ZHANG Xiaoqian,ZHANG Wei,LU Yaping,et al.The inhibitory effect of the fermentation filtrate of Brevibacillus brevis on Microcystis aeruginosa[J].Journal of Nanjing Agricultural University,2017,(4):625-631.[doi:10.7685/jnau.201610003]





The inhibitory effect of the fermentation filtrate of Brevibacillus brevis on Microcystis aeruginosa
张小倩 张炜 卢亚萍 高胜玲 汪瑾
南京农业大学生命科学学院, 江苏 南京 210095
ZHANG Xiaoqian ZHANG Wei LU Yaping GAO Shengling WANG Jin
College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China
Brevibacillus brevisMicrocystis aeruginosaalgicidecyanobacterial inhibition effect
[目的]微囊藻水华给湖泊生态系统及人类健康带来很大危害,本研究旨在探寻高效环保的微生物抑藻材料。[方法]通过流式细胞仪测定短短芽孢杆菌B15不同发酵时间及添加量下无菌发酵上清液的抑藻率,通过激光共聚焦显微镜和电子显微镜观察处理后的藻细胞形态,并通过叶绿素荧光仪测定处理前、后藻细胞光合作用的变化。另外,检测了发酵上清液在不同温度、pH值和蛋白酶处理下的活性变化。[结果]0.3%、0.7%、1.0%和2.0%添加量的发酵液处理6 d后抑藻率分别为51.89%、79.89%、89.47%和86.94%。发酵液发酵48和72 h抑藻活性无明显差异,但略优于发酵24 h。发酵上清液处理6 d后,藻细胞Fv/FmETRmax值均显著下降,表明其光合作用受到抑制。激光共聚焦显微镜观察发现:许多细胞停留在分裂末期,呈2细胞或4细胞状态。1.0%发酵上清液处理6 d后藻液中2细胞和4细胞所占比例分别达到62.23%和14.95%,表明藻细胞分裂增殖严重受阻。透射电镜观察结果显示:经处理后藻细胞严重受损,出现质壁分离、类囊体结构及细胞膜断裂的现象。发酵上清液超滤结果显示抑藻活性物质相对分子质量小于10×103。将抑藻物质在4、25和37 ℃处理6 h后,抑藻活性和对照相比无显著差异,说明该活性物质在低温下有较好的稳定性。另外,在pH值2~12的范围内,抑藻物质都保持很好的活性。抑藻物质对胰蛋白酶和蛋白酶K均不敏感。[结论]短短芽孢杆菌B15通过分泌小分子化合物高效而持久地抑制铜绿微囊藻的生长繁殖,降低其光合作用效率,并阻遏细胞的分裂。
[Objectives]The frequent outbreaks of algal-bloom have done great harm to ecosystems and human life. The aim of this study was to explore a high-effective and eco-friendly algicide from microbes. [Methods]The inhibitory rates of the sterile filtrate at different fermentation time and different concentrations were determined by flow cytometry. The morphology of the cyanobacterial cells was observed by laser scanning confocal microscope and electron microscope. The changes of photosynthetic activities between control and treated cells were measured by using a pulse amplitude-modulated fluorescence monitoring system. In addition,the changes of the algicidal activities of the sterile filtrate under different temperature,pH value and protease treatment were detected. [Results]The sterile filtrate with the added amount of 0.3%,0.7%,1.0% and 2.0% had an inhibitory rate of 51.89%,79.89%,89.47% and 86.94%,respectively. There was no significant difference between the inhibitory activity of 48 and 72 h fermentation filtrate,both of which were better than that of 24 h fermentation. After 6 days’ treatment,Fv/Fm and ETRmax value decreased significantly,which suggested the inhibition of photosynthesis. Under the laser scanning confocal microscope,many cells were found to remain at the end of cell division,forming 2- or 4-cell aggregates. The proportion of 2- and 4-cell after treatment for 6 d by 1.0% of fermentation filtrate was 62.23% and 14.95%,respectively,showing the cell division was severely blocked. Transmission electron microscope showed that the algal cells were seriously damaged,with the phenomenon of plasmolysis,the rupture of the thylakoid and cytomembrane. The molecular weight of the main active substances was speculated to be less than 10×103 by ultrafiltration of the sterile filtrate. After treatment for 6 h at 4,25 and 37 ℃,the inhibitory activity had no marked difference from the control,which showed that the active substances were stable at these temperatures. In addition,after treatment at pH value 2-12,the active substances remained good inhibitory activity. The fermentation filtrate was not sensitive to trypsin or protease K. [Conclusions]B15 can inhibit the growth and reproduction of M.aeruginosa by secreting small molecules,which reduce its photosynthetic efficiency and repress cell division,with a high and lasting inhibitory rate.


[1] Anderson D M. Turning back the harmful red tide[J]. Nature,1997,388:513-514.
[2] Lu Y P,Wang J,Yu Y,et al. Inhibition of Camellia sinensis(L.)O.Kuntze on Microcystis aeruginosa and isolation of the inhibition factors[J]. Biotechnology Letters,2013,35(7):1029-1034.
[3] Li Y,Liu L,Xu Y,et al. First evidence of Altererythrobacter sp. LY02 with indirect algicidalactivity on the toxic dinoflagellate,Alexandrium tamarense[J]. Current Microbiology,2016,73(4):550-560.
[4] Li Z,Lin S,Liu X,et al. A freshwater bacterial strain,Shewanella sp.Lzh-2,isolated from Lake Taihu and its two algicidal active substances,hexahydropyrrolo[1,2-a]pyrazine-1,4-dione and 2,3-indolinedione[J]. Applied Microbiology and Biotechnology,2014,98(10):4737-4748.
[5] Nobuyuki N,Kazunori N,Norio S,et al. A novel cyanobacteriolytic bacterium,Bacillus cereus,isolated from a eutrophic lake[J]. Journal of Bioscience and Bioengineering,2003,95(2):179-184.
[6] 汪瑾,杜明勇,于玉凤,等. 几种植物浸提液对铜绿微囊藻的抑制作用及抑藻特性[J]. 南京农业大学学报,2014,37(4):91-96. DOI:10.7685/j.issn.1000-2030.2014.04.013.Wang J,Du M Y,Yu Y F,et al. The inhibitory effect of plant extracts on Microcystis aeruginosa and its algal inhibiting characteristics[J]. Journal of Nanjing Agricultural University,2014,37(4):91-96(in Chinese with English abstract).
[7] Li Z,Geng M,Yang H. Algicidal activity of Bacillus sp.Lzh-5 and its algicidal compounds against Microcystis aeruginosa[J]. Applied Microbiology and Biotechnology,2015,99(2):981-990.
[8] Zhou S,Yin H,Tang S,et al. Physiological responses of Microcystis aeruginosa against the algicidal bacterium Pseudomonas aeruginosa[J]. Ecotoxicology and Environmental Safety,2016,127:214-221.
[9] Luo J,Wang Y,Tang S,et al. Isolation and identification of algicidal compound from Streptomyces and algicidal mechanism to Microcystis aeruginosa[J]. PLoS ONE,2013,8(10):e76444.
[10] Li Y,Yi W H,Komatsu M,et al. Isolation and characterization of bacterial isolates algicidal against a harmful bloom-forming cyanobacterium Microcystis aeruginosa[J]. Biocontrol Science,2012,3(17):107-114.
[11] Edwards S G,Seddon B. Mode of antagonism of Brevibacillus brevis against Botrytis cinerea in vitro[J]. Journal of Applied Microbiology,2001,91(4):652-659.
[12] Li S,Zhao Z A,Li M,et al. Purification and characterization of a novel chitinase from Bacillus brevis[J]. Acta Biochimica et Biophysica Sinica,2002,34(6):690-696.
[13] Che J M,Ye S W,Liu B,et al. Effects of Brevibacillus brevis FJAT-1501-BPA on growth performance,faecalmicroflora,faecal enzyme activities and blood parameters of weaned piglets[J]. Antonie van Leeuwenhoek,2016,109(12):1545-1553.
[14] 王宇婷,易有金,夏菠,等. 内生短短芽胞杆菌011菌发酵滤液抑菌活性研究[J]. 植物保护,2013,39(4):29-33.Wang Y T,Yi Y J,Xia B,et al. Antibiosis of the ferment of the endophyte Brevibacillu sbrevis strain 011[J]. Plant Protection,2013,39(4):29-33(in Chinese with English abstract).
[15] Majumdar S,Sarmah B,Gogoi D,et al. Characterization,mechanism of anticoagulant action,and assessment of therapeutic potential of afibrinolytic serine protease(Brevithrombolase)purified from Brevibacillu sbrevis strain FF02B[J]. Biochimie,2014,103(1):50-60.
[16] 张晓舟,徐剑宏,李顺鹏.植病生防芽孢杆菌的分离筛选与初步鉴定[J]. 土壤,2005,37(1):85-88.Zhang X Z,Xu J H,Li S P. Isolation,screening and preliminary identification of plant pathogenic fungi biocontrol strains of Bacillus spp.[J]. Soils,2005,37(1):85-88(in Chinese with English abstract).
[17] Rippka R,Desrulles J,Waterbury J B,et al. Generic assignment,strain histories and properties of pure cultures of cyanobacteria[J]. Journal of General Microbiology,1979,111:1-61.
[18] Jung S W,Kim B H,Katano T,et al. Pseudomonas fluorescens HYK0210-SK09 offers species-specific biological control of winter algal blooms caused byfreshwater diatom Stephano discushantzschii[J]. Journal of Applied Microbiology,2008,105:186-195.
[19] Paul C,Pohnert G. Induction of protease release of the resistant diatom Chaetoceros didymusin response to lytic enzymes from an algicidal bacterium[J]. PLoS ONE,2013,8(3):e57577.
[20] Yoshikawa K,Adachi K,Nishijima M,et al. β-cyanoalanine production by marine bacteria on cyanide-free medium and its specific inhibitory activitytoward cyanobacteria[J]. Applied and Environmental Microbiology,2000,66(2):718-722.
[21] Zhang H,Peng Y,Zhang S,et al. Algicidal effects of prodigiosin on the harmful algae Phaeocystis globosa[J]. Frontiers in Microbiology,2016,7:602.
[22] Park S C,Lee J K,Kim S W,et al. Selective algicidal action of peptides against harmful algal bloom species[J]. PLoS ONE,2011,6(10):e26733.
[23] Dakhama A,Noüe J D L,Lavoie M C.Isolation and identification of antialgal substances produced by Pseudomonas aeruginosa[J]. Journal of Applied Phycology,1993,5:297-306.
[24] Jeong S Y,Ishida K,Ito Y,et al. Bacillamide,a novel algicide from the marine bacterium,Bacillus sp.SY-1,against the harmful dinoflagellate,Cochlodinium polykrikoides[J]. Tetrahedron Letters,2003,44(43):8005-8007.
[25] Che J M,Liu B,Chen Z,et al. Identification of ethylparaben as the antimicrobial substance produced by Brevibacillus brevis FJAT-0809-GLX[J]. Microbiological Research,2015,172:48-56.
[26] Westman E L,Yan M,Waglechner N,et al. Self-resistance to the atypical cationic antimicrobial peptide edeine of Brevibacillus brevis Vm4 by the N-acetyltransferase EdeQ[J]. Chemistry and Biology,2013,20(8):983-990.
[27] 贾雯,李长玲,黄翔鹄.侧孢短芽孢杆菌溶藻活性代谢物部分特性的研究[J]. 应用海洋学学报,2014,33(1):68-77.Jia W,Li C L,Huang X H. Research on some characteristics of the extracellular algae-lysing components from Brevibacillus laterosporus[J]. Journal of Applied Oceanography,2014,33(1):68-77(in Chinese with English abstract).
[28] Zhang H,Zhang S,Peng Y,et al. Effectiveness and toxicity of a novel isolated actinomycete strain Streptomyces sp.JS01 on a harmful alga Phaeocystis globosa[J]. Applied Microbiology and Biotechnology,2015,99(11):4807-4814.
[29] Li Y,Zhu H,Zhang H,et al. Toxicity of algicidal extracts from Mangrovimonas yunxiaonensis strain LY01 on a HAB causing Alexandrium tamarense[J]. Journal of Hazardous Materials,2014,278:372-381.


 CHEN Qiao-ling,HU Jiang*,WANG Han-cheng,et al.Effects of bio-organic fertilizer application on population of Ralstonia solanacearum and Brevibacillus brevis in tobacco rhizosphere[J].Journal of Nanjing Agricultural University,2012,35(4):75.[doi:10.7685/j.issn.1000-2030.2012.01.013]
 WANG Jin,DU Mingyong,YU Yufeng,et al.The inhibitory effect of plant extracts on Microcystis aeruginosa and its algal inhibiting characteristics[J].Journal of Nanjing Agricultural University,2014,37(4):91.[doi:10.7685/j.issn.1000-2030.2014.04.013]
 WU Yingchuan,ZOU Lin,WANG Jin,et al.The inhibitory effect and mechanism of EGCG on Microcystis aeruginosa[J].Journal of Nanjing Agricultural University,2016,39(4):78.[doi:10.7685/jnau.201506007]


更新日期/Last Update: 2017-07-07