sludge) were used in this experiment, which lead to aelative low power density. In fact, most studies which are underuch basic circumstances without any catalyst, the power output ist the similar level as ours (Rismani-Yazdi et al., 2010; Pant et al.,010).

.1.1. Effect of temperatureLow temperature had great effect on power output in MFCs,

hich reduced the value of the maximum voltage by 70% for all thehree different anodes. Temperature coefficient Q10 (Xiao, 2000)as used to analyze the low temperature effect among the three

nodes.

10 =(

R1

R2

)10/(T2−T1)

tures on different anodes for the three runs.

The maximum voltage of MFCs was the rate (R), Q10 was cal-culated when T1 = 8 ◦C, T2 = 34 ◦C. The value of Q10 was 0.61, 0.65,0.63 for unmodified, PANI and PAOA anode separately, i.e. low tem-perature had very similar effect on the maximum voltage of thethree MFCs. In this experiment, Q10 was lower than the value of1.6 obtained by Larrosa-Guerrero et al., 2010, which may causedby different inoculation and feed of MFC.

Further, under low temperature, there were fewer sorts of bac-teria on the all three kinds of anodes than those under middletemperature (Fig. 5).

4.1.2. Effect of anode materialConductive polymers were used to coat anodes in MFCs, which

prompted the speed of start-up and improved the performance ofMFC by 35% (PANI) and 18% (PAOA), compared with unmodifiedMFC.

This improvement may benefit from the more biocompatibilityand electrocatalytic activity of the anode conductive polymers coatbrings.

The comparison of the three was: PANI (27.4 mW/m2) > PAOA(23.8 mW/m2) > unmodified (20.2 mW/m2), which coincided withthe low temperature operation. The result means that the copoly-mer PAOA, with phenolic hydroxyl group introduction to PANI,reduced the performance of MFC compared with PANI.

Qiao et al. (2007) used pure culture (E. coli) as bacterial bio-catalyst and applied carbon nanotubes to further modify MFCanode besides PANI, which obtain a maximum power output of42 mW/m2. This result indicated that the conductive filler mate-rial (such as CNT) could further increase the performance based onPANI modified MFC. In order to study the independent influenceof conductive polymers, any other factors which can improve MFCanodes were not applied in our experiment.

4.1.3. Repeatability analysisThe repeatability of MFC operation is very important. Experi-

ments were conducted under middle temperature to analyze therepeatability, especially for power output and anode biodiversity.

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C. Li et al. / Biosensors and Bi

Fig. 2 showed that the operation and maximum voltage of thehree runs. The P value was 0.73 according to t-test, which showedhat the difference of MFC performance between run 2 and run 3ere not statistically significant. Little deviation can be observed

etween the two middle temperature runs in our experimentalonditions.

Fig. 5 showed that the bacterial diversity was very similarn run 2 and 3, even they based on different operation time39 days and 62 days). According to DGGE, only some quan-ity deviation for B4, B5, B6 existed, and this may be regardeds the further evolution of anode biofilm in later operationeriod.

.2. Conductive polymers versus bacteria

The anode biofilm is the key factor in MFC performance sincet directly affects the electron transfer from bacteria to anode andhe performance of anodic half-cell (Cheng et al., 2008). Conduc-ive polymers such as PANI benefits from its highly conjugatedouble-bonded, electron transfer via current carrier in the PANIackbone. As the MFC anode coat, they can directly improve thenode performance, or indirectly, affect the anode biofilm andhen improve anode performance. The relationship between con-uctive polymers and anode biofilm can embody in the threespects:

1) Adsorption: PANI (PAOA) coat provides a new selective environ-ment, with higher anode surface and more biocompatibility,for the microorganism adhesion. According to the result of thisexperiment, they have the function of helping the MFC anodesto adsorb and create a more robust and stable biofilm with notonly increasing number of bacteria (as Fig. 4 showed), but alsoa higher biodiversity in biofilm (Fig. 5), since the number ofdetectable DGGE bands increased from 12 for unmodified to 15for modified.

2) Electron transfer mediator: The conductive polymer materialthemselves can role as the artificial electrochemical media-tors such as neutral red, methylene blue (Park and Zeikus,2000; Schroder et al., 2003), which can change the electrontransfer process from bacteria to anode, and help the bacte-ria which have no (or little) ability of transferring electronto anode, to achieve this process. However, superior to thesesoluble electrochemical mediators (neutral red), conductivepolymers are firmly fixed on anode as a coat and can henceenrich this kind of bacteria on anode. Different from the tra-ditional electricity producing bacteria, we named this kindof bacteria “potential electrochemically active bacteria” (PEB)which refers to the bacteria that transfer the electron to anodeonly with the help of conductive polymer material. In thisexperiment, the bacteria specially enriched on the conductivepolymers modified anodes (Fig. 5), are likely to be the PEB.These enriched bacteria mainly belong to Desulfurellaceae andClostridiales, but differ from the traditional electricity produc-ing bacteria found in the two species before (Ieropoulos et al.,2005; Niessen et al., 2004). The enrichment of these PEB maybe the reason of enhanced power generation by conductivepolymers.

3) Efficient in electrocatalysis: The PANI (PAOA) coat, as a conduct-ing transporting matrix, can helps to improve the oxidation oforganic molecules in the biofilm (Han and Furukawa, 2006),and further increase the activity (Hoa et al., 2010) of the anodebacteria and improve MFC power generation. These materials

distribute the bacteria and substrates within the convenientelectron conducting matrix, which will help to efficiently trans-port charge to the reaction site. Nevertheless, the originalcarbon felt anode in this experiment lacks this catalytic surface.

ronics 26 (2011) 4169–4176 4175

4.3. Effect of conductive polymers on electron transfer mechanism

The process of electron transfer from bacteria to anode is thekey factor of MFC anode performance. Unfortunately, our under-standing of the process is still not complete (Cheng et al., 2008).Recent study had proposed several electrons transfer mechanismfrom bacteria to the anode surface, which includes: via membraneactive proteins (Bond and Lovley, 2003); via conducting nanowires(Gorby et al., 2006); via exogenous (artificial) redox mediators (Parkand Zeikus, 2000); via primary or secondary metabolites (as medi-ators) (Rabaey et al., 2005).

The mixed culture was inoculated in two-chamber MFC in thisexperiment, so the mechanism we discussed is the synergistic effectof all kinds of processes above. Compare with the unmodified, con-ductive polymer (PANI and PAOA) coat, can improve the electrontransfer process of the whole biofilm to anode, which can more effi-ciently harvest electrons. For PAOA, the introduced –OH can furtheraffect the electron transfer in the anode, or from bacteria to anode,compare with PANI.

4.3.1. Diversity of electron transfer process on modified anodeThe higher biodiversity on the modified anode, according to this

experiment, indicated the more diversified electron transfer pro-cess in the biofilm. The mediator less electron transfer bacteria areusually gram-negative, and need a series of extracellular matrixand outer-membrane complexes such as the transporting pro-tein, membrane-bound cytochromes (Rabaey and Rozendal, 2010).These bacteria are more suitable for enriching on the hydrophilicsupport. The protonic acid doped PANI and PAOA can provide MFCanode a more hydrophilic coat to meet this requirement. Besides,conductive polymers can be regarded as the fixed electron transfermediators. Therefore, the processes which electron transfer finalto carbon anode become more diversity under the help of conduc-tive polymers. Especially the two kinds of transfer processes, whichrely on membrane protein or via exogenous redox mediators, hadbeen enhanced and increased. This facilitates the electron transferin more various ways from biofilm to anode, which can improveelectronic transfer efficiency.

4.3.2. Phenolic hydroxyl group effectIn PAOA, electron withdrawing group such as –OH, has electro-

static interaction with aniline radical cations in the doped PANIchains, which impose restrictions on the spread of current car-rier within the copolymer chain. Also the existing of –OH groupscreate space obstacles, which increase the distance between eachchain, and further block direct electron transfer process amongthese copolymer chains. So, the electron transfer ability of PAOAis relative lower than the parent PANI and showed lower powergeneration of MFC in this experiment.

Moreover, the polarity and space structure effect of –OH bring anew integral structure to the polymers coat, which is in the form ofa sparse array of chains compared with PANI. Besides, –OH in thecopolymer chains can be oxidized to quinine and reduced again inorder to exchange proton between the material and the solution(Zhang et al., 2006), which provide a pH adjusting microenviron-ment for the MFC anode. Therefore, the biofilm growth on modifiedanode will be further affected by the –OH functional group. Accord-ing to the biodiversity analysis in this experiment, compared withPANI, Clostridiales, which is usually with much flagella, more tendto adhere on the surface of PAOA. It seems that relative more sparseand porous structure of polymers is apt for adsorption of bacterialsurface appendages such as flagella, which can also, indirectly affect

the MFC power generation.

However, more research needs to be conducted, since differ-ent groups introduction or even different molar ratio (1:10 foraminophenol and aniline in PAOA in our experiment) can greatly

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176 C. Li et al. / Biosensors and Bi

hange the properties of original PANI (Liu et al., 2006), and itrobably has much different result in anode biodiversity and MFCerformance.

. Conclusions

1) The PANI and PAOA modified MFCs, have faster increasingspeed during the start-up period, compare with the unmodi-fied one, and lead to a higher power density by 35% and 18% forPANI and PAOA separately.

2) PAOA, a new conductive polymer material first applied in MFC,whose maximum power density was 23.8 mW/m2, can lead toa better performance than unmodified one. But compare withPANI (27.3 mW/m2), PAOA cannot further improve MFC perfor-mance by introducing phenolic hydroxyl to PANI.

3) Both the amount and species of bacteria adhere on anodes canbe influenced by these two conductive polymer material. Withthe help of PANI and PAOA, the amount of bacteria on anodeincreased, and the microbial diversity became higher (from 12to 15 bacterial species according to DGGE), which may providea more stable biofilm on anode and more benefit for poweroutput of MFCs.

4) Even possessing the similar structure of PANI backbone, theperformance and biodiversity of the two anodes are differentbecause of the influence of phenolic hydroxyl introduction.

cknowledgement

This work was supported by National High Technology Researchnd Development Program of China (no. 2009AA063903) andational Water Pollution Control and Management Science andechnology Breakthrough Program (no. 2009ZX07106-004).

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.bios.2011.04.018.

ronics 26 (2011) 4169–4176

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