WO2020083071A1 - 微藻纯化培养基及分离纯化微藻的方法 - Google Patents

微藻纯化培养基及分离纯化微藻的方法 Download PDF

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WO2020083071A1
WO2020083071A1 PCT/CN2019/111229 CN2019111229W WO2020083071A1 WO 2020083071 A1 WO2020083071 A1 WO 2020083071A1 CN 2019111229 W CN2019111229 W CN 2019111229W WO 2020083071 A1 WO2020083071 A1 WO 2020083071A1
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microalgae
medium
purification
agarose
purifying
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李健
彭滟茹
李勇
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攀枝花学院
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
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  • the invention relates to a microalgae purification medium and a method for sterilization, separation and purification of microalgae, which belongs to the field of microalgae biotechnology.
  • Microalgae are a type of autotrophic plants that are widely distributed on land and in the ocean, are rich in nutrients, and have high photosynthetic efficiency. Polysaccharides, proteins, and pigments produced by cell metabolism make them useful in the fields of food, medicine, genetic engineering, and liquid fuels Very good development prospects.
  • the pure-breeding of microalgae is indispensable for studying the nutrition, physiology and biochemistry of marine microalgae.
  • microalgae are usually contaminated by other microorganisms, making their growth rate and cultivation Density is affected.
  • Sterile pure algae is the basis for in-depth research on algae physiology and genetics, so pure-breeding and preservation are the basis and key link of its application, and it is also indispensable to study its physiology, biochemistry, nutritional value, pharmacology, toxicology Fewer steps. It has been proved that certain bacteria can promote the aging and sinking of microalgae cells, thereby limiting the growth and reproduction of microalgae. Therefore, preventing contamination by other microorganisms is an important part of microalgae biotechnology research and development.
  • the irradiation method has the disadvantages of cumbersomeness, heavy workload, low success rate, and narrow application range.
  • the physiological characteristics of the irradiated algae body are easily affected and are prone to lethal effects. Strain, it is difficult to obtain ideal results, such as Geng Yuhuan and so on.
  • Antibiotic method It uses the characteristics of antibiotics to kill bacteria. This is the most simple and widely used method. The disadvantage is that different antibiotics have different bacteria. Some antibiotics cannot completely kill bacteria, and some antibiotics are at the same time. It can also cause damage to algae. Zhou Wenjun et al. Sterile treatment of marine microalgae and its influence on its growth characteristics and biochemical composition [J] .Acta Oceanologica Sinica (Chinese Edition), 2012,34 (06): 177-186.
  • the first technical problem to be solved by the present invention is to provide a new microalgae purification sterilization medium.
  • the preparation method of the microalgae purification medium of the present invention includes the following steps:
  • a microalgae liquid inorganic medium is prepared, and then 1 to 1.5 wt% of high-purity agarose is added to the liquid inorganic medium to obtain a mixed liquid, and the mixed liquid is sterilized to prepare a microalgae solid medium.
  • the high-purity agarose is agarose that has been purified by chromatographic column cleaning.
  • the method of chromatographic column cleaning and purification is to put agarose in the chromatographic column and use 0.6 to 1.0 ml of water per minute The flow rate per gram of agarose is washed for more than 10 hours.
  • the water is organic-free water; preferably ultrapure water, high-purity water, distilled water or deionized water.
  • the liquid inorganic medium is a medium that does not contain any organic components, preferably BBM liquid basal medium.
  • the second technical problem to be solved by the present invention is to provide the use of the above-mentioned microalgae purification medium in the separation and purification of microalgae.
  • the above microalgae purification medium is applied to isolate and purify microalgae.
  • the third technical problem to be solved by the present invention is to provide a method for separating and purifying microalgae, which is simple and practical, with high efficiency and good effect.
  • the method for separating and purifying microalgae includes inoculating the microalgae sample to be purified on the microalgae purification medium, and cultivating the purified monoclonal microalgae under light.
  • the method of inoculation is coating inoculation.
  • the inoculation is performed in a sterile operating station.
  • the purified monoclonal microalgae are small green dots.
  • the culturing is to place the solid medium upside down under light to cultivate.
  • the microalgae is Haematococcus pluvialis.
  • the microalgae separation and sterilization method of the present invention is to use high-purity agarose instead of the conventional method of agar to make an inorganic solid medium to cultivate the microalgae.
  • the principle of this method is to use high-purity agarose , So that the organic matter in the medium is greatly reduced, reducing the risk of bacterial infection during the cultivation process, while not harming the algae.
  • the present invention has the following advantages: it does not adversely affect the microalgae itself, and the present invention is simple to operate, does not require multiple cultivation of the microalgae, and has high working efficiency and low cost.
  • Figure 1 is Comparative Example 1 Haematococcus pluvialis cultured using BBM solid medium made of ordinary agar;
  • FIG. 2 shows Haematococcus pluvialis cultured in BBM solid medium made of purified agarose in Example 1.
  • FIG. 2 shows Haematococcus pluvialis cultured in BBM solid medium made of purified agarose in Example 1.
  • the preparation method of the microalgae purification medium of the present invention includes the following steps:
  • a microalgae liquid inorganic medium is prepared, and then 1 to 1.5 wt% of high-purity agarose is added to the liquid inorganic medium to obtain a mixed liquid, and the mixed liquid is sterilized to prepare a microalgae solid medium.
  • the high-purity agarose is agarose that has been purified by chromatographic column cleaning.
  • the method of chromatographic column cleaning and purification is to put agarose in the chromatographic column and use 0.6 to 1.0 ml of water per minute The flow rate per gram of agarose is washed for more than 10 hours.
  • the water is organic-free water; preferably ultrapure water, high-purity water, distilled water or deionized water.
  • the liquid inorganic medium is a medium that does not contain any organic components, preferably BBM liquid basal medium.
  • the above microalgae purification medium is applied to isolate and purify microalgae.
  • the method for separating and purifying microalgae includes inoculating the microalgae sample to be purified on the microalgae purification medium, and cultivating the purified monoclonal microalgae under light.
  • the method of inoculation is coating inoculation.
  • the inoculation is performed in a sterile operating station.
  • the purified monoclonal microalgae are small green dots.
  • the culturing is to place the solid medium upside down under light to cultivate.
  • the microalgae is Haematococcus pluvialis.
  • Step 1 Weigh 3.0g of agarose and put it into an empty glass chromatographic column with a size of ⁇ 2cmX20cm. Continuously drop purified water into the column at a flow rate of 2.5ml / min. Wash the agarose overnight.
  • Step 2 Prepare 200ml of BBM liquid culture medium according to the conventional method
  • Step 3 Add 1.5w% of the agarose washed in Step 1 to the 200ml BBM liquid medium prepared in Step 2, ie, about 3.0g, and sterilize the plate to obtain BBM solid medium;
  • Step 4 The inoculation operation is carried out on a sterile operating table.
  • the method of coating inoculation is adopted.
  • the algae solution used for inoculation is Haematococcus haematococcus (Pluvialis) FACHB-712.
  • the inoculation volume is 100 ⁇ l;
  • Step 5 Place the inoculated solid medium upside down and place it under light for 24 hours of light to observe the growth on BBM solid medium. If green dots appear on BBM solid medium, it is the purified monoclonal Haematococcus pluvialis. See Figure 2 for details. In Figure 2, there are no mixed bacterial colonies around the green colonies formed by Haematococcus pluvialis.
  • Example 1 The only difference from Example 1 is that Haematococcus pluvialis cultured in a conventional method using agar instead of agarose as the matrix of the BBM solid medium, and the agar used does not need to be chromatographic column cleaning.
  • the other steps are the same as in Example 1 See Figure 1.
  • Figure 1 a large number of milky white bacteria colonies appear around the green colonies formed by Haematococcus pluvialis.

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Abstract

一种微藻纯化培养基及除菌、分离和纯化微藻的方法，属于微藻生物技术领域。微藻纯化培养基的制备方法包括如下步骤：配制微藻液体无机培养基，然后在所述液体无机培养基中加入1～1.5wt％的高纯琼脂糖得到混合液，将所述混合液灭菌后制得微藻固体培养基。微藻纯化培养基用于纯化微藻不会对微藻本身产生不良影响，操作简单，无需对微藻进行多次培养。

Description

微藻纯化培养基及分离纯化微藻的方法 技术领域
本发明涉及一种微藻纯化培养基及除菌、分离和纯化微藻的方法，属于微藻生物技术领域。
背景技术
微藻是一类在陆地、海洋分布广泛，营养丰富、光合利用度高的自养植物，细胞代谢产生的多糖、蛋白质、色素等，使其在食品、医药、基因工程、液体燃料等领域具有很好的开发前景。微藻的纯种培养对研究海洋微藻的营养学、生理学及生物化学等方面是必不可少的，但在其培养过程中，微藻通常会受到其他微生物的污染，使得其生长速度和培养密度受到影响。无菌纯藻是深入开展藻类生理学和遗传学研究的基础,故而纯种培养和保存是其应用的基础和关键性环节，也是研究其生理、生化、营养价值、药理学、毒理学等必不可少的步骤。经证明，某些细菌可促使微藻细胞老化和下沉附底，从而限制了微藻的生长繁殖。因此，预防被其他微生物污染是微藻生物技术研发的一个重要环节。
微藻分离纯化除菌有多种方法：1.物理方法：①离心洗涤法。采用反复离心的方法，能在一定程度上消除细菌，增加分离的可能性，但是不同的微藻能承受的离心力不同，故而采用此方法的时候需要根据微藻的大小确定合适的离心力。②稀释、滤过法：该方法简单容易实施，且能够大范围使用，但是不适用于具有胶质鞘的微藻，同时操作繁琐，耗时长，除菌的质量没有有效的保证。③辐照法：张培玉等.UV-B辐射增强对海洋大型藻与微型藻种群生长关系的影响[J].生态学报,2005(12):3335-3342.许复华等.UV-B辐射对孔石莼与青岛大扁藻的生长影响[J].青岛大学学报(工程技术版),2006(02):49-53公开利用微藻与杂菌对射线抗性的差异，采用紫外线等射线进行辐照处理，杀死杂菌，可达到无菌化的目的。在无菌化研究中，辐照法存在繁琐、工作量大、成功率低、适用范围窄等缺点，辐照的藻体生理特性易受影响易出现致死效应，不能保证筛选到自然状态的藻株，难以获得理想的结果，例如耿予欢等.UV-B辐射对极地雪藻Chlamydomonas nivalis的生物学效应[J].华南理工大学学报(自然科学版),2006(03):106-110.公开.UV-B辐射对极地雪藻有一定的致死效应。④毛细吸管显微分离法。即用无菌毛吸管在解剖镜或者显微镜下把微藻从一滴培养液移至另一滴培养基液，连续转移，直至藻类细胞无污染，但是该方法对设备的要求较高，操作要求熟练。
2.化学方法：①抗生素法：即利用抗生素的特性杀死细菌，这是最为简便且广泛使用的方法，缺点是不同抗生素其作用的细菌不同，有些抗生素并不能完全杀死细菌，有些抗生素 同时也会对藻造成伤害。周文俊等.海洋微藻的无菌化处理及对其生长特性和生化组成的影响[J].海洋学报(中文版),2012,34(06):177-186.的研究表明中高浓度(≥100mg/dm 3)的单种抗生素或抗生素组合可抑制微藻的生长,个别低浓度(50mg/dm 3)的抗生素或抗生素组合可促进微藻的生长。②化学消毒法。利用化学物质杀灭细菌，但是化学物质同时也可能会对藻造成危害。
3.利用其他生理特性法。利用藻类具备的一些特殊的生理特性，采用特殊的纯化方法，可以达到除去杂菌的目的，如趋向性、耐高盐性，但是该方法适用范围窄，工作繁琐，周期长。
张虎、温小斌等.一株富含碳水化合物微藻的筛选和分子鉴定.植物科学学报2014,32(6):645～654公开了一种采用平板划线法纯化藻种。将单种培养的微藻在BG-11(1.5％琼脂糖)固体平板培养基上反复划线纯化，直到获得无菌纯藻种。其实质上是传统的平板划线纯化法，纯化过程复杂，需要反复划线培养。
发明内容
本发明要解决的第一个技术问题是提供一种新的微藻纯化除菌培养基。
为了解决本发明的第一个技术问题，本发明的微藻纯化培养基的制备方法包括如下步骤：
配制微藻液体无机培养基，然后在所述液体无机培养基中加入1～1.5wt％的高纯琼脂糖得到混合液，将所述混合液灭菌后制得微藻固体培养基。
优选的，所述的高纯琼脂糖为经过色谱柱清洗纯化过的琼脂糖，优选所述的色谱柱清洗纯化的方法为：将琼脂糖放入色谱柱内，用水以0.6～1.0毫升每分钟每克琼脂糖的流速清洗10小时以上，所述水为不含有机质的水；优选为超纯水、高纯水、蒸馏水或去离子水。
优选的，所述的液体无机培养基为不含任何有机成分的培养基，优选为BBM液体基础培养基。
本发明要解决的第二个技术问题是提供上述的微藻纯化培养基在分离纯化微藻中的用途。
为了解决本发明的第二个技术问题，将上述的微藻纯化培养基应用于分离纯化微藻。
本发明要解决的第三个技术问题是提供一种分离纯化微藻的方法，该方法简单实用，效率高，效果好。
为了解决本发明的第三个技术问题，所述分离纯化微藻的方法包括将待纯化的微藻样本接种在上述的微藻纯化培养基上,光照培养得到纯化的单克隆的微藻。
优选的，所述接种的方法为涂布接种。
优选的，所述接种在无菌操作台中进行。
优选的，所述的纯化的单克隆的微藻为小绿点。
优选的，所述的培养为将固体培养基倒置放置在光照下培养。
优选的，所述微藻为雨生红球藻。
有益效果：本发明的微藻分离除菌方法是通过使用高纯度的琼脂糖而不是常规方法的琼脂制作无机固体培养基对微藻进行培养，该种方法的原理是通过使用高纯的琼脂糖，从而使得培养基内的有机物质大大减少，降低了在培养过程中感染细菌的风险，同时不会对藻造成伤害。
与现有技术相比，本发明所具有的优点为：不会对微藻本身产生不良影响，且本发明操作简单，无需对微藻进行多次培养，工作效率高，成本较低。
附图说明
图1为对比例1使用普通琼脂制作的BBM固体培养基培养的雨生红球藻；
图2为实施例1使用经过纯化的琼脂糖制作的BBM固体培养基培养的雨生红球藻。
具体实施方式
为了解决本发明的第一个技术问题，本发明的微藻纯化培养基的制备方法包括如下步骤：
称取相当数量的琼脂糖，放入玻璃色谱柱，连续滴入纯净水清洗过夜备用。
配制微藻液体无机培养基，然后在所述液体无机培养基中加入1～1.5wt％的高纯琼脂糖得到混合液，将所述混合液灭菌后制得微藻固体培养基。
优选的，所述的高纯琼脂糖为经过色谱柱清洗纯化过的琼脂糖，优选所述的色谱柱清洗纯化的方法为：将琼脂糖放入色谱柱内，用水以0.6～1.0毫升每分钟每克琼脂糖的流速清洗10小时以上，所述水为不含有机质的水；优选为超纯水、高纯水、蒸馏水或去离子水。
优选的，所述的液体无机培养基为不含任何有机成分的培养基，优选为BBM液体基础培养基。
为了解决本发明的第二个技术问题，将上述的微藻纯化培养基应用于分离纯化微藻。
为了解决本发明的第三个技术问题，所述分离纯化微藻的方法包括将待纯化的微藻样本接种在上述的微藻纯化培养基上,光照培养得到纯化的单克隆的微藻。
优选的，所述接种的方法为涂布接种。
优选的，所述接种在无菌操作台中进行。
优选的，所述的纯化的单克隆的微藻为小绿点。
优选的，所述的培养为将固体培养基倒置放置在光照下培养。
优选的，所述微藻为雨生红球藻。
下面结合实施例对本发明的具体实施方式做进一步的描述，并不因此将本发明限制在所述的实施例范围之中。
实施例1
用BBM基础固体培养基纯化雨生红球藻的方法：
步骤一：称取3.0g的琼脂糖，放入到一只大小为φ2cmX20cm空的玻璃色谱柱内,向色谱柱连续滴入纯净水，流速2.5ml/min，清洗琼脂糖过夜备用。
步骤二：按照常规方法配制BBM液体培养基200ml；
步骤三：在步骤二配制好的200mlBBM液体培养基中加入1.5w％，即大约3.0g的步骤一清洗过的琼脂糖，经过灭菌倒平板操作从而制得BBM固体培养基；
步骤四：在无菌操作台进行接种操作，采用涂布接种的方法，接种采用的藻种液为购买自中科院水生生物研究所藻种库的雨生红球藻(Haematococcus pluvialis)FACHB-712，接种量为100μl；
步骤五：将接种好的固体培养基倒置放置在光照下培养，给予24h光照，观察BBM固体培养基上的生长情况，若BBM固体培养基上出现绿色的小点，即为已经纯化的单克隆的雨生红球藻。详见图2，在图2中，雨生红球藻形成的绿色藻落周围没有出现杂菌菌落。
对比例1
与实施例1唯一不同的是常规方法用琼脂而不是琼脂糖作为BBM固体培养基的基质培养的雨生红球藻，其所用琼脂也不用色谱柱清洗，其它步骤与实施例1相同，结果详见图1，在图1中，雨生红球藻形成的绿色藻落周围均出现了大量乳白色的杂菌菌落。

Claims (10)

  1. 微藻纯化培养基，其特征在于，所述培养基的制备方法包括如下步骤：
    配制微藻液体无机培养基，然后在所述液体无机培养基中加入1～1.5wt％的高纯琼脂糖得到混合液，将所述混合液灭菌后制得微藻固体培养基。
  2. 根据权利要求1所述的微藻纯化培养基，其特征在于，所述的高纯琼脂糖为经过色谱柱清洗纯化过的琼脂糖，优选所述的色谱柱清洗纯化的方法为：将琼脂糖放入色谱柱内，用水以0.6～1.0毫升每分钟每克琼脂糖的流速清洗10小时以上，所述水为不含有机质的水；优选为超纯水、高纯水、蒸馏水或去离子水。
  3. 根据权利要求1或2所述的微藻纯化培养基，其特征在于，所述的液体无机培养基为不含任何有机成分的培养基，优选为BBM液体基础培养基。
  4. 权利要求1～3任一项所述的微藻纯化培养基在分离纯化微藻中的用途。
  5. 分离纯化微藻的方法，其特征在于，所述方法包括将待纯化的微藻样本接种在权利要求1～3任一项所述的微藻纯化培养基上,光照自养培养微藻得到纯化的单克隆藻株。
  6. 根据权利要求5所述的分离纯化微藻的方法，其特征在于，所述接种的方法为涂布接种。
  7. 根据权利要求5或6所述的分离纯化微藻的方法，其特征在于，所述接种在无菌操作台中进行。
  8. 根据权利要求5～7任一项所述的分离纯化微藻的方法，其特征在于，所述的纯化的单克隆的藻株为小绿点。
  9. 根据权利要求5～8任一项所述的分离纯化微藻的方法，其特征在于，所述的培养为将固体培养基倒置放置在光照下培养。
  10. 根据权利要求5～9任一项所述的分离纯化微藻的方法，其特征在于，所述微藻为雨生红球藻。
PCT/CN2019/111229 2018-10-25 2019-10-15 微藻纯化培养基及分离纯化微藻的方法 WO2020083071A1 (zh)

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