【作者】 吴强；

【导师】 李朋富；

【作者基本信息】 南京大学 ， 生物学， 2020， 博士

【摘要】 探讨微囊藻藻际菌群的分布规律将有助于阐明细菌在蓝藻水华形成过程中所发挥的作用。为了避免野外环境因子对藻际菌群的影响,本文在实验室培养群体形态的微囊藻,对微囊藻附生菌群和游离菌群的组成,以及不同大小群体微囊藻的附生菌群组成进行比较分析。实验结果显示,在对数生长期,变形菌（Proteobacteria）是附生菌群中最主要门类细菌,而拟杆菌（Bacteroidetes）是游离菌群中最主要门类细菌,变形菌在附生菌群中的相对丰度要高于在相对应的游离菌群中的相对丰度,而拟杆菌在附生菌群中的相对丰度要低于在相对应的游离菌群中的相对丰度。α变形菌（Alphaproteobacteria）在附生菌群中的相对丰度要高于在相对应的游离菌群中的相对丰度,而纤维粘网菌（Cytophagia）和鞘脂杆菌（Sphingobacteriia）在附生菌群中的相对丰度要低于在相对应的游离菌群中的相对丰度。3个OTU（分别是属于德沃斯氏菌Devosia的OTU#2、属于中慢生根瘤菌Mesorhizobium的OTU#17和属于根瘤菌Rhizobiaceae的OTU#29）在附生菌群中的相对丰度要高于在相对应的游离菌群中的相对丰度,2个OTU（分别是属于鞘脂杆菌Sphingobacteriales的OTU#55和属于里德拜特氏菌Leadbetterella的OTU#27）在游离菌群中的相对丰度要高于在相对应的附生菌群中的相对丰度。PCA模型和Bray-Curtis相异度指数分析结果表明,附生菌群和游离菌群之间的差异在对数生长期更大,在静止期更小。变性梯度凝胶电泳（DGGE）图谱显示不同大小群体微囊藻的附生菌群组成具有差异。在对数生长期,附生菌群中变形菌的相对丰度随微囊藻群体大小的减小而减小,而附生菌群中拟杆菌的相对丰度则增大。在对数生长期,附生菌群中主要纲类细菌α变形菌的相对丰度随群体大小减小而减小,而附生菌群中另外三种主要纲类细菌（分别是γ变形菌、鞘脂杆菌和纤维粘网菌）的相对丰度随群体大小的增大而减小。在对数生长期,3个OTU（分别是属于假单胞菌的OTU#9、属于里德拜特氏菌的OTU#27和属于鞘脂杆菌的OTU#55）的相对丰度随群体大小的减小而增大,而属于中慢生根瘤菌的OTU#17的相对丰度随群体大小的减小而减小。在对数生长期,不同大小微囊藻群体之间的附生菌群组成差异按照以下顺序排列:>100μm和<50μm群体之间的差异最大,50-100μm和<50μm群体之间的差异次之,>100μm和50-100μm群体之间的差异最小。这些研究结果表明,微囊藻附生菌群组成不同于游离菌群组成,微囊藻群体大小在构建微囊藻的附生菌群组成方面起到了重要作用。细菌和藻类的相互作用在水华的形成和发展过程中可能发挥了重要作用。微囊藻是一种分布最广泛的水华蓝藻,在自然水体中通常是以群体形态存在。因此,研究细菌和群体微囊藻的相互作用就显得很有必要。本文从微囊藻群体中分离附生细菌,分析附生细菌对微囊藻生长、群体大小和光合活性的影响。结果显示,从太湖采集的铜绿微囊藻群体中共分离得到了11株细菌。在这些细菌中,7株细菌显著影响铜绿微囊藻群体大小,4株细菌显著影响铜绿微囊藻生长率。4株细菌（分别是隶属于微小杆菌Exiguobacterium、戴尔福特菌Delftia、芽孢杆菌Bacillus和寡养单胞菌Stenotrophomonas）显著减小铜绿微囊藻群体大小,减小的范围是36-51%,2株细菌（分别是隶属于金黄杆菌Chryseobacterium和假单胞菌Pseudomonas chengduensis）显著增大铜绿微囊藻群体大小,分别增大了89%和63%,然而这6株细菌对铜绿微囊藻的生长率没有影响。2株细菌（分别是隶属于莱茵海默氏菌Rheinheimera和假单胞菌Pseudomonas）显著降低铜绿微囊藻的生长率,分别降低了47%和36%,1株细菌（隶属于气单胞菌Aeromonas）提高了铜绿微囊藻的生长率,提高了22%,然而这3株细菌对铜绿微囊藻的群体大小没有影响。1株细菌（隶属于鞘氨醇单胞菌Sphingomonas）显著增大铜绿微囊藻群体大小,增大了80%,而且降低铜绿微囊藻的生长率,降低了21%。光合活性分析结果显示,11株细菌对铜绿微囊藻的最大光化学量子产量（F_v/F_m）没有影响,同时对铜绿微囊藻的最大相对电子传递速率(r ETR_max)也没有影响,这些结果表明11株细菌对铜绿微囊藻的光系统II（PSII）不产生影响。PCR-DGGE分析结果表明,所有分离菌株都能够定殖于铜绿微囊藻群体。在不同氮磷浓度条件下,菌株QW12（隶属于金黄杆菌）和QW15（隶属于假单胞菌）对铜绿微囊藻群体大小的影响都没有显著性差异,表明菌株QW12和QW15对铜绿微囊藻群体大小的影响可能不受水体中氮和磷浓度的影响。上述研究结果表明,微囊藻附生细菌可能影响微囊藻群体大小和生长,并进而影响微囊藻水华的形成和发展。为了探讨附生细菌影响微囊藻群体大小的机制,本文挑选对铜绿微囊藻群体大小影响非常显著的两株细菌QW12和QW15进行进一步研究。结果显示,菌株QW12和QW15培养滤液显著影响铜绿微囊藻的群体大小。菌株QW12和QW15培养滤液对铜绿微囊藻的生长率和胞外多糖产率都没有影响,但是都显著增加铜绿微囊藻细胞疏水性,分别增加了28%和16%。这些研究结果表明,附生细菌QW12和QW15通过产生胞外代谢产物影响微囊藻细胞疏水性,从而影响微囊藻群体大小。综上,本文揭示了微囊藻附生菌群组成不同于游离菌群组成,微囊藻群体大小在构建微囊藻的附生菌群组成方面起到了重要作用。另外,本文首次报道了微囊藻群体中的附生细菌能够影响微囊藻群体大小和群体微囊藻生长。研究结果将有助于深入揭示蓝藻水华的暴发机制,也为利用生物操纵理论控制蓝藻水华提供重要的科学参考依据。更多还原

【Abstract】 A better understanding of the distribution pattern of bacterial community in the Microcystis phycosphere will aid in elucidating the role of bacteria in the formation of cyanobacterial bloom.In this study,the bacterial community composition between Microcystis colony-attached and free-living bacteria,as well as among bacteria attached with Microcystis colonies of various sizes were compared using the laboratory-grown Microcystis colonies in order to overcome the interference of the natural ecosystem.In the exponentially growing cyanobacterial cultures,Proteobacteria was the most dominant phylum in each colony-attached bacterial community,whereas Bacteroidetes was the most dominant phylum in each free-living bacterial community.In the Microcystis cultures of exponential phase,the relative abundance of Proteobacteria in each colony-attached bacterial community was higher than that in its corresponding free-living bacterial community,while the relative abundance of Bacteroidetes in each colony-attached bacterial community was lower than that in its corresponding free-living bacterial community.Further comparison at the class level indicated that in both exponential and stationary cyanobacterial cultures,the relative abundance of Alphaproteobacteria in each colony-attached bacterial community was higher compared with its corresponding free-living bacterial community.Contrarily,the relative abundance of Cytophagia and Sphingobacteriia in each colony-attached bacterial community was lower compared with its corresponding free-living bacterial community.Three OTUs（OTU#2 belonging toDevosia,OTU#17 belonging to Mesorhizobium and OTU#29 belonging to Rhizobiaceae）were more abundant in the colony-attached bacterial communities than in their free-living counterparts.Two OTUs（OTU#55 assigned to Sphingobacteriales and OTU#27 assigned to Leadbetterella）were more abundant in the free-living bacterial communities compared with colony-attached bacterial communities.The analysis using an indirect PCA model and Bray–Curtis dissimilarity index indicated that the dissimilarity between colony-attached and free-living bacterial communities was greater in the exponentially growing cyanobacterial cultures,and it became smaller in the stationary cultures of Microcystis.The denaturing gradient gel electrophoresis（DGGE）profiles showed that the differential distribution of bacterial communities occurred within Microcystis colonies of various sizes for each species of Microcystis.In the exponential growth phase of Microcystis,the relative abundance of Proteobacteria in colony-attached bacterial communities tended to decrease with decreasing colony size,whereas the relative abundance of Bacteroidetes in colony-attached bacterial communities tended to increase.In the exponential growth phase of Microcystis,the relative abundance of the dominant class Alphaproteobacteria attached with colonies tended to decrease with decreasing colony size.In contrast,the relative abundance of the three dominant classes（Gammaproteobacteria,Sphingobacteriia and Cytophagia）attached with colonies tended to decrease with increasing colony size.In the exponential growth phase of Microcystis,the relative abundance of three OTUs（OTU#9 related to Pseudomonas,OTU#27 assigned to Leadbetterella and OTU#55 assigned to Sphingobacteriales）tended to increase with decreasing colony size,while the relative abundance of OTU#17 affiliated with Mesorhizobium tended to decrease with decreasing colony size.In the exponential growth phase of Microcystis,the community composition dissimilarity among bacteria attached with Microcystis colonies of various sizes could be ranked in a descending order as follows:>100μm versus<50μm;50–100μm versus<50μm;and>100μm versus 50–100μm.These data indicated that the community composition of Microcystis colony-attached bacteria was different from that of free-living bacteria,and the colony size of Microcystis played an important role in structuring the community composition of Microcystis-attached bacteria.The interactions between bacteria and algae may play a significant part in the formation and development of algal blooms.The bloom-forming cyanobacterium Microcystis occurs mainly as colonial form in natural waters,and thus it is necessary to study the interaction between bacteria and colonial Microcystis.In this study,bacterial strains were isolated from Microcystis colonies.We investigated the effects of the cultivable bacteria attached to Microcystis colonies on the colony size,growth and photosynthetic activities of Microcystis.Eleven bacterial strains were isolated from M.aeruginosa colonies collected from Lake Taihu.Among these bacteria,seven bacterial isolates significantly influenced the colony size of M.aeruginosa,and four bacterial isolates significantly influenced the growth rate of M.aeruginosa.Four isolates,related to the Exiguobacterium,Delftia,Bacillus and Stenotrophomonas,significantly decreased the colony size of M.aeruginosa by 36-51%,and two isolates,assigned to the Chryseobacterium and Pseudomonas chengduensis,significantly increased the M.aeruginosa colony size by 89%and 63%,respectively,while these isolates had no effect on the cyanobacterial growth rate.Two isolates,belonged to the Rheinheimera and Pseudomonas,significantly decreased the growth rate of M.aeruginosa by 47%and 36%,respectively,and one bacterial strain related to Aeromonas increased the cyanobacterial growth rate by 22%,while these isolates had no effect on the cyanobacterial colony size.One isolate belonged to Sphingomonas was found to significantly increase the colony size of M.aeruginosa by 80%and significantly decrease the growth rate of M.aeruginosa by 21%.Eleven bacterial isolates had no effect on the maximum optical quantum yield of PSII（F_v/F_m）and the maximum relative electron transport rate(r ETR_max)of M.aeruginosa,suggesting that these isolates had no effect on the structure of PSII in M.aeruginosa.PCR-DGGE analysis showed that all of the bacterial isolates were able to colonize M.aeruginosa colonies.No significant difference was observed in terms of the influence of two bacterial isolates（QW12 assigned to the Chryseobacterium and QW15 related to Pseudomonas）on the colony size of M.aeruginosa among all the groups at different nitrogen and phosphorus levels,indicating that the concentrations of nitrogen and phosphorus in the lake water had no effect on the influence of these isolates on the cyanobacterial colony size.These data suggested that the bacteria attached to Microcystis colonies might influence the cyanobacterial colony size and growth,and thus influence the formation and development of Microcystis blooms.To further study the mechanism of the influence of bacteria attached to Microcystis colonies on the colony size of Microcystis,two bacterial strains QW12and QW15,exhibiting stronger effect on the cyanobacterial colony size,were selected.The filtrates of cultures of the two bacterial strains were shown to significantly influence the colony size of M.aeruginosa.The filtrates had no effect on the growth rate and extracellular polysaccharide（EPS）content of M.aeruginosa,but significantly increased the cell hydrophobicity of M.aeruginosa by 28%and 16%,respectively.These data suggested that two bacterial isolates（QW12 and QW15）influenced the cell hydrophobicity of M.aeruginosa through producing extracellular compounds,and in turn influenced the colony size of Microcystis.In conclusion,this dissertation revealed that the community composition of Microcystis colony-attached bacteria was different from that of free-living bacteria,and the colony size of Microcystis played an important role in structuring the community composition of Microcystis-attached bacteria.In addition,it is the first time to demonstrate that the bacteria attached to the colonies of Microcystis were able to influence the growth of colonial Microcystis and its colony size.These results would help to further reveal the mechanism of formation of cyanobacterial blooms,and provided a basis for controlling cyanobacterial blooms based on the theory of biomanipulation.更多还原