生化需氧量传感器生物膜电极的初步研究

2013年度本科生毕业论文生化需氧量传感器生物膜电极的初步研究学院：理工学院专业：环境工程年级：2009级学生姓名：刘永福学号：09222069导师及职称：胡锐(讲师)2013年5月 StudyonefficientmicrobialmembrancewithmarineyeastappliedinBODsensorforfisheryseawateroffshoreDepartment:SchoolofSicenceandTechnologyMajor:EnvironmentEngineeringGrade:2009Student’sName:YongfuLiuStudentNo.:09222069Tutor:LecturerRuiHuFinishedbyMay,2013 毕业论文原创性声明本人所呈交的毕业论文是我在导师的指导下进行的研究工作及取得的研究成果。据我所知，除文中已经注明引用的内容外，本论文不包含其他个人已经发表或撰写过的研究成果。对本论文的研究做出重要贡献的个人和集体，均已在文中作了明确说明并表示谢意。作者签名：日期：毕业论文授权使用说明本论文作者完全了解琼州学院有关保留、使用毕业论文的规定，学校有权保留论文并向相关部门送交论文的电子版和纸质版。有权将论文用于非赢利目的的少量复制并允许论文进入学校图书馆被查阅。学校可以公布论文的全部或部分内容。保密的论文在解密后适用本规定。 作者签名：指导教师签名：日期：日期： 刘永福毕业论文答辩委员会(答辩小组)成员名单姓名职称单位备注秦玉春教授理工学院主席（组长）李丽蓉讲师理工学院薛长风副教授理工学院王玉杰讲师理工学院 琼州学院本科生毕业论文摘要生化需氧量(BOD)是监测水体中可生物降解的有机物含量的一个重要参数,是目前最常用的水体有机污染综合指标之一。其标准测定方法为五日法,以被检验的水样在20±1℃下,5天内的耗氧量为代表,称其为5日生化需氧量,简称BOD5。但该方法操作较复杂,耗时长,结果准确度及重现性差,无法满足当前环境监测中快速测定的要求。而日益得到广泛应用的微生物传感器法能够快速、准确测定水体中BOD,并可实现在线连续测定,更加适应于现代环境监测和管理的要求。本文阐述了BOD微生物传感器技术的研究进展及应用。分析了微生物传感器的工作原理及其在水质测定中的优点,重点研究了其核心部分微生物膜的研究现状以及制备过程中存在的问题。针对微生物膜的研究现状,以菌种选育和固定化方法的选择作为本次实验研究的重点。本文综述了微生物固定化方法在各类废水处理中的研究现状,对固定化方法进行了分类,并对其性能作了比较。通过比较三种微生物膜电极的底物适用范围、线性范围及其测量结果的准度和精密度，选择了对数生长期的汉逊德巴利海洋酵母Debaryomyces hanseniiHXY-09构建的微生物膜电极作为实验所用电极。进一步对近海养殖水体BOD含量的检测结果有显著影响的待测样品底液pH、电极工作温度和底液离子浓度3个因素为自变量，以传感器的响应电流为响应值，研究了外部检测条件的变化对实验结果的影响，为该复合功能菌群进一步研究近海养殖水体BOD传感器奠定基础。关键词：生化需氧量；微生物膜；微生物固定化；微生物传感器；溶氧电极 琼州学院本科生毕业论文ABSTRACTBiochemicaloxygendemand(BOD)isanimportantparameteroftheorganicmattercontentofbiodegradablemonitoringinwater,isoneofthecomprehensiveindexoforganicpollutioninwateristhemostcommonlyusedatpresent.Thestandarddeterminationmethodforfivedays,towatersamplestestedat20±1℃,oxygenconsumptionof5daysastherepresentative,calledthe5daybiochemicaloxygendemand,referredtoasBOD5.Butthemethodiscomplicated,time-consuming,accuracyandreproducibilityispoor,unabletomeettherapiddeterminationofpresentenvironmentalmonitoringrequirements.Ithasbeenwidelyusedinmicrobialsensormethodcanfast,accuratedeterminationofBODinwater,andcanrealizecontinuousonlinemeasurement,moresuitableformodernenvironmentalmonitoringandmanagementrequirements.Inthispaper,theresearchprogressandapplicationofBODmicrobialsensortechnology.Analysisoftheprincipleofmicrobialsensoranditsadvantagesinmeasurementinwater,hasfocusedonitscorepartofbiofilmresearchstatusandproblemsintheprocessofpreparation.Accordingtotheresearchstatusofmicrobialmembrane,tothebreedingandtheimmobilizedmethodchosenasthekeyresearchofthisexperiment.Thispapersummarizedthepresentsituationofresearchonmicrobialimmobilizationmethodinallkindsofwastewaterdisposal,theimmobilizationmethodsareclassified,andcompareditsperformance.TheDebaryomyceshanseniibasedbiosensorischaracterizedbythehighestsensitivityand,accordingly,theminimallowerlimitoflinearrange.Thus,theDebaryomyceshanseniibiosensorhasthebestcharacteristicsofthosetested.ResponseSurfaceMethodology(RSM)wasusedtoinvestigatetheeffectsofpH,Temperatureandconcentrationbuffersontheresults.A33factorialdesignwasperformedtooptimizetheconditions.Theexperimentalresultsshowedthattheoptimalconditionsare:pHof7.7,temperature20℃andconcentrationbuffers40mmol/L,andthesmallrelativeerrorof1%betweenbiosensorandBOD5.Thisstudy 琼州学院本科生毕业论文indicatesthatDebaryomyceshanseniibasedbiosensorcanbeusedasareliablemethodtodetermineBODinseawater,thelinearfluctuantcoefficients(R2)intherangeof9.3~422mg/dm3whenaglucose/glutamateBODstandardwasapplied.Suchdeviceprovidesalow-cost,easy-operated,fast-response,sensitiveandreliablemethodtomeasureBODinseawater,andalsoissuitableforon-linemeasurements.Keywords:BiochemicalOxygenDemand(BOD);MicrobialFilm;ImmobilizedMicroorganism;MicrobialSensor;DissolvedOxygenEletrode 琼州学院本科生毕业论文目录第一章绪论11.1问题的提出11.1.1传统测量方法存在的问题11.1.2BOD微生物传感器的产生11.2课题研究的目的和意义21.3课题研究的内容3第二章实验材料和方法42.1材料42.1.1菌种与来源42.1.2培养基42.1.3试剂和仪器42.2海洋酵母活菌总数与生长周期[10]42.3BOD标准溶液的配制52.4微生物膜电极的制备52.5近海养殖水体中BOD微生物传感系统的组装62.6样品的测定8第三章结论和分析93.1三株海洋酵母菌株在不同时期的相关酶活力的变化93.2三株海洋酵母菌株构建的微生物膜电极底物适用性93.3缓冲溶液PH值对响应电流影响103.4温度对响应电流影响123.5不同缓冲溶液体系对响应电流影响123.6缓冲溶液离子强度对响应电流影响13 琼州学院本科生毕业论文第四章小结15参考文献16致谢18 琼州学院本科生毕业论文第一章绪论1.1问题的提出1.1.1传统测量方法存在的问题生物化学需氧量(BOD)是表示水中有机物等需氧污染物质含量的一个综合指标，它说明水中有机物由于微生物的生化作用进行氧化分解，使之无机化或气体化时所消耗水中溶解氧的总数量，其单位以mg/L表示，其值越高，说明水中有机污染物质越多,污染也就越严重。以悬浮或溶解状态存在于生活污水和制糖、食品、造纸、纤维等工业废水中的碳氢化合物、蛋白质、油脂、木质素等均为有机污染物，可经过好氧细菌的生物化学作用而分解，由于在分解过程中消耗氧气，故亦称需氧污染物质。若这类污染物质排入水体过多,将造成水中溶解氧缺乏，同时有机物又通过水中厌氧菌的分解引起腐败现象，产生甲烷、硫化氢、硫醇和氨等恶臭气体，使水体变质发臭。污水中各种有机物得到完全氧化分解的时间，总共约需100天，为了缩短检测时间，一般生化需氧量以被检验的水样在20℃下，5天内的耗氧量为代表,称其为5日生化需氧量,简称BOD5，对生活污水来说,它约等于完全氧化分解耗氧量的70%。生化需氧量的经典测定方法是稀释接种法,此外还有检压式库仑法、短时日法、坪台值法、相关估算法和瓦勃呼吸法等[1]。但是无论库仑滴定法、差压法和瓦勃呼吸计法还是标准稀释测定法均有许多不足之处，例如操作复杂、重现性差、耗时耗力、干扰性大、不宜现场监测等，不能及时为环境管理和决策、科研、事故污染鉴定等提供科学依据。因此，快速、准确测定水体中BOD一直是环境监测中的一大难题[2]。1.1.2BOD微生物传感器的产生环境监测中快速测定的要求迫切需要一种操作简单、准确、快速、自动化程度高和适用范围广的新方法来测定BOD，因此生物传感器监测BOD的技术和方法应运而生，与传统的标准稀释测定法通过绝对耗氧量计算BOD不同，1 琼州学院本科生毕业论文生物传感器测定BOD只涉及到初始氧化速率，两者之间的相关性可以通过对标准溶液的测定获得，这就可以将测定时间缩短到lh以内。1977年，Kurabe等研制了第一台BOD微生物传感器[8],它由固定化活性污泥与氧电极构成,仅几十分钟就可完成BOD测量,大大减少了BOD的分析时间,降低了分析成本,实现了BOD快速测定。Kurabe最初研制的传感器寿命仅有10天，后改用多孔醋酸纤维膜固定酵母细胞制成的微生物传感器，可连续使用17天。Strand等(1984)[9]研制BOD传感器,用于城市污水测定,成功工作了20多天，后来在微生物、换能器、测量原理方面有了许多改进，出现了各种各样的BOD传感器,我们国家在20世纪80年代起也有一批学者进行着不断地研究和探索。如河北科技大学的孙裕生[3]。复旦大学的邓家棋[4]。天津环境监测中心的魏恩棋[5]等人均发表过BOD微生物传感器的研究论文。日本在世界上率先研制成以微生物电极为传感器的工业快速BOD测定仪,并于1990年颁布了微生物电极法工业标准（JISK3602-1990）[6]。我国也于2002年颁布了该方法的行业标准（HJ/T86）[7],为该类仪器的市场准入制定了工作标准。随着研究的不断深入，不同的BOD快速检测仪问世，在一定程度上弥补了传统方法测定水中BOD的不足，并逐渐出现了可用于测定废水中BOD值的生物传感器和适于现场测定的便携式测定仪。随着BOD决速测定研究的不断深入，还有研究发现BODst(快速BOD测定值)还可作为在线监测生物处理过程的一个重要参数，因此，生物传感器监测BOD对于水质的实时在线监测以及提高水质污染的监测水平和废水处理过程的控制水平，均有十分重要的意义。对BOD决速测定仪的研究也成为水质检测科技发展的方向。至今，普通废水BOD传感器已经相对成熟，并己市场化，但选择不同的微生物膜，适宜的仪器及适宜的分析条件，一直是环境监测者竞相探讨的重点问题[16]。1.2课题研究的目的和意义生物传感器缩短了水体BOD测定周期，节省了人力、物力、财力，具有高选择性，高灵敏度，较好的稳定性，低成本特点。更重要的是能及时为管理和决策部门为掌握地表水，工业废水和生活污水排放现状（尤其是污染事故的发生）提供科学决策的依据，同时也为工业企业污水治理，污水处理厂的污水治理工艺设计、控制、处理效率及时提供参数。可以预见，该仪器的完善。成功研制将带1 琼州学院本科生毕业论文来较大的环境效益和社会效益,将会成为最具潜力的环境监测工具。而微生物膜是微生物传感器的核心部分，所选用的菌株种类、数量及其固定化方法决定了微生物传感器的响应特性和使用寿命，因此高效微生物膜的制备是研究的重点[12.13]。本课题组主要对目前微生物传感器中微生物膜制备这一核心技术进行研究，旨在确定微生物膜制作的适宜菌种。固定化方法及材料，确定切实可行的微生物膜制备工艺，以得到性能稳定、活化周期短、对待测溶液浓度变化适应性强。使用寿命长的微生物膜，满足微生物传感器的使用。1.3课题研究的内容本研究首次从三亚近海养殖水体污染严重的水域分离纯化到三株耐盐、耐高压的海洋酵母菌，通过相关理化实验、发酵谱和碳源同化谱TheYeast,ATaxonomicstudy(Kurtzman&Fell,2000)书中的标准菌株进行比对，初步确认获得的三株海洋酵母菌为DebaryomyceshanseniiHXY-09,PichiaguillermondiiHXY-04和CandidaparapsilosisHXY-06[4]。进一步采用夹膜法将上述三株目标菌株玻璃纤维滤纸直接固定化制备成微生物膜，安装于Clark溶氧电极初步构建近海养殖水体BOD微生物传感系统，分析了各种因素对传感系统的响应特性的影响，优化了传感器的监测条件，为实现近海水体BOD测定的数据处理自动化控制奠定基础。1 琼州学院本科生毕业论文第二章实验材料和方法2.1材料2.1.1菌种与来源酵母菌DebaryomyceshanseniiHXY-09，PichiaguilliermondiiHXY-04和CandidaparapsilosisHXY-06三株菌分别以高分子有机污染物为唯一碳源，从海南省三亚海宝近海养殖专业合作社养殖场（拐点坐标（N=18°14′08.608671″，E=109°21′03.218849″）水面下约0.5m）中多轮分离筛选获得（具体筛选和鉴定实验另见文章报道）。2.1.2培养基酵母菌培养基：葡萄糖10g/L、胰蛋白胨5g/L、酵母膏0.5g/L、(NH4)2HPO410g/L、KH2PO420g/L、MgSO4.7H2O0.5g/L、CaCl2.2H2O0.3g/L，海水配制，8磅15min灭菌。2.1.3试剂和仪器聚乙烯醇(PVA1799±50)、氧气(99.99%)由三亚市人民医院提供，其它化学试剂均为国产分析纯；GC9800型气相色谱仪购自上海科创色谱有限公司，PascoCI-6542溶解氧传感器和ScienceWorkshop500数据处理系统（PascoScientific,Roseville,CA），Potentiostat/Galvanostat283A型电化学测试系统，M270数据处理软件，TGL-16G型台式离心机购自上海安亭科学仪器厂[14]。2.2海洋酵母活菌总数与生长周期[10]无菌操作取事先已灭菌的15cm玻璃试管8支，依次编号1~8号，分别加入9mL灭菌空白培养基，用1mL的无菌微量移液器吸取DebaryomyceshanseniiHXY-091 琼州学院本科生毕业论文菌悬液，沿试管壁缓慢注于盛有9mL空白培养基的1号管中（注意吸头尖端不要触及稀释液面），用涡旋式振荡器（德国，Heidolph）充分混合均匀，制成10倍稀释的发酵液，按照上一步骤的操作方法，依次制备10倍系列稀释的2-8号管，每递增稀释1次换用1支无菌吸头；从5-8号管中各取0.2mL稀释液入固体培养基中，用经火焰灭菌的玻璃棒涂抹均匀，每个稀释度做2个平皿。同时分别吸取0.2mL空白培养基入2个无菌平皿内作空白对照。选取菌落数在30-300CFU（Colony-FormingUnits，菌落计数单位）之间、无蔓延菌落生长的平板计算菌落总数。低于30CFU的平板可记录具体菌落数，大于300CFU的平板记录为多不可计。每个稀释度的菌落数可以取2个平板的平均数，若只有一个稀释度平板上的菌落数在适宜计数范围内，计算2个平板菌落数的平均值，用平均值乘以相应稀释倍数后再乘以5，作为每毫升发酵液中活菌总数结果。若有2个连续稀释度的平板菌落数在适宜计数范围内时，则按下式计算，N=∑C/(n1+0.1n2)5d。式中：N为活菌总数，∑C为平板（含适宜范围菌落数）菌落数之和，n1为第一稀释度（低稀释倍数）平板个数，n2为第二稀释度（高稀释倍数）平板个数，d为稀释倍数（第一稀释度）。并以菌株的培养时间为横坐标，以菌株的生长速率为纵坐标作图，既得该菌株的生长曲线。2.3BOD标准溶液的配制实验采用的标准溶液为GGA溶液[8]，即：150mg/L谷氨酸和150mg/L葡萄糖，15mg/LKH2PO4，30mg/L(NH4)2SO4，50mg/LMgSO4.7H2O，3.75mg/LCaCl2，0.25mg/LFeCl3.6H2O，5.0mg/LMnSO4.H2O，105mg/LNaHCO3，10mL/L微量元素。微量元素成分为：1.5g/L氨三乙酸,0.1g/LFeSO4.7H2O,0.1g/L，MnCl2.4H2O,0.17g/LCoCl2.6H2O,0.1g/LCaCl2.2H2O,0.1g/LZnCl2,0.02g/LCuCl2.2H2O,0.01g/LH3BO3,0.01g/L钠钼酸盐,0.017g/LNa2SeO3,0.026g/LNiSO4.6H2O,1g/LNaCl,0.1g/LNa2WO4.7H2O。该标准溶液的BOD为(200±10)mg/L,通过稀释得到不同BOD值的标准溶液,用磷酸缓冲液调节pH至7.0±0.1,-20℃冷藏备用。1 琼州学院本科生毕业论文2.4微生物膜电极的制备高速冷冻离心机2000rpm/min离心收集液体培养物菌体10min，用磷酸盐缓冲溶液（20mmol/L，pH6.8）洗涤菌体洗涤菌体3次，用Tris-Buffer(三羟甲基氨基甲烷缓冲溶液)以150mg/cm3溶解菌体保存备用。量取3μl上述菌液用玻璃纤维滤纸（WhatmanGF/A:1.6μm，美国Sigma）固定化制备复合功能菌群生物膜（3×3mm2），并于空气中20℃干燥15min备用。再将已切成适于装配尺寸的圆形复合功能菌群生物膜用磷酸盐缓冲溶液润湿，用镊子轻轻地夹起放在Clark氧电极的塑料薄膜上，装上带有垫圈的电极帽并旋紧。2.5近海养殖水体中BOD微生物传感系统的组装图2-2为实验中构建的近海养殖水体BOD微生物传感系统示意图，将溶解氧电极的内腔注满电解液（氯化钾-四硼酸钠溶液），在阴极表面覆盖一层聚四氟乙烯薄膜，再将已切成适于装配尺寸的圆形微生物膜用磷酸盐缓冲溶液润湿，用镊子轻轻地夹起放在氧电极的塑料薄膜上，装上带有垫圈的电极帽并旋紧，保证两层膜紧密接触，将装配好的电极插入含有10mL的50mmol/L磷酸盐缓冲液的检测室中，通过流量计控制待测溶液进入检测室中。如图2-1所示，BOD传感器的微生物膜电极一般是由固定化的微生物膜与氧电极紧密结合而组成。当传感器处于氧饱和的磷酸盐缓冲缓冲溶液中时，微生物处于内源呼吸阶段，其呼吸活性是恒定的，当溶解氧扩散进入氧电极表面的速率达到恒定时，BOD电极输出的电流达到稳定，此时的电流值达到最大值。当含有一定浓度缓冲液的BOD标样（或水样）加入时，水样中溶解性可生化降解的有机物被微生物作为营养源所利用，同时微生物呼吸活性加强，消耗溶液中的溶解氧，相应的其扩散进入电极表面的速率减小，输出电流值降低，并在几分钟内达到新的稳定态，上述两种稳定电流值之差，与被测试样浓度呈线性关系，藉此可以进行BOD值的测定。1 琼州学院本科生毕业论文图2-1近海养殖水体BOD生物传感系统示意图图2-2近海养殖水体中BOD微生物传感器生物膜的工作机理1 琼州学院本科生毕业论文2.6样品的测定测试时以缓冲溶液为空白溶液，活化电极10~15min，此时微生物处于内源呼吸阶段，由溶液扩散到氧电极的氧达到扩散平衡时，BOD电极输出的电流初值达到稳态，切换至测试样品状态，微生物降解样品中有机物导致溶液溶解氧浓度降低，BOD电极输出的电流随之降低，一定时间后电流差值达到稳态。在一定条件下电流差值与被测试样浓度呈线性关系，由已知标准样品求出两者之间的线性关系,可测得样品的BOD浓度。1 琼州学院本科生毕业论文第三章结论和分析3.1三株海洋酵母菌株在不同时期的相关酶活力的变化BOD生物传感器应用于近海养殖水体污染的监测时，结果的准确性和精密度取决于微生物膜电极上固定化海洋酵母菌株胞内或胞外相关酶氧化活力大小，而菌株在不同的生长周期中酶活力存在差异。因此，为了构建高效、灵敏的生物膜电极，比较了三株海洋酵母菌株在不同时期的相关酶活力的变化，结果如表1所示。表3-1三株酵母生长时期的酶活性出现的时期表生长时期毕赤海洋酵母菌株汉逊德巴利海洋酵母菌平滑假丝酵母适应期(h)0-140-180-8指数生长期(h)14-3818-308-20平稳期(h)38-4230-4020-30衰亡期(h)42-4840-4830-48最大酶活期(h)182414据表3-1可知，毕赤海洋酵母菌株、汉逊德巴利海洋酵母和平滑假丝酵母存在不同的生长周期，，毕赤海洋酵母菌株和汉逊德巴利海洋酵母较平滑假丝酵母晚进入稳定期，具有较长的滞后期和对数生长期。表1说明海洋酵母菌株细胞内外最大酶活出现的时间不同表明，并非细胞生长越旺盛，菌株相关酶活力就越大，3株菌的最大酶活均出现在对数生长期。因此，在固定化微生物细胞构建生物膜电极时，我们应该选择在活化培养基中培养18h的赤海洋酵母菌株、24h的汉逊德巴利海洋酵母和14h的平滑假丝酵母的海洋酵母细胞固定化构建微生物膜电极。3.2三株海洋酵母菌株构建的微生物膜电极底物适用性以生物传感器为例，电极的选择性是其生物膜上的固定化微生物细胞对于底物的专一性决定的。而对于近海养殖水体的BOD1 琼州学院本科生毕业论文传感器来说，如果其微生物膜电极具有广泛的底物适用范围，有利于增加检测结果的准确性和精密度。因此，我们选择了20种容易造成近海养殖水体富营养化的有机污染物作为底物[10]，考察了3株固定化海洋酵母菌株构建的生物膜电极的底物适用范围，据图1可知，只有汉逊德巴利海洋酵母Debaryomyces hanseniiHXY-09构建的BOD传感器能够对20中底物均具有响应电流，因此选择汉逊德巴利海洋酵母Debaryomyces hanseniiHXY-09作为目标菌株，固定化构建近海养殖水体BOD生物传感器的生物膜电极。图3-1株海洋酵母构建BOD传感器生物膜电极底物适用范围（底物浓度，0.09mol/dm3）3.3缓冲溶液pH值对响应电流影响为了确定检测体系的最佳pH值，在缓冲溶液pH为3~12的范围内考察了BOD生物传感器的固定化微生物膜电极对标准BOD溶液的响应特性，实验结果如图2所示。1 琼州学院本科生毕业论文图3-2不同pH值对DebaryomyceshanseniiHXY-09微生物膜电极响应电流的影响（GGA3g/dm3）传感器的电流输出值随着体系pH的增高而增大，直至7.2时达到最大。当继续增大pH值，响应电流迅速下降。这是因为不同的酶，氨基组成不同，则酶分子中可解离的基团性质不同，过高或过低的pH值会影响酶活性中心构象及其解离状态，在海洋酵母细胞相关酶的最适pH值范围内，酶分子上活性基团的解离状态最适于酶与底物的结合。而高于或低于最适pH值范围酶活性部位基团解离状态均不利于酶与底物的结合[18]。1 琼州学院本科生毕业论文3.4温度对响应电流影响图3-3不同温度值对DebaryomyceshanseniiHXY-09微生物膜电极响应电流的影响（GGA3g/dm3）温度对酶的活性有较大的影响，因此在20℃-55℃之间研究温度对传感器的影响。据图3-3可知，随着温度的升高，溶液中溶解氧浓度下降值增大，在40℃响应值下降，可能的原因是酶在较高的温度下，能够获得较高的反应活性，从而加快了酶促反应过程中氧的消耗速率，从而得到一个较大的氧浓度改变信号，既响应电流达到最大值。尽管升高温度有利于加快酶促反应的速率，但酶是蛋白质，在高温时容易变性。这样一来会降低传感器使用寿命。同时温度对氧气在溶液中的溶解度有较大的影响，温度高时溶解度会下降。因此，增加温度会导致溶解氧浓度的降低以至于产生较窄的线性范围。3.5不同缓冲溶液体系对响应电流影响不同的缓冲体系往往影响酶的活性[11]。为了测试缓冲体系对生物传感器的影响，在pH均为7、体积均为10mL、浓度均为70mmol/L的磷酸缓冲体系、醋酸盐缓冲体系和柠檬酸缓冲体系中分别加入等量的BOD标准溶液，得到传感器响应电流曲线，如图3-4所示。1 琼州学院本科生毕业论文图3-4不同缓冲溶液体系对DebaryomyceshanseniiHXY-09微生物膜电极响应电流的影响（GGA3g/dm3）在磷酸盐缓冲体系中传感器获得最大响应电流，可能的原因是醋酸盐缓冲溶液和柠檬酸缓冲体系与生物传感器中海洋酵母产生的相关底物的氧化酶的活性中心铜离子发生络合反应，从而降低了酶的生物活性[15]，故本实验选用磷酸盐体系做底液。3.6缓冲溶液离子强度对响应电流影响室温下，在pH值为7.0、体积均为10mL、浓度为1、25、50、70、100、200、300、400、500mmol/L磷酸缓冲溶液中分别加入等量的BOD标准溶液，得到传感器响应电流曲线。由图3-5可知：随着离子强度的增大，生物传感器的响应逐渐增大，但当缓冲溶液的浓度到达70mmol/L，响应电流开始下降。1 琼州学院本科生毕业论文01020304050600100200300400500缓冲溶液浓度mmol/L响应电流（nA/min）图3-5缓冲溶液中离子强度对DebaryomyceshanseniiHXY-09微生物膜电极响应电流的影响（GGA3g/dm3）1 琼州学院本科生毕业论文第四章小结采用汉逊德巴利海洋酵母微生物膜固定化溶氧电极上面构建的微生物传感器有一个比较好的响应电流，检测池中的底物要有适宜的温度、PH值，适宜的缓冲溶液离子强度。各种因素对传感系统的响应特性的影响，优化了传感器的监测条件，为实现近海水体BOD测定的数据处理自动化控制奠定基础。用纤维滤纸固定化的微生物膜具有周期性短、敏感程度高、准确度高和寿命长。比传统的稀释法测定污水节省了很多工作、人力和财力，而且更自动化。1 琼州学院本科生毕业论文参考文献[1]卢文,杨荣,任孝芬"伏安式BOD生物传感器的研究[J].化学传感器，1993，13(4):54-58.[2]HihaRastogi.BODanalysisofindustrialeffiuents:5daysto5min[J].CurrentApliedPhysies,2003,(3):191-194[3]孔裕生，刘献梅，崔健升.用于低浓度BOD微生物传感器的研究[J].环境科学.1992.13:59-63.[4]邓家棋，孔继烈，姜忠宝，等.伏安式快速测定BOD的微生物传感器研制[J].上海环境科学.1991.10:25-27.[5]魏恩棋，伊立志，袁秀文，等.用微生物传感器测定BOD的研究[J].城市环境与城市生态.199811(4):40-49.[6]JapaneseindustrialstandardcommitteeJISK3602,JapanesegoodstandardsAssociation,Tokyo,1990.[7]王德龙.微生物传感器快速测定BOD的标准方法颁布实施[J].中国给水排水，2003,19(5):75-76.[8]李花子(HuaziLi)，张悦(YueZhang)，施汉昌(HanchanShi)，王建龙(JianlongWang).ApplicationofBODbiosensorformarinemonitoring[J].MarineEnvironmentalScience（海洋环境科学）,2002,21(3):14-17.[9]王晓辉，史佩红，崔建升等.海水中BOD5的自动监测仪的研究[J].环境工程学报,2004,22(5):62-63.[10]KongLZ,ZhanHZ,ChenYL.EnvironmentalChemistryExperiment.NanJing,China:NanJinguniversitypress,1989.[11]CHOIMMF,WONGPS.ApplicationofaDataloggerinBiosensing:AGlucoseBiosensor[J].JournalofChemicalEducation,2002,79:982-984.[12]王学江，张峰华，丁钰力等.毒性分析微生物传感器电极固定方法的研究[J].同济大学学报自然科学版,2009,37(11):1497-1501.[13]刘蕊，孟庆庆.微生物传感器快速测定水中BOD含量的研究[J].黑龙江环1 琼州学院本科生毕业论文境通报,2008,32(8):28-29.[14]水质生化需氧量(BOD)的测定微生物传感器快速法,HJ/T86-2002.[15]国家环保总局《水和废水监测分析方法》编委会.水和废水监测分析方法(第四版)(M).北京:中国环境科学出版社,2002.[16]李洛娜,周颖,蔡琦,等.BOD微生物传感器检测仪(BODs)研究进展[J].净水技术,2007,26(6):54~56.[17]李捷,宋志文,孙新成,等.新型BOD微生物传感流动注射检测仪的研制[J].环境污染治理技术与设备,2005,6(3):91-9.[18]王里奥,崔志强,钱宗琴,等.微生物固定化的发展及在废水处理中的应[J].重庆大学学报,2004,27(3):125-129.[19]谢东海,韩奇,唐文浩.微生物固定化技术在污水处理中的应用[J].环境与可持续发展,2006,(4):48-50.1 琼州学院本科生毕业论文致谢时光飞快，转眼间，我们即将告别美好的大学生活，回首走过的岁月，还算充实，在这短暂的四年里，我对环境科学和环境工程及其相关问题进行了深入地学习，使我能够及时将学到的新知识融入实践中，动手能力有了一定的提高。四年的学习生活中，很多人给了我各方面的帮助，在此，向曾经给予我帮助的人致以最诚挚的感谢。首先感谢我的论文指导老师胡老师，在百忙的教学工作之后，还能帮我审查、修改我的论文。他渊博的专业知识，严谨的治学态度，一丝不苟的作风。不仅是我树立了远大的学习目标，还使我明白了许多待人接物处事的道理。再次感谢我大学四年中我的各位代课老师，老师们的循循善诱、不拘一格的思路和悉心的指导，使我顺利完成了我的学业，这些都是我今后工作和学习的榜样。最后感谢，我的那些给予我生活莫大帮助和在我最无助的时候，给我建议、鼓励同学，你们都是我大学生中最美好的回忆。即将完成学业，走出校园，我将珍惜在琼州学院学习的美好时光，将所学到的知识运用到实际工作和生活中，不辜负众位老师和自己四年来的努力，相信我的未来充满阳光。刘永福2013年5月1 1 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