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1、Effect of external resistance on the sensitivity of microbial fuel cellbiosensor for detection of different types of pollutantsYue Yia,b,Beizhen Xiea,b,Ting Zhaoa,b,Zhaoming Lia,b,Devard Stomc,Hong Liua,baInstitute of Environmental Biology and Life Support Technology,School of Biological Science and

2、 Medical Engineering,Beihang University,Beijing 100191,ChinabInternational Joint Research Center of Aerospace Biotechnology&Medical Engineering,Beihang University,Beijing 100191,ChinacInstitute of Biology,Irkutsk State University,Irkutsk 664025,Russiaa b s t r a c ta r t i c l ei n f oArticle histor

3、y:Received 18 May 2018Received in revised form 5 September 2018Accepted 6 September 2018Available online 17 September 2018The relatively poor sensitivity is the main bottleneck restricting the application of microbial fuel cell biosensor(MFC-biosensor)for toxicity monitoring.Previous studies have sh

4、own that external resistance(Rext)had an ob-viouseffectonsensorsensitivity.However,thesestudiesreporteddifferentresultsandthereasonofthisdiscrep-ancy was not clear.The objective of this research was to observe the effect of Rexton sensor sensitivity whendetecting different types of pollutants and re

5、veal its microbiological mechanism.Results demonstrated thatthe optimal Rextof MFC-biosensor varied with the type of pollutants.The optimal values for detectingavermectins,tetracyclines and heavy metals were 100,330 and 680,respectively.This discrepancy wasmainly due to the visible differences in an

6、odic microbial communities at different Rextsettings.Both Azospirillumand Acinetobacter were susceptible to Cd and Pb,occuping 19.20%of the anodic microbial population in 680 MFC-biosensor.Pseudomonas accounted for 10.73%in 330 MFC-biosensor and possessed the sensitivity totetracyclines.As for 100 M

7、FC-biosensor,the avermectin-intolerant Ocillibacter made up 2.55%of the anodicmicrobial community.This study indicated that the Rextof MFC-biosensor should be optimized according tothe potential pollutants.2018 Elsevier B.V.All rights reserved.Keywords:Microbial fuel cellBiosensorHeavy metalAntibiot

8、icExternal resistanceMicrobial community1.IntroductionWater supply safety is critical for urban construction and social sta-bility.With the continuous development of global industrialization,the type and quantity of toxic pollutants in aquatic environment are in-creasing annually.Reliable methods of

9、 water quality monitoring are in-dispensable for the guarantee of water supply safety.Physicochemicalmethods,which are broadly applied in conventional monitoring ofwater quality,are highly sensitive and accurate.However,thesemethods are inadequate to detect unconventional pollutants and failto provi

10、de a timely warning of water pollution.Moreover,thesemethodscannotgiveanindicationofbiologicaltoxicity.Genericbiosen-sors can compensate for the deficiencies of physicochemical methods.Fish,algae,invertebrates and bacteria are suggested as common indica-tor organisms 1.Amongvarious types of biosenso

11、rs,microbialfuel cellbiosensors(MFC-biosensors)have attracted growing interest in thearea of water quality alert in the last decade.Compared with other bio-sensors,MFC-biosensor can be self-sustainable without the require-ments of additional signal transducer devices and special chemicals,which make

12、s it a promising application in water quality monitoring2,3.MFC is a device which utilizes electricigens as catalysts and simulta-neously converts chemical energy from organic matter into electricity4.Presence of toxic agents can inhibit the activity of electricigens,and further leads to a decay in

13、output signal.Hence water quality canbe monitoredin real-timebymeasuringthechange of MFC outputvolt-age.The first biomonitoring system using MFC was reported by Kimet al.5.They installed the system at the inlet of a wastewater treat-ment plant and found that toxic substances including Pb2+and Cd2+co

14、uld inhibit MFC current,demonstrating the feasibility of monitoringbiotoxicitybyMFC.Sincethen,aseriesofconstructionalandoperationalparameters including external resistance(Rext)6,configuration 7,electrode material 8,contact time 9,shear rate 10 and flow mode11 were optimized to enhance sensor sensit

15、ivity.Among these parameters,Rextis considered to be an important fac-tor which directly impacts the power generation of MFC-biosensorand further affects sensor sensitivity 12,13.An early study reportedthat increasing Rextsettings(100,470,1000)had an adverse ef-fect on the sensitivity of MFC-biosens

16、or for detecting sodium dodecylsulfate 6.When monitoring a heavy metal shock induced by Cu2+,Jiang et al.11 observed that the sensitivity increased firstly,and thenreduced with the decrease of Rextfrom 3000 to 50.Another studyBioelectrochemistry 125(2019)7178 Corresponding author.E-mail address:(B.X

17、ie).https:/doi.org/10.1016/j.bioelechem.2018.09.0031567-5394/2018 Elsevier B.V.All rights reserved.Contents lists available at ScienceDirectBioelectrochemistryjournal homepage: by Yi et al.14 revealed that MFC-biosensor designed fortesting Cd2+obtained the highest sensitivity when Rextwas equal to50

18、0,andhigherorlowerRextsettingswouldbothdeterioratethesen-sitivity.Interestingly,these studies reported inconsistent effects of Rexton sensor sensitivity when monitoring different types of pollutants.This phenomenon suggested that the optimal Rextmight vary with thetype of pollutants but a comparison

19、 about the optimal Rextfor organicand inorganic pollutants has not been revealed yet.Besides,theinfluencing mechanism of Rextwas not clear.It was found in all the pre-viousstudiesthatRexthadsimilareffectsonMFCpowergeneration.Thiscould not explain the different effects of Rexton sensor sensitivity wh

20、endetecting various pollutants.Therefore,the mechanism needed to befurther studied.From the microbiological perspective,Rexthad a significant effect onanodic community structure 15,16.The species and distribution ofelectricigens had obvious differences under different Rextvalues17,18.Considering the

21、 discrepancies in microbial resistance to differ-ent types of toxic pollutants,it could be deduced that the optimal Rextmight vary with the type of pollutants.That is to say,Rextcould impactthemicrobialcommunitystructureofMFC-biosensorandfurtheraffectsits sensitivity.In this research,our objective w

22、as to investigate the effect of Rextonsensor sensitivity when detecting different types of pollutants and re-veal its microbiological mechanism.For this purpose,MFC-biosensorsloaded with different Rextvalues were constructed.After steady-operation,toxic pollutant tests were performed to study the re

23、lation-ship between optimal Rextand pollutant type.Three types of pollutants,including heavy metals,tetracyclines and avermectins,were tested andeach type was represented by two pollutants.Then,two types of com-bined contaminations represented by the binary metal mixture andmetal-tetracycline compos

24、ite pollutant were examined to assess thechange of optimal Rextinduced by combined contamination.Finally,high-throughput sequencing technology was employed to investigatetheanodicmicrobialcommunitiesestablishedunderoptimalRextvaluesfor different pollutants and the changes of community structures tot

25、oxic shocks.2.Materials and methods2.1.Chemicals and reagentsAnalytical reagents 3CdSO48H2O and PbCl2were purchased fromBeijing Lanyi Chemical Products Co.,Ltd.,China.Chlortetracycline hy-drochloride(CTC)and oxytetracycline hydrochloride(OTC)were ac-quired from Dalian Ronghai Biological Technology C

26、o.,Ltd.,China.Avermectin(AVM)and ivermectin(IVM)were obtained fromWuhan Bioland Biological Technology Co.,Ltd.,China.3CdSO48H2O,PbCl2,CTC,OTC were individually dissolved in deionized water to pre-pare stock solutions while AVM and IVM were dissolved in absolutemethanol respectively.The concentration

27、 of the stock solution foreach toxic pollutant was 1.0 g/L and all were preserved in 4 C refrig-erator for further use.Anolyte and catholyte were prepared prior touse.The fresh anolyte used in this study consisted of 5.85 g NaCl,0.13gKCl,0.31gNH4Cl,6.08gNaH2PO42H2O,21.83gNa2HPO412H2O,12.50 mL trace

28、minerals solution and 5.00 mL vita-min solution in 1.00 L deionized water 19.The catholyte consistedof 5.85 g NaCl,6.08 g NaH2PO42H2O and 21.83 g Na2HPO412H2O in1.00 L deionized water.During the start-up process,sodium acetate(NaAc)was added to fresh anolyte as carbon source to achieve afinal concen

29、tration of 0.82 g/L.When toxic pollutants tests were per-formed,the concentrations of NaAc in non-toxic and toxic anolytewere switched to 0.082 g/L to enhance sensor sensitivity 20.Toxicand non-toxic anolyte was prepared by adding a certain volume oftoxic stock solution and the same volume of solven

30、t in fresh anolyte,respectively.2.2.MFC construction and start upFifteen MFCs(MFC115)of optimized configuration(Fig.A.1)wereconstructed and divided into two groups.MFC19 with different exter-nal loads(100,330,680),were constructed to optimize Rextforvarious pollutants.For each Rextvalue,three MFCs w

31、ere set to test thereproducibility of results and perform statistics analysis.The othergroup(MFC1015)was used for subsequent analysis of anodic micro-bial community.All parameters except for Rextwere consistent in eachMFC.The working volume of the anodic chamber was 13 mL while thecathodic chamber w

32、as 25 mL.Proton exchange membrane(Nafion117,Hesen Electric Corporation,China)was used to divide the twochambers.A3.0cm2.5cmpieceofcarboncloth(HCP330,HesenElec-tric Corporation,China)treated by ammonia 21 was used as anode.Cathode consisted of two 2.0 cm 2.0 cm pieces of carbon paper con-taining 0.5

33、mg/cm2Pt catalyst.Air was sparged into the catholyte toallow oxygen reduction on the cathode.To investigate the effect ofRexton start-up process,the incubation conditions kept identical andthe anolyte of each MFC was refreshed simultaneously.All the MFCswere inoculated with the effluent taken from a

34、n acetate-fed MFCusing the same inoculum size of 30%(v/v).Each feed replenishmentwas performed by pumping anoxic fresh anolyte containing 0.82 g/LNaAc with a volume of 28.00 mL into anodic chamber.During thestart-up process,the anolyte was forced to flow through the anodicelectrode 11 with the appli

35、cation of self-circulation at a flow rate of2 mL/min.All the MFCs were maintained at a temperature of 25.0 0.5 C during all experiments.2.3.Toxic pollutants testsAfter the start-up process,the MFC-biosensors(MFC19)were fedwith a continuous flow of influent and challenged with a series oftoxicpolluta

36、nts.Theflowratewasmaintainedat2mL/min.Theconcen-trations of organic pollutants(tetracyclines and avermectins)were1.0 mg/L with reference to a previous study 5.The concentration ofCd2+as well as Pb2+was 1.5 mg/L according to a previous studywhich indicated that electricigens were tolerant to heavy me

37、tals to acertain extent 22.Combined contaminations consisted of two binarymixtures:1.5 mg/L Pb2+was mixed with 1.5 mg/L Cd2+and 1.0 mg/LOTC respectively.The detection procedure comprised of three steps.Firstly,non-toxic anolyte was continuously pumped into the anodicchamber.Secondly,whentheoutputvol

38、tageofeachMFC-biosensorsta-bilized for more than two hours,the influent was switched to the toxicanolyte containing a certain concentration of target toxic pollutant andthe exposure time was one hour.Finally,toxic anolyte was dischargedfrom the anodic chamber and simultaneously non-toxic anolyte was

39、continuously pumped to remove residual toxic pollutants inside thebiofilm.EachtoxicshockwasperformedintriplicateforoneRextsetting.2.4.Microbial community analysisTo investigate the biological mechanism of the discrepancy in opti-mal Rextvalues for various pollutants,six MFCs(MFC1015)were con-structe

40、d at the optimal Rextsettings of the six pollutants above,respectively.After the start-up process,non-toxic anolyte containing0.082 g/L NaAc was continuously pumped into the anodic chamber for15 days.Then sterilized scalpel was used to collect a 3.0 cm 0.3 cmpiece of carbon cloth sample from the sam

41、e position in the anode ofeach MFC.All the samples were stored at 80 C refrigerator afterquick-frozen with liquid nitrogen.Then each MFC was challengedwith the toxic anolyte containing a certain concentration of targettoxic pollutant in the next seven days.The concentrations used herewere identical

42、to the above description.After exposure to toxic pollut-ants for seven days,anodic biofilms were all sampled again.Samplingtechnique and storage method were mentioned above.72Y.Yi et al./Bioelectrochemistry 125(2019)7178DNA extraction of all the samples and the quality control of DNAsamples were per

43、formed according to Xie et al.23.The hypervariableregion V3-V4 of the 16S rRNA gene of all the samples was amplified bypolymerase chain reaction using primers 338F(5-ACTCCTACGGGAGGCAGCA-3)and 806R(5-GGACTACHVGGGTWTCTAAT-3).Each samplewas sequenced by Illumina Hiseq platform at Biomarker Technologies

44、Co.,Ltd.,China.Raw sequence data were filtered firstly and then high-quality sequences were clustered into operational taxonomic units(OTUs)at a similarity threshold value of 97%by QIIME software.Atthe classification level of OTU,Principal Coordinates Analysis(PCoA)was performed using the binary-jac

45、card algorithm by R software(3.5.1,The R Foundation for Statistical Computing,www.r-project.org)to analyze the differences among microbial communities formedunder different Rextsettings.2.5.AnalysisThe outputvoltage of MFC was recordedat a samplingintervalof 1 susinga data acquisition system(USB-160

46、8FS,Measurement ComputingCorporation,USA).The internal resistance of MFC was characterized byelectrochemical impedance spectroscopy(EIS).Before EIS measure-ment,the MFC was disconnected with the external resistor for at least3 h to obtain a stable open circuit voltage 24.EIS was measured inthe dual-

47、electrode mode using an electrochemical workstation(Zennium E,Zahner Company,Germany)at open circuit in a frequencyrange of 100 mHz to 10 KHz with a signal of 10 mV amplitude.EIS datawas fitted with an equivalent circuit model(Rs+CPE1/R1+CPE2/R2)according to a previous study 25.Rsrepresented the ohm

48、ic resistance.R1and R2referred to the polarization resistance of cathode(Rc)andanode(Ra)respectively.Two constant phase elements(CPE1andCPE2)were used to simulate nonideal capacitors.The inhibition ratio(IR)of output voltage was used to assess the sensitivity of MFC-biosensor in order to eliminate t

49、heinterference of baselinesignal in dif-ferent MFCs 9,26.It was defined as the relative change of output volt-age after toxic shock and calculated according to eq.(1):IR VnVt.Vn?100%1where Vnwas the voltage before thetoxic shockandVtwasthevolt-age after exposure to the toxic pollutant for one hour.O

50、ptimal Rextwasdetermined when the maximum IR was picked out in the MFC-biosensors with different Rextsettings.Statistical analysis was con-ducted via SPSS Statistics 22.Significance test was used to analyze theresponses of MFC-biosensors operated at different Rextsettings to eachtoxic pollutant via