CN110982747A - 人工海水组合物、其培养的海洋希瓦氏菌和应用 - Google Patents
人工海水组合物、其培养的海洋希瓦氏菌和应用 Download PDFInfo
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Abstract
本发明公开了人工海水组合物、其培养的海洋希瓦氏菌和应用，该组合物以蒸馏水1000mL来计算，还包含：氯化钠28g、七水硫酸镁7g、六水氯化镁4g、氯化钾0.70g、氯化钙1.11g、硝酸钠0.10g、磷酸钾0.01g、β‑五水甘油磷酸二钠盐0.014g、九水硅酸钠0.15g、2μg/mL的维生素B12 0.10mL、1μg/mL的维生素B7 1mL、100μg/mL维生素B1 1mL、pⅡMetals 10mL、S2Metals 10mL、Tris 1g和氨基三乙酸0.10g，由其培养的海洋希瓦氏菌形成的生物被膜诱导厚壳贻贝稚贝附着效果与自然海水相近，对厚壳贻贝培育与养殖具有指导作用。
Description
技术领域
本发明涉及人工海水组合物的配方，具体涉及一种用于替代自然海水培养海洋细菌的人工海水组合物，特别用于培养海洋希瓦氏菌(Shewanella loihica)，并验证该海洋希瓦细菌的生物被膜对厚壳贻贝稚贝附着的诱导作用。
背景技术
厚壳贻贝(Mytilus coruscus)，隶属于软体动物门(Mollusca)，双壳纲(Bivalvia)，贻贝目(Mytilodia)，贻贝科(Mytilidae)，分布于黄海、渤海和东海沿岸，是我国重要的海产贝类养殖品种。厚壳贻贝是从受精卵发育到眼点幼虫后，再经过附着变态这一过程，发育成稚贝，并最终发育为成贝。在由稚贝向成贝生长发育过程中，当环境发生变化时，厚壳贻贝稚贝能够自行切断足丝，开始爬行并重新选择适宜的附着基再次附着。
近年来，由于海水污染严重以及海水养殖面积增长过快，考虑用人工海水替代自然海水，以此来排除海水污染问题，并且可以将海水养殖转移至陆地上，减少海洋中海水养殖面积，恢复海洋生态。但是，以往实验发现人工海水养殖的文昌鱼、对虾等产品的生长能力显著低于天然海水养殖，且目前还没有用人工海水替代自然海水培养海洋细菌生物被膜以及诱导稚贝附着的相关报道。
发明内容
本发明的目的在于提供一种用于替代自然海水培养海洋细菌的人工海水组合物，以蒸馏水1000mL来计算，还包含以下重量的组分：氯化钠28g、七水硫酸镁7g、六水氯化镁4g、氯化钾0.70g、氯化钙1.11g、硝酸钠0.10g、磷酸钾0.01g、β-五水甘油磷酸二钠盐0.014g、九水硅酸钠0.15g、2μg/mL的维生素B12 0.10mL、1μg/mL的维生素B7 1mL、100μg/mL维生素B1 1mL、pⅡMetals 10mL、S2Metals 10mL、Tris 1g和氨基三乙酸0.10g。
本发明还涉及上述人工海水组合物用于替代自然海水培养海洋希瓦氏菌(Shewanella loihica)。
上述人工海水组合物培养的海洋希瓦氏菌在诱导厚壳贻贝稚贝附着上的用途，通过形成所述海洋希瓦氏菌生物被膜诱导厚壳贻贝稚贝附着实现。
在某些实施方案中，所述海洋希瓦氏菌的初始菌液密度为1×106～9×108cells/mL，优选为5×108cells/mL。
与现有技术相比，本发明复配出的人工海水组合物可用于替代自然海水培养海洋细菌，特别是培养海洋希瓦氏菌生物被膜以及诱导稚贝附着，填补该领域空白。通过验证该海洋细菌的生物被膜对厚壳贻贝稚贝附着的诱导作用，效果与自然海水相近，可以替代自然海水在海水养殖方面使用，并且使用人工海水可以依据需求调节其中各种离子浓度以达到更好的养殖效果，为人工海水养殖提供理论依据。
附图说明
图1是本发明中人工海水组合物与自然海水分别培养海洋希瓦氏菌形成的生物被膜对厚壳贻贝稚贝附着的诱导作用。
具体实施方式
以下结合具体实施例进一步解释本发明的技术方案，但不限于此。
本发明的海洋希瓦氏菌(Shewanella loihica)与中国专利ZL201410741373.4保藏的海洋希瓦氏细菌(Shewanella sp.ECSMB56)相同，来自于浙江省嵊泗县枸杞岛(122°46′E 30°43′N)的自然生物被膜表面，在NCBI上的序列号为KM369861，其保藏信息为：该细菌菌株保藏于中国普通微生物菌种保藏管理中心，保藏日期为2014年10月20日，保藏编号为CGMCC No:9798，保藏单位地址为北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所。
以下实施例所用的人工海水组合物由以下物质复配得到，其由蒸馏水1000mL、氯化钠28g、七水硫酸镁7g、六水氯化镁4g、氯化钾0.70g、氯化钙1.11g、硝酸钠0.10g、磷酸钾0.01g、β-五水甘油磷酸二钠盐0.014g、九水硅酸钠0.15g、2μg/mL的维生素B12 0.10mL、1μg/mL的维生素B7 1mL、100μg/mL维生素B1 1mL、pⅡMetals 10mL、S2Metals 10mL、Tris 1g和氨基三乙酸0.10g组成，且可以按照与上述配方倍数放大或缩小。
实施例1人工海水组合物培养海洋希瓦氏菌
先将海洋希瓦氏菌的纯种菌株在2216E固体培养基进行涂板和划线得到的单菌落，转接到Zobell 2216E液体培养基中扩大培养，再离心洗涤制成悬浮液。然后用吖啶橙染色并在荧光显微镜1000倍镜头下随机选取10个点进行计数，确定菌液总密度为2.1×109cells/mL。
实施例2海洋希瓦氏菌对厚壳贻贝稚贝附着的诱导实验
选用实施例1中初始菌液密度分别为1×106cells/mL，1×107cells/Ml，1×108cells/mL和5×108cells/mL的海洋希瓦氏菌液，在灭菌培养皿中放入一个灭菌的载玻片，分别加入灭菌的自然海水和本发明中配置的人工海水，使培养皿中液体总体积为20mL，在18℃、黑暗条件下培养48h形成生物被膜。将一枚附有生物被膜的载玻片转移到新的含有灭菌自然海水的培养皿中，在载玻片左右均匀放入10只厚壳贻贝稚贝(壳长0.68±0.04mm、壳高0.46±0.02mm)，记录24h时的稚贝附着率，即载玻片上附着的稚贝个数占该培养皿中稚贝总个数的百分比，结果如图1所示。
由图1可知，在初始菌液密度为1×106cells/mL时，人工海水和自然海水培养的生物被膜的诱导活性均为31.1％；在初始菌液密度为1×107cells/mL时，人工海水和自然海水培养的生物被膜的诱导活性为30％和32.2％；在初始菌液密度为1×108cells/mL时，人工海水和自然海水培养的生物被膜的诱导活性为43.3％和46.7％。在初始菌液密度为5×108cells/mL时，人工海水和自然海水培养的生物被膜的诱导活性为44.4％和43.3％。由此可见，该人工海水与自然海水在相同初始菌液密度下对海洋希瓦氏菌生物被膜诱导厚壳贻贝稚贝附着的诱导活性相近，并且使用人工海水可以依据需求调节其中各种离子浓度以达到更好的养殖效果，对于探究厚壳贻贝稚贝附着机制和厚壳贻贝培育与养殖均具有指导作用，为人工海水养殖提供理论依据。
Claims (5)
1.人工海水组合物，其特征在于，以蒸馏水1000mL来计算，还包含以下重量的组分：氯化钠28g、七水硫酸镁7g、六水氯化镁4g、氯化钾0.70g、氯化钙1.11g、硝酸钠0.10g、磷酸钾0.01g、β-五水甘油磷酸二钠盐0.014g、九水硅酸钠0.15g、2μg/mL的维生素B12 0.10mL、1μg/mL的维生素B7 1mL、100μg/mL维生素B1 1mL、pⅡMetals 10mL、S2Metals 10mL、Tris 1g和氨基三乙酸0.10g。
2.权利要求1所述人工海水组合物在替代自然海水培养海洋希瓦氏菌(Shewanellaloihica)上的用途。
3.根据权利要求2所述的用途，其特征在于，所述海洋希瓦氏菌诱导厚壳贻贝稚贝附着，且通过所述人工海水组合物培养所述海洋希瓦氏菌形成生物被膜诱导厚壳贻贝稚贝附着实现。
4.根据权利要求2或3所述的用途，其特征在于，所述海洋希瓦氏菌菌株保藏于中国普通微生物菌种保藏管理中心，保藏编号为CGMCC No:9798，且所述海洋希瓦氏菌的初始菌液密度为1×106～9×108cells/mL。
5.根据权利要求4所述的用途，其特征在于，所述海洋希瓦氏菌的初始菌液密度为5×108cells/mL。
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