Chryseobaterium sp.S7溶藻过程光谱学特性及机理研究
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摘要
藻类的大量繁殖对饮用水源、养殖业、旅游业以及人类健康造成了极大的影响。溶藻细菌作为一种生物控制手段,在控制藻类爆发方面显示出了极大的潜力。课题组前期分离获得一株金黄杆菌属溶藻菌Chryseobaterium sp.S7,研究发现该菌株具有明显的溶藻作用,作用方式为通过分泌溶藻物质进行间接溶藻,为进一步揭示该菌的溶藻特征及机理,以铜绿微囊藻为目标藻种,运用UV-Vis, EEMs, FTIR和FCM技术,分析Chryseobaterium sp.S7溶藻过程的光谱特性。实验结论如下:将菌株发酵液与藻液共培养7 d,利用UV-Vis和EEMs技术对藻细胞Chla含量与PC荧光值变化趋势进行分析,结果显示:藻细胞Chla含量在第1 d便开始下降,表明在短时间内,细菌胞外溶藻物质便可快速作用于藻细胞,第7 d时Chla去除率为59.37%。藻细胞PC荧光值也呈现下降趋势,与Chla变化趋势表现为一致性,表明在溶藻过程中伴有Chla和PC的减少。FTIR分析结果显示:藻细胞结构中的CO, C—H, O—H键分别在1 647, 2 927和3 475～3 437 cm~(-1)处的吸收峰强度明显减弱,表明藻细胞内的多糖物质和蛋白质结构可能被破坏,处于2 500～1 700 cm~(-1)范围的若干小吸收峰则进一步表明藻细胞解体的现象。分别在共培养第3 d和第7 d时对藻液进行PI特异染色,应用FCM对藻细胞PI特异性荧光和Chla, PC自发荧光特性进行分析,结果显示,在细菌S7的溶藻过程中,藻细胞PI特异性荧光逐渐增强, Chla、 PC自发荧光呈下降趋势、表明藻细胞膜、 Chla、 PC三者破坏程度在溶藻过程中具有紧密的内在联系和较高的一致性。溶藻过程中藻细胞表现为多种形式的损伤,且损伤处于动态变化过程中,由Q1(Q5)区细胞按顺序逐步向Q4(Q8)区细胞移动。推测Chryseobaterium sp.S7可能的溶藻过程为:细菌将溶藻活性物质释放到细胞外,溶藻活性物质通过破坏铜绿微囊藻细胞膜中的多糖和蛋白质的结构,增加膜的通透性,进一步破坏胞体内的Chla, PC和DNA/RNA等物质,使藻体裂解死亡,最终形成细胞碎片。通过对Chryseobaterium sp.S7溶藻过程藻细胞的光谱学特性的分析,初步揭示了Chryseobaterium sp.S7的溶藻机理,为微生物控藻及修复技术提供了理论依据。
The proliferation of algae has had a major impact on drinking water sources, aquaculture, tourism and human health. As a kind of biological control, algae-lysing bacteria, have shown great potential in controlling algal blooms. The research group isolated a strain of Chrysosporium sp. S7 in the early stage and found that, by secreting algae-dissolving substances, the strain had obvious algae-dissolving effect on algae in an indirect way. In order to reveal its algae-dissolving characteristics and mechanism, this study, with the Microcystis aeruginosa as the target algae species, employed S7 UV-Vis, EEMs, FTIR and FCM techniques to analyze the spectral characteristics of the algae-dissolving process of Chryseobaterium sp. S7. By co-culturing the fermentation broth of the strain with the solution of algae for 7 days and by analyzing the change trend of Chla content and PC fluorescence value of algae cells through UV-Vis and EEMs techniques, the research group got the following results: the content of Chla in algae cells began to decrease on the 1 st day, which indicated that the extracellular algae-dissolving substances of bacteria could quickly act on algae cells in short time, and the removal rate of Chla was 59.37% on the 7 th day. In addition, the fluorescence value of PC cells in algae cells also showed a similar downward trend with the trend of Chla, indicating a decrease in Chla and PC during the algae-dissolving process. The research group found that the absorption peaks of CO, C—H and O—H bonds in the algal cell structure showed a significant downtrend at 1 647, 2 927 and 3 475～3 437 cm~(-1) respectively, which suggested that the polysaccharide content in algae cells and the protein structure might be destroyed, while several small absorption peaks in the range of 2 500～1 700 cm~(-1) further indicated the phenomenon of disintegration of algae cells. The research group also carried out PI-specific staining of algae liquid on the 3 rd and 7 th day, and analyzed the PI-specific fluorescence of algae cells and the auto fluorescence characteristics of Chla and PC by FCM technique. The results showed that, the PI-specific fluorescence of cells increased gradually in the algae-dissolving process of bacteria S7, and the autofluorescence of Chla and PC showed a downward trend, indicating that the damage degree of algal cell membrane, Chla and PC had a close internal relationship with each other and high consistency in the algae-dissolving process. During the algae-dissolving process, the algae cells showed various forms of damage, and the damage was in the process of dynamic change, with the Q1(Q5) quadrant cells gradually moving to the Q4(Q8) quadrant cells in sequence. Therefore, the possible algae-dissolving process of Chryseobaterium sp. S7 could be speculated as follows: The bacteria release the algae-dissolving active substance to the outside of the cell, and the algae-removing active substance changes the structure and permeability of the algal cell membrane by destroying the structure of the polysaccharide and protein in the cell membrane of Microcystis aeruginosa, which will further destroy Chla in the cell body, PC, DNA/RNA and other substances. All these will cause the algae to lyse and die, eventually forming cell debris. This study, by analyzing the crystallization characteristics of algae cells in the algae process of Chryseobaterium sp. S7, reveals the algae-dissolving mechanism of algae-lysing bacteria, and thus provides a theoretical basis for microbial algae control and restoration technology.
The proliferation of algae has had a major impact on drinking water sources, aquaculture, tourism and human health. As a kind of biological control, algae-lysing bacteria, have shown great potential in controlling algal blooms. The research group isolated a strain of Chrysosporium sp. S7 in the early stage and found that, by secreting algae-dissolving substances, the strain had obvious algae-dissolving effect on algae in an indirect way. In order to reveal its algae-dissolving characteristics and mechanism, this study, with the Microcystis aeruginosa as the target algae species, employed S7 UV-Vis, EEMs, FTIR and FCM techniques to analyze the spectral characteristics of the algae-dissolving process of Chryseobaterium sp. S7. By co-culturing the fermentation broth of the strain with the solution of algae for 7 days and by analyzing the change trend of Chla content and PC fluorescence value of algae cells through UV-Vis and EEMs techniques, the research group got the following results: the content of Chla in algae cells began to decrease on the 1 st day, which indicated that the extracellular algae-dissolving substances of bacteria could quickly act on algae cells in short time, and the removal rate of Chla was 59.37% on the 7 th day. In addition, the fluorescence value of PC cells in algae cells also showed a similar downward trend with the trend of Chla, indicating a decrease in Chla and PC during the algae-dissolving process. The research group found that the absorption peaks of CO, C—H and O—H bonds in the algal cell structure showed a significant downtrend at 1 647, 2 927 and 3 475～3 437 cm~(-1) respectively, which suggested that the polysaccharide content in algae cells and the protein structure might be destroyed, while several small absorption peaks in the range of 2 500～1 700 cm~(-1) further indicated the phenomenon of disintegration of algae cells. The research group also carried out PI-specific staining of algae liquid on the 3 rd and 7 th day, and analyzed the PI-specific fluorescence of algae cells and the auto fluorescence characteristics of Chla and PC by FCM technique. The results showed that, the PI-specific fluorescence of cells increased gradually in the algae-dissolving process of bacteria S7, and the autofluorescence of Chla and PC showed a downward trend, indicating that the damage degree of algal cell membrane, Chla and PC had a close internal relationship with each other and high consistency in the algae-dissolving process. During the algae-dissolving process, the algae cells showed various forms of damage, and the damage was in the process of dynamic change, with the Q1(Q5) quadrant cells gradually moving to the Q4(Q8) quadrant cells in sequence. Therefore, the possible algae-dissolving process of Chryseobaterium sp. S7 could be speculated as follows: The bacteria release the algae-dissolving active substance to the outside of the cell, and the algae-removing active substance changes the structure and permeability of the algal cell membrane by destroying the structure of the polysaccharide and protein in the cell membrane of Microcystis aeruginosa, which will further destroy Chla in the cell body, PC, DNA/RNA and other substances. All these will cause the algae to lyse and die, eventually forming cell debris. This study, by analyzing the crystallization characteristics of algae cells in the algae process of Chryseobaterium sp. S7, reveals the algae-dissolving mechanism of algae-lysing bacteria, and thus provides a theoretical basis for microbial algae control and restoration technology.
引文
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[4] Inaba N,Trainer V L,Onishi Y,et al.Harmful Algae,2017,62:136.
[5] Liao C L,Liu X B.Environmental Science and Pollution Research,2016,23(6):5925.
[6] CHAI Tian,LIU Xiao-yu,XU Peng-cheng,et al(柴天,刘晓宇,许鹏成,等).Journal of Natural Disasters(自然灾害学报),2015,24(5):237.
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[11] Su J F,Shao S C,Ma F,et al.Chemical Engineering Journal,2016,293:139.
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[14] Tan S,Hu X L,Yin P H,et al.Journal of Microbiology,2016,54(5):364.
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