波吉卵囊藻常温保存的研究.doc

分类号：s984.4 密 级 ：公开u d c ：632 学 号 ：201050013117 广 东 海 洋 大 学 硕士学位论文 波吉卵囊藻常温保存的研究 何震寰 指 导 教 师：黄翔鹄 教授 申 请 学 位 类 别：理学硕士 专 业 名 称：海洋生物学 研 究 方 向：海洋环境与生物资源保护 学 院 名 称：水产学院 中国湛江 2013 年 6 月学位论文答辩委员会组成 主席： 委员： 导师： 时间： 2013 年 6 月 4 日 广 东 海 洋 大 学 学位论文原创性声明 本人郑重声明：所呈交的论文是本人在导师的指导下独立进行研究所取得的研究成果。除了文中特别加以标注引用的内容外，本论文不包含任何其他个人或集体已经发表或撰写的成果作品。对本文的研究做出重要贡献的个人和集体，均已在文中以明确方式标明。本人完全意识到本声明的法律后果由本人承担。 作者签名： 何震寰 2013 年 6 月 4 日 学位论文版权使用授权书 本学位论文作者完全了解学校有关保留、使用学位论文的规定，同意学校保留并向国家有关部门或机构送交论文的复印件和电子版，允许论文被查阅和借阅。本人授权广东海洋大学可以将本学位论文的全部或部分内容编入有关数据库进行检索，可以采用影印、缩印或扫描等复制手段保存和汇编本学位论文。 本学位论文属于 1、保密，在_年解密后适用本授权书。 2、不保密。 （请在以上相应方框内打“”）作者签名： 何震寰 2013 年 6 月 4 日导师签名： 2013 年 6 月 4 日 波吉卵囊藻常温保存的研究 摘要 波吉卵囊藻（oocystis borgei）作为一种对虾高位池种群稳定，分布广，适应能力强，具有改善养殖水质，增强对虾抗病能力等功能的优良藻种，无疑是实现对虾养殖生态调控防病的首选。采用波吉卵囊藻进行生态调控可减少病害的发生、药物的使用以及药品残留所带来的二次污染。目前未曾有关于波吉卵囊藻常温保存的研究。为推动波吉卵囊藻在水产养殖业的应用，减少养殖水环境的恶化，研究波吉卵囊藻在常温下的保存效果不仅为常温下微藻的保存提供理论依据，还能找出一定时间内在室内环境下保存波吉卵囊藻的最佳方法，实现波吉卵囊藻的简易保存便于短时间内的市场流通与对虾养殖的直接投放使用。本文研究了不同浓度二甲基亚砜、甘油、山梨酸钾以及沼泽红假单胞菌（rhodopseudomonas palustris）在不同温度条件下对波吉卵囊藻保存效果的影响；通过正交试验探讨二甲基亚砜、甘油和山梨酸钾保存波吉卵囊藻的最优组合，同时研究了不同藻浓度对波吉卵囊藻保存效果的影响，并在最佳保存浓度下将二甲基亚砜、甘油和山梨酸钾的最优组合与沼泽红假单胞菌对波吉卵囊藻保存效果的影响作对比，以求波吉卵囊藻常温下保存的最佳方案。实验结果如下： 1、 在 不同二甲基亚砜浓度对波吉卵囊藻保存效果的影响结果显示： 25 、 30和 35 下当二甲基亚砜浓度为 4.5 时波吉卵囊藻保存效果最好； 下添加二甲基 25亚砜浓度为 4.5 时，波吉卵囊藻存活率达到 86.89，叶绿素含量为 27.13 g/l，经恢复培养后，比增长率为 0.416，叶绿素含量为 52.61 g/l。30 下添加二甲基亚砜浓度为 4.5 时，波吉卵囊藻存活率达到 73.41 ，叶绿素含量为 22.54 g/l，经恢复培养后，比增长率为 0.408，叶绿素含量为 38.02 g/l；35 下添加二甲基亚砜浓度为 4.5 时，波吉卵囊藻存活率达到 50.56 ，叶绿素含量为 12.25 g/l，经恢复培养后，比增长率为 0.320，叶绿素含量为 21.94 g/l。 2、 在 不同浓度甘油对波吉卵囊藻保存效果的影响结果显示， 25 、 和 35 30下当甘油浓度为 6 时波吉卵囊藻保存效果最好。25 下添加甘油浓度为 6 时，波吉卵囊藻存活率达到 91.43 ，叶绿素含量为 30.58 g/l，经恢复培养后，比增长率为 0.409，叶绿素含量为 55.82 g/l；30 下添加甘油浓度为 6 时，波吉卵囊藻存活率达到 74.46 ，叶绿素含量为 25.72 g/l，经恢复培养后，比增长率为 0.387，叶绿素含量为 45.66 g/l；35 下添加甘油浓度为 6 时，波吉卵囊藻存活率达到53.65 ，叶绿素含量为 16.84 g/l，经恢复培养后，比增长率为 0.341，叶绿素含量为 20.97 g/l。 3、不同浓度山梨酸钾对波吉卵囊藻保存效果的影响结果显示，在 25 、30 和 35 下当山梨峒嘏任?0.1g/l 时波吉卵囊藻保存效果最好。 下添加山梨酸 25钾浓度为 0.1 g/l 时，波吉卵囊藻存活率达到 89.49 ，叶绿素含量为 27.07 g/l，经恢复培养后，比增长率为 0.444，叶绿素含量为 58.72 g/l；30 下添加山梨酸钾浓度为 0.1 g/l 时，波吉卵囊藻存活率达到 75.38 ，叶绿素含量为 22.68 g/l，经恢复培养后，比增长率为 0.430，叶绿素含量为 43.21 g/l；35 下添加山梨酸钾浓度为0.1 g/l 时，波吉卵囊藻存活率达到 50.75 ，叶绿素含量为 12.93 g/l，经恢复培养后，比增长率为 0.372，叶绿素含量为 17.98 g/l。 4、正交实验结果显示，25 下当二甲基亚砜浓度为 3 ，甘油浓度为 4 ，山梨酸钾浓度为 0.1 g/l 时对波吉卵囊藻的保存效果最好。存活率达到 89.30 ，叶绿素含量为 27.63 g/l，经恢复培养后，比增长率为 0.432，叶绿素含量为 51.41 g/l。 5、不同沼泽红假单胞菌浓度对波吉卵囊藻保存效果的影响结果显示，在 25 、30 和 35 下波吉卵囊藻保存的存活率、叶绿素含量，以及恢复培养后的比增长率和叶绿素含量与沼泽红假单胞菌添加浓度呈正相关，1.94 6 个/ml 为最佳浓度。 1025 下添加沼泽红假单胞菌浓度为 1.94 6 个/ml 时，存活率达到 87.5 ，叶绿素 10含量为 31.14 g/l，经恢复培养后，比增长率为 0.424，叶绿素含量为 55.94 g/l。 30下添加沼泽红假单胞菌浓度为 1.94 6 个/ml 时，存活率达到 76.13 ，叶绿素含量 10为 27.59 g/l，经恢复培养后，比增长率为 0.432，叶绿素含量为 48.3 g/l。35 下添加沼泽红假单胞菌浓度为 1.94 6 个/ml 时，波吉卵囊藻存活率达到 45.43 ，叶 10绿素含量为 12.27 g/l，经恢复培养后，比增长率为 0.388，叶绿素含量为 20.85 g/l。 6、在 25下研究不同藻浓度对波吉卵囊藻保存效果的影响，实验结果表明，当波吉卵囊藻藻浓度为 241.34 4 个/ml 时保存效果最好，波吉卵囊藻存活率达到 1087.84 ，叶绿素含量为 104.01 g/l，经恢复培养后，比增长率为 0.381，叶绿素含量为 52.96 g/l。 7、在 25、藻浓度为 258.54 4 个/ml 下研究添加二甲基亚砜、甘油和山梨酸 10钾的混合保护剂与沼泽红假单胞菌（浓度为 1.94 6 个/ml）对波吉卵囊藻保存效果 10的差异，实验结果显示，添加沼泽红假单胞菌的保存效果最好，波吉卵囊藻存活率达到 90.77 ，叶绿素含量为 112.15 g/l，经恢复培养后，比增长率为 0.434，叶绿素含量为 55.22 g/l。关键词：波吉卵囊藻，保存，二甲基亚砜，甘油，山梨酸钾，沼泽红假单胞菌，藻浓度 the storage of oocystis borgei at normal temperature abstract oocystis borgei is a dominant algae in subtropical prawn farming and plays animportant role in stabling pood ecological structure and regulating water quality.there isno doubt that o.borgei is the first choice for ecological requlation and disease preventionof prawn farming. using o.borgei for water ecological requlation can reduce diseaseoccurs reduce drug used and drug residue of secondary pollution. there is few reportabout how to preserve o.borgei at normal temperature so far. for promoting aquaculturewirh o.borgei to reduce water environment degradationstudying the storage of o.borgei atnormal temperature not only can offer theoretical foundation of how to preservemiroalgae at normal temperaturebut also can find a best way to preserve o.borgei in anindoor environment so that the market circulation of o.borgei can be within a time ofresignation and direct use in subtropical prawn farming. the storage of o.borgei wasstudied in this paper through adding different concentration of dmso glycerolpotassium sorbate and rhodopseudomonas palustris palustris at 25 30 and 35 through orthogonal experiment dicuss the best combination of dmso glycerolpotassium sorbate for the efftect of o.borgei storage also studied the effect of o.borgeistorage with different algae concentration then make comparisons of the effect ofo.borgei storage with adding hybrid protective agent and r.palustris with the best preservetemperature and the best algae concentration so that find the best way to preserveo.borgei at the normal tempetature.experiment resukts are as follows: 1.the effect of preserve o.borgei with adding different concentration of dmso wasstudied at 25 and 35 .it showed thatadding 4.5 dmso could get the besteffect of preserve o.borgei at all temperature. it showed that: when the temeprature was25 ，the survival rate of the storage o.borgei was 86.89 the chlorophyll a content ofstorage o.borgei was 27.13 g/l after re-cultduring the pecific growth rate of o.borgeiwas 0.416 the chlorophyll a of o.borgei was 52.61 g/l.when the temperature was30 the survival rate of the storage o.borgei was 73.41 the chlorophyll a content ofstorage o.borgei was 22.54 g/l after re-cultduring the pecific growth rate of o.borgeiwas 0.408 the chlorophyll a of o.borgei was 38.02 g/l. when the temperature was35 the survival rate of the storage o.borgei was 50.56 the chlorophyll a content ofstorage o.borgei was12.25 g/l after re-cultduring the pecific growth rate of o.borgeiwas 0.320 the chlorophyll a of o.borgei was 21.94 g/l. 2.the effect of preserve o.borgei with adding different concentration of glycerol wasstudied at 25 ，30 and 35 .it showed thatadding 6 glycerol could get the besteffect of preserve o.borgei at all temperature it showed that: when the temperature was25 the survival rate of the storage o.borgei was 91.43 the chlorophyll a content ofstorage o.borgei was 30.58 g/l after re-cultduring the pecific growth rate of o.borgeiwas 0.409 the chlorophyll a of o.borgei was 55.82 g/l. when the temperature was30 the survival rate of the storage o.borgei was 74.46 the chlorophyll a content ofstorage o.borgei was 25.72 g/l after re-cultduring the pecific growth rate of o.borgeiwas 0.387 the chlorophyll a of o.borgei was 45.66 g/l. when the temperature was35 the survival rate of the storage o.borgei was 53.65 the chlorophyll a content ofstorage o.borgei was16.84 g/l after re-cultduring the pecific growth rate of o.borgeiwas 0.341 the chlorophyll a of o.borgei was 20.97 g/l. 3.the effect of preserve o.borgei with adding different concentration of potassiumsorbate was studied at 25 ，30 and 35 . adding 0.1 g/l potassium sorbate could getthe best effect of preserve o.borgei at all temperature it showed that: when thetemperature was 25 ，the survival rate of the storage o.borgei was 89.49 thechlorophyll a content of storage o.borgei was 27.07 g/l after re-cultduring the pecificgrowth rate of o.borgei was 0.444 the chlorophyll a of o.borgei was 58.72 g/l. whenthe temperature was 30 the survival rate of the storage o.borgei was 75.38 thechlorophyll a content of storage o.borgei was 22.68 g/l after re-cultduring the pecificgrowth rate of o.borgei was 0.430 the chlorophyll a of o.borgei was 43.21 g/l. whenthe temperature was 35 the survival rate of the storage o.borgei was 50.75 thechlorophyll a content of storage o.borgei was12.93 g/l after re-cultduring the pecificgrowth rate of o.borgei was 0.372 the chlorophyll a of o.borgei was 17.98 g/l. 4.the result of orthogonal test showed that:3 dmso4 glycerol and 0.08 g/lpotassium sorbate was the most suitable for preserving o.borgei at 25 . it showed that:the survival rate of the storage o.borgei was 89.30 the chlorophyll a content of storageo.borgei was 27.63 g/l after re-cultduring the pecific growth rate of o.borgei was0.432 the chlorophyll a of o.borgei was 51.41 g/l. 5. the effect of rhodopseudomonas palustrwas with different concentration on thestorage of oocystwas borgei was studied at 25 30 and 35 . the concentration ofr.palustrwas and surivival rate chlorophyll a content of the storage of o.borgei pecificgrowth rate and chlorophyll a content of after re-cultduring are positively correlated thebest concentration of r.palustrwas was 1.94 6 cells/ml at all temperature. it showed 10that: when the temperature was 25 the survival rate of the storage o.borgei was 87.5 the chlorophyll a content of storage o.borgei was 31.14 g/l after re-cultduring thepecific growth rate of o.borgei was 0.424 the chlorophyll a of o.borgei was 55.94 g/l.when the temperature was 30 the survival rate of the storage o.borgei was 76.13 the chlorophyll a content of storage o.borgei was 27.59 g/l after re-cultduring thepecific growth rate of o.borgei was 0.432 the chlorophyll a of o.borgei was 48.32 g/l.when the temperature was 35 the survival rate of the storage o.borgei was 45.43 the chlorophyll a content of storage o.borgei was 12.27 g/l after re-cultduring thepecific growth rate of o.borgei was 0.388 the chlorophyll a of o.borgei was 20.85 g/l. 6. the effect of preserve o.borgei with different algae concentration was studied at25 .when the algae concentration of o.borgei was 241.34 4 cells/mlthe effect of 10o.borgei storage was the best.it showed that: the survival rate of the storage o.borgei was87.84 the ch