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为探究雨生红球藻(Haematococcus pluvialis)藻际可培养细菌多样性及可培养细菌对雨生红球藻生长和虾青素积累的影响,本实验采用培养分离法,在乙酸钠兼养条件下,共分离到8株雨生红球藻藻际细菌,通过16S rDNA测序鉴定,并在NCBI网站比较序列发现,编号为DBQ、HS、BTH、DBB、XBL、HB、FH、JH的菌株分别与Pseudomonas sp.、Methylobacterium sp.、Chryseobacterium sp.、Bacillus sp.、Paracoccus sp.、Planctopirus hydrillae、Rhodobacter vinaykumarii、Brevundimonas vesicularis的16S rDNA序列具有最高的相似性。分别将8株细菌与通过抗生素处理获得的纯种雨生红球藻共培养,测定了8株藻际细菌对雨生红球藻细胞密度、藻液pH、叶绿素及虾青素含量的影响。实验发现,HS处理组在绿色阶段培养第9天获得最高细胞密度,为3.65×10~5cells/mL,且显著高于对照组;DBB处理组虽然能够促进藻细胞密度增加,但与对照组并无显著差异;DBQ处理组藻细胞密度显著低于对照组及其他处理组。第1～7天,藻液叶绿素浓度的变化与藻细胞密度变化趋势有一定相似性,绿色阶段后期,叶绿素浓度呈现下降趋势,也说明藻细胞内部叶绿素向其他色素转变,同时标志着绿色阶段的结束。绿色阶段培养过程中,藻液pH变化幅度较大,由初始值7.0左右上升到9.30左右,但是培养后期处理组与对照组藻液pH并无显著差异。在测定藻液虾青素含量时,仅HS处理组虾青素含量略高于对照组,但并无显著差异;通过计算单个藻细胞虾青素含量发现DBQ处理组单个藻细胞虾青素含量显著高于对照组。
Abstract：In order to explore the diversity of cultivable bacteria in Haematococcus pluvialis phycosphere and their effects on the growth of H. pluvialis and its astaxanthin accumulation, we used the traditional culture separation method to isolate 8 bacterial strains in the mixotrophic culture of H. pluvialis. Based on 16 S rRNA gene sequences, strains(DBQ, HS, BTH, DBB, XBL, HB, FH and JH) were found to be highly similar with Pseudomonas sp., Methylobacterium sp., Chryseobacterium sp., Bacillus sp., Paracoccus sp., Planctopirus hydrillae, Rhodobacter vinaykumarii and Brevundimonas vesicularis, respectively. We used 8 strains as the additives, and studied their effects on cell density, algal pH, and chlorophyll and astaxanthin contents of H. pluvialis purified ahead with antibiotics. We found that the HS obtained the highest cell density on the 9 th day, which was 3.65×105 cells/mL, significantly higher than that of control. DBB increased the cell density; however no significant difference was found from that of control. From day 1 to day 7, the change in chlorophyll concentration was the same as that in algal cell density. At the end of green phase, the concentration of chlorophyll showed a decreasing trend, indicating that the chlorophyll in algal cells changed to other pigments and marked the end of green phase. At the green stage, the pH of algal liquid changed greatly, from the initial of 7.0 to about 9.30. However, there was no significant difference from that of control at the end of culture. The astaxanthin content of HS was slightly higher than that of control; however, no significant difference from that of control. It was also found that the content of astaxanthin in single algal cells of DBQ was significantly higher than that of control.
[1] 熊玉琴,杨锐,杨华田,等.藻际芽孢杆菌对坛紫菜生理的影响[J].生物学杂志,2018,35(1):37-41.Xiong Y Q,Yang R,Yang H T,et al.Effects of phycosphere Bacillus sp.WPy SW2 on physiology of Pyropia haitanensis[J].Journal of Biology,2018,35(1):37-41.
[2] Croft M T,Lawrence A D,Raux-Deery E,et al.Algae acquire vitamin B12 through a symbiotic relationship with bacteria[J].Nature (London),2005,438(7064):90-93.
[3] Amin S A,Hmelo L R,Van Tol H M,et al.Interaction and signalling between a cosmopolitan phytoplankton and associated bacteria[J].Nature,2015,522(7554):98-101.
[4] Amin S A,Green D H,Hart M C,et al.Photolysis of iron-siderophore chelates promotes bacterial-algal mutualism[J].Proceedings of the National Academy of Sciences,2009,106(40):17071-17076.
[5] Amavizca E,Bashan Y,Ryu C M,et al.Enhanced performance of the microalga Chlorella sorokiniana remotely induced by the plant growth-promoting bacteria Azospirillum brasilense and Bacillus pumilus[J].Scientific Reports,2017,7:41310.
[6] Mouget J L,Dakhama A,Lavoie M C,et al.Algal growth enhancement by bacteria:Is consumption of photosynthetic oxygen involved?[J].Fems Microbiology Ecology,2010,18(1):3-5.
[7] Paul C,Pohnert G.Interactions of the algicidal bacterium Kordia algicida with diatoms:Regulated protease excretion for specific algal lysis[J].PLOS ONE,2011,6(6):21032.
[8] Scott P,Kenneth F.Growth inhibition of Nannochloropsis species by Bacillus pumilus[J].Algal Research,2016,20:70-76.
[9] Seyedsayamdost M R,Case R J,Kolter R,et al.The Jekyll-and-Hyde chemistry of Phaeobacter gallaeciensis[J].Nature Chemistry,2011,3(4):331-335.
[10] Hui W,Tomasch Jürgen,Victoria M,et al.Identification of genetic modules mediating the jekyll and hyde interaction of dinoroseobacter shibae with the dinoflagellate Prorocentrum minimum[J].Frontiers in Microbiology,2015,6:1262.
[11] Wang H,Tomasch J,Jarek M,et al.A dual-species co-cultivation system to study the interactions between Roseobacters and Dinoflagellates[J].Frontiers in Microbiology,2014,5:311.
[12] Tanabe,Yuuhiko Okazaki,Yusuke Yoshida,et al.A novel alphaproteobacterial ectosymbiont promotes the growth of the hydrocarbon-rich green alga Botryococcus brauni[J].Scientific Reports,2015,5(10):10467.
[13] Rivas M O,Riquelme V C E.Interactions of Botryococcus braunii cultures with bacterial biofilms[J].Microbial Ecology,2010,60(3):628-635.
[14] 褚椒江,朱鹏,严小军.海带藻际微生物抗菌活性及其多样性的初步研究[J].水生生物学报,2011,35(6):972-979.Chu J J,Zhu P,Yan X J.Antimicrobila activity and diversity of marine bacteria in phycosphere of Laminaria japonica[J].Acta Hydrobiologica Sinica,2011,35(6):972-979.
[15] 方文雅.紫菜与藻际微生物互作研究平台的初步研究[D].宁波:宁波大学,2010.Fang W Y.Preliminary Study of the Plantform to Study the Interacion of Porphyra spp.and Their Phycoshperes[D].Ningbo:Ningbo University,2010.
[16] 沈梅丽,杨锐,骆其君,等.坛紫菜养殖周期中的藻际微生物多样性[J].微生物学报,2013,53(10):1087-1102.Shen M L,Yang R,Luo Q J,et al.Microbial diversity of Pyropia haitanensis phycosphere during cultivation[J].Acta Microbiologica Sinica,2013,53(10):1087-1102.
[17] 王剑.典型赤潮藻类可培养藻际细菌的分子鉴定与藻菌关系研究[D].广州:暨南大学,2014.Wang J.Studies on the Relationships Between Microalgae and Phycosphere Bacterial and Molecular Identification of Culturable Bacterial Isolated from Typical Red Tid Microalgae[D].Guangzhou:Jinan University,2014.
[18] 连玉武,王艳丽,郑天凌,等.赤潮科学中藻菌关系研究的若干进展[J].海洋科学,1999(1):35-38.Lian Y W,Wang Y L,Zheng T L,et al.Advance in the research in interaction between red tid algae and bacterial[J].Marine Sciences,1999(1):35-38.
[19] 王晴晴,高金伟,时晓婷,等.海洋饵料微藻与共栖细菌相互关系研究进展[J].农业与技术,2016,36(7):11-12.Wang Q Q,Gao J W,Shi X T,et al.Advances in research on the relationship between marine bait-microalgae and commensal bacteria[J].Agriculture and Technology,2016,36(7):11-12.
[20] 黄振华,陈星星,柯爱英,等.2种海洋饵料微藻藻际环境的细菌多样性研究[J].水生态学杂志,2018,39(1):44-49.Huang Zh H,Chen X X,Ke A Y,et al.Bacterial diversity in the phycosphere of two marine bait-microalgae[J].Journal of Hydroecology,2018,39(1):44-49.
[21] 张怀瑾.雨生红球藻培养优化及藻际微生物的研究[D].北京:清华大学,2016.Zhang H J.Optimization of Haematococcus pluvialis Cultivation and the Bacteria in Its Phycophere Environment[D].Beijing:Tsinghua University,2016.
[22] 张睿钦,管斌,孔青,等.雨生红球藻异养转化产虾青素的条件研究[J].浙江大学学报(农业与生命科学版),2011,37(6):624-630.Zhang R Q,Guan B,Kong Q,et al.Study on astaxanthin accumulation by heterotrophic transformation in Haematococcus pluvialis[J]Journal of Zhejiang University(Agric&Life Sci),2011,37(6):624-630.
[23] 孙慧洁,黄惠琴,朱军,等.2株海绵细菌的分离与鉴定[J].微生物学杂志,2010,30(3):1-4.Sun H J,Huang H Q,Zhu J,et al.Isolation and identification of two sponge bacteria from marine sponge[J].Journal of Microbiology,2010,30(3):1-4.
[24] 崔丹丹,杨柳,孙雪,等.玉米素和水杨酸对雨生红球藻(Haematococcus pluvialis)生长及虾青素积累的影响[J].海洋与湖沼,2018,49(3):682-691.Cui D D,Yang L,Sun X,et al.Effects of zeatin and salicylic acid on growth and astaxantin accumulation Haematococcus pluvialis[J].Oceanologia et Limnologia Sinica,2018,49(3):682-691.
[25] 李富超,周婕,秦松.元基因组用于未培养微生物的研究[J].海洋科学,2010,34(5):79-82,88.Li F Ch,Zhou J,Qing S.Metagenomics research on uncultured microorganisms[J].Marine Sciences,2010,34(5):79-82,88.
[26] 范念斯,齐嵘,杨敏.未培养微生物的培养方法进展[J].应用与环境生物学报,2016,22(3):524-530.Fan N S,Qi R,Yang M.Current technical progresses in the cultivation for uncultured microorganism[J].Chinese Journal of Applied and Environmental Biology,2016,22(3):524-530.
[27] 黄振华,陈星星,柯爱英,等.2种海洋饵料微藻藻际环境的细菌多样性研究[J].水生态学杂志,2018,39(1):44-49.Huang Zh H,Cheng X X,Ke A Y,et al.Bacterial diversity in the phycosphere of two marine bait-microalgae[J].Journal of Hydroecology,2018,39(1):44-49.
[28] 苗祯,杜宗军,李会荣,等.5株北极微藻藻际环境的细菌多样性[J].生态学报,2015,35(5):1587-1600.Miao Zh,Du Z J,Li H R,et al.Analysis of bacterial diversity in the phycosphere of five arctic microalgae[J].Acta Ecologica Sinica,2015,35(5):1587-1600.
[29] 杨小茹,苏建强,郑小伟,等.基于分子技术的1株产毒藻藻际细菌多样性分析[J].环境科学,2009,30(1):271-279.Yang X R,Su J Q,Zheng X Q,et al.16S rDNA clone library analysis of microbial diversity associated with the PSP-producing dinoflagellate alexandrium tamarense[J].Environmental Science,2009,30(1):271-279.
[30] 郑艳,姚婷.PCR-DGGE分析甘薯酸浆自然发酵过程中细菌多样性[J].食品科学,2016,37(7):99-103.Zheng Y,Yao T.Analysis of bacterial diversity during natural fermenation of sweet potato sour liquid by PCR-DGGE[J].Food Science,2016,37(7):99-103.
[31] 白玉.天山冻土微生物的系统多样性分析及生长特性的研究[D].兰州:兰州大学,2007.Yu B.Systematic Diversity Analysis and Growth Characteristics of Microbes in the Frozen Soil of Tianshan Mountains[D].Lanzhou:Lanzhou University,2007.
[32] 司文攻,吕志刚,许超.耐受高浓度氨氮异养硝化菌的筛选及其脱氮条件优化[J].环境科学,2011,32(11):3448-3454.Si W G,Lv Zh G,Xu Ch.Isolation of heterotrophic nitrifiers which can tolerate high concentration of ammonia-nitrogen and the optimization of their nitrogen removal efficiency in wastewater[J].Environmental Science,2011,32(11):3448-3454.
[33] 毛健,骆永明,滕应,等.一株高分子量多环芳烃降解菌的筛选、鉴定及降解特性研究[J].微生物学通报,2008(7):1011-1015.Mao J,Luo Y M,Teng Y,et al.Isolation and characterization of a high-molecular-weight (HMW) PAHs degrading bacterial strain[J].Microbiology China,2008(7):1011-1015.
[34] 季金殿.羽毛角蛋白质降解菌的分离、鉴定与角蛋白酶基因的克隆、表达[D].泰安:山东农业大学,2009.Ji J D.Isolation,Identification of Feather Keratin Degrading Bacteria and Cloning,Expression of Keratinase Gene[D].Taian:Shandong Agricultural University,2009.
[35] 韩吉昌,王松,张琳,等.一种利用高浓度联合抗生素批量纯化微藻的方法[J].中国海洋湖沼学报,2016,34(1):79-85.Han J Ch,Wang S,Zhang L,et al.A method of batch-purifying microalgae with multiple antibiotics at extremely high concentrations[J].Chinese Journal of Oceanology and Limnology,2016,34(1):79-85.
[36] 王旎,管斌,孔青,等.利用控pH流加培养法提高雨生红球藻的生物量及虾青素产量[J].食品与发酵工业,2016,42(2):120-124.Wang N,Guan B,Kun Q,et al.Enhancing biomass of Haematococcus pluvialis and astaxanthin production by pH-controlled fed-batch culture[J].Food and Fermentation Industries,2016,42(2):120-124.
[37] Cheirsilp B,Torpee S.Enhanced growth and lipid production of microalgae under mixotrophic culture condition:Effect of light intensity,glucose concentration and fed-batch cultivation[J].Bioresource Technology,2012,110:510-516.
[38] Pang N,Chen S.Effects of C5 organic carbon and light on growth and cell activity of Haematococcus pluvialis under mixotrophic conditions[J].Algal Research,2017,21:227-235.
[39] 孟春晓,高政权,王依涛,等.影响雨生红球藻中虾青素积累条件的研究进展[J].水产科学,2011,30(2):118-121.Meng X Ch,Gao Zh Q,Wang Y T,et al.Progress of factors affecting accumulation of astaxanthin in green alga Haematococcus pluvialis[J].Fisheries Science,2011,30(2):118-121.
[40] 王群,桑敏,李爱芬,等.雨生红球藻培养过程中色素动态变化与光合生理特性研究[J].天然产物研究与开发,2010,22(5):850-854.Wang Q,Sang M,Li A F,et al.The dynamic change of pigments and photosynthetic physiological characteristics of Haematococcus pluvialis cultures[J].Natural Product Research and Development,2010,22(5):850-854.
[41] 廖兴辉,王明兹,李小妹,等.高产虾青素的雨生红球藻胁迫条件和中试研究[J].食品与发酵工业,2015,41(6):29-34.Liao X H,Wang M i,Li X M,et al.Research on the stress induced conditions and pilot test of astaxanthin-rich Haematococcus pluvialis[J].Food and Fermentation Industries,2015,41(6):29-34.
[42] Wan M,Zhang J,Hou D,et al.The effect of temperature on cell growth and astaxanthin accumulation of Haematococcus pluvialis during a light-dark cyclic cultivation[J].Bioresource Technology,2014,167(3):276-283.
[43] 林伟,陈騳,刘秀云.海洋微藻除菌及除菌与自然带菌微藻生长特点比较[J].海洋与湖沼,2000(6):647-652.Lin W,Chen D,Liu X Y.Marine microalgal axenation and comparison of growth characteristics between natural and axenic marine microalgaea[J].Oceanologia et Limnologia Sinic,2000(6):647-652.
[44] 王群,桑敏,李爱芬,等.雨生红球藻培养过程中色素动态变化与光合生理特性研究[J].天然产物研究与开发,2010,22(5):850-854.Wang Q,Sang M,Li A F,et al.The dynamic change of pigments and photosynthetic physiological characteristics of Haematococcus pluvialis cultures[J].Natural Product Research and Development,2010,22(5):850-854.
[45] 周进,林光辉,蔡中华.微生物在藻际环境中的物质循环作用[J].应用生态学报,2016,27(8):2708-2716.Zhou J,Lin G H,Cai Zh H.Roles of microbes in matter cycles in phycosphere niche[J].Journal of Applied Ecology,2016,27(8):2708-2716.
[46] 巩风英.雨生红球藻中虾青素合成及几何异构体的分析研究[D].青岛:中国科学院大学(中国科学院海洋研究所),2017.Gong F Y.A Study on the Systhesis of Astaxanthin and the Analysis of Its Geometric Isomers in Haematococcus pluvialis[D].Qingdao:University of Chinese Academy of Sciences (Institute of Oceanology,Chinese Academy of Sciences),2017.
[47] 应巧兰,叶勇.影响雨生红球藻797株生长和虾青素积累的某些因素[J].应用与环境生物学报,2002,8(1):56-60.Ying Q L,Ye Y.Some factors affceting growth and astaxanthin accumulation of Haematococcus Pluvialis strain 797[J].Journal of Applied and Environmental Biology,2002,8(1):56-60.
[48] 孟春晓.雨生红球藻中虾青素合成关键酶基因的顺式作用元件研究[D].青岛:中国科学院研究生院(海洋研究所),2005.Meng X Ch.Characterization of Cis-acting Elements of Genes Encoding Two Key Enzymes in Astaxanthin Biosynthesis in Haematococcus pluvialis[D].Qingdao:Graduate School of the Chinese Academy of Sciences (Ocean Institute),2005.
基本信息:
DOI：10.16441/j.cnki.hdxb.20190124
中图分类号:Q945
引用信息:
[1]李芸,刘发龙,王巧晗,等.藻际可培养细菌对雨生红球藻生长和虾青素积累的影响[J],2020,50(07):44-55.DOI:10.16441/j.cnki.hdxb.20190124.
基金信息:
青岛海洋经济发展示范城市项目资助~~