【摘要】：隨著現(xiàn)代社會的快速發(fā)展,全球范圍內(nèi)水生態(tài)系統(tǒng)藻類水華尤其是有害藍(lán)藻水華(Harmful cyanobacterial algal blooms,Cyano HABs)的爆發(fā)頻率和影響程度逐年遞增。微生物控藻技術(shù)因為具有成本低、操作簡單和環(huán)境友好等特點而備受關(guān)注。本文以分布廣泛、危害最大的水華藍(lán)藻銅綠微囊藻(Microcystis aeruginosa)為控制對象,從重慶紫色土中分離出一批具有高效溶藻活性的溶藻菌,以溶藻效果最好的放線菌G3、G9和細(xì)菌AF-1為研究對象,對藻-菌相互作用關(guān)系、溶藻特性和溶藻機制進(jìn)行了深入研究,主要結(jié)果如下所述:1.溶藻放線菌的分離與鑒定。本部分研究從紫色土中分離得到了10株高效溶藻放線菌,其中放線菌G3和G9的溶藻效果最好(溶藻效率分別為84%、87%,t=5 d),結(jié)合它們的形態(tài)學(xué)特征以及16S r RNA基因序列分析,G3和G9被鑒定為鏈霉菌屬(Streptomyces)。2.Streptomyces sp.G3和G9溶藻特性的研究。G3和G9溶藻具有廣譜性,不僅對銅綠微囊藻表現(xiàn)出高效的溶藻效果,對其它常見水華藍(lán)藻,如水華魚腥藻、顫藻和席藻等也具有很好的溶藻效果,溶藻效率均達(dá)到了60%以上(t=5 d),并且能夠造成絲狀藍(lán)藻藻絲的斷裂。這兩株菌的溶藻效率隨銅綠微囊藻的濃度增加而降低,控制對數(shù)生長期藻的效果好于穩(wěn)定期的藻,但隨著培養(yǎng)時間的延長最終都能導(dǎo)致藻的黃化死亡。透析袋培養(yǎng)實驗結(jié)果表明,G3和G9是通過釋放某種溶藻活性物質(zhì)間接溶藻。3.多功能溶藻菌的分離鑒定及其溶藻特性的研究。利用平板對峙法從土壤中分離了一株具有禾谷鐮刀菌(Fusarium graminearum)強拮抗活性的細(xì)菌,命名為AF-1。該菌在平板上抑制禾谷鐮刀菌生長,形成明顯的抑菌圈,并且導(dǎo)致真菌菌絲發(fā)生明顯的形態(tài)變化。根據(jù)菌體細(xì)胞和菌落形態(tài)學(xué)特征及16S r RNA基因序列分析將AF-1鑒定為芽孢桿菌屬(Bacillus)細(xì)菌。AF-1對銅綠微囊藻有強烈的殺滅作用,5 d的溶藻效率可達(dá)93%。AF-1的有效溶藻細(xì)胞密度低至1.56×103 cfu/m L。此外,該菌株還能利用銅綠微囊藻釋放的營養(yǎng)物質(zhì)進(jìn)行生長發(fā)揮溶藻活性。AF-1的溶藻效率與銅綠微囊藻的生長時期有關(guān),幾乎不受自身生長時期的影響;AF-1對銅綠微囊藻的溶藻作用具有種屬特異性,對絲狀藍(lán)藻和真核綠藻的生長沒有抑制作用。4.Bacillus sp.AF-1溶藻活性物質(zhì)及溶藻機制的研究。AF-1的溶藻方式為分泌活性物質(zhì)間接溶藻。分泌的溶藻活性物質(zhì)具有耐熱、耐酸堿和蛋白酶的特點,表明活性物質(zhì)不是蛋白質(zhì),是性質(zhì)穩(wěn)定的化合物;采用不同極性的有機物抽提溶藻活性物質(zhì),發(fā)現(xiàn)AF-1分泌的溶藻活性物質(zhì)親水性更強。對溶藻過程中藻細(xì)胞的形態(tài)變化和ROS積累水平研究結(jié)果表明,在AF-1的培養(yǎng)液上清作用下,銅綠微囊藻細(xì)胞內(nèi)積累高水平的ROS,進(jìn)而造成藻細(xì)胞的氧化脅迫,導(dǎo)致藻細(xì)胞完整性受到嚴(yán)重破壞,最終導(dǎo)致藻細(xì)胞的裂解死亡。用RT-q PCR研究藻細(xì)胞重要基因差異表達(dá)的結(jié)果表明,AF-1無菌濾液造成藻細(xì)胞的DNA損傷,影響藻細(xì)胞的正常分裂,同時嚴(yán)重影響銅綠微囊藻的光合系統(tǒng)及抑制微囊藻毒素的合成。
[Abstract]:With the rapid development of modern society, algae blooms, especially harmful cyanobacterial algal blooms (Cyano HABs) in aquatic ecosystems worldwide are increasing year by year. Microcystis aeruginosa, the most harmful bloom cyanobacteria, is widely distributed in Chongqing. A number of algae-lysing bacteria with high algae-lysing activity were isolated from purple soil of Chongqing. Actinobacteria G3, G9 and bacteria AF-1 with the best algae-lysing effect were selected as the research objects to study the algae-bacteria interaction, algae-lysing characteristics and algae-lysing mechanism. The main results are as follows: 1. Isolation and identification of alginolytic actinomycetes. In this study, 10 strains of high efficient alginolytic actinomycetes were isolated from purple soil. Among them, actinomycetes G3 and G9 had the best alginolytic effect (alginolytic efficiency was 84%, 87%, t = 5 d), combined with their morphological characteristics and 16S R RNA gene sequence analysis, G3 and G. Streptomyces sp. G3 and G9 were identified as Streptomyces. 2. Study on the algae-dissolving characteristics of Streptomyces sp. G3 and G9. G3 and G9 have a broad spectrum of algae-dissolving properties. They not only have high algae-dissolving effect on Microcystis aeruginosa, but also have good algae-dissolving effect on other common bloom cyanobacteria, such as Anabaena algae, Vibrio algae and Seaweed. The algae-dissolving efficiency of G3 and G9 The algae lysis efficiency of the two strains decreased with the increase of the concentration of Microcystis aeruginosa. The algae in logarithmic growth period was better than that in stable period, but could eventually lead to algae yellowing death. Isolation and identification of multi-functional algicidal bacteria and their algicidal characteristics. A strain of Fusarium graminearum with strong antagonistic activity was isolated from soil by plate confrontation method. It was named AF-1. The strain inhibited the growth of Fusarium graminearum on the plate and formed obvious growth of Fusarium graminearum. AF-1 was identified as Bacillus according to the morphological characteristics of cell and colony and 16S R RNA gene sequence analysis. AF-1 had a strong killing effect on Microcystis aeruginosa, and the effective algicidal cell density of AF-1 was as low as 1.56%. The efficiency of AF-1 was related to the growth period of Microcystis aeruginosa, and was hardly affected by its own growth period. AF-1 had species-specific algae-lysis on Microcystis aeruginosa and on filamentous cyanobacteria and eukaryotic green algae. Bacillus sp. AF-1 algae-lysing active substances and algae-lysing mechanism. AF-1 algae-lysing way is secreting active substances indirect algae-lysing. The secreted algae-lysing active substances have the characteristics of heat-resistant, acid-resistant and protease-resistant, indicating that the active substances are not proteins, but stable compounds with different polarities. It was found that AF-1 secreted more hydrophilic algae-lysing active substances by extracting algae-lysing active substances. The morphological changes and ROS accumulation level of algae cells in the process of algae lysis were studied. The results showed that under the supernatant of AF-1 culture medium, high levels of ROS were accumulated in the cells of Microcystis aeruginosa, resulting in oxidative stress of algae cells, resulting in the end of algae cells. The results of RT-q PCR showed that AF-1 aseptic filtrate caused DNA damage in algae cells, affected the normal division of algae cells, and seriously affected the photosynthetic system of Microcystis aeruginosa and inhibited the synthesis of microcystins.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：X52;X172

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