一株微杆菌CBA01对球形棕囊藻的溶藻特性与生理响应研究

doi:10.13560/j.cnki.biotech.bull.1985.2021-0161

摘要/Abstract

摘要：

赤潮给海洋生态环境和海水养殖业造成了严重危害,溶藻细菌是目前生物防治赤潮的有效手段之一。旨在探究从蓝细菌Cyanobacterium sp SCSIO-45682分离的4株共附生细菌中得到的一株对球形棕囊藻具有明显溶藻效应的微杆菌属菌株Microbacterium sp. CBA01的溶藻特性和初步的作用机理。采用细菌发酵液与棕囊藻共培养模式测定菌株CBA01的溶藻效应,并分别研究去除菌体、不同温度、不同pH条件下的溶藻特性,进一步测定藻细胞丙二醛（MDA）和抗氧化相关酶系统的响应规律。结果显示,菌株CBA01发酵菌液和无菌上清液对棕囊藻有明显的溶藻作用,而菌体本身不具有溶藻活性;其溶藻效应随着菌液发酵时间延长、菌液添加量增多以及藻液初始浓度降低而增强。经CBA01无菌上清液处理,棕囊藻生长初期的细胞丙二醛（MDA）含量大幅度上升,超氧化物歧化酶（SOD）和过氧化氢酶（POD）活性显著提高。菌株CBA01通过胞外分泌溶藻物质经间接溶藻作用而导致球形棕囊藻的裂解,其分泌的溶藻活性物质具有一定耐高温性、耐酸不耐强碱性;而该菌株产生的溶藻物质,可能具有较强烈的氧化活性,或产生诱导藻细胞产生过量氧自由基的活性,这可能是其形成溶藻效应的重要机制。

关键词: Microbacterium sp. CBA01, 赤潮, 球形棕囊藻, 溶藻特性, 生理响应

Abstract:

The harmful algae blooms have seriously affected marine ecological environment and mariculture industry,and the exploitation of algae-lying bacteria is one of the effective biological methods to control the harmful algae blooms. This work aims to investigate the algicidal characteristics and preliminary mechanism of a strain of Microbacterium sp. CBA01,one of the 4 strains of epiphytic bacterium isolated from Cyanobacterium sp. SCSIO-45682,with strong algicidal effect on Phaeocystis globosa. The co-culture mode of bacterial fermentation broth and P. globosa was used to determine the algae-lying effect of the strain CBA01,and the algicidal characteristics were studied by removing bacteria,different temperature and different pH,respectively. Furthermore,algal cells about the response rules of malondialdehyde（MDA）and antioxidant related enzyme system were determined. The strain CBA01 fermentation broth and sterile supernatant had obvious algicidal effect on P. globosa,but the mycelia itself showed no algicidal activity. The algicidal effect enhanced with the increase of fermentation time and the addition amount of fermentation broth,and the decrease of the initial concentration of algae. After adding the CBA01 sterile supernatant,the MDA content and the activities of superoxide dismutase（SOD）and peroxidase（POD）significantly increased in the early growth stage of P. globosa. In conclusion,the strain CBA01 caused the lysis of P. globosa by secreting extracellular algicidal substances. The active algicidal substances are of certain high temperature resistance,acid resistance but weak alkaline resistance. And the algicidal substances produced by the strain CBA01 may either have strong oxidation activity or induce the algal cells to produce excess reactive oxygen species（ROS）,which might be an important mechanism of alginolytic effect.

Key words: Microbacterium sp. CBA01, harmful algae blooms, Phaeocystis globosa, algicidal characteristics, physiological response

王灵, 向文洲, 卫华宁, 吕金亭, 吴华莲, 吴后波. 一株微杆菌CBA01对球形棕囊藻的溶藻特性与生理响应研究[J]. 生物技术通报, 2021, 37(10): 91-99.

WANG Ling, XIANG Wen-zhou, WEI Hua-ning, LV Jin-ting, WU Hua-lian, WU Hou-bo. Study on the Algicidal Characteristics and Physiological Response of Microbacterium sp. CBA01 to Phaeocystis globosa[J]. Biotechnology Bulletin, 2021, 37(10): 91-99.

图/表 6

图1 细菌CAB01的生长曲线

Fig. 1 Growth curve of symbiotic bacteria CAB01

图2 菌液不同发酵时间（A）、菌液不同添加量（B）及藻液不同初始浓度（C）对溶藻活性的影响

Fig. 2 The effect of different fermentation time (A) and addition amount of bacterial solution (B) and different initial concentration of P. globosa (C) on algicidal activity

图3 菌液不同处理对溶藻活性的影响

Fig. 3 The effect of different addition treatment of bacter-ial solution on algicidal activity

图4 不同温度（A）和酸碱（B）条件处理后的CBA01无菌上清液对球形棕囊藻的溶藻效果

Fig. 4 Algicidal efficiency of CBA01 sterile filtrate trea-ted with different temperature (A) and pH (B) conditions on P. globosa

图5 无菌上清液对球形棕囊藻细胞抗氧化系统相关酶活性的影响 **表示与对照组相比差异极显著（P<0.01）,下同

Fig. 5 Effect of sterile supernatant on the activity of antioxidant enzymes of P. globosa cells ** indicates a very significantly difference from the control group（P<0.01）, the same below

图6 无菌上清液对球形棕囊藻细胞叶绿素a含量的影响

Fig 6 Effect of sterile supernatant on chlorophyll a acti-vity of P. globosa cells

参考文献 32

[1]	石新国, 李悦, 郑文煌, 等. 一株中肋骨条藻特异溶藻菌的分离鉴定及溶藻特性[J]. 微生物学通报, 2020, 47(11):3527-3538.
	Shi XG, Li Y, Zheng WH, et al. Lysis effect and algicidal characteristics of a specific algicidal bacterium on Skeletonema costatum[J]. Microbiology China, 2020, 47(11):3527-3538.
[2]	Imai I, Sunahara T, Nishikawa T, et al. Fluctuations of the red tide flagellates Chatonella spp. (Raphidophyceae)and the algicidal bacterium Cytophaga sp. In the Seto Inland Sea, Japan[J]. Marine Biology, 2001, 138:1043-1049. doi: 10.1007/s002270000513 URL
[3]	Tang KW, Jakobsen HH, Visser AW. Phaeocystis globosa(Prymnesiophyceae)and the planktonic food web:Feeding, growth, and trophic interactions among grazers[J]. Limnology and Oceanography, 2001, 46(8):1860-1870. doi: 10.4319/lo.2001.46.8.1860 URL
[4]	Schoemann V, Becquevort S, Stefels J, et al. Phaeocystis blooms in the global ocean and their controlling mechanisms:A review[J]. Journal of Sea Research, 2005, 53:43-66. doi: 10.1016/j.seares.2004.01.008 URL
[5]	Manage PM, Kawabata Z, Nakano S. Algicidal effect of the bacterium Alcaligenes denitrificans on Microcystis spp[J]. Aquatic Microbial Ecology, 2000, 22:111-117. doi: 10.3354/ame022111 URL
[6]	席宇, 朱大恒, 黄进勇, 等. 溶藻细菌的生态学作用及其生物量检测方法[J]. 微生物学杂志, 2005(5):62-67.
	Xi Y, Zhu DH, Huang JY, et al. Ecological role and biomass detecting methods of algae-lysing bacteria[J]. Journal of Microbiology, 2005(5):62-67.
[7]	赵玲, 陈淼银, 等. 细菌B1胞外活性物质对球形棕囊藻的溶藻机制初探[J]. 环境科学学报, 2013, 33(5):1286-1291.
	Zhao L, Chen MY, Yin PH, et al. Preliminary investigation on algicidal mechanism of extracellular active substances from an algae-lysing bacterium B1 to Phaeocystis globosa[J]. Acta Scientiae Circumstantiae, 2013, 33(5):1286-1291.
[8]	Tilney CL, Pokrzywinski KL, et al. Effects of a bacterial algicide, IRI-160AA, on dinoflagellates and the microbial community in microcosm experiments[J]. Harmful Algae, 2014, 39:210-222. doi: 10.1016/j.hal.2014.08.001 URL
[9]	Zhao L, Chen LN, Yin PH. Algicidal metabolites produced by Bacillus sp. strain B1 against Phaeocystis globosa[J]. Journal of Industrial Microbiology & Biotechnology, 2014, 41(3):593-599.
[10]	Zhang CC, Massey IY, Liu Y, et al. Identification and characterization of a novel indigenous algicidal bacterium Chryseobacterium species against Microcystis aeruginosa[J]. Journal of Toxicology & Environmental Health Part A, 2019, 82(15):845-853.
[11]	杨冰洁, 向文洲, 等. 一株溶藻菌CBA02的分离、鉴定及溶藻特性研究[J]. 生物技术通报, 2020, 36(11):55-62.
	Yang BJ, Xiang WZ, Jin XJ, et al. Isolation and identification of an algicidal bacteria CBA02 and it’ s algae-lytic characteristic[J]. Biotechnology Bulletin, 2020, 36(11):55-62.
[12]	Xu LY, Zheng L, Han XP, et al. Screening of microalgae associated bacteria with quorum sensing system and their algicidal activity[J]. Oceanologia et Limnologia Sinica, 2012, 43(6):1149-1155.
[13]	Tang MX, Tan L, Wu HL, et al. Gelatiniphilus marinus gen. nov. sp. nov. a novel bacterium from the culture broth of a microalga, Picochlorum sp. 122, and emended description of the genus Hwangdonia[J]. International Journal of Systematic and Evolutionary Microbiology, 2016, 66(8):2893-2898. doi: 10.1099/ijsem.0.001074 URL
[14]	Guillard RRL, Ryther JH. Studies on marine planktonic diatoms. 1. Cyclotella nana Hustedt and Detonula confervacea(Cleve)Gran[J]. Canadian Journal of Microbiology, 1962, 8:220-239.
[15]	GB17378.7, 海洋监测规范, 第7部分: 近海污染生态调查和生物监测[S]. 国家市场监督管理总局, 2007.
	GB17378.7, The specification for marine monitoring, Part 7: Ecological survey for offshore pollution and biological monitoring[S]. State Administration for Market Regulation, 2007.
[16]	Jeffrey SW, Humphrey GF. New spectrophotometric equations for determining chlorophylls a, b, c1 and c2 in higher plants, algae and natural phytoplankton[J]. Biochemie und Physiologie der Pflanzen, 1975, 167(2):191-194. doi: 10.1016/S0015-3796(17)30778-3 URL
[17]	韩贝贝, 张书飞, 吴风霞, 等. 一株嗜盐杆菌对中肋骨条藻的溶藻作用机理研究[J]. 生态科学, 2020, 39(1):1-9.
	Han BB, Zhang SF, Wu FX, et al. Study of algicidal mechanism on Skeletonema costatum by Halobacillus bacteria[J]. Ecological Science, 2020, 39(1):1-9.
[18]	Tan S, Hu XL, et al. Photosynthetic inhibition and oxidative stress to the toxic Phaeocystis globosa caused by a diketopiperazine isolated from products of algicidal bacterium metabolism[J]. Journal of Microbiology, 2016, 54(5):364-375. doi: 10.1007/s12275-016-6012-0 URL
[19]	黄洪辉, 韩贝贝, 张书飞, 等. 海洋溶藻菌的研究进展[J]. 南方水产科学, 2019, 15(5):126-132.
	Huang HH, Han BB, Zhang SF, et al. Advance in marine algicidal bacteria research[J]. South China Fisheries Science, 2019, 15(5):126-132.
[20]	焦彦凯, 严小军, 李小兵. 溶藻细菌及溶藻化合物研究进展[J]. 工业微生物, 2018, 48(4):56-62.
	Jiao YK, Yan XJ, Li XB. Research progress of alginolytic bacteria and alginolytic compounds[J]. Industrial Microbiology, 2018, 48(4):56-62.
[21]	黄珺, 黄洪辉, 等. 嗜盐杆菌HSQAY1对中肋骨条藻的溶藻物质特性[J]. 环境工程学报, 2016, 10(4):2077-2082.
	Huang J, Huang HH, Wu FX, et al. Characteristics of algicidal substance against Skeletonema costatum produced by Halobacillus sp. HSQAY1[J]. Chinese Journal of Environmental Engineering, 2016, 10(4):2077-2082.
[22]	Zhang BZ, Cai GJ, Wang HT, et al. Streptomyces alboflavus RPS and its novel and high algicidal activity against harmful algal bloom species Phaeocystis globosa[J]. PLoS One, 2014, 9(3):e92907. doi: 10.1371/journal.pone.0092907 URL
[23]	陈丽娜. 芽孢杆菌B1胞外活性物质的分离鉴定及溶藻机制的研究[D]. 广州:暨南大学, 2014.
	Chen LN. Isolation and identification of algicidal compounds from Bacillus sp. strain B1 and algicidal mechanisms to the harmful algae[D]. Guangzhou:Ji’nan University, 2014.
[24]	孙军德, 魏雅冬, 佟德利, 等. 溶藻细菌对青苔的防除效果研究[J]. 沈阳农业大学学报, 2009, 40(4):439-443.
	Sun JD, Wei YD, Tong DL, et al. The controlling effects of algae-lysing bacteria on moss[J]. Journal of Shenyang Agricultural University, 2009, 40(4):439-443.
[25]	Liu J, Yang C, Chi Y, et al. Algicidal characterization and mechanism of Bacillus licheniformis Sp34 against Microcystis aeruginosa in Dianchi Lake[J]. Journal of Basic Microbiology, 2019, 59(11):1112-1124. doi: 10.1002/jobm.v59.11 URL
[26]	Lee YK, Ahn CY, Kim HS, et al. Cyanobactericidal effect of Rhodococcus sp. isolated from eutrophic lake on Microcystis sp.[J]. Biotechnology Letters, 2010, 32:1673-1678. doi: 10.1007/s10529-010-0350-5 URL
[27]	Imai I, Ishida Y, Hata Y. Killing of marine phytoplankton by a gliding bacterium Cytophaga sp. isolated from the coastal sea of Japan[J]. Marine Biology, 1993, 116(4):527-532. doi: 10.1007/BF00355470 URL
[28]	Li Y, Zhu H, Guan C, et al. Towards molecular, physiological, and biochemical understanding of photosynthetic inhibition and oxidative stress in the toxic Alexandrium tamarense induced by a marine bacterium[J]. Applied Microbiology and Biotechnology, 2014, 98(10):4637-4652. doi: 10.1007/s00253-014-5578-x URL
[29]	史顺玉, 沈银武, 李敦海, 等. 溶藻细菌DC21的分离、鉴定及其溶藻特性[J]. 中国环境科学, 2006(5):77-80.
	Shi SY, Shen YW, Li DH, et al. Isolation, identification and algae-lytic characteristic of a bacterium Staphylococcus sp.[J]. China Environmental Science, 2006(5):77-80.
[30]	牛丹丹, 郑青松, 刘兆普, 等. 溶藻细菌YZ对铜绿微囊藻的溶藻特性研究[J]. 中国环境科学, 2011(2):321-326.
	Niu DD, Zheng QS, Liu ZP, et al. Algicidal effect of an algae-lysing bacterium YZ on Microcystis aeruginosa[J]. China Environmental Science, 2011(2):321-326.
[31]	马浩. 一株短波单胞菌的溶藻特性及溶藻物质的分离纯化[D]. 青岛:山东大学, 2017.
	Ma H. Algicidal characteristics of Brevundimonas sp. and purification of the algicidal substances[D]. Qingdao:Shandong University, 2017.
[32]	Hu XL, Yin PH, Zhao L, et al. Characterization of cell viability in Phaeocystis globosa cultures exposed to marine algicidal bacteria[J]. Biotechnology & Bioprocess Engineering, 2015, 20(1):58-66.