铜绿微囊藻溶藻菌的分离鉴定、溶藻特性及溶藻机制研究

发布时间：2018-09-06 13:08

【摘要】：随着现代社会的快速发展,全球范围内水生态系统藻类水华尤其是有害蓝藻水华(Harmful cyanobacterial algal blooms,Cyano HABs)的爆发频率和影响程度逐年递增。微生物控藻技术因为具有成本低、操作简单和环境友好等特点而备受关注。本文以分布广泛、危害最大的水华蓝藻铜绿微囊藻(Microcystis aeruginosa)为控制对象,从重庆紫色土中分离出一批具有高效溶藻活性的溶藻菌,以溶藻效果最好的放线菌G3、G9和细菌AF-1为研究对象,对藻-菌相互作用关系、溶藻特性和溶藻机制进行了深入研究,主要结果如下所述:1.溶藻放线菌的分离与鉴定。本部分研究从紫色土中分离得到了10株高效溶藻放线菌,其中放线菌G3和G9的溶藻效果最好(溶藻效率分别为84%、87%,t=5 d),结合它们的形态学特征以及16S r RNA基因序列分析,G3和G9被鉴定为链霉菌属(Streptomyces)。2.Streptomyces sp.G3和G9溶藻特性的研究。G3和G9溶藻具有广谱性,不仅对铜绿微囊藻表现出高效的溶藻效果,对其它常见水华蓝藻,如水华鱼腥藻、颤藻和席藻等也具有很好的溶藻效果,溶藻效率均达到了60%以上(t=5 d),并且能够造成丝状蓝藻藻丝的断裂。这两株菌的溶藻效率随铜绿微囊藻的浓度增加而降低,控制对数生长期藻的效果好于稳定期的藻,但随着培养时间的延长最终都能导致藻的黄化死亡。透析袋培养实验结果表明,G3和G9是通过释放某种溶藻活性物质间接溶藻。3.多功能溶藻菌的分离鉴定及其溶藻特性的研究。利用平板对峙法从土壤中分离了一株具有禾谷镰刀菌(Fusarium graminearum)强拮抗活性的细菌,命名为AF-1。该菌在平板上抑制禾谷镰刀菌生长,形成明显的抑菌圈,并且导致真菌菌丝发生明显的形态变化。根据菌体细胞和菌落形态学特征及16S r RNA基因序列分析将AF-1鉴定为芽孢杆菌属(Bacillus)细菌。AF-1对铜绿微囊藻有强烈的杀灭作用,5 d的溶藻效率可达93%。AF-1的有效溶藻细胞密度低至1.56×103 cfu/m L。此外,该菌株还能利用铜绿微囊藻释放的营养物质进行生长发挥溶藻活性。AF-1的溶藻效率与铜绿微囊藻的生长时期有关,几乎不受自身生长时期的影响;AF-1对铜绿微囊藻的溶藻作用具有种属特异性,对丝状蓝藻和真核绿藻的生长没有抑制作用。4.Bacillus sp.AF-1溶藻活性物质及溶藻机制的研究。AF-1的溶藻方式为分泌活性物质间接溶藻。分泌的溶藻活性物质具有耐热、耐酸碱和蛋白酶的特点,表明活性物质不是蛋白质,是性质稳定的化合物;采用不同极性的有机物抽提溶藻活性物质,发现AF-1分泌的溶藻活性物质亲水性更强。对溶藻过程中藻细胞的形态变化和ROS积累水平研究结果表明,在AF-1的培养液上清作用下,铜绿微囊藻细胞内积累高水平的ROS,进而造成藻细胞的氧化胁迫,导致藻细胞完整性受到严重破坏,最终导致藻细胞的裂解死亡。用RT-q PCR研究藻细胞重要基因差异表达的结果表明,AF-1无菌滤液造成藻细胞的DNA损伤,影响藻细胞的正常分裂,同时严重影响铜绿微囊藻的光合系统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.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X52;X172

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