红树林沉积物中磺胺间甲氧嘧啶（SMMX）降解菌的筛选及其降解性能研究

发布时间：2018-05-09 10:32

本文选题：红树林 + 磺胺间甲氧嘧啶　；参考：《广东海洋大学》2015年硕士论文

【摘要】：磺胺间甲氧嘧啶(sulfamonomethoxine,SMMX)属于广谱、高效和高选择性的磺胺类抗生素,广泛应用于医药、畜禽与水产养殖业中的细菌、真菌或原生生物引起的感染疾病治疗。市政废水排放入海和海水养殖用药,造成抗生素类药物在包括红树林在内的滨海湿地药物残留,可能削弱微生物的分解作用从而降低生态系统的功能。本文采集了湛江观海长廊人工红树林区的沉积物样品,以筛选SMMX为单一碳源的抗生素降解细菌为目标,采用驯化方法筛选到了6株具有降解潜力的初筛菌株,运用经典和现代分子生物学方法对降解菌株进行了系统分类鉴定,同时采用超高效液相色谱仪(UPLC)检测方法结合正交试验设计在实验室内研究了降解菌株(Aeromonas caviae,HA-J1)对SMMX的生物降解性能,以获得最优降解条件,为后续构建生物修复工程菌株奠定基础。具体研究结果如下:1、应用摇瓶富集驯化的方法,通过荧光显微计数法测定驯化菌株在培养前后的数量变化,从湛江观海长廊人工红树林区的沉积物样品筛选分离到6株以兽药SMMX抗生素为单一碳源的初筛菌株,分别编号为HA-J1、HA-J4、HA-J5、HA-J7、HA-J8和HA-J9。在相同培养条件下,随兽药浓度的增大6株菌的数量先增加后减少,在800mg/L时,生长最适宜。通过生长曲线测定,菌株在15~24h处于生长旺盛的阶段,可以作为测定生物降解量末端的合适取样时间。2、根据细菌形态学观察、生理生化试验和细菌16s rDNA扩增对6株SMMX降解菌进行了系统分类鉴定,结果表明,HA-J1、HA-J4、HA-J5、HA-J8为气单胞菌属(Aeromonas),HA-J7、HA-J9为克雷伯氏菌属(Klebsiella),再分别通过看家基因gyr B/gyr A基因进一步鉴定,结果表明HA-J1、HA-J4、HA-J5、HA-J8为豚鼠气单胞菌(Aeromonas caviae),HA-J7、HA-J9为肺炎克雷伯氏菌(Klebsiella pneumoniae)。3、通过在无机盐培养液中添加色谱纯SMMX室内摇瓶培养研究6株降解菌进行浓度递增复筛,采用UPLC测定降解菌培养前后的SMMX的降解率,结合荧光显微镜测定培养前后SMMX降解菌的数量变化,获得复筛结果,6株菌HA-J1、HA-J4、HA-J5、HA-J7、HA-J8与HA-J9的有效生物降解率依次为24.13%、6.8%、1.4%、4%、13%、3.75%,由此将HA-J1菌株确定为SMMX高效生物降解菌株。通过Pearson相关性分析6株初筛菌对磺胺间甲氧嘧啶降解率与降解前后细菌的增量,发现相关性不显著(p0.05)。4、通过正交试验设计测定了HA-J1菌株在不同pH值、温度、盐度、SMMX浓度的四因素五水平的25组实验组合条件下对SMMX降解率及降解菌数量变化,获得HA-J1菌株最优降解条件组合为温度25℃、pH5、盐度为15、抗生素浓度为200mg/L,最优组合条件下的生物降解率达14.47%。多因素方差分析结果显示各因素间无显著性差异(p0.05)。通过Pearson相关性分析,发现各组合下的降解率与降解前后的细菌增量有显著的正相关(r=0.735,p0.05)。在最优正交组合下测定细菌最佳接种量为16%,降解率为19.29%。5、为探讨降解菌株对其他磺胺类药物的降解性能,研究了6株初筛菌株在相同培养条件下分别在以100mg/L的磺胺间甲氧嘧啶(SMMX)和磺胺甲VA唑(SMX)为单一碳源的无机盐培养液中的生物降解效率,HA-J1、HA-J4、HA-J5、HA-J7、HA-J8与HA-J9对SMMX的有效生物降解率依次为14.11%、1.55%、1.47%、4.87%、8.49%、3.33%,对SMX的生物降解率分别为0.64%、0.32%、0.47%、0.45%、0.14%。配对T检验结果表明,降解菌对SMX的降解率显著低于其对SMMX的降解率(p0.05)。6、研究了添加100mg/L的葡萄糖、甲醇、蔗糖等不同碳源后高效降解菌株HA-J1对相同质量体积浓度SMMX抗生素(即也为100mg/L)的降解率,结果表明,添加葡萄糖、甲醇、蔗糖这三种碳源后的降解效率依次分别为23.02%、18.36%、14.61%,无外加碳源的对照组降解率为13.45%。添加外加碳源不同程度的提高了高效降解菌株对SMMX的降解率,但两者之间无显著差异(ANOVA,p0.05)。本文的研究结果表明从红树林区沉积物中可以筛选得到高效降解磺胺类药物磺胺间甲氧嘧啶的单一菌株,并鉴定为豚鼠气单胞菌(Aeromonas caviae),但其降解功能基因及降解途径尚不清楚,还有待于进一步研究。
[Abstract]:Sulfamonomethoxine (SMMX) is a broad-spectrum, highly efficient and highly selective sulfonamide, widely used in medicine, in livestock and in the aquaculture industry, for the treatment of infectious diseases caused by bacteria, fungi, or protists. Municipal wastewater is discharged into the sea and aquaculture, causing antibiotic drugs in mangroves, including mangroves. The residues of drugs in the coastal wetlands may weaken the decomposition of microbes and reduce the function of the ecosystem. In this paper, the sediment samples from the artificial mangrove area of Zhanjiang Guan Hai corridor were collected in order to screen SMMX as a single carbon source of antibiotic degrading bacteria, and the biodegradation potential of 6 strains of screening were screened by acclimatization. The strain was systematically classified and identified by classical and modern molecular biology methods. At the same time, the biodegradability of the degraded strain (Aeromonas caviae, HA-J1) to SMMX was studied in the laboratory by the ultra high performance liquid chromatograph (UPLC) detection method combined with the orthogonal design in order to obtain the optimal degradation conditions for the subsequent construction. The basis of the bioremediation engineering strain was laid. 1, 1, using the method of shaking bottle enrichment and acclimation, the quantity changes of the domesticated strains before and after the culture were measured by the fluorescence microscopy, and the screening and separation of the sediment samples from the artificial mangrove area of the Zhanjiang Guan Hai corridor were screened and separated to the initial screening of the single carbon source with the veterinary drug SMMX antibiotics. The strains were numbered as HA-J1, HA-J4, HA-J5, HA-J7, HA-J8 and HA-J9. in the same culture conditions. The number of 6 strains increased first and then decreased with the increase of the concentration of veterinary drugs. In 800mg/L, the growth was the most suitable. By measuring the growth curve, the strain was at the stage of vigorous growth, which could be used as a suitable sampling time for the determination of the end of biodegradation. .2, according to bacterial morphological observation, physiological and biochemical tests and bacterial 16S rDNA amplification, 6 strains of SMMX degrading bacteria were systematically classified and identified. The results showed that HA-J1, HA-J4, HA-J5, HA-J8 were Aeromonas (Aeromonas), HA-J7, HA-J9 were Klebsiella (Klebsiella), and further identified by the family gene. The results showed that HA-J1, HA-J4, HA-J5, HA-J8 were Aeromonas of guinea pig (Aeromonas caviae), HA-J7 and HA-J9 were Klebsiella pneumoniae (Klebsiella pneumoniae).3. The concentration of 6 strains of degraded bacteria were studied by adding a chromatographic pure SMMX chamber shake flask culture in the inorganic salt culture solution. The number of SMMX degradation bacteria before and after culture was measured by fluorescence microscopy, and the results of rescreening were obtained. The effective biodegradation rates of 6 strains of bacteria HA-J1, HA-J4, HA-J5, HA-J7, HA-J8 and HA-J9 were 24.13%, 6.8%, 1.4%, 4%, 13%, 3.75%. Thus, HA-J1 strain was identified as a SMMX high efficiency biodegrading strain. The 6 strains of screening bacteria were analyzed by Pearson correlation. The correlation between the degradation rate of sulfadimethoxine and the increment of bacteria before and after degradation was not significant (P0.05).4. Through the orthogonal design, the SMMX reduction rate and the number of degrading bacteria were measured under the four factors and five levels of the pH value, temperature, salinity and SMMX concentration, and the optimal drop of the HA-J1 strain was obtained. The combination of the solution conditions was 25 C, pH5, the salinity 15, the antibiotic concentration of 200mg/L, and the biodegradation rate of 14.47%. under the optimal combination conditions was up to 14.47%.. The results showed that there was no significant difference between the factors (P0.05). The degradation rate of each combination and the increment of bacteria before and after degradation were found to be significant positive phase through Pearson correlation analysis. (r=0.735, P0.05). Under the optimal orthogonal combination, the optimum inoculation amount of bacteria was 16% and the degradation rate was 19.29%.5. In order to investigate the degradation performance of the degrading strains to other sulfonamides, the 6 initial screening strains were used as the single carbon source of sulfadiazine (SMMX) and sulfamethoxazole (SMX) under the same culture conditions. The biodegradability of HA-J1, HA-J4, HA-J5, HA-J7, HA-J8 and HA-J9 to SMMX in the inorganic salt medium was 14.11%, 1.55%, 1.47%, 4.87%, 8.49%, 3.33%. The biodegradation rate of SMX, respectively, 0.64%, 0.32%, 0.47%, 0.45%, and 0.14%., respectively, showed that the degradation rate of the degrading bacteria to SMX was significantly lower than that of SMMX. Degradation rate (P0.05).6, the degradation rate of High Efficient Degradation Strain HA-J1 for the same mass volume concentration SMMX antibiotic (also 100mg/L) was studied after different carbon sources, such as glucose, methanol and sucrose were added to 100mg/L, and the results showed that the degradation efficiency of the three carbon sources after adding glucose, methanol and sucrose, respectively, was 23.02%, 18.36%, 14.61%, respectively. The degradation rate of the carbon source in the control group was 13.45%. adding additional carbon source to improve the degradation rate of SMMX, but there was no significant difference between the two groups (ANOVA, P0.05). The results of this study showed that a single strain of highly efficient sulfadiazine sulfadiazine can be screened from the mangrove area sediments. It is identified as Aeromonas caviae, but its degradation function genes and degradation pathways are not yet clear.

【学位授予单位】：广东海洋大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X172

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