喷气燃料特征污染微生物裂解剂探索
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摘要
微生物的有效裂解以获取三磷酸腺苷(ATP)是利用ATP生物荧光法检测喷气燃料微生物污染的兲键。为了获得良好的微生物裂解效果,以五种喷气燃料特征污染真菌的混合菌为研究对象,研究了BAB和BAC两种季铵盐类阳离子表面活性剂对真菌的裂解效果及荧光素酶活性的影响。研究结果表明,BAB比BAC在裂解特征污染真菌方面更有效,且BAB的最佳使用浓度为0.05%;研究了Cu2+和pH等理化因子对BAB裂解效果的影响,发现Cu2+浓度为0.015%,pH值为8.5时,可以达到较好的裂解效果;通过对荧光素酶-荧光素反应体系的优化,使得混合菌菌落数与相对发光强度之间达到了较好的线性相兲度(R2=0.999 5),可用于喷气燃料微生物检测。相比于传统的培养法和HY-LITE JET A1燃料试验,该裂解剂及优化的荧光素酶-荧光素反应体系,具有检测效率显著提高、检测成本大幅减低等优点。
Effective microbial extraction to obtain adenosine triphosphate(ATP) is the key to detect microbial contamination of jet fuel by ATP bioluminescence assay. In order to obtain a better microbial extraction, the effect of BAB and BAC,two kinds of quaternary ammonium salt cationic surfactants, on the extraction effect of fungi and luciferase activity was studied by taking the five characteristic fungi as the subjects. The results showed that BAB was more effective than BAC in extracting characteristic fungi, and the optimal concentration of BAB was 0.05%. The effect of physicochemical factors such as Cu2+ and pH on the lysis effect of BAB was studied. It was found that when the concentration of Cu2+ was 0.015%, and the pH value was 8.5, better lysis effect could be achieved. By optimizing the luciferase-fluorescein reaction system, a good linear correlation(R2=0.999 5) between the number of mixed bacteria colonies and the relative luminescence intensity was obtained and could be used for microbial detection of jet fuel. Compared with the traditional culture method and the HY-LITE JET A1 fuel test, the cracking agent and the optimized luciferase-luciferin reaction system had the advantages of significantly higher detection efficiency and significantly lower detection cost.
Effective microbial extraction to obtain adenosine triphosphate(ATP) is the key to detect microbial contamination of jet fuel by ATP bioluminescence assay. In order to obtain a better microbial extraction, the effect of BAB and BAC,two kinds of quaternary ammonium salt cationic surfactants, on the extraction effect of fungi and luciferase activity was studied by taking the five characteristic fungi as the subjects. The results showed that BAB was more effective than BAC in extracting characteristic fungi, and the optimal concentration of BAB was 0.05%. The effect of physicochemical factors such as Cu2+ and pH on the lysis effect of BAB was studied. It was found that when the concentration of Cu2+ was 0.015%, and the pH value was 8.5, better lysis effect could be achieved. By optimizing the luciferase-fluorescein reaction system, a good linear correlation(R2=0.999 5) between the number of mixed bacteria colonies and the relative luminescence intensity was obtained and could be used for microbial detection of jet fuel. Compared with the traditional culture method and the HY-LITE JET A1 fuel test, the cracking agent and the optimized luciferase-luciferin reaction system had the advantages of significantly higher detection efficiency and significantly lower detection cost.
引文
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[13]赵奕良,杨慧文,宋浩亮,陈倩瑜.三氯乙酸(TCA)提取法与微波提取法检测活性污泥中三磷酸腺苷(ATP)[J].微生物学通报,2017(44):2714-2721.
[14]M.Siro,H.Romar,T.Lovegren,Continuous flow method for extraction and bioluminescence assay of ATP in baker’s yeast[J].Microbiol Biotechnol,1982(15):258-264.
[15]王志惠,谭玉静,杨雪霞,赵曙辉.纯棉织物上黑曲霉胞内ATP提取方法探索[J].东华大学学报(自然科学版),2015(40):324-328.
[16]S.E.Kolehmainen,V.Tarkkanen.Selective determination of nucleotides in viable somatic and microbial cells:US Patent,4,303,752[P].1981.
[17]W.A.Hamilton,W.B.Hugo.Inhibition and destruction of the microbial cell[J].Academic Press,1977:77-93
[18]程侣柏.精细化工产品的合成及应用[M].第二版.大连理工大学出版社,1995:140-144
[19]轻工业部织物调理技术考察组.赴西德、荷兰考察织物调理剂技术报告[J].日用化学工业,1988(5):77-93.
[2]HILL E C,Hill G C.Microbial Contamination and Associated Corrosion in Fuels,during Storage,Distribution and Use[J].Advanced Materials Research,2008,38:257-268.
[3]HILL T.Microbial growth in aviation fuel[J].Aircraft Engineering&Aerospace Technology,2003,75(5):497-502.
[4]舒柏华,赵志耀,徐顺清,周宜开.利用生物发光分析技术快速检测微生物的方法学研究[J].发光学报,1999(01):79-82.
[5]Mcelroy W D,Stremer B L.Factors influencing the Response of the Bioluminescent to Adenosine Triphosphate[J].Arch.Bioch-enl,1949,22:420-433.
[6]Mcelroy W D,Stremer B L.Factors influencing the Response of the Bioluminescent to Adenosine Triphosphate[J].Arch.Bioch-enl,1949,22:420-433.
[7]Marlene Deluca,William D.Mcelroy.Kinetics of the Firefly Luciferase Catalyzed Reactions[J].Biochemistry,1974,13(5):921-925.
[8]LaRossa R A.Methods in Molecular Biology,Vol 102:Biolumi-nescence Methods and protocols[M],Totowa,New Jersey:Humana press,1998:3-20.
[9]Sevin BU,Peng Z I,Perras J P,et al.Application of an ATP bioluminescence assay in human tumor chemosensitivity testing[J].Gynecol oncol,1988,31(1):191-204.
[10]Sevin B U,Peng Z I,Averette H E,et al.Chemosensitivity testing in ovarian cancer[J].Cancer,1993,71(4):1613-1620.
[11]张菊梅,吴清平,吴慧清,郭伟鹏,李程思.微生物细胞ATP释放条件研究[J].微生物学通报,2006(04):100-105.
[12]黄彬,余元善,唐道邦,徐玉娟,刘忠义,吴继军.CTAB提取ATP的生物荧光法快速检测细菌总数准确性研究[J].现代食品科技,2014(30):269-273.
[13]赵奕良,杨慧文,宋浩亮,陈倩瑜.三氯乙酸(TCA)提取法与微波提取法检测活性污泥中三磷酸腺苷(ATP)[J].微生物学通报,2017(44):2714-2721.
[14]M.Siro,H.Romar,T.Lovegren,Continuous flow method for extraction and bioluminescence assay of ATP in baker’s yeast[J].Microbiol Biotechnol,1982(15):258-264.
[15]王志惠,谭玉静,杨雪霞,赵曙辉.纯棉织物上黑曲霉胞内ATP提取方法探索[J].东华大学学报(自然科学版),2015(40):324-328.
[16]S.E.Kolehmainen,V.Tarkkanen.Selective determination of nucleotides in viable somatic and microbial cells:US Patent,4,303,752[P].1981.
[17]W.A.Hamilton,W.B.Hugo.Inhibition and destruction of the microbial cell[J].Academic Press,1977:77-93
[18]程侣柏.精细化工产品的合成及应用[M].第二版.大连理工大学出版社,1995:140-144
[19]轻工业部织物调理技术考察组.赴西德、荷兰考察织物调理剂技术报告[J].日用化学工业,1988(5):77-93.