發(fā)布時(shí)間：2018-03-23 16:35

  本文選題：靈菌紅素　切入點(diǎn)：粘質(zhì)沙雷氏菌　出處：《東南大學(xué)》2016年碩士論文

【摘要】：我國(guó)水體富營(yíng)養(yǎng)化問(wèn)題日趨嚴(yán)重,由此引發(fā)的有毒有害藻類水華及其分泌的藻毒素等嚴(yán)重威脅著人類和環(huán)境的安全。微生物防治具有成本低、效率高、不易產(chǎn)生二次污染和安全性強(qiáng)等優(yōu)點(diǎn),在藻和藻毒素的治理上更具有應(yīng)用前景。本論文首先探究靈菌紅素對(duì)銅綠微囊藻的作用特性和作用機(jī)制。其次成功分離出藻毒素降解純種菌株a7,并研究其降解藻毒素的途徑與機(jī)制。最后初步探索靈菌紅素-藻毒素降解菌和粘質(zhì)沙雷氏菌-藻毒素降解菌協(xié)同作用體系在抑制藻類和降解藻毒素上的綜合效果。一、靈菌紅素對(duì)銅綠微囊藻的作用及機(jī)制研究培養(yǎng)銅綠微囊藻至對(duì)數(shù)期后染毒,應(yīng)用細(xì)胞計(jì)數(shù)結(jié)合流式細(xì)胞儀分析不同劑量靈菌紅素對(duì)銅綠微囊藻生長(zhǎng)抑制作用。1.25、2.50、5.00μg/mL靈菌紅素染毒24h、48h,研究靈菌紅素對(duì)ROS、SOD、DNA含量以及細(xì)胞內(nèi)、外MCs的影響。靈菌紅素染毒15d,觀測(cè)藻密度及胞內(nèi)、外MCs的變化。結(jié)果顯示,靈菌紅素染毒24h對(duì)銅綠微囊藻的ECso為2.76μg/mL。不同濃度的靈菌紅素染毒24h后銅綠微囊藻的直徑均明顯大于正常細(xì)胞,細(xì)胞DNA含量均明顯高于正常。2.50、5.00μg/mL靈菌紅素染毒24h后,ROS顯著升高,SOD顯著降低。染毒后的銅綠微囊藻細(xì)胞膜的完整性存在不同程度的受損。靈菌紅素抑制MC-LR的產(chǎn)生,2.50μg/mL時(shí)抑制效果最顯著。染毒時(shí)間增加后,靈菌紅素的抑制作用隨之增強(qiáng),胞外MC-LR濃度也逐漸升高。綜上,靈菌紅素能誘導(dǎo)細(xì)胞氧化損傷和增殖抑制,抑制銅綠微囊藻生長(zhǎng)。靈菌紅素短期抑制MC-LR的產(chǎn)生,長(zhǎng)期作用后亦增加胞外MC-LR的釋放量。二、太湖土著藻毒素降解菌的篩選及降解特性研究篩選藻毒素降解菌群,并對(duì)其16S rDNA V4區(qū)進(jìn)行高通量測(cè)序,分析菌相構(gòu)成及變化。通過(guò)16S rDNA鑒定分離獲得的菌株a7。提取菌株a7的不同細(xì)胞物質(zhì),判斷其降解物質(zhì)來(lái)源。PCR擴(kuò)增并測(cè)序菌株a7的mlr基因簇。通過(guò)HPLC和LC-TOF-MS分析MC-LR降解過(guò)程中的主要中間產(chǎn)物,初步探討降解途徑。結(jié)果顯示,藻毒素的馴化培養(yǎng)中,具有藻毒素降解功能的細(xì)菌逐漸成為優(yōu)勢(shì)菌種。藻毒素降解菌株a7對(duì)藻毒素粗提液和標(biāo)準(zhǔn)品中MC-LR的平均降解速率分別約為0.60μg/(mLμh)和3.33μg/(mLμh)。經(jīng)鑒定,菌株a7屬于鞘氨醇單胞菌屬(Sphingopyxis sp.)。菌株a7降解MC-LR的活性物質(zhì)位于胞內(nèi),且不耐熱。PCR擴(kuò)增到mlrA、mlrC、mlrD基因。LC-TOF-MS檢測(cè)到615.3398([M+H]+),315.1955 ([M+H-NH3]+)和283.1700 ([M+H-NH3-MeOH]+)的中間產(chǎn)物質(zhì)子化離子。綜上,菌株a7含有mlrA、mlrC、mlrD同源基因,降解活性物質(zhì)可能是胞內(nèi)酶,降解產(chǎn)物中存有Tetrapeptide、Adda和其他離子碎片。三、靈菌紅素與太湖土著藻毒素降解菌協(xié)同控藻作用研究培養(yǎng)銅綠微囊藻至對(duì)數(shù)期,加入2μg/mL靈菌紅素和1/5(v/v)藻毒素降解菌群,檢測(cè)藻密度和胞外藻毒素,研究靈菌紅素-藻毒素降解菌群的協(xié)同作用。繼而,分別研究藻毒素降解菌群含量、靈菌紅素濃度、溫度、加入時(shí)間間隔對(duì)協(xié)同作用的影響。加入2.5μg/mL靈菌紅素和不同體積菌株a7,研究二者協(xié)同作用效果及菌量影響因素。構(gòu)建粘質(zhì)沙雷氏菌-藻毒素降解菌群,粘質(zhì)沙雷氏菌-菌株a7,粘質(zhì)沙雷氏菌和LB試驗(yàn)及對(duì)照組,驗(yàn)證協(xié)同作用體系在模擬湖水中的作用效果。結(jié)果顯示,靈菌紅素和藻毒素降解菌群協(xié)同作用時(shí),藻密度值最低,胞外MC-LR濃度逐步降低至檢測(cè)限以下。1/5(v/v)藻毒素降解菌群含量,適宜的靈菌紅素濃度,28-37℃,同時(shí)或優(yōu)先加入藻毒素降解菌群能更高效地發(fā)揮協(xié)同作用。靈菌紅素與菌株a7協(xié)同作用時(shí),藻密度值最低,胞外MC-LR于第2天低于檢測(cè)限,且菌株a7的用量很少。在驗(yàn)證體系中,LB組的藻密度明顯高于其他組,各組的胞外MC-LR均逐漸降低至檢測(cè)限以下。各組的TN、TP、TOC均呈現(xiàn)下降趨勢(shì)。綜上,靈菌紅素或粘質(zhì)沙雷氏菌與藻毒素降解菌協(xié)同作用,既能抑制藻類生長(zhǎng)又能降解藻類釋放的藻毒素。
[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.

【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：X52;X172

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