Inocula selection in microbial fuel cells based on anodic biofilm abundance of Geobacter sulfurreducens

Guotao Sun ,Diogo de Sacadura Rodrigues ,Anders Thygesen ,*,Geoffrey Daniel,Dinesh Fernando ,Anne S.Meyer

1 Center for Bio Process Engineering,Department of Chemical and Biochemical Engineering,Technical University of Denmark,S?ltofts Plads 229,DK-2800 Kgs.Lyngby,Denmark

2 Department of Forest Products,Swedish University of Agricultural Sciences,SE-75651 Uppsala,Sweden

1.Introduction

A microbial fuel cell(MFC)encompasses anode and cathode reactions to drive redox processes that result in production of electricity.The core principles of the electricity generation are similar to those in chemical fuel cells,but in MFCs,the reactions rely on bacterial metabolism based on a microbial biofilm on the anode electrode[1].Fermentative bacteria are needed to convert complex substrates(e.g.glucose)into carboxylic acids including acetate,which can then be digested by electrogenic bacteria[2,3].Geobacter sulfurreducens,is an electrogenic bacterium widely found in nature,which means that it can directly transfer electrons to the electrode[4,5].The performance of MFCs depends therefore on the type and abundance of the microbial consortium in the anode chamber and notably in the anode biofilm.The inoculum source of electrogenic and fermentative bacteria is therefore important in the establishment of the anodic biofilm.

Inocula sources that have been studied in MFCs include pure bacteria[5],domestic wastewater(DW)[6-8]and biogas sludge(BS)[9].Nevinet al.reported that pure cultures of electrogenic bacteria can produce higher maximum power density(MPD=1900 mW·m-2)than mixed communities(1600 mW·m-2)with acetate as feed[5].Holmeset al.[10]operated MFCs inoculated with marine sediment,salt-marsh sediment and freshwater sediment and showed that the power output was linked to electrogenic bacteria regardless of the salinity.Yateset al.[7]examined the microbial community in two-chamber H-shape MFCs inoculated with DW(two sources tested)and lake sediment(LS).They found that the cell voltage reached similar values[(470±20)mV]after 20 operational cycles and that the anodic biofilm community were dominated byGeobactersp.

Previous studies have shown that external resistance(Rext)and substrate concentration affect the power generation and microbial community composition[11-13].It is known that in a mixed culture,the electrogenic bacteria compete for substrate with fermentative non-electrogenic bacteria[13].From the available literature,it is clear that a decaying microbiota is required for the MFC to convert organic substrates to electric currentviaelectrogenic bacteria,but it is unclear whether the frequently tested DW may be surpassed by denser inocula such as BS and LS.Abetter understanding of the evolution of electrogenicversusfermentative bacteria will aid in improving MFC performance.

The objective of this work is to assess the electrochemical performance,stability and microbial consortium development using three inocula including DW,BS and LS,respectively.It was expected that a denser inoculum would allow an increase in power generation and make the process more robust to substrate changes.Based on the optimal inocula,the effect on the microbial evolution of a variation ofRextand substrate loading(Lsub)was examined to improve MFCs performance.The process analysis was performed with thorough microbial analysis,chemical analysis and electrochemical impedance spectroscopy(EIS).

2.Materials and Methods

2.1.MFCs configuration

The H-shaped reactors used in this study were constructed by two cylindrical acrylic glass bottles with a volume of 300 cm3for each of the compartments(220 cm3liquid),which were connected with a tube with an inner diameter of 30 mm[6].A proton exchange membrane(Na fion?N117,Dupont Co.,USA)with an area of 7.1 cm2was placed between the chambers.The two chambers were tightened with rubberrings.Both anode and cathode electrode were made of paralleled carbon paper sheets(TGPH-020,Fuel Cells Etc,USA)of 3 cm×8 cm(A=24 cm2)and a thickness of 0.35 mm.

2.2.Inoculation and operational conditions

The basic anolyte consisted of M9 medium containing per litre:6 g Na2HPO4,3 g KH2PO4,1 g NaHCO3,1 g NH4Cl,0.5 g NaCl,0.247 g MgSO4·7H2O,0.0147 g CaCl2and 1 cm3trace element solution[6].pH could be maintained at7.0 due to the high buffer capacity of the M9 medium(64 mmol·dm-3of phosphate buffer+12 mmol·dm-3of carbonate buffer).The carbon source(sodium acetate or xylose)was added to the medium.The cathode solution was 100 mmol·dm-3of K3Fe(CN)6and 100 mmol·dm-3of phosphate buffer(pH 6.7)and was replaced at the beginning of each cycle.All the MFCs were operated at 30°C in an incubator with magnetic stirring[6].

Reactors(triplicates)were inoculated with three types of inocula:D Wobtained after the fine separation process on a domestic wastewater treatment plant(Lyngby Taarb?k Community,Denmark);LS collected from Sor?lake(55°25′20.8″N,11°32′22.7″E);and BS from Hash?jBiogas(Dalmose,Denmark).The LS sample was on March 11th,2014,collected at 40 cm water depth and was a suspension of anaerobic surface sediment and water.pH,electric conductivity(EC),dry matter(DM)and chemical oxygen demand(COD)of these inocula are shown in Table 1.The reactors were inoculated in a 1:1 ratio of medium to inocula and fed with sodium acetate(1 g·dm-3of COD)usingRextof 1000 Ω.Feeding was done every 5 days(equal to one cycle)with fresh medium and corresponding substrates.Due to start up time,the first cycle lasted for 7 days.After 2 to 3 batch cycles,stable power generation was obtained in all the reactors.The acetate substrate was at beginning of Cycle 4 changed to xylose to study the adaptability of the microbial community to a fermentative substrate still using 1 g·dm-3of COD content.

Table 1Chemical parameters of the inocula including pH,electric conductivity(EC),dry matter(DM)and chemical oxygen demand(COD)

Based on the inocula test,four reactors(duplicate)inoculated with the optimal inoculum(LS)were operated in batch mode testingRextof 200,500,800 and 1000 Ω.Anode solution was replaced every 5 days,which equals to one cycle.From second cycle,all the reactors were fed with fresh medium and sodium acetate.After 3 batch cycles,stable power generation was obtained and differentLsub(0.5,1,1.5 and 2 g·dm-3of COD)were tested in the MFCs.Operational cycles and correspondingRextandLsubare outlined in Table 2.

Table 2Overview of the operational parameters in 4 MFCs(duplicates)testing R ext and L sub

2.3.Microbial community analysis

Biofilm samples from the anode chamber were obtained by cutting 0.5 cm2of the anode electrode at the end of each cycle[6].Genomic DNA extraction followed by polymerase chain reaction(PCR)and denaturing gradient gel electrophoresis(DGGE)were conducted as previously described[6,14].Similarity between the samples was analysed by using BioNumerics software v.7.1(Applied Maths,Sint-Martens Latem,Belgium)[6].

A clone library for providing a phylogenetic affiliation of the DGGE bands was constructed and resulting sequences have been submitted to EMBL Nucleotide Sequence Database(Accession No.LN650984-LN651064)by Sun et al.[6].Subsequently the unique clones were amplified by PCR as described above.The PCR products were then run in a DGGE gel to identify the bands formed by biofilm samples[6].

2.4.Scanning electron microscopy

In order to examine biofilms on the anode surfaces,the anodic electrode(～1 cm2)was removed without touching its surface.Small samples(1 cm × 1 cm)were fixed in 50 dm3·m-3glutaraldehyde+20 dm3·m-3paraformaldehyde in 0.1 mol·dm-3Na-acetate in deionized water(pH 7.2).After fixation,the samples were dehydrated in aqueous ethanol using:20%,40%,60%,80%,90%and 100%for 20 min in each solution.Subsequent dehydration was performed in 33%,66%and 100%acetone in ethanol before samples were critical point dried using Agar E3000 critical point dryer(Agar Scientific,Stansted,UK)with liquid CO2as drying agent.Following coating with gold using an Emitech E5000 sputtercoater,samples were observed using a Philips XL30 ESEM scanning electron microscope at 50 to 10000 times of magnification[15].

2.5.Chemical,electrochemical and statistical analysis

The COD concentration and dry matter content were measured similar to Sunet al.[6].Concentrations of monomeric sugars and volatile fatty acids(VFA)were measured by HPLC(high-performance liquid chromatography)[6].pH and electrical conductivity were tested by multimeter(Multi 3430,WTW,Germany).

Electric current was recorded every 15 min by a data logger(Model 2700,Keithley Inc.).In polarisation tests,Rextwas varied between 30 Ω and 50 kΩ.The current density(I)and maximum current density(Imax)were calculated by dividing the current with the electrode surface area(A=48 cm2)including both sides.EIS was carried out with a potentiostat(SP-150,BioLogic,France).The anode polarisation resistance was measured by connecting the MFCs to the potentiostat in the three-electrode mode within the frequency range from 10 kHz to 0.1 Hz with amplitude of 10 μA.Lower frequencies were not tested since it can disturb the microbial process due to a long test period(＞1 h).The anode and cathode were used as working electrode and counter electrode,respectively.The third lead was attached to a reference electrode(Ag/AgCl;#MF2079;Bioanalytical Systems Inc.)inserted in the anode chamber.Zview(Scribmer Associates Inc.)was used for EIS data fitting.Coulombic efficiency(CE)was calculated as the ratio of accumulative charges produced from the MFCs to the charges released from substrate degradation.Statistics analysis by ANOVA(one-way;p＜0.05)was done by using Minitab 16 www.minitab.com and means were compared using Turkey's multiple range procedure.The significant difference between the values was indicated by letters A-D.

3.Results and Discussion

3.1.Electricity generation in MFCs using the three inocula

The currentdensity outputs ofthe DW-,LS-and BS-inoculated MFCs are shown in Fig.1.During cycle 1,DW-inoculated MFCs needed shorter lag time(2 days)to achieve stable current than LS-inoculated MFCs(4 days)and BS-inoculated MFCs(5 days).The short lag time of DW-inoculated MFCs indicated rapid start-up compared with previous studies of7 days by Lietal.[16]and 9.5 days by Zhangetal.[8].After 2 cycles of MFC operation,the average current density[Iave=(138±2)mA·m-2]in LS-inoculated MFCs was slightly higher(2%-5%)than in DW-and BS-inoculated MFCs.When xylose was added to all the MFCs(cycle 3),they needed one day to recover to stable current generation.Adaptation of the MFCs to xylose also resulted in a 20%drop inIave.In particular for DW-inoculated MFCs(Fig.1A),Iaveshowed earlier drop at the end of cycles 4 and 5,but after 3 cycles,all MFCs converged to a similarIave[(140±2)mA·m-2].Thereby DW showed the shortest lag time while LS gave the highestIave.However,Iavewas similar with the three inocula after shifting to xylose(cycle 5).

Fig.1.Current density in MFCs inoculated with DW(A),LS(B)and BS(C),respectively.The arrows indicate the substrate replacement.pH was found constant on 7 during the experimental cycles.

Imaxis a key factor demonstrating the capability of power generation that MFCs can produce(Table 3).Imaxin all the MFCs increased from cycles 2 to 6,which can be explained by the study of Readet al.[3]showing that a stronger biofilm can be formed when the MFCs run for longer time.With acetate,LS-inoculated MFCs showed the highestImax[cycle 3;(690 ±30)mA·m-2]compared with DW[(440 ± 50)mA·m-2]and BS[(370±30)mA·m-2].After addition of xylose(cycle 6),LS-inoculated MFCs still generated higherImax[(1690 ± 40)mA·m-2],than DW and BS with(1330±10)and(930±50)mA·m-2,respectively.The differentiation inImaxproved that the inocula had a significant effect on electricity generation and that LS-inoculated MFCs performed best.

3.2.Substrate conversion and efficiency using the three inocula

For the acetate fed MFCs,the utilisation of acetate and current generation are shown in Fig.2A,B,C(cycle 3).Acetate removal rates in the range of 58%-61%were achieved after 5 days of current generation(Iave=131-138 mA·m-2)with the three inocula.For the xylose fed MFCs,the utilisation of xylose and formation of acetate and propionate are shown in Fig.2D,E,F(cycle 5).Xylose was completely degraded with all the inocula after the first day with accumulation of acetate and propionate as by-products.The accumulation of acetate[(5.2±0.2)mmol·dm-3]in DW-inoculated MFCs was higher than with LS[(4.7±0.4)mmol·dm-3]and with BS[(3.7 ±0.2)mmol·dm-3].The high formation of acetate with DW indicates a large abundance of xylose-fermenting bacteria since acetate is produced faster than it is utilized in the electrogenic bacteria[2].

CE was calculated based on the accumulated charge produced from the MFCs divided by the charge released from substrate degradation as shown in Table 3.LS showed the highest CE of29%±1%when acetate was used as substrate(cycle 3).The higher CE is due to the high current density and low COD removal.After xylose was added to the MFCs(cycle 4),CE dropped dramatically to 14%±2%,18%±1%and 17%±0.1%for DW,LS and BS,respectively.However,the CE increased to 17%±3%,23%±1%and 21%±1%respectively after 3 cycle of operation(cycle 6).The highest CE(23%)andImax(1690 mA·m-2)were thereby obtained in the LS-inoculated MFCs.

3.3.Anode polarisation resistance using the three inocula

In an MFC,the biofilm,which is attached to the anode,serves as biocatalyst for electricity generation.The metabolism of bacteria in MFCs is one of the limiting factors for power generation which can be represented by the polarisation resistance of the anode.EIS is an efficient nondestructive technique to determine the anode polarisation resistance[17].Measurements were conducted by connecting the MFC to a potentiostat in three-electrode mode.The impedance of the anode is presented in Fig.3 and was used to calculate anode polarisation resistance(Rp)by fitting the impedance data to Randles circuit(Fig.3D).The anode polarisation resistance for DW-,LS-and BS-inoculated MFCs were 94 Ω,119 Ω and 87 Ω,respectively,before MFCs started work.The differentiation of the resistance at this time is due to the different EC in the inocula(Table 1).Resistance decreased after the MFCs achieved stable current generation to 51 Ω (DW),30 Ω (LS)and 40 Ω(BS),respectively.The decrease in resistance indicated that the biofilm formed on the anode surface activated the electrochemical reaction and that LS-inoculated MFCs can generate higherImaxthan DW and BS.Furthermore,when the more complicated substrate(xylose)was added to all the MFCs,LS-inoculated MFCs performed with lower anode resistance(24 Ω)than DW(41 Ω)and BS(35 Ω).These results are corroborated by Fanet al.[18]that the lower anode resistance with LS contribute to higher power generation(Table 3).

Table 3I max and CE generated in MFCs inoculated with DW,LS and BS,respectively.Batch no.is corresponding to the batch test in Fig.1.Letters A-D indicate column wise significant difference

Fig.2.I ave and substrate degradation as function of time in MFCs enriched with DW(A,D),LS(B,E)and BS(C,F)respectively.The substrate used in(A,B,C)and(D,E,F)are acetate and xylose,respectively.The initial concentration for each substrate was 1 g·dm-3 of COD.

3.4.Effects of Rext and Lsub on electricity generation

Four MFCs(duplicate),with differentRext(200,500,800 and 1000 Ω),were evaluated from cycle 1 to 3 forIaveandImax(Table 4).The reactors with 200 Ω needed 1.5 days before notable current generation was obtained,while the reactors at 500-1000 Ω needed 2.5 days.The MFCs with lowerRextperformed thereby a better start-up in agreement with a previous study[10].After stable current was observed,Iaveranged from(145 ± 10)mA·m-2(1000 Ω)to(555 ± 8)mA·m-2(200 Ω).Differences ofImaxamong these reactors with different Rextwere also noted.The MFCs with 200 Ω produced highestImaxof(1780 ± 30)mA·m-2,while 1000 Ω only generated(570 ±10)mA·m-2.After all MFCs changed to use 200 Ω (cycle 4),similarIave[(557±13)mA·m-2]andImax[(1800±20)mA·m-2]were generated.AtRextof200Ω(cycle 5),theLsubshowed no significant effect onIaveandImaxexcepting theLsubof 0.5 g COD·dm-3,which generated lowerIave[(419 ± 28)mA·m-2]than the higherLsub(555 mA·m-2).This can be explained by previous research,which reported that only at low resistances or at near maximum current the increasedLsubcan result in increased electricity generation[10].

Fig.3.The impedance of the anode in MFCs inoculated with DW,LS and BS respectively.(A)Beginning MFC operation;(B)MFCs using acetate as substrate;(C)MFCs using xylose as substrate.(D)Schematic of Randles equivalent circuit to model charge transfer:ohmic resistance(R s),polarisation resistance(R p)and constant phase element(CPE).

Table 4Average current density(I ave),maximum current density(I max),COD removal rate(CODrr)and coulombic efficiency(CE)in the MFCs using different external resistance(R ext)and substrate loading(L sub)

Table 4 also reported COD removal rate(CODrr)and CE in the MFCs with differentRextandLsub.The MFCs with lowerRextshowed both higher CODrr[(152 ± 1)g·m-3·d-1]and higher CE(58% ± 1%),which can be attributed to the higher rate of elecrogenesis resulting in higher current generation.Comparatively,the decreasingLsubresulted in lower CODrr[(92 ± 6)g·m-3·d-1]and higher CE(61% ± 2%).A previous study,using the same MFC design,also reported that the increasingLsubfrom 0.25 to 2 g·dm-3of COD resulted in a decrease of CE from 37%to 16%[19].HighIaveand high CE were thereby obtained at lowRext(200 Ω)and a relatively lowLsubof 1 g·dm-3of COD.

3.5.Microbial community:Effect of inocula

SEM analysis of the micro-and ultrastructure of anode electrode biofilms after the 6 cycles of MFC operation showed considerable differences as shown in Fig.4.The control showed no bacterial colonisation over the surface of the electrodes(Fig.4A).The electrode rods had clean,smooth and homogeneous surfaces(Fig.4A,inset top right)with even diameter of 8 μm.

BS:Not dense unevenly distributed bacteria and only low biofilm slime formation was observed(Fig.4B).Sometimes,rods were observed with areas of non-colonized clear surfaces(Fig.4B,inset top right).In addition,a diverse bacterial community(e.g.long rod types(arrowhead,Fig.4C)and oval shaped ones(arrows,Fig.4C))was apparent(Fig.4C).These characteristics agree the lowImaxof 930 mA·m2(Table 3).

DW:Electrode rods had unclean surfaces with several inhomogeneous particles(arrows,Fig.4D).A close-up view showed condensed colonies of mostly rod shaped bacteria with infrequent presence of slimy material(inset top right,Fig.4D and E).Different bacterial morphology was also found(Fig.4F)and the bacteria were attached to each other(Fig.4E and F).In addition,there was infrequently observed nano-threads like structures from bacteria(arrows,Fig.4G)and all these characteristics of the biofilm should collectively contribute to the 43%higherImax(Table 3).

LS:An even higher and thick colonisation of the electrode surfaces were seen(Fig.4H)with more frequent particles of varying sizes densely distributed over electrodes(arrows,Fig.4H).The large particles were thick highly concentrated bacterial colonies(inset top right,Fig.4H)that are thought to contribute for higher electricity production.In addition,morphology of the biofilm indicated less diverse bacterial communities where long rod-shaped bacteria were more commonly observed(Fig.4I).Interestingly,nano threads-like appendages ranging from 70-120 nm in width and extending tens of μm long were often seen associated with rod-shaped bacteria(arrowheads,Fig.4J)presumably representing bacterial nanowires.G.sulfurreducensare known to produce nanowires that are highly conductive and have potential for long-range exocellular electron transfer across biofilm[20,21].These characteristics lead to 82%higherImaxthan with BS(Table 3)and suggesthigh abundance and activity ofelectrogenicGeobactersp.asevident from DGGE analysis(Fig.5).

Fig.4.Scanning electron micrographs ofthe electrode withoutbio film(A)and electrodes in MFCs showing theirmicro-and ultrastructure of biofilms formed afterinoculated with BS(B,C),DW(D-G)and LS(H-J),respectively.Bars:A,B,D,H,100 μm;C,3 μm;E,10 μm;F,G,I,J,2 μm.

3.5.2.Molecular determination of microbial community

In order to provide greater insight into microbial diversity of the biofilm samples,bacterial gene libraries were examined using full length 16S rRNA(Table 5).The bacterial species identified included the electrogenic speciesG.sulfurreducens[5]and the fermenting speciesBacteroides graminisolvens[22],Arcobacter butzleri[23],Paludibacter propionicigenes[24],Thermanaerovibrio acidaminovorans[25],Enterobactercancerogenus[26],Citrobacterbraakii[27]andPropionisporahippie[28].

The anodic biofilms in the three types of inoculated MFCs were sampled at the end of each batch test(from cycle 2 to 5)as shown in Fig.1.The microbial community of the biofilm samples were analysed with 16S rRNA-based DGGE in combination with a clone library as summarized in Fig.5A.The band patterns of the biofilm in all the MFCs became stable after 7 days of enrichment with inocula and acetate(cycle 1 in Fig.1).The similarities between the lanes comparing cycle 2 and 3 were higher than 88%for the 3 inocula.However,the band patterns in cycle 2 varied significantly between the three types of inoculated MFCs with 59%for LS compared to DW(LS_2:DW_2)and with 33%for LS compared to BS(LS_2:BS_2).

The patterns of the bands also changed after switching substrate from acetate to xylose,with similarities from cycle 3 to 4 of 46%,40%and 4%for LS,DW and BS,respectively.After short acclimation of the MFCs to xylose,stable band patterns were observed in all the biofilm samples with similarities above 80%(LS_4,LS_5;DW_4,DW_5;and BS_4,BS_5).The distinct similarities among the inocula and substrates demonstrated that they are key factors affecting anodic microbial community in MFCs.

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When acetate was used in MFCs,G.sulfurreducenswas predominant with all the inocula.In addition,T.acidaminovoranswas dominant with DW,ShigellaflexneriandAzonexus caeniwere dominant with LS andS.flexneriwas dominant with BS(comparing cycle 2 and 3).Among these species,onlyG.sulfurreducenshas the potential to electricity generation as a metal-reducing bacterium[4,5,29].

The change to use xylose as substrate resulted also in a more diverse microbial community.LS-inoculated MFCs became dominated byE.cancerogenus,G.sulfurreducens,C.braakiiandP.hippie.The presence of a more diverse microbial community after addition of xylose further illustrated why it took a short adaptation time for the MFCs to enrich fermentative bacteria to convert complex substrates(xylose)to nonfermentable substrates(e.g.acetate and propionate)[8].

Fig.5.Bacterial 16S rRNADGGE profiles(A)and relative abundance of G.sulfurreducens in MFCs inoculated with DW,LS and BS respectively(B).The numbers(2,3,4 and 5)in lanes name(DW_2,DW_3,……,BS_4,BS_5)means the samples were taken at the end of 2nd,3rd,4th and 5th cycle,respectively.The identified bands(1-11)are presented in Table 4.UB indicates bands not identified by cloning.Letters A-C indicates significant difference at 95% confidence limit.

3.5.3.Quantification of G.sulfurreducens

Composite analysis ofthe DGGE bands showed different proportions ofG.sulfurreducensin the biofilm community(Fig.5B).When acetate was added to MFCs(cycle 2),LS-inoculated MFCs had the highest percentage ofG.sulfurreducens(18%±1%)compared to DW and BS with 12%±0.4%and 11%±3%,respectively.The high proportion ofG.sulfurreducensin LS-inoculated MFCs may further explain the higherImax(Table 3).These results are also corroborated by Liet al.showing that DW-inoculated MFCs produced much higher MPD(33 mW·m-2)than activated sludge inoculated MFCs(23 mW·m-2)with the predominance ofGeobacter pickeringiiandMagnetospirillumsp.in the wastewater inoculated MFCs[16].However,the abundance of these species has not been quantified.

After xylose was added to the MFCs(cycle 4),the proportion ofG.sulfurreducensdecreased to 6%-11%.This may be due to that xylose boosts the growth of fermentative bacteria,which resulted in a significant drop in CE(Table 3).However,the concentration ofG.sulfurreducensincreased after two cycles of MFC operation to 13%±0.3%in LS-inoculated MFCs,which was higher than with DW(11%±0.2%)and BS(10%±0.3%).These results show thatImaxincreased versus the abundance of electrogenic bacteria(mostG.sulfurreducenswas found with the LS inoculum).

3.6.Effects of Rext and Lsub on microbial community and current generation

Based on DGGE band intensities in Fig.6A,the abundance ofG.sulfurreducensin the biofilm communities was estimated(Fig.6B).After 3 batches,the MFCs withRextof 200-Ω showed highestproportion ofG.sulfurreducens(21%±0.7%),followed by 18%±0.4%,16%±0.4%and 16% ± 0.4%for resistances of 500,800 and 1000 Ω,respectively.The higher abundance ofG.sulfurreducensin 200-Ω MFCs explains why they generated higherImaxand CE(Table 4).The results also indicated that the lowerRextassist the enrichment ofG.sulfurreducens,as explained as that lowerRextresults in higher electrode potential[11],which is favoured byG.sulfurreducensgrowth.When all the MFCs changed to useRextof 200 Ω,no significant difference in the proportion ofG.sulfurreducens(22%-23%)was observed.

The increase in MFC performanceversusthe abundance ofG.sulfurreducensis also reflected byIavein the MFCs with differentLsub(Table 4).The maximumIavewas(557 ± 13)mA·m-2at 200 Ω,which is almost two times higher thanIave[(285 ± 6)mA·m-2]at 150 Ω reported by Jung and Regan[13].Whereas an increase inLsubfrom 0.5 to 1.0 g·dm-3of COD had no measureable effect on the abundance ofG.sulfurreducens.In general,increasedLsubsignificantly decreased the abundance ofG.sulfurreducens(20%?12%)(Fig.6B).The increasedLsubboosted thereby enrichment of fermenting bacteria,which in turn significantly decreased CE.The increased abundance ofG.sulfurreducensresulted in an increase of CE regardless ofRextandLsub,which demonstrated that CE increasedversusthe abundance of electrogenic bacteria.The results show that lowRextand lowLsubincreased the abundance ofG.sulfurreducens,which in turn gave higherIave.

Overall SEM microscopy(Fig.4)showed dense,less diverse and highly active bacterial communities and DGGE showed high dominance ofG.sulfurreducensfor the LS inoculum(Fig.5).Both of these resultsconfirm the hypothesis that high current generation is linked to dominance ofG.sulfurreducens(Table 3).

Table 5DGGE 16S rRNA gene band identification and characterisation of the bacterial species

Fig.6.Bacterial 16S rRNA gene-derived DGGE profiles(A)and relative abundance of G.sulfurreducens with different R ext and L sub(B).The letters a-d indicating the MFCs started with 200,500,800 and 1000 Ω respectively.The numbers(3,4 and 5)in lanes name(a_3,a_4,……,c_5,d_5)means the sample were taken at end of the batch cycle 3,4 and 5 respectively.The identified bands(1-11)are presented in Table 4.UB indicates bands not identified by cloning.Letters A-C indicates significant difference.

4.Conclusions

This study showed that the lake sediment(LS)inoculated MFCs yielded higherImaxup to 1690 mA·m-2and CE up to 23%± 1%atRextof 1000 Ω.A decrease ofRextsignificantly increasedImaxand CE to 1800 mA·m-2and 59% ± 1%,respectively,while an increase ofLsubonly showed effect on CE with a decrease.On the basis of electrochemical performance and microbial community analysis,the higher abundance ofG.sulfurreducensresulted in higher MFCs performance with emphasis onImaxand CE.Elucidating the positive correlation between microbial community and electrochemical performance will assist in optimization of MFCs technology for practical application.

Acknowledgements

The authors are grateful to Danida Fellowship Centre for supporting the research project(Biobased electricity in developing countries,DFC No.11-091 Ris?).The financial support from China Scholarship Council(CSC No.2011635051)for Guotao Sun is gratefully acknowledged.Annette E.Jensen,DTU is thanked for technical support.

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