CN113980815B - 一种广盐性的小球藻及其应用 - Google Patents
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Abstract
本发明涉及微生物技术领域，特别涉及一株广盐性的小球藻及其应用。本发明从水样中分离获得一株小球藻(Chlorella sp.)MEM‑A‑411，于2021年8月25日保藏在中国典型培养物保藏中心，保藏编号为CCTCC NO:M20211079。该藻株具有较高的耐盐性，在盐度为18‰～105‰的环境中依然能够保持较高的生长速度，生长速度显著高于现有的小球藻。
Description
技术领域
本发明涉及微生物技术领域，特别涉及一种广盐性的小球藻及其应用。
背景技术
小球藻是一种单细胞绿藻，细胞为球形或者广椭圆形，其直径为3-12μm，小球藻种类丰富，生态类型多种多样，是我国为数不多的可以进行大规模培养的藻种之一。我国国内普通小球藻、椭圆小球藻以及蛋白核小球藻比较普遍。小球藻含有丰富的营养物质，比如蛋白质，氨基酸、脂肪酸、多糖，维生素，类胡萝卜素，DHA，EPA，虾青素等，广泛应用于制造饲料、生产食品、提炼生物柴油以及治理环境等领域。在普通培养环境下，其生存环境极有可能被其他微生物抢占，而高盐环境中，大部分微生物很难生存，进而减少了在微藻培养过程中染菌的风险，因此探究盐胁迫对于藻细胞生长的影响是有意义的。小球藻有高于其它植物十倍以上的光合作用能力。一般情况下池塘中70％以上的氧气来源于光合作用。溶氧是池塘最重要的指标，可以提高饵料的转化率、快速转化水体中的有害物质、提高水产动物的抵抗力、能促进虾蟹的蜕壳生长，尤其是提高虾蟹蜕壳后的增长倍数，节省养殖成本。在水产养殖行业，小球藻养殖多是小规模的尝试，生产效率远远不能满足育苗需求，且多采用藻池或天然水体进行粗放培养，对自然环境依赖强，常导致供应不及时，与育苗进度脱节，在生产技术和应用方法上也没有既定标准，造成微藻浓度低、饵料效价低等，研发难度大，生产工艺门槛高等问题。
针对于沿海等地，小球藻的培养一直是个难题。因沿海地区，海水资源丰富，淡水资源十分珍贵，而海水盐度高于小球藻的生长温度。普通的小球藻的生长盐度在20‰，因此不适于在海水环境下进行快速增长,大大限制大大小球藻的产业化生产。因此，提供一种可用于沿海地区广盐性养殖用水生产的广盐性水产养殖用小球藻，为当地水产养殖环境提供有效的微生态治理服务，具有重要的现实意义。
发明内容
有鉴于此，本发明提供了一种广盐性的小球藻及其应用，该藻株具有较高的耐盐性，在盐度为18‰～105‰的环境中依然能够保持较高的生长速度。
本发明从海口市户外养殖水体中分离获得一株小球藻(Chlorella sp.)，命名为MEM-A-411，其保藏编号为CCTCC NO:M20211079。
所述小球藻MEM-A-411的18S rRNA基因片段的序列如SEQ ID NO.1所示。
所述小球藻采用平板划线和涂布法分离获得。
其中，所述平板划线和涂布法采用的培养基包括如下组分：
每200ml所述营养液母液的组成为：
硝酸钠 40g
一水合磷酸二氢钠 2.66g
水 余量；
每100ml所述所述维生素母液的组成为：
每200ml所述微量元素溶液的组成为：
每10ml所述微量元素溶液的组成为：
氨苄青霉素钠 1g；
头孢氨噻肟 1g；
水 余量。
所述小球藻的耐盐性为18‰～105‰。
本发明还提供了所述的小球藻在制备动物饲料中的应用。
本发明还提供了所述的小球藻在制备保健品、食品或护肤品中的应用。
本发明还提供了所述小球藻在环境治理中的应用，所述环境包括土壤环境和水体环境。
本发明还提供了所述的小球藻在制备生物柴油中的应用。
本发明提供的小球藻MEM-A-411具有较高的耐盐性，在盐度为18‰～105‰的环境中依然能够保持较高的生长速度，生长速度显著高于现有的小球藻。
生物保藏说明
小球藻MEM-A-411(Chlorella sp.MEM-A-411)，于2021年8月25日保藏在中国典型培养物保藏中心，地址为:中国，武汉，武汉大学，保藏编号为CCTCCNO:M20211079。
附图说明
图1为在高盐条件下比较小球藻MEM-A-411与普通小球藻的生长速率；
图2为在高盐条件下比较小球藻MEM-A-411与普通小球藻的光合能力。
具体实施方式
本发明公开了一种广盐性的小球藻及其应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
下面结合实施例，进一步阐述本发明：
实施例1
1、从户外养殖水体中，采集微藻藻液，通过平板划线及涂布法，分离出较纯的单藻落(纯度80％以上)。
平板培养基配置如下：
表1营养液母液配方(200mL)
表2维生素母液配方(100mL)：
表3微量元素溶液母液配方(200mL)
表4抗生素配方(10mL)
| 物料名称 | 分子式 | 级别 | 称量量(g) |
| 氨苄青霉素钠 | C16H18N3NaO4S | AR | 1 |
| 头孢氨噻肟 | C16H16N5O7S2Na | AR | 1 |
将15g琼脂溶入1L纯化水中，加入35gNaCL，加热溶解后放入灭菌锅中121℃，灭菌20min。灭菌完成后趁热在超净工作台上用移液器吸取5mL营养液母液，0.5mL维生素母液，1mL微量元素溶液，1mL抗生素加入琼脂液中，摇匀，封口，待冷却(营养液母液及微量元素溶液均需要提前灭菌，121℃，20min，然后0.22膜过滤除菌)。
2、将平板中的单藻落，挑选至30ml F/2液体培养基中进行培养，每个单藻落挑选入一个50ml锥形瓶中。培养所需培养基为液体，与平板培养基基本一致，不加琼脂。将挑选好的试管放置在光照培养箱中进行培养。试管培养温度；25(±1)℃，光照强度50μmol·m-2·s-1。培养5-10天。
3、将培养一段时间后的藻体离心收集，取藻泥分别接种于盐度为18‰、35‰、70‰、105‰的无菌F/2液体培养基，置于温度25℃、光强50μmol·m-2·s-1、在光照时间24h的培养箱中培养。
4、于2、4、6、8d测定各试验组的D750nm(见图1)；于0、1、3、6、12、24、48、96h测定各组的Fv/Fm，结果见图2。
由图1～2可知，本发明小球藻MEM-A-411具有较高的耐盐性，在盐度为18‰～105‰的环境中依然能够保持较高的生长速度，生长速度显著高于现有的小球藻。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 海南大学
<120> 一种广盐性的小球藻及其应用
<130> MP21025384
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1025
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
cagtcctagt tactgcttta tactgtgaac tgcgaatggc tcattaaatc agttatagtt 60
tatttgatgg taccttacta ccggataacc gtagtaattc tagagctaat acgtgcgtaa 120
accccgactc ctggaagggg cgtatttatt agatttaagg ccgacccggc tctgccggtc 180
tcgcggtgaa tcatgataac ttcacgaatc gcatggcctt gtgccggcga tgtttcattc 240
aaatttctgc cctatcaact ttcgatggta ggatagaggc ctaccatggt ggtaacgggt 300
gacggaggat tagggttcga ttccggagag ggagcctgag aaacggctac cacatccaag 360
gaaggcagca ggcgcgcaaa ttacccaatc ctgacacagg gaggtagtga caataaataa 420
caataccggg ccttttcagg tctggtaatt ggaatgagta caatctaaac cccttaacga 480
ggatcaattg gagggcaagt ctggtgccag cagccgcggt aattccagct ccaatagcgt 540
atatttaagt tgctgcagtt aaaaagctcg tagttggatt tcgggcgggg cctgccggtc 600
cgccgtttcg gtgtgcactg gccgggcccg ccttgttgcc ggggacgggc tcctgggctt 660
cactgtccgg gactcggagt cggcgctgtt actttgagta aattagagtg ttcaaagcag 720
gcctacgctc tgaatacatt agcatggaat aacacgatag gactctggcc tatcctgttg 780
gtctgtagga ccggagtaat gattaagagg gacagtcggg ggcattcgta tttcattgtc 840
agaggtgaaa ttcttggatt tatgaaagac gaactactgc gaaagcattt gccaaggatg 900
ttttcattaa tcaagaacga aagttggggg ctcgaagacg attagatacc gtcctagtct 960
caaccataaa cgatgccgac tagggatcgg cggatgtttt cttcgatgac tccgccggca 1020
cctat 1025
Claims (9)
1.保藏编号为CCTCC NO:M20211079的广盐性的小球藻(Chlorella sp.)。
2.根据权利要求1所述的小球藻，其特征在于，其18S rRNA基因片段的序列如SEQ IDNO.1所示。
3.权利要求1所述的小球藻，其特征在于，所述小球藻采用平板划线和涂布法分离获得。
4.根据权利要求3所述的小球藻，其特征在于，所述平板划线和涂布法采用的培养基包括如下组分：
每200ml所述营养液母液的组成为：
硝酸钠40g
一水合磷酸二氢钠2.66g
水余量；
每100ml所述所述维生素母液的组成为：
每200ml所述微量元素溶液的组成为：
5.根据权利要求1～4任一项所述小球藻，其耐盐性为18‰～105‰。
6.权利要求1～4任一项所述的小球藻在制备动物饲料中的应用。
7.权利要求1～4任一项所述的小球藻在制备保健品、食品或护肤品中的应用。
8.权利要求1～4任一项所述的小球藻在环境治理中的应用，所述环境包括土壤环境和水体环境。
9.权利要求1～4任一项所述的小球藻在制备生物柴油中的应用。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000036084A1 (fr) * | 1998-12-17 | 2000-06-22 | Kirin Beer Kabushiki Kaisha | Chlorelle riche en chlorophylle et resistant au sel |
| CN102294227A (zh) * | 2011-06-30 | 2011-12-28 | 国家海洋局天津海水淡化与综合利用研究所 | 小球藻海水净化生物吸附剂的制备方法及其应用方法 |
| WO2019078398A2 (ko) * | 2017-10-17 | 2019-04-25 | 재단법인 탄소순환형 차세대 바이오매스 생산전환 기술연구단 | 고농도의 이산화탄소 및 고염 조건에서 지질 생산성 및 세포 생장률이 우수한 클로렐라 속 abc-001 균주 및 이의 용도 |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000036084A1 (fr) * | 1998-12-17 | 2000-06-22 | Kirin Beer Kabushiki Kaisha | Chlorelle riche en chlorophylle et resistant au sel |
| CN102294227A (zh) * | 2011-06-30 | 2011-12-28 | 国家海洋局天津海水淡化与综合利用研究所 | 小球藻海水净化生物吸附剂的制备方法及其应用方法 |
| WO2019078398A2 (ko) * | 2017-10-17 | 2019-04-25 | 재단법인 탄소순환형 차세대 바이오매스 생산전환 기술연구단 | 고농도의 이산화탄소 및 고염 조건에서 지질 생산성 및 세포 생장률이 우수한 클로렐라 속 abc-001 균주 및 이의 용도 |
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