Preliminary Study on E. coli Microbial Fuel Cell and On-electrode ...

第 8 卷 第 6 期 过 程 工 程 学 报 Vol.8 No.62008 年 12 月 The Chinese Journal of Process Engineering Dec. 2008<strong>Preliminary</strong> <strong>Study</strong> on E. coli Microbial Fuel Cell andOn-electrode Taming of the BiocatalystXI Ming-yue ( 郗 名 悦 ), SUN Yan-ping ( 孙 彦 平 )(Department of Chemical Engineering, Taiyuan University of Technology, Taiyuan, Shanxi 030024, China)Abstract: A mediator microbial fuel cell (MFC) was constructed by using E. coli as biocatalyst and new methylene blue aselectron mediator. E. coli cells were carried out in anaerobic growth prior to inoculating them into the MFC in order topre-adapt bacterial metabolism in an anaerobic environment, the electricity generation of MFC was tested, its maximumpower density reached 263.94 mW/m 2 with the corresponding current density 1287.50 mA/m 2 , the internal resistance of MFCwas 200 Ω, and capability of the MFC was even better than those reported so far. Moreover, on-electrode taming method wasadopted to improve electrochemical activity of E. coli, namely a combination of E. coli taming and electricity generationsimultaneously in the same MFC without scraping off the biofilm of MFC, after the 4 th on-electrode taming, the tamed E. coliMFC showed a 54% improvement in peak current density, being 612.50 mA/m 2 , and a 64% improvement in the maximumpower output, being 166.67 mW/m 2 , compared with that of parental E. coli MFC. And the maturation time of tamed biofilmwas obviously reduced to 240 min, quickening up 1 times compared with that of parental E. coli biofilm.Key words: microbial fuel cell; E. coli; taming; biofilmCLC No.: Q417; Q503 文 献 标 识 码 :A 文 章 编 号 :1009−606X(2008)06−1179−061 INTRODUCTIONMicrobial fuel cell (MFC) can be considered as abioreactor using bacteria as catalysts, which convertschemical energy stored in biomass to electricity [1,2] .Based on the different ways that bacteria transferelectrons in MFC [3,4] , bacteria can be divided intomediator-less and mediator MFC bacteria. Mediator-lessMFC bacteria can directly transfer electrons to anode,which can be constructed as mediator-less MFC,mediator MFC bacteria must transfer electrons to anodevia electron mediators, which can be constructed asmediator MFC. Because most of bacteria in nature aremediator MFC bacteria [5,6] , on the other hand, theelectricity generation of mediator MFCs reported wassignificantly higher than that of mediator-less MFCs [6−8] ,so that research on cheap and efficient electron acceptornew methylene blue (NMB) once again attracts moreattention in exploitation of mediator MFCs [9] , andcorresponding work is more meaningful in this aspect.The electrochemical activity of biocatalyst is thekey influential factor in electricity generation ofMFC [5,10] , biocatalyst must be tamed to acquireacceptable electrochemical activity [4,5,11,12] .Cho et al. [11] tamed of single mediator-less MFCbiocatalyst via serial transfer, which only needs to joinelectron donor and acceptor into bacterial liquidmedium and anaerobically incubate diluted bacterialsuspension. This method was operated simply and couldimprove bacterial anaerobic adaptability, but did notobviously improve bacterial electrochemicalactivity [11,12] . Rabaey et al. [12] started with anaerobicsludge and tamed of a mixed bacterial culture in themediator-less MFC via repeatedly scraping biofilm, thismethod had obvious taming effect, unfortunately, itsoperation was complex. In contrast to many studies thathave focused on taming of mediator-less MFC bacteria,little has been reported on taming of mediator MFCbacteria.In this work, easy-culture and well-security singlebacterium E. coli was selected as biocatalyst, newmethylene blue as electron mediator to construct amediator MFC, the electricity generation of MFC wastested and checked, meantime, a simple taming methodcombining electricity generation of MFC and bacterialtaming was exploited, namely a taming method thatbiofilm was always conserved at the electrode, whichwas named after on-electrode taming method for short.The electricity generation of MFC was tested toinvestigate the effect that the on-electrode tamingmethod improves electrochemical activity of biocatalystand assess practical feasibility that this method wasReceived date: 2008−07−14; Accepted date: 2008−10−23Foundation item: Supported by Natural Science Foundation of China (No.20776091)Biography: XI Ming-yue (1983−), female, native of Hulun Buir City, Inner Mongolia Autonomous Region, master student, major in electrochemical reactionengineering; SUN Yan-ping, corresponding author, Tel.: 0351-6010070, E-mail: ypsun@tyut.edu.cn.

1180 过 程 工 程 学 报 第 8 卷applied to industrialization.2 EXPERIMENTAL2.1 E. coli and Culture ConditionsE. coli 1.0487 was obtained from the ChinaGeneral Microbiological Culture Collection Center(CGMCC).Aerobic growth of E. coli cells was carried out insterilized Luria–Bertani (LB) medium (10 g/L glucose,10 g/L peptone, 5 g/L yeast, 10 g/L NaCl, and pH=7). E.coli cells were inoculated into the liquid medium, thengrew at 30℃ in a constant temperature incubator. After24 h, the E. coli cells were carried out the anaerobicgrowth prior to inoculating them into the MFC topre-adapt bacterial metabolism in an anaerobicenvironment, and electrochemical activity of E. coli wasfurther improved to some extent. The LB medium wascontinually aerated with N 2 for 30 min to remove O 2 ,finally vitamin C and olefin were added to insurecompletely anaerobic conditions in culture process. theE. coli cells were incubated for 24 h at 30℃.2.2 MFC SystemNafion112 membrane. It was obtained fromAmerican Du Pont Company. Before use, the membranewas boiled in 3% H 2 O 2 for 1 h, then treated with 80℃deionized water and 0.5 mol/L H 2 SO 4 , again treatedwith 80℃ deionized water twice, and finally kept indeionized water [9] .Carbon paper. It was obtained from TorayCorporation, Japan. New electrodes were soaked in 1mol/L HCl solution for 1 d to remove possible metalcontamination. After each use, the electrodes werewashed with 1 mol/L NaOH solution to treat theattached cells [13] .MFC. A dual-chambered cell was constructed,using 4.0 cm 2 carbon paper as the anode, 1.0 cm 2platinum gauze (50 mesh) as the cathode. Connectionswere made with copper wire screwed into holes drilleddirectly into the electrodes (the hole connected the wireand the carbon paper was sealed with epoxy resin toavoid corrosion of copper wire [7,9] ). The chambers wereseparated with a Nafion112 membrane. Each chamberhad 60 mL volume in dimensions of 3 cm×4 cm×5 cm,the exposure area of membrane was 1.0 cm 2 . Prior to theinoculation of E. coli cells, N 2 was aerated into theanode chamber to remove O 2 in the medium, whichguaranteed the strict anoxic conditions [9] .Electrolyte. The anode solution contains LBmedium, buffer solutions (10 g/L NaHCO 3 and 8.5 g/LNaH 2 PO 4 ), electron mediator (1.5 g/L NMB) with thepH value at 7. The cathode solution contains 1 mol/LK 3 Fe(CN) 6 , 0.1 mol/L phosphate buffer, and 10 g/LNaCl with the pH value at 7 [14−16] .35 mL fresh anode solution was added in the anodechamber of sterilized MFC (UV light: 256 nm,overnight), then N 2 was aerated for 30 min, and finallyvitamin C and olefin were added to insure completelyanaerobic conditions. 40 mL catholyte was added intothe cathode chamber. 5 mL E. coli cell suspensionanaerobically cultured were rapidly inoculated into theanode chamber,then the electrochemical test of MFCwas carried out at 30℃.2.3 Methods2.3.1 Open circuit voltage (OCV)After 5 mL E. coli cell suspension was added intothe MFC, the relationship of OCV of MFC with timewas tested until the OCV had been stable for 30 min.2.3.2 Polarization curve of MFCAfter the OCV was stable, the resistance betweenthe electrodes was lowered stepwise, pausing at eachresistance setting for about 3∼5 min [17,18] , current (I, mA)and voltage (U, V) were recorded at each resistance,Power (P) was calculated according to P=IV. Currentand power were normalized by the cross-sectional area(projected) of the anode.2.3.3 Peak current densityThe relationship between current density of theMFC with time was tested under a fixed load of 1000Ω. During the growth of E. coli cells, the currentdensity of MFC would gradually rise, and finallyreached a peak current density, namely the biofilmattached to the electrode had matured [3,6,19,20] .3 RESULTS AND DISCUSSION3.1 Working Mechanism of MFCTaking glucose as the fuel, E. coli metabolizedglucose, then released electron and proton, electron wastransferred to anode by mediator (NMB), then tocathode by external circuit, meantime, proton moved tocathode chamber through proton exchange membrane,then at the cathode it combined with electron andoxygen to form water. The detailed transfer process isshown in Fig.1 (Switches 1 and 2 were off when thecircuit was open, switch 1 was on and switch 2 off whenthe circuit was closed, and switch 1 was off and switch2 on when a circuit with external variable resistance wasused). The related reactions are expressed in Eqs.(1~4).Glucose is completely oxidized to CO 2 on anode:C 6 H 12 O 6 +6H 2 O ⎯ E. ⎯→coli ⎯ 6CO +24H + +24e − . (1)2

第 6 期 XI Ming-yue, et al.: <strong>Preliminary</strong> <strong>Study</strong> on E. coli Microbial Fuel Cell and On-electrode Taming of the Biocatalyst 1181In the cathod chamber, [Fe(CN) 6 ] 3− is reduced to[Fe(CN) 4 ] 2− , then [Fe(CN) 4 ] 2− oxidized to [Fe(CN) 6 ] 3− :4[Fe(CN) 6 ] 3− +4e −4[Fe(CN) 6 ] 4− +4H + +O 2The MFC reaction is expressed as:C 6 H 12 O 6 +6O 2⎯→4[Fe(CN) 6 ] 4− , (2)Pt⎯⎯→ 4[Fe(CN) 6 ] 3− +2H 2 O. (3)⎯→6CO 2 +6H 2 O. (4)shown in Fig.3. Along with the outer resistanceincreasing, the power output first increased thendecreased, there was a maximum value of power output,being 263.94 mW/m 2 , based on the ohm law, the outerresistance is equal to the inner resistance when poweroutput attains the maximum, so we can know that theinner resistance of MFC in this work was about 200 Ω.250Power density (mW/m 2 )200150100500 2000 4000 6000 8000 10000Resistance (Ω)Fig.1 Illustration of the MFC3.2 Electrochemical Measurement3.2.1 OCV test of MFCFigure 2 shows the open circuit voltage of MFCover time. After E. coli cells were inoculated into theanode solution, the stable time of MFC′s OCV needed65 min, and the value was 0.598 V. Zou et al. [9] reporteda parental E. coli MFC, its stable time was 300 min,which is much higher than that of the present work.Open circuit voltage (V)0.600.580.560.540.520.500.480.460.440.4220 40 60 80 100 120 140Time (min)Fig.2 Open circuit voltage of the MFC with time3.2.2 Polarization curve of MFCWhen the OCV of MFC attained the plateau phase,the resistance (0∼10000 Ω) between the electrodes waslowered stepwise to select proper outer resistance forbenefiting the power output of MFC. The relationshipbetween resistance and power density for the MFC isFig.3 Relationship between power density andresistance of microbial fuel cellFigure 4 shows the cell voltage and power outputdensity in the MFC as a function of current density.When the outer resistance decreased, then the currentdensity increased gradually and the cell voltagedecreased gradually, the maximum power density ofMFC was 263.94 mW/m 2 with the correspondingcurrent density of 1287.50 mA/m 2 .Cell voltage (V)0.452500.400.352000.301500.250.201000.15500.100 200 400 600 800 1000120014001600Current density (mA/m 2 )Fig.4 Cell voltage and power output density inthe MFC as a function of current densityThe capabilities of MFC in this work (stable time,current density, and power output) were even better thanthose reported in the literature so far for E. coliMFCs [9,16] . The reason was because of less innerresistance of the MFC [18,21] . Additionally, in theexperimental process, E. coli cells were carried outunder anaerobic growth prior to inoculating them intoPower output density (mW/m 2 )

1182 过 程 工 程 学 报 第 8 卷the MFC, anaerobic experiments with E. coli cells wereinitiated in order to pre-adapt bacterial metabolism in ananaerobic environment, then E. coli′s electrochemicalactivity was improved to some extent. So MFC couldrapidly generate electricity and attain stable state.3.2 On-electrode Taming MethodsBiofilm attached to electrode is developed asfollows: bacterial attachment to electrode, earlymaturation and acquisition of biofilm structure, maturebiofilm, and bacterial detachment [22] . The electricitygeneration of MFC is extremely related to the formedmature biofilm attached to the electrode [6,19,20] . However,most of MFC bacterial taming methods usually needrepeatedly scrap biofilm to select and optimize bacteria,but the operation of scraping biofilm can result in theloss of electrochemical activity of biofilm andpostponing the time that biofilm again matures.On-electrode taming method judges the maturation ofbiofilm according to the voltage−ampere curve of MFC,which means that the current density of MFC justattains the plateau phase (reaching the peak currentdensity) when the biofilm has matured [3,6,19,20] , theoperations of on-electrode taming without scraping offof biofilm can simply and rapidly achieve the bacterialselection and optimization.During the experiments, when the MFC attainedthe plateau of current density for the first time, thebiofilm attached to carbon paper had matured, and theelectrochemically activated E. coli biomass wasaccumulated, meantime, the anode solution wasremoved out to eliminate the infection of accumulatedoutgrowth and detached cells in the anode chamber afterthe MFC run for a long time [6,8] , and the anode chamberwas rapidly added with 40 mL medium to anaerobicallyculture the mature E. coli biofilm attached to theelectrode, and the electrochemical activity of E. colibiofilm was conserved. When MFC was again addedwith anolyte to carry out the electrochemical test, itspeak current density was increased, and the time toreach the peak current value was reduced. Successiveanaerobic cultivation and electrochemical test werecarried out to tame of E. coli biofilm and graduallyimprove the electrochemical activity of E. coli biofilm.As shown in Fig.5, after the 4 th taming (n=4), themaximum current density of E. coli MFC and theparental (n=0) E. coli MFC was respectively 612.5 and397.5 mA/m 2 , namely the biofilms attached to theelectrodes had matured. We can see from theexperimental data that the maturation time of tamedbiofilm was obviously reduced to 240 min, quickeningup 1 times compared with that of parental E. coli,meantime, the maximum current density of the tamed E.coli MFC was increased 0.54 times compared with thatof parental E. coli MFC.Current density (mA/m 2 )400650390 (a) Before taming600(b) After the 4th taming380370550360500350450340400330350320200 250 300 350 400 450 500 550300200 250 300 350 400 450 500Time (min)Time (min)Fig.5 Relationship between current density and time in MFCs before and after taming of E. coliFigure 6 shows the relationship of cell voltage andpower density with current density of MFC in thetaming process under n=0, 2, 4. After the 4 thon-electrode taming (n=4), the maximum power densityof tamed E. coli MFC reached 166.67 mW/m 2 ,increasing 64% compared with the maximum powerdensity 101.81 mW/m 2 of the parental E. coli MFC(n=0).As summarized in Fig.7, after the 4 th on-electrodetaming, the electricity generation of tamed E. coli MFCwas remarkably improved compared with the electricityCurret density (mA/m 2 )generation of parental E. coli MFC, the tamed E. coliMFC showed a 54% improvement in peak currentdensity, reaching 612.50 mA/m 2 , a 64% improvement inmaximum power output, reaching166.67 mW/m 2 . Zou etal. [9] reported a parental E. coli MFC, its maximum poweroutput was 70% of that of the MFC in this work, being116 mW/m 2 , and the initial concentration of E. coli inthe literature was 220% of that in this work, being 2.73mg/mL.Because taming of mediator MFC bacteria byserial transfer was not reported in the literature, in the

第 6 期 XI Ming-yue, et al.: <strong>Preliminary</strong> <strong>Study</strong> on E. coli Microbial Fuel Cell and On-electrode Taming of the Biocatalyst 1183Cell voltage (V)0.451800.401600.351400.30120n0.250 1000.2028040.15600.10400.05200.000 300 600 900 1200 1500 1800Power density (mW/m 2 )Peak current density (mA/m 2 )6005505004504000 2 4 6170160150140130120110100Maximum power density (mW/m) 2 )Current density (mA/m 2 )Taming timesFig.6 Relationships of current density with voltageand power density in taming processesFig.7 Relationships of taming times with the maximumcurrent and power densitypresent experiments, we ever attempted to adopt theoperating steps of literature [11] to tame E. coli. Theelectron mediator NMB was added for taming ofmediator MFC bacteria, however, plenty of black-greendeposition was found in diluent, which led to theremarkable fluctuation of experimental data. Whether ornot this method can be adopted for taming of mediatorMFC bacteria still needs more research.Owing to benefiting environmental protection,electricity generation with sludge and sewage hasimportant significance to development of MFC. Rabaeyet al. [12] tamed of a mixed bacterial culture in themediator-less MFC via repeatedly scraping biofilm, butthis method had tamed for 7 times and 63 d. Theon-electrode taming method reported in this work hasno special requirement to the types of bacteria and doesnot need to scrap off biofilm, so the advantages ofsimple operation and short time make on-electrodetaming method a highly promising candidate forapplication in microbially aggressive environments likesewage or sludge.4 CONCLUSIONS(1) A mediator microbial fuel cell (MFC) wasconstructed with E. coli in this work. The capabilities ofthe MFC were even better than those reported in theliterature so far for E. coli MFCs. E. coli cells werecarried out in anaerobic growth prior to inoculatingthem into the MFC in order to pre–adapt bacterialmetabolism in an anaerobic environment, the anaerobicgrowth operates simply, and may apply to improveelectrochemical activity of all kinds of MFCbiocatalysts. The electricity generation of E. coli MFCis characterized with the following data: when the initialconcentration of E. coli cells was 3.41 mg/mL, themaximum power density of MFC reached 263.94mW/m 2 with the corresponding current density of1287.50 mA/m 2 . The internal resistance of MFC was200 Ω.(2) After the 4 th on-electrode taming, the electricitygeneration of tamed E. coli MFC was compared withthe electricity generation of parental E. coli MFC whenthe initial concentration of E. coli cells was 1.24 mg/mL,the tamed E. coli MFC showed a 54% improvement inpeak current density, being 612.50 mA/m 2 ; a 64%improvement in maximum power output, reaching166.67 mW/m 2 . In the meantime, the maturation time oftamed biofilm was obviously reduced to 240 min,quickening 1 times compared with that of parental E.coli biofilm. The on-electrode taming method describedhere does not need to scrap off biofilm, and has theobvious advantages of the less taming times and shortertaming time.REFERENCES:[1] Kim J R, Jung S H, Regan J M, et al. Electricity Generation andMicrobial Community Analysis of Alcohol Powered Microbial FuelCells [J]. Bioresour. Technol., 2007, 98(13): 2568−2577.[2] Frank D, S′eamus P J Higson. Biofuel Cells—Recent Advances andApplications [J]. Biosens. Bioelectron., 2007, 22(7): 1224−1235.[3] Lovley D R. Microbial Fuel Cell: Novel Microbial Physiologies andEngineering Approaches [J]. Curr. Opin. Biotechnol., 2006, 17(3):327–332.[4] Kong X Y, Li L H, Sun Y M, et al. Fundamental <strong>Study</strong> on MicrobialFuel Cell and Influent Factors on Output Power [J]. Modern Chem.Ind., 2007, 27(2): 282−286 (in Chinese).[5] Hong Y G, Guo J, Sun G P. Recent Progress in Electricigens andMicrobial Fuel Cell [J]. Acta Microbiologica Sinica, 2007, 47(1):173−177 (in Chinese).[6] Lian J, Feng Y L, Li H R, et al. Construction and <strong>Preliminary</strong> Studieson the Direct Microbial Fuel Cell [J]. The Chinese Journal of ProcessEngineering, 2006, 6(3): 408−412 (in Chinese).[7] Kim H J, Park H S, Hyun M S, et al. A Mediator-less Microbial FuelCell Using a Metal Reducing Bacterium, Shewanella utrefacien [J].Enzyme Microb. Technol., 2002, 30(2): 145–152.[8] Park D H, Zeikus J G. Electricity Generation in Microbial Fuel CellsUsing Neutral Red as an Electronophore [J]. Appl. Environ.

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