BOD微生物传感检测仪中生物敏感材料的研究

发布时间：2018-03-23 07:12

本文选题：微生物传感器　切入点：微生物膜　出处：《青岛理工大学》2015年硕士论文　论文类型：学位论文

【摘要】：生化需氧量(BOD)是表征水体中有机物等需氧污染物质含量的重要指标之一,现在普遍用于测定生物化学需氧量的标准方法是五日生化需氧量法,即样品在20℃±1℃的温度下培养5d,分别测定样品培养前后的溶解氧,二者之差即为5d的生化需氧量。此方法是十分复杂的生物化学过程,受多种因素的影响,测试周期过于长,效率低,测试结果不稳定,重现性差,所以方法本身存在很多的局限性。微生物传感器快速测定法克服了上述测定法的缺点,使整个测定周期大大缩短,测试结果直接打印出来,方便快捷,并且可以实现在线监测,从而日益得到广泛的应用。本文阐述了微生物传感器的产生、微生物传感器的原理及组成、微生物传感器的研究进展包括微生物传感器的类型、微生物的类型及固定化方法,对微生物传感器的应用BOD快速检测仪进行了简单介绍及运用。重点对核心部分敏感材料做了研究,包括敏感材料微生物膜的研究现状、制备过程中遇到的问题及敏感材料微生物小球性能分析。本研究的重点为菌种的选择、菌种培养温度的选择及固定化方法的选择。1.本文敏感材料的制备首先选用传统的生物膜法。选择四个影响因素,分别是菌种、包埋剂、培养温度、包埋剂与菌种配比,每个因素分为三个水平,即菌种:1#、2#、3#;温度:25℃、30℃、35℃;包埋剂:聚乙烯醇、聚丙烯酰胺、海藻酸钠;包埋剂与菌种配比:1:2、2:1、1:1。使用正交设计对这四因素三水平进行实验安排,根据实验安排进行微生物膜的制作,使用BOD快速检测仪对标准溶液(BOD=2000±160mg/L)BOD值进行测试。然后进行正交分析,包括极差分析和方差分析。得出各因素对实验指标的影响程度,按影响大小排序为:菌种、培养温度、包埋剂、包埋剂与菌体配比。其中菌种的选择和培养温度对实验结果影响明显,其余两个次之。进一步分析出来的最优方案即选用菌种3#、培养温度35℃、包埋剂聚乙烯醇、固定化材料与菌体配比1:2。最后对其进行实验验证,分别对BOD值为2000±160mg/L的标准溶液及实际水样进行测定,证明最优方案是符合要求的并对于不同来源水样测定均具有较好的准确性。在最优方案所对应的实验条件下,使用课题组的专利技术,用微生物小球作为敏感材料,使用BOD快速检测仪分别对BOD值为2000±160mg/L的标准溶液进行测试并进行数据分析,结果表明:相同的实验条件、实验材料下,使用微生物小球作为敏感材料的效果要好于使用微生物膜。数值上下波动幅度更小,测试结果很稳定,重现性好。用其测定的水样的BOD值与5日培养法无显著差异,这表明该生物小球对于不同来源水样测定均具有较好的准确性。筛选出最优方案中对有机物降解能力强的3#菌种进行菌种鉴定,首先革兰氏染色,然后利用16S r DNA序列同源性分析技术,进行DNA提取、PCR扩增后,送往上海桑尼生物科技有限公司完成测序反应。菌种最终鉴定结果:细菌(Bacteria)厚壁菌门(Firmicutes)、芽孢杆菌纲(Bacilli)、芽孢杆菌目(Bacillales)、芽孢杆菌科(Bacillaceae)、芽孢杆菌属(Bacillus.sp)。
[Abstract]:Biochemical oxygen demand (BOD) is one of the important indexes in water organic pollutants such as aerobic content, now commonly used standard method for the determination of BOD is Bod five, which were cultured in 5D 20 C + 1 DEG C temperature, dissolved oxygen samples were measured before and after training two, the difference is the biochemical oxygen demand 5D. This method is the biochemical process is very complex, affected by many factors, the test cycle is too long, low efficiency, unstable test results, poor reproducibility, so the method itself has many limitations. The rapid determination to overcome the above determination the shortcomings of microbial sensor, measurement period is greatly shortened, convenient test results directly printed, and can realize online monitoring, which has been widely used. This paper expounds the microbial sensor, micro The principle and composition of biosensor, research progress of microbial sensor types including microbial sensor, microorganism types and immobilization methods, the application of BOD rapid detection of microbial sensor are introduced and used. Focuses on the research on the core part of sensitive materials, including the research status of sensitive material of biofilm, analysis the preparation process and the problems in the sensitive materials for microbial ball performance. The focus of this study is the choice of culture, this paper select the.1. temperature sensitive materials and the selection of immobilized bacteria culture method in the preparation of the first selection of biofilm in the traditional method. Four factors are bacteria, entrapment agent, culture temperature, embedding agent and strain ratio, each factor is divided into three levels, namely: 1#, 2#, 3# strains; temperature: 25 C, 30 C, 35 C; embedding agent: polyvinyl alcohol, polyacrylamide, alginate Sodium; embedding agent and strain ratio: 1:2,2:1,1:1. using orthogonal design of the four factors and three levels of experimental arrangement, according to the experimental design and production of microbial membrane, the standard solution using BOD fast detector (BOD=2000 + 160mg/L) BOD values were tested. Then the orthogonal analysis, including the analysis of range and variance of. Various factors on the extent of the index, according to the influence of the order of strain, culture temperature, embedding agent, embedding agent and cell ratio. One species selection and cultivation of the temperature effect on the experimental results, the remaining two times. The optimal solution is selected out for further analysis of strain 3#, culture temperature 35 polyvinyl alcohol, embedding agent, immobilized cell material and the ratio of 1:2. finally carries out experiments on it, respectively. The value of BOD as the standard solution of 2000 + 160mg/L and the actual water samples were measured, it is proved that the optimal solution is To meet the requirements for different sources of water samples were measured and has good accuracy. The experimental conditions corresponding to the optimal scheme, using the research group of the patented technology, using microorganism balls as a sensitive material, the use of BOD rapid detection instrument respectively. The value of BOD was 2000 + 160mg/L Standard Test and data analysis, the quasi solution results show that the same experimental conditions, experimental materials, the use of microbial balls as better sensitive materials in the use of microbial membrane. The numerical fluctuation amplitude is smaller, the test result is stable, with good reproducibility. And the 5 day culture method had no significant difference with the determination of the BOD value of water sample, which indicates that the bio beads were has good accuracy for different sources of water samples were determined. Screened strains were identified on 3# strains of organic matter degradation ability of the optimal scheme, firstly, Gram staining, and then use 16S R Sequence analysis technology DNA, DNA extraction, PCR amplification, sent to Shanghai Sonny biological technology Limited completed the sequencing reaction. The final identification results: bacterial strains (Bacteria) Firmicutes bacilli (Firmicutes), (Bacilli), bacillales (Bacillales), Bacillaceae (Bacillaceae). The genus Bacillus (Bacillus.sp).

【学位授予单位】：青岛理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X853;TP212.3

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