灵菌红素与太湖土著藻毒素降解菌协同控藻作用研究

发布时间：2018-03-23 16:35

本文选题：灵菌红素　切入点：粘质沙雷氏菌　出处：《东南大学》2016年硕士论文

【摘要】：我国水体富营养化问题日趋严重,由此引发的有毒有害藻类水华及其分泌的藻毒素等严重威胁着人类和环境的安全。微生物防治具有成本低、效率高、不易产生二次污染和安全性强等优点,在藻和藻毒素的治理上更具有应用前景。本论文首先探究灵菌红素对铜绿微囊藻的作用特性和作用机制。其次成功分离出藻毒素降解纯种菌株a7,并研究其降解藻毒素的途径与机制。最后初步探索灵菌红素-藻毒素降解菌和粘质沙雷氏菌-藻毒素降解菌协同作用体系在抑制藻类和降解藻毒素上的综合效果。一、灵菌红素对铜绿微囊藻的作用及机制研究培养铜绿微囊藻至对数期后染毒,应用细胞计数结合流式细胞仪分析不同剂量灵菌红素对铜绿微囊藻生长抑制作用。1.25、2.50、5.00μg/mL灵菌红素染毒24h、48h,研究灵菌红素对ROS、SOD、DNA含量以及细胞内、外MCs的影响。灵菌红素染毒15d,观测藻密度及胞内、外MCs的变化。结果显示,灵菌红素染毒24h对铜绿微囊藻的ECso为2.76μg/mL。不同浓度的灵菌红素染毒24h后铜绿微囊藻的直径均明显大于正常细胞,细胞DNA含量均明显高于正常。2.50、5.00μg/mL灵菌红素染毒24h后,ROS显著升高,SOD显著降低。染毒后的铜绿微囊藻细胞膜的完整性存在不同程度的受损。灵菌红素抑制MC-LR的产生,2.50μg/mL时抑制效果最显著。染毒时间增加后,灵菌红素的抑制作用随之增强,胞外MC-LR浓度也逐渐升高。综上,灵菌红素能诱导细胞氧化损伤和增殖抑制,抑制铜绿微囊藻生长。灵菌红素短期抑制MC-LR的产生,长期作用后亦增加胞外MC-LR的释放量。二、太湖土著藻毒素降解菌的筛选及降解特性研究筛选藻毒素降解菌群,并对其16S rDNA V4区进行高通量测序,分析菌相构成及变化。通过16S rDNA鉴定分离获得的菌株a7。提取菌株a7的不同细胞物质,判断其降解物质来源。PCR扩增并测序菌株a7的mlr基因簇。通过HPLC和LC-TOF-MS分析MC-LR降解过程中的主要中间产物,初步探讨降解途径。结果显示,藻毒素的驯化培养中,具有藻毒素降解功能的细菌逐渐成为优势菌种。藻毒素降解菌株a7对藻毒素粗提液和标准品中MC-LR的平均降解速率分别约为0.60μg/(mLμh)和3.33μg/(mLμh)。经鉴定,菌株a7属于鞘氨醇单胞菌属(Sphingopyxis sp.)。菌株a7降解MC-LR的活性物质位于胞内,且不耐热。PCR扩增到mlrA、mlrC、mlrD基因。LC-TOF-MS检测到615.3398([M+H]+),315.1955 ([M+H-NH3]+)和283.1700 ([M+H-NH3-MeOH]+)的中间产物质子化离子。综上,菌株a7含有mlrA、mlrC、mlrD同源基因,降解活性物质可能是胞内酶,降解产物中存有Tetrapeptide、Adda和其他离子碎片。三、灵菌红素与太湖土著藻毒素降解菌协同控藻作用研究培养铜绿微囊藻至对数期,加入2μg/mL灵菌红素和1/5(v/v)藻毒素降解菌群,检测藻密度和胞外藻毒素,研究灵菌红素-藻毒素降解菌群的协同作用。继而,分别研究藻毒素降解菌群含量、灵菌红素浓度、温度、加入时间间隔对协同作用的影响。加入2.5μg/mL灵菌红素和不同体积菌株a7,研究二者协同作用效果及菌量影响因素。构建粘质沙雷氏菌-藻毒素降解菌群,粘质沙雷氏菌-菌株a7,粘质沙雷氏菌和LB试验及对照组,验证协同作用体系在模拟湖水中的作用效果。结果显示,灵菌红素和藻毒素降解菌群协同作用时,藻密度值最低,胞外MC-LR浓度逐步降低至检测限以下。1/5(v/v)藻毒素降解菌群含量,适宜的灵菌红素浓度,28-37℃,同时或优先加入藻毒素降解菌群能更高效地发挥协同作用。灵菌红素与菌株a7协同作用时,藻密度值最低,胞外MC-LR于第2天低于检测限,且菌株a7的用量很少。在验证体系中,LB组的藻密度明显高于其他组,各组的胞外MC-LR均逐渐降低至检测限以下。各组的TN、TP、TOC均呈现下降趋势。综上,灵菌红素或粘质沙雷氏菌与藻毒素降解菌协同作用,既能抑制藻类生长又能降解藻类释放的藻毒素。
[Abstract]:China's water eutrophication is becoming a serious problem, toxic and harmful algal blooms caused by the secretion of toxins and other serious threat to human and environmental safety. Microbial control has the advantages of low cost, high efficiency, easy to produce the advantages of two pollution and safety and has more application prospects in the treatment of algae and algae toxin on. This paper first explores the prodigiosin on Microcystis properties and mechanism of action. Then successfully isolated degrading microcystins purebred strain A7, and to study the reduction approach and mechanism of algal toxin solution. Finally, a preliminary exploration of prodigiosin - microcystin degrading bacteria and Serratia marcescens algae toxin degradation bacteria synergy system in inhibiting algae and degradation of microcystins on the comprehensive effect of prodigiosin. A study on effect and mechanism of Microcystis aeruginosa and Microcystis aeruginosa cultured to logarithmic phase after exposure, using cell counting Combined with the analysis of different doses of prodigiosin on Microcystis aeruginosa growth inhibitory effect of.1.25,2.50,5.00 g/mL prodigiosins by 24h 48h, flow cytometry, study of prodigiosin on ROS, SOD, DNA content and intracellular MCs. Effect of prodigiosin 15d exposure, algal density and cell observation in the change of MCs. The results showed that the prodigiosin 24h exposure on Microcystis aeruginosa ECso 2.76 g/mL. different concentrations of prodigiosin after 24h exposure of Microcystis aeruginosa in diameter were significantly higher than those in normal cells, DNA cells were significantly higher than normal.2.50,5.00 g/mL prodigiosine after 24h exposure ROS, significantly increased, SOD decreased significantly. The integrity of Microcystis cell membrane after exposure to the presence of varying degrees of damage. Prodigiosin inhibited the production of MC-LR, 2.50 g/mL the most significant inhibitory effect. The exposure time increased after prodigiosus red pigment inhibition with enhanced cell The concentration of MC-LR was gradually increased. In conclusion, prodigiosin induced oxidative damage and cell proliferation, inhibit the growth of Microcystis aeruginosa. Prodigiosin short-term inhibited the production of MC-LR, the long-term effects after increased release of extracellular MC-LR. Two, study on screening and degradation characteristics of Taihu native microcystin degrading bacteria the screening of microcystin degrading bacteria, and high-throughput sequencing of the 16S rDNA V4 District, and changes of microflora. Strain a7. was isolated by 16S rDNA identification extraction in different cell material strain A7, determine its source material degradation of MLR gene cluster.PCR amplification and sequencing. The main intermediate strain A7 HPLC and LC-TOF-MS analysis of MC-LR products in the degradation process, preliminary Study on degradation pathway. The results showed that microcystins acclimation, algae toxin degradation function has gradually become the dominant strains of bacteria. Microcystin degrading strain A7 on algae The average degradation rate of crude toxin solution and MC-LR standard respectively in about 0.60 g/ (mL h) and 3.33 g/ (mL h). After identification, the strain A7 belongs to the genus Sphingomonas (Sphingopyxis sp.). The active substances of strain A7 degrading MC-LR in intracellular, and heat the amplification of.PCR to mlrA, mlrC, mlrD gene.LC-TOF-MS was detected in 615.3398 ([M+H]+), 315.1955 ([M+H-NH3]+) and 283.1700 ([M+H-NH3-MeOH]+) of the intermediate product of the protonated ions. To sum up, the strain A7 containing mlrA, mlrC, mlrD gene, degradation of active substances may be endoenzyme and degradation products are Tetrapeptide, Adda and other debris ion. Three, prodigiosin and Taihu native microcystin degrading bacteria of cooperative control effect on algal culture of Microcystis aeruginosa to logarithmic phase, adding 2 g/mL of prodigiosin and 1/5 (v/v) of microcystin degrading bacteria, detection of algae density and extracellular microcystins, of prodigiosin - toxin Synergistic effect of degrading bacteria. Then, microcystin degrading bacteria were studied, prodigiosin concentration, temperature, influence of time intervals for synergy. Adding 2.5 g/mL of prodigiosin and different volume of two A7 strains, the effect of synergy effect and the amount of bacteria factors. Construction of clay Serratia - microcystin degrading bacteria, Serratia marcescens strain A7, Serratia marcescens and LB test and control group. The effect of synergistic effect in the simulation verification system in the water. The results showed that the prodigiosin and microcystin degrading bacteria synergistic effect when the algae density was the lowest cell the concentration of MC-LR gradually decreased to below the detection limit of.1/5 (v/v) of microcystin degrading bacteria content, prodigiosus red suitable concentration, 28-37 DEG C, at the same time or first priority to join the microcystin degrading bacteria can be more efficient. The synergistic effect of prodigiosin and strain A7 synergistic effect when the algae density The lowest value, the extracellular MC-LR in second days and below the detection limit of strain A7 in small amounts. In the verification system, the density of algae in LB group was significantly higher than the other groups, each group of extracellular MC-LR gradually decreased to below the detection limit. Each group of TN, TP, TOC showed a downward trend. In summary, the spirit bacterioruberin or Serratia marcescens and microcystin degrading bacteria synergistic effect, can inhibit the growth of algae and algae degradation of algal toxin release.

【学位授予单位】：东南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：X52;X172

【相似文献】