WO2023040761A1 - 一种难培养微生物新物种的分离培养方法 - Google Patents

一种难培养微生物新物种的分离培养方法 Download PDF

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WO2023040761A1
WO2023040761A1 PCT/CN2022/118037 CN2022118037W WO2023040761A1 WO 2023040761 A1 WO2023040761 A1 WO 2023040761A1 CN 2022118037 W CN2022118037 W CN 2022118037W WO 2023040761 A1 WO2023040761 A1 WO 2023040761A1
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dmap
difficult
final concentration
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microorganisms
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何山
郑多运
徐恩永
石煜彤
刘力伟
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宁波大学
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  • the invention belongs to the technical field of microorganisms, and in particular relates to a method for isolating and cultivating difficult-to-cultivate new microorganism species.
  • New microbial resources obtained from diverse environments are reliable sources for natural product discovery. These natural products are diverse and of great biotechnological value, including antibiotics for therapeutic purposes, biopesticides and biofertilizers in agriculture, secondary metabolites for bioremediation, and biofuels.
  • the pure cultivation of novel microbial strains is essential for the efficient discovery of these valuable natural products.
  • the prior art has disclosed a variety of isolation techniques to increase the diversity of cultured microorganisms, such as separating cells by physical means to change growth conditions to avoid competition between microorganisms or to reduce inhibitors of microbial growth (Connon and Giovannoni, 2002; Zengler et al., 2002), simulated natural environment (Kaeberlein et al., 2002; Nichols et al. 2010), extended culture time (Davis et al., 2005), reduced nutrient concentration (Janssen et al., 2002) and the use of alternative gelling agents ( Tamaki et al., 2005), the application of these methods has made it possible to cultivate some microbial species that were difficult to cultivate before.
  • the existing standard culture technology lacks the necessary chemical factors for the growth of many new species of difficult-to-cultivate microorganisms. Therefore, it is particularly important to develop new methods for isolating and cultivating difficult-to-culture microorganisms.
  • the purpose of the present invention is to overcome the defects of the prior art and provide a method for isolating and cultivating difficult-to-cultivate new species of microorganisms.
  • Another object of the present invention is to provide the application of 6-DMAP in the preparation of culture medium for new difficult-to-cultivate microorganisms.
  • Another object of the present invention is to provide a culture medium for difficult-to-cultivate new species of microorganisms.
  • 6-DMAP is a serine threonine protein kinase inhibitor known to accelerate and enhance the formation of prokaryotic
  • the present invention now proposes the use of 6-DMAP to isolate new bacteria that were not cultured before. The applicant found that,
  • 6-DMAP can facilitate the recovery of some non-growing microorganisms that were not previously cultured by standard culture methods.
  • a method for isolating and cultivating new species of difficult-to-cultivate microorganisms adding the 6-DMAP solution sterilized by PTEE filter to the sterilized liquid agar medium until the final concentration of 6-DMAP is 1-3 ⁇ g /mL, and then solidified to obtain a solid medium, and the natural samples were inoculated in the solid medium for cultivation.
  • the final concentration of 6-DMAP is 2-3 ⁇ g/mL.
  • the final concentration of 6-DMAP is 2.5 ⁇ g/mL.
  • 6-DMAP Application of 6-DMAP in the preparation of culture medium for new difficult-to-cultivate microorganisms.
  • the final concentration of 6-DMAP is 1-3 ⁇ g/mL.
  • the final concentration of 6-DMAP is 2-3 ⁇ g/mL.
  • the final concentration of the 6-DMAP is 2.5 ⁇ g/mL.
  • a culture medium for new difficult-to-cultivate microorganisms includes an agar medium containing 6-DMAP at a final concentration of 1-3 ⁇ g/mL.
  • the final concentration of 6-DMAP is 2-3 ⁇ g/mL.
  • the final concentration of 6-DMAP is 2.5 ⁇ g/mL.
  • the present invention is simple in operation, good in economy and wide in application.
  • the efficiency of isolating new microbial species resources of the present invention is very high, and about 50% of the bacterial strains obtained through culture are potential new species.
  • Fig. 1 is a schematic diagram of the preparation process of Example 1 of the present invention.
  • FIG. 2 is a graph showing the experimental results of Example 2 of the present invention.
  • FIG. 3 is a graph showing the experimental results of Example 3 of the present invention.
  • Fig. 4 is one of the experimental result diagrams of Example 4 of the present invention.
  • Fig. 5 is the second diagram of the experimental results of Example 4 of the present invention.
  • the preparation method of difficult-to-cultivate microorganism new species culture medium of the present invention comprises:
  • NBU2953 (its 16S rRNA sequence is 97% similar to the closest known model strain Erythromicrobium ramosum)
  • NBU2952 (its 16S rRNA sequence is the closest to the known model strain) Pseudooceanicola nitratireducens 97% similarity)
  • NBU2954 (its 16S rRNA sequence is 97% similar to the closest known type strain Formosa maritima)
  • NBU2956 its 16S rRNA sequence is the closest known type strain Marinicella sediminis
  • NBU2955 (its 16S rRNA sequence is 97% similar to the closest known type strain Hyphobacterium vulgare).
  • the samples from the environment were cultured with the medium prepared in Example 1, thereby screening out the above-mentioned Bacteroides and Proteus.
  • the effect of different concentrations of 6-DMAP was tested by calculating the colony formation efficiency ratio of the medium containing 6-DMAP and the medium without 6-DMAP corresponding to the above microorganisms.
  • these strains were cultured in 5 mL of 1:10 diluted R2A medium containing 2% artificial sea salt (10% of the recommended concentration, Hopebio, Qingdao, China) for 3-5 days (cells were collected at the beginning of the stationary phase).
  • the collected strains were diluted with artificial seawater (1:100), and then cultured at 5°C for 3 days to reduce the activity of microorganisms.
  • Escherichia coli and Pseudomonas aeruginosa were used to detect the influence of the culture medium of difficult-to-cultivate new species of microorganisms prepared in Example 1 corresponding to Example 2 on the growth of microorganisms.
  • the above two test strains were cultured in 5 mL of 1:10 diluted R2A medium containing 2% artificial sea salt (10% of the recommended concentration, Hopebio, Qingdao, China) at 26°C for 3-5d (collected at the beginning of the stationary phase) cell).
  • the collected strains were diluted with PBS (phosphate buffered saline), and then cultured at 5° C. for 3 days to reduce the microbial viability.
  • the liquid cultures with different dilutions were inoculated in triplicate on the following medium: 1:10 diluted R2A agar medium (6-DMAP final concentration was 2.5 ⁇ g/mL) and corresponding agar medium without 6-DMAP .
  • the environment sample was cultured with the hard-to-cultivate new species culture medium prepared in Example 1 corresponding to Example 2.
  • the basic medium used in this example is diluted R2A medium (1:10).
  • the isolation culture method using 6-DMAP was applied to the following environmental samples: marine sediment, seawater, lake water and soil.
  • the proportion of the new species of bacterial strains obtained by the method of this embodiment was compared with the corresponding results of the standard culture method (without 6-DMAP) , to test the efficiency of the method of this embodiment.
  • each 1 g sample was placed in a 15 mL conical tube containing 10 mL of sterilized artificial seawater (corresponding to marine sediment samples) or PBS (corresponding to soil samples), and then homogenized with a vortex machine. Quality 10min. Seawater samples and lake water samples were directly used as inoculum. Each sample was diluted 10 -2 to 10 -5 with sterilized artificial seawater or PBS, and then inoculated on agar medium.
  • the invention discloses a method for isolating and cultivating new difficult-to-cultivate microorganisms.
  • the 6-dimethylaminopurine (6-DMAP) solution filtered and sterilized by a PTEE filter is added to a sterilized liquid agar medium , until the final concentration of 6-DMAP is 1-3 ⁇ g/mL, and then solidified to obtain a solid medium, and the natural samples are inoculated in the solid medium for culture.
  • the invention has a very high efficiency of isolating new microbial species resources, about 50% of the pure cultured strains obtained by culture are potential new species, and has industrial applicability.

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Abstract

提供一种难培养微生物新物种的分离培养方法，将经PTEE滤网过滤除菌的6-二甲氨基嘌呤(6-DMAP)溶液加入到灭菌后且呈液态的琼脂培养基中，至6-DMAP的终浓度为1-3 μg/mL，然后凝固获得固体培养基，将自然界样本接种于该固体培养基中进行培养。所提供的分离新微生物物种资源的效率非常高，大约50％培养所获得的纯培养菌株是潜在新种。

Description

一种难培养微生物新物种的分离培养方法 技术领域
本发明属于微生物技术领域，具体涉及一种难培养微生物新物种的分离培养方法。
背景技术
从不同的环境中获得的新的微生物资源是天然产物发现的可靠来源。这些种类繁多且具有重要生物技术价值的天然产物，包括用于治疗目的的抗生素、农业中的生物农药和生物肥料、用于生物修复的次级代谢产物和生物燃料。新型微生物菌种的纯培养对于有效发现这些有价值的天然产物至关重要。
尽管如此，环境中的大多数微生物无法在标准培养基中培养出来。在自然样本中的可培养细胞的数目仅占总数的大约1％。大多数的细菌菌种是难以用标准的分离技术分离出来的，因为相比于其他快速生长的细菌(例如实验室条件下非常容易分离得到的Pseudomonas spp.或Bacillus spp.)，这些难以分离的细菌菌种往往不适应标准培养条件。
现有技术已经公开了多种分离技术以增加培养出的微生物的多样性，如通过物理方法分离细胞以改变生长条件来避免微生物之间竞争或减少微生物生长的抑制剂(Connon和Giovannoni,2002；Zengler等,2002)、模拟自然环境(Kaeberlein等.,2002；Nichols等.2010)、延长培养时间(Davis等.,2005)、降低营养素浓度(Janssen等,2002)以及替代凝胶剂的使用(Tamaki等,2005)，以上这些方法的应用使得一些以前难以培养的微生物种类的培养成为可能。但是大多数的微生物仍然无法得到培养。这些方法中的大部分需要特殊的材料和复杂的步骤，以至于难以在实验室和商业条件下大规模应用，如Connon和Giovannoni的方法需要昂贵的设备和先进的技术(例如需要显微观察或荧光激活细胞分选FACS)，模拟自然环境则需要特殊材料的使用和复杂设备的操作。这些方法中的其他方法相对简单，但由于筛选效率低而无法商业化，如Tamaki等使用结冷胶作为替代的凝胶剂，虽然应用不复杂，但由于其效率低而未广泛使用。
综上，现有的标准培养技术缺少许多难培养微生物新物种生长的必要化学因子， 所以，发展分离培养难微生物的新方法尤为重要。
发明内容
本发明的目的在于克服现有技术缺陷，提供一种难培养微生物新物种的分离培养方法。
本发明的另一目的在于提供6-DMAP在制备难培养微生物新物种培养基中的应用。
本发明的再一目的在于提供一种难培养微生物新物种培养基。
6-DMAP是一种丝氨酸苏氨酸蛋白激酶抑制剂，已知它能加速和增强原核的形
成。本发明现在提出使用6-DMAP来分离以前未培养的新细菌。申请者发现，
6-DMAP可以促进恢复一些之前未被标准培养方法培养的非生长微生物。
本发明的技术方案如下：
一种难培养微生物新物种的分离培养方法，将经PTEE滤网过滤除菌的6-DMAP溶液加入到灭菌后且呈液态的琼脂培养基中，至6-DMAP的终浓度为1-3μg/mL，然后凝固获得固体培养基，将自然界样本接种于该固体培养基中进行培养。
在本发明的一个优选实施方案中，所述6-DMAP的终浓度为2-3μg/mL。
进一步优选的，所述6-DMAP的终浓度为2.5μg/mL。
本发明的另一技术方案如下：
6-DMAP在制备难培养微生物新物种培养基中的应用。
在本发明的一个优选实施方案中，所述6-DMAP的终浓度为1-3μg/mL。
进一步优选的，所述6-DMAP的终浓度为2-3μg/mL。
更进一步优选的，所述6-DMAP的终浓度为2.5μg/mL。
本发明的再一技术方案如下：
一种难培养微生物新物种培养基，包括琼脂培养基，该琼脂培养基中含有终浓度为1-3μg/mL的6-DMAP。
在本发明的一个优选实施方案中，所述6-DMAP的终浓度为2-3μg/mL。
进一步优选的，所述6-DMAP的终浓度为2.5μg/mL。
本发明的有益效果是：
1.与之前的标准方法相比，本发明操作简单、经济性好，适用面广。
2.本发明的分离新微生物物种资源的效率非常高，大约50％培养获得的菌株是潜在新种。
附图说明
图1为本发明实施例1的制备过程示意图。
图2为本发明实施例2的实验结果图。
图3为本发明实施例3的实验结果图。
图4为本发明实施例4的实验结果图之一。
图5为本发明实施例4的实验结果图之二。
具体实施方式
以下通过具体实施方式结合附图对本发明的技术方案进行进一步的说明和描述。
实施例1
如图1所示，本发明的难培养微生物新物种培养基的制备方法包括：
(1)用孔径为0.2μm的PTEE滤膜对6-DMAP溶液进行过滤；
(2)将琼脂培养基进行高压灭菌；
(3)在50℃的温度下将过滤后的6-DMAP溶液加入经高压灭菌的琼脂培养基中充分混合；然后自然冷却至室温，待琼脂糖凝固，即成。
实施例2
本实施例涉及的拟杆菌和变形菌为：NBU2953(其16S rRNA序列与已知最接近的模式菌株Erythromicrobium ramosum的相似度为97％)、NBU2952(其16S rRNA序列与已知最接近的模式菌株Pseudooceanicola nitratireducens的相似度为97％)、NBU2954(其16S rRNA序列与已知最接近的模式菌株Formosa maritima的相似度为97％)、NBU2956(其16S rRNA序列与已知最接近的模式菌株Marinicella sediminis的相似度为97％)和NBU2955(其16S rRNA序列与已知最接近的模式菌株 Hyphobacterium vulgare的相似度为97％)。
本实施例用以上拟杆菌和变形菌来优化实施例1制得的难培养微生物新物种培养基中的6-DMAP的最佳浓度。
将来自环境的样本用实施例1制得的培养基进行培养，从而筛选出上述各拟杆菌和变形菌。通过计算上述各微生物对应的含有6-DMAP的培养基和不含6-DMAP的培养基的菌落形成效率比来检验不同浓度的6-DMAP的效果。具体的，这些菌株均在5mL的1:10稀释的含有2％人造海盐的R2A培养基(建议浓度的10％，中国青岛Hopebio)中培养3-5d(在稳定期开始的时候收集细胞)。将收集的菌株用人造海水稀释(1:100)，接着于5℃培养3d以降低微生物的活性。然后将不同稀释度的液体培养物一式三份接种于实施例1制得的难培养微生物新物种培养基(1:10稀释的R2A琼脂培养基，含有终浓度为0.25μg/mL、2.5μg/mL和25μg/mL的6-DMAP)和不含6-DMAP的相应琼脂培养基中培养。26℃培养5d后，计算每个琼脂平板的CFU。选取产生30-500CFU(菌落形成单位)的稀释度进行菌落计数。针对不同6-DMAP的应用浓度，测定含有和不含有6-DMAP的培养基之间菌落数比。如图2所示，除了NBU2952以外，多数菌株在b条件下(6-DMAP终浓度为2.5μg/mL)显示出最高的效率。
实施例3
本实施例用大肠杆菌(Escherichia coli)和铜绿假单胞菌(Pseudomonas aeruginosa)检测了对应实施例2的实施例1制得的难培养微生物新物种培养基对微生物的生长的影响。
将上述两种检测菌株分别在5mL的1:10稀释的含有2％人造海盐的R2A培养基(建议浓度的10％，中国青岛Hopebio)中26℃培养3-5d(在稳定期开始的时候收集细胞)。将收集的菌株用PBS(磷酸盐缓冲液)稀释，接着于5℃培养3d以降低微生物活力。然后将不同稀释度的液体培养物一式三份接种于如下培养基：1:10稀释的R2A琼脂培养基(6-DMAP终浓度为2.5μg/mL)和不含6-DMAP的相应琼脂培养基。26℃培养5d后，计算每个琼脂平板的CFU。针对每个检测菌株，测定含有和不含有6-DMAP的培养基之间菌落数比。结果如图3所示，在培养基中添加特定浓度的6-DMAP能够提高检测菌株的生长能力。
实施例4
本实施例用对应实施例2的实施例1制得的难培养微生物新物种培养基对环境样本进行细菌培养。本实施例使用的基本培养基为稀释的R2A培养基(1:10)。使用6-DMAP的分离培养方法被应用于如下环境样本：海洋沉积物、海水、湖水和土壤。本实施例的方法所获得新种菌株(其16S rRNA序列与已知最接近的模式菌株的相似度小于等于97％)的比例与标准培养方法(不含6-DMAP)的相应结果进行了比较，以检测本实施例方法的效率。
为了准备海洋沉积物和土壤样本中的接种物，每1g样本置于含有10mL灭菌人造海水(对应海洋沉积物样本)或PBS(对应土壤样本)的15mL锥形管中，然后用涡流机均质10min。海水样本和湖水样本则直接作为接种物。每个样本用灭菌人造海水或PBS稀释10 -2至10 -5，然后接种于琼脂培养基。
为了检测含有和不含有6-DMAP条件下的微生物菌落生成情况，每个样本的细菌数量均在显微镜下进行了计数，并在琼脂平板上进行了CFU对比。对于所有环境样本来说，含有6-DMAP的培养基的菌落数量更高，并且超过了采用标准培养方法的对照组的菌落数量1.5-2倍(如图4所示)。结果表明，6-DMAP的添加能够获得传统标准培养方法难以培养的一些微生物。
下一步，在含有和不含6-DMAP的条件下，对应每个样本，从琼脂平板上随机选取100个菌株在新的琼脂培养基(1:10稀释的R2A培养基)上进行纯培养。培养一周之后，将长大的菌落作为PCR的模板，对其16SrRNA基因的750bp片段测序以进行分类鉴定。根据厂商(SangonBiotech，中国)推荐的PCR体系，以27F(5'-agagtttgatcctggctcag-3'，SEQ ID NO.01)和1492R(5'-ggttaccttgttacgactt-3'，SEQ ID NO.02)作为通用引物进行16srRNA基因的扩增。PCR产物则通过商业(SangonBiotech，中国)荧光染料终止子测序方法进行测序。将所测得的序列与EzBioCloud(https://www.ezbiocloud.net)中的数据库进行比对，测定与最接近的模式菌株序列的相似度。
如图5所示，采用本发明的添加6-DMAP的方法分离的菌株中，对应海洋沉积物、海水、淡水和土壤，其中依次有57％、35％、24％和16％的菌株是新物种(与 GenBank中的最接近的16S rRNA近亲序列的相似性≤97％为标准)；作为对比，未添加6-DMAP的对照组中，依次只有4％、9％、2％和3％为新物种。结果表明，本发明应用6-DMAP的新的分离培养方法相比标准培养方法能培养获得显著更多的微生物新物种，这些新的细菌种类是标准培养方法所难以培养的。
以上所述，仅为本发明的较佳实施例而已，故不能依此限定本发明实施的范围，即依本发明专利范围及说明书内容所作的等效变化与修饰，皆应仍属本发明涵盖的范围内。
工业实用性
本发明公开了一种难培养微生物新物种的分离培养方法，将经PTEE滤网过滤除菌的6-二甲氨基嘌呤(6-DMAP)溶液加入到灭菌后且呈液态的琼脂培养基中，至6-DMAP的终浓度为1-3μg/mL，然后凝固获得固体培养基，将自然界样本接种于该固体培养基中进行培养。本发明的分离新微生物物种资源的效率非常高，大约50％培养所获得的纯培养菌株是潜在新种,具有工业实用性。

Claims (11)

  1. 一种难培养微生物新物种的分离培养方法，其特征在于：将经PTEE滤网过滤除菌的6-DMAP溶液加入到灭菌后且呈液态的琼脂培养基中，至6-DMAP的终浓度为1-3μg/mL，然后凝固获得固体培养基，将自然界样本接种于该固体培养基中进行培养。
  2. 如权利要求1所述的分离培养方法，其特征在于：所述6-DMAP的终浓度为2-3μg/mL。
  3. 如权利要求2所述的分离培养方法，其特征在于：所述6-DMAP的终浓度为2.5μg/mL。
  4. 6-DMAP在制备难培养微生物新物种培养基中的应用。
  5. 如权利要求4所述的应用，其特征在于：所述6-DMAP的终浓度为1-3μg/mL。
  6. 如权利要求5所述的应用，其特征在于：所述6-DMAP的终浓度为2-3μg/mL。
  7. 如权利要求6所述的应用，其特征在于：所述6-DMAP的终浓度为2.5μg/mL。
  8. 如权利要求4或5所述的应用，其特征在于：所述培养基为琼脂。
  9. 一种难培养微生物新物种培养基，其特征在于：包括琼脂培养基，该琼脂培养基中含有终浓度为1-3μg/mL的6-DMAP。
  10. 如权利要求9所述的一种难培养微生物新物种培养基，其特征在于：所述6-DMAP的终浓度为2-3μg/mL。
  11. 如权利要求10所述的一种难培养微生物新物种培养基，其特征在于：所述6-DMAP的终浓度为2.5μg/mL。
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