WO2018192059A1 - 一种基于纸质培养装置的微藻培养方法 - Google Patents

一种基于纸质培养装置的微藻培养方法 Download PDF

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WO2018192059A1
WO2018192059A1 PCT/CN2017/085869 CN2017085869W WO2018192059A1 WO 2018192059 A1 WO2018192059 A1 WO 2018192059A1 CN 2017085869 W CN2017085869 W CN 2017085869W WO 2018192059 A1 WO2018192059 A1 WO 2018192059A1
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microalgae
paper
culture
agar
culture device
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陈冠益
张道虹
颜蓓蓓
胡毡
齐云
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Tianjin University
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Tianjin University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/02—Photobioreactors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/04—Flat or tray type, drawers
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/20—Material Coatings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes

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  • the invention belongs to a microalgae cultivation method, in particular to a microalgae cultivation method based on a paper culture device.
  • biodiesel is non-toxic, has good lubricity, safety and combustion properties, and is biodegradable and environmentally friendly. Therefore, countries all over the world are vigorously promoting research on biodiesel and biomass energy utilization.
  • Microalgae is considered to be the only raw material for biodiesel that can completely replace fossil fuels.
  • the potential of microalgae is huge, the use of microalgae as a bioenergy is still limited by its size and cost.
  • microalgae generally uses a solid medium, and the selection of culture conditions and oil accumulation conditions is often carried out in a liquid medium.
  • Large-scale culture conditions require a certain amount of resources to select experiments.
  • Common microalgae culture methods include open large-cell culture, closed photobioreactor, heterotrophic culture, etc. These culture methods require a large space and a large amount of human and material resources.
  • the microalgae are small, and the concentration in the culture solution is very low, and the harvesting is very difficult.
  • the methods for harvesting microalgae include centrifugation, flocculation, foam separation, and microfiltration. However, the cost of harvesting still accounts for 20% to 30% of the cost of breeding. Due to the cultivation and harvesting of microalgae, the resulting water and environmental pollution also requires further treatment to increase the cost of cultivation.
  • microalgae The immobilized production of microalgae can avoid shortcomings such as small growth density, large volume, and difficulty in harvesting, and has very good prospects.
  • the immobilization technology of microalgae is still not perfect, and there are disadvantages such as high cost of immobilized materials. Therefore, there is a need for an experiment that requires low cost, small experimental space, and a small amount of microalgae to select culture conditions and oil accumulation conditions.
  • the cultured microalgae have a high density, and there is no need for post-harvesting, and the microalgae cultivation method does not pollute the environment.
  • the object of the present invention is to overcome the deficiencies of the prior art and to provide a microalgae culture method based on a paper culture device which is environmentally friendly and can reduce experimental research and production costs.
  • a microalgae cultivation method based on a paper culture device comprising the following steps:
  • step 1) using a wax spray printer, according to the design of step 1), spray printing on the filter paper, the printed wax is located in the rectangle except the circular portion (2);
  • the first agar aqueous solution is dropped on the circular surface of the surface of the paper culture device obtained in the step 3); the second agar aqueous solution to which the microalgae is added is dropped on the paper obtained in the step 3)
  • the abalone on the A side of the mass culture device, and the agar on the A and B sides are condensed;
  • the first aqueous solution of agar is prepared by the following method: taking 0.8-1.5 g of agar and adding water to 100 mL, mixing, and heating and dissolving;
  • the second agar aqueous solution was prepared by adding 0.1-0.5 g of agar to water to 100 mL, mixing, and dissolving by heating.
  • the number of circles is 6-96, preferably 6, 12, 24, 48 or 96.
  • the filter paper is preferably a qualitative filter paper or a quantitative filter paper.
  • the conditions of the step (4) culture are: at 20-35 ° C, and the light is 4-8 days.
  • the selection of culture conditions and oil accumulation conditions can be carried out using a small amount of microalgae.
  • microalgae is fixed and cultured, no need to harvest, and the yield per unit area is large.
  • the technology used in the present invention can be applied to all marine microalgae and freshwater microalgae, and the like, and only need to use different density gels for fixing according to the size of different microalgae.
  • Figure 1 is a diagram of a 24 circular culture device.
  • Fig. 2 is a schematic view showing the cultivation of microalgae by a paper culture device.
  • Fig. 3 shows the results of culture on day 0 and day 5 of microalgae culture on a paper culture apparatus.
  • Figure 4 shows the effect of a single salt on the growth of microalgae.
  • the present invention does not limit the wax spray printer, and the spray printer used includes: Xerox ColorQube 8580, Xerox ColorQube 8700, Xerox ColorQube 8880 or Xerox ColorQube 8900.
  • the Xerox ColorQube 8700 is used in various embodiments of the invention.
  • the microalgae Desmodesmus brasiliensis was purchased from the Aquatic Institute of the Chinese Academy of Sciences in October 2015.
  • the present invention uses Desmodesmus brasiliensis as an example of microalgae, but this microalgae does not limit the present invention, and experiments have shown that the use of other microalgae can also be cultured using the apparatus of the present invention.
  • the culture device graphic has 24 circular 1 distributed rectangles in the middle, and the circular diameter is 5.0 mm;
  • step 2) using a wax spray printer, according to the design of step 1), spray printing on the qualitative filter paper, the printed wax is located in the rectangle except the circular portion 2;
  • the filter paper Place the filter paper after the wax spray printing on the heating plate, heat it at 140 °C for 3 minutes, make the wax penetrating filter paper into a hydrophobic area, and the circular area is a hydrophilic area, and obtain a paper culture device to spray the filter paper.
  • the printed side is called the A side and the other side is called the B side;
  • the surface of the device B obtained in the step 4) was immersed in a 90 mm glass culture dish containing 10 mL of BG-11 culture solution, and cultured at 25 ° C, light intensity of 2000 lx, and light for 5 days, and the microalgae were harvested (24 were taken out)
  • the circular paper culture apparatus is dried to obtain an OD of more than 8.0, as shown in FIG.
  • the first aqueous solution of agar was prepared by the following method: adding 1.0 g of agar to water to 100 mL, mixing, and heating to dissolve;
  • the second agar aqueous solution was prepared by adding 0.3 g of agar to water to 100 mL, mixing, and heating to dissolve.
  • the microalgae have a high density and are attached to the agar gel, and the microalgae is harvested without centrifugation or flocculation. Preventing the pollution caused by the use of flocculants to the water body while saving the harvesting cost. At the same time, since the microalgae is only present in the gel, the culture solution can be reused.
  • Each circle in the culture device pattern is equivalent to a conical flask cultured in a conventional conical flask; a 24 circular paper culture device is equivalent to 24 conical flasks. Therefore, the method of the present invention can greatly save the cultivation space and material consumption.
  • a microalgae cultivation method based on a paper culture device comprising the following steps:
  • the culture device graphic has six circularly distributed rectangles in the middle; the diameter of the circular shape is 7.8 mm;
  • step 1) Using a wax spray printer, according to the design of step 1), spray wax on the qualitative filter paper, the printed wax is located in a portion other than the rectangle except the circle;
  • the surface of the device B obtained in the step 4) was immersed in a 90 mm glass culture dish containing 10 mL of BG-11 culture solution, and cultured at 20 ° C, light intensity of 2000 lx, and light for 8 days, and the microalgae were harvested (6 were taken out)
  • the circular paper culture apparatus was dried to obtain an OD of more than 8.0.
  • the first aqueous solution of agar was prepared by the following method: adding 0.8 g of agar to water to 100 mL, mixing, and heating to dissolve;
  • the second agar aqueous solution was prepared by adding 0.1 g of agar to water to 100 mL, mixing, and heating to dissolve.
  • a microalgae cultivation method based on a paper culture device comprising the following steps:
  • the culture device graphic has 96 circularly distributed rectangles in the middle; the diameter of the circular shape is 5.0 mm;
  • step 1) Using a wax spray printer, according to the design of step 1), spray wax on the quantitative filter paper, the printed wax is located in a portion other than the rectangle except the circle;
  • the filter paper Place the filter paper after the wax spray printing on the heating plate, heat at 150 ° C for 0.5 minutes, make the wax penetrating filter paper into a hydrophobic area, and the circular area is a hydrophilic area, and obtain a paper culture device to spray the filter paper.
  • the printed side is called the A side and the other side is called the B side;
  • the surface of the device B obtained in the step 4) was immersed in a 90 mm glass culture dish containing 10 mL of BG-11 culture solution, and cultured at 35 ° C, light intensity of 2000 lx, and light for 4 days, and the microalgae were harvested (96 samples were taken out).
  • the circular paper culture apparatus was dried to obtain an OD of more than 8.0.
  • the first aqueous solution of agar was prepared by the following method: adding 1.5 g of agar to water to 100 mL, mixing, and heating to dissolve;
  • the second agar aqueous solution was prepared by adding 0.5 g of agar to water to 100 mL, mixing, and heating to dissolve.
  • Step 3 Culture of microalgae (Desmodesmus brasiliensis): 20 ⁇ L of the first aqueous agar solution was dropped on the circular surface of the B side of the paper culture apparatus obtained in the step 3); the microalgae liquid cultured in the BG-11 culture solution was subjected to After centrifugation, the culture solution was removed, and the microalgae were resuspended in ultrapure water to obtain an OD 4.0 microalgae solution, and the microalgae solution and the second agar aqueous solution were mixed at a volume ratio of 1:4, and 10 ⁇ L of the solution was dropped.
  • Step 3 The circular portion of the surface of the paper culture device obtained is obtained, and then a saline solution is added to the colloidal microalgae colloid, and the volume of the drop is 4 ⁇ L.
  • the surface of the device B obtained in the step 4) is immersed in a 90 mm glass petri dish containing 10 mL of BG-11 ultrapure water, cultured at 25 ° C, light intensity of 2000 lx, light for 5 days; harvesting of the microalgae (removing the paper) The mass culture apparatus was dried), and the obtained OD was more than 8.0.
  • the first aqueous solution of agar was prepared by the following method: adding 1.0 g of agar to water to 100 mL, mixing, and heating to dissolve;
  • the second agar aqueous solution was prepared by adding 0.3 g of agar to water to 100 mL, mixing, and heating to dissolve.
  • microalgae culture results are shown in Figure 4.
  • the microalgae added with NaNO 3 grows fastest, so the N source is the essential nutrient for the growth of the microalgae; 2
  • the microalgae of CO 3 grew faster in the early stage and slowed down in the later stage, which may be due to the faster consumption of C source; the addition of single P source did not promote the growth of microalgae.
  • microalgae CaCl 2 Two kinds of trace elements Ca and Mg contrast, microalgae CaCl 2 was added, the growth speed is slower than the negative control, may be due to excessive growth of the single Ca ions microalgae has been suppressed, or promote lipid accumulation; addition of MgSO 4 The growth rate of 7H 2 O microalgae is also slower than that of the negative control, which may result in the accumulation of chlorophyll due to the addition of Mg, thereby inhibiting the growth of microalgae.
  • the positive control added 5 salts and the fastest growth rate.
  • volume ratio of the microalgae and the second agar aqueous solution of each embodiment may also be 1:5, 1:6 or 1:7, and the like.

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Abstract

基于纸质培养装置的微藻培养方法，包括：1）设计培养装置图形为中间有圆形分布的矩形；2）向滤纸上喷蜡打印，打印的蜡是位于矩形除圆形以外的部分；3）加热，使蜡穿透滤纸，得纸质培养装置，将喷蜡打印的一面称为A面，另一面为B面；4）将第一琼脂水溶液滴在B面的圆形处，将加入微藻的第二琼脂水溶液滴在A面的圆形处；5）将B面浸于培养液中培养。

Description

一种基于纸质培养装置的微藻培养方法 技术领域
本发明属于微藻培养方法，具体地涉及一种基于纸质培养装置的微藻培养方法。
背景技术
化石燃料替代能源的研发，是全世界范围内科学研究的一个重要领域。作为新型燃料中重要的一员，生物柴油无毒，具有良好的润滑性、安全性能和燃烧性能，并可生物降解，对环境友好。因此世界各国都在大力推进生物柴油和生物质能利用的研究。
微藻被认为是唯一可以完全替代化石燃料的生物柴油的原料。利用微藻获得生物柴油，包括微藻的筛选、培养和采收加工等三个重要步骤。虽然微藻的潜力巨大，但使用微藻作为生物能源，仍制约于其规模和成本。
微藻的筛选一般使用固体培养基，其培养条件和油脂积累条件的选择，则常在液体培养基中进行。大批量的培养条件选择实验需要耗费一定的资源。常见的微藻培养方法有开放式大池培养、密闭式光生物反应器、异养培养等，这些培养方法都需要占用较大空间和大量人力物力。同时，微藻个体微小，且在培养液中的浓度很低，采收难度非常大。微藻的采收方法有离心法、絮凝法、泡沫分离法、微滤法等。但其采收的成本仍占其养殖成成本的20％～30％。由于培养和采收微藻，产生的水体和环境污染，也需要进行进一步治理，增加培养成本。
而微藻的固定化生产，可以避免其生长密度小、体积大、采收困难等缺点，有非常良好的前景。但微藻的固定化技术仍不完善，有固定化材料造价高等缺点。因此亟需一种成本低廉，使用很少的实验空间和少量微藻，就可进行培养条件和油脂积累条件选择等实验。同时，培养出的微藻密度很高，无需后期采收，对环境不造成污染的微藻培养方法。
发明内容
本发明的目的是克服现有技术的不足，提供一种对环境友好，可减少实验研究和生产成本的基于纸质培养装置的微藻培养方法。
本发明的技术方案概述如下：
一种基于纸质培养装置的微藻培养方法，包括如下步骤：
1)设计培养装置图形，培养装置图形为中间有圆形(1)分布的矩形；
2)使用喷蜡打印机，按照步骤1)的设计，向滤纸上喷蜡打印，打印的蜡是位于矩形除圆形处以外的部分(2)；
3)将喷蜡打印后的滤纸放加热，使蜡穿透滤纸，得纸质培养装置，将滤纸的喷蜡打印的一面 称为A面，另一面称为B面；
4)微藻的培养：将第一种琼脂水溶液滴在步骤3)获得的纸质培养装置B面的圆形处；将加入了微藻的第二种琼脂水溶液滴在步骤3)获得的纸质培养装置A面的圆形处，A和B面的琼脂凝结；
5)将步骤4)获得的装置B面浸于含有BG-11培养液的玻璃培养皿中，培养；
所述第一种琼脂水溶液用下述方法制成：取0.8-1.5g琼脂加水至100mL，混匀，加热溶解；
所述第二种琼脂水溶液用下述方法制成：取0.1-0.5g琼脂加水至100mL，混匀，加热溶解。
圆形的个数为6-96个，优选6、12、24、48或96个。
滤纸优选定性滤纸或定量滤纸。
步骤(4)培养的条件为：在20-35℃，光照4-8天。
本发明的优点：
1.利用纸质的培养装置对微藻进行培养，简化操作，减少所需空间，并降低成本。
2.使用少量微藻，即可进行培养条件和油脂积累条件的选择。
3.便于进行重复性实验。
4.微藻固定化培养，无需采收，单位面积产量大。
5.本发明所用技术可用于所有海洋微藻及淡水微藻等原料，只需根据不同微藻的大小，使用不同密度的凝胶进行固定。
6.本方法对环境不造成污染。
附图说明
图1为有24个圆形的培养装置图。
图2为纸质培养装置对微藻进行培养示意图。
图3为在纸质培养装置上进行微藻培养第0天和第5天的培养结果。
图4为单种盐对微藻生长的影响。
具体实施方式
为了使本发明的技术方案及特点更加清晰，以下结合附图和具体实施方式，对本发明作进一步说明。此处描述的具体实施例仅用于解释本发明，但并不限定本发明的保护范围。
本发明对喷蜡打印机不进行限定，所使用的喷蜡打印机包括：Xerox ColorQube 8580，Xerox ColorQube 8700，Xerox ColorQube 8880或Xerox ColorQube 8900。本发明各实施例使用的是Xerox ColorQube 8700。
微藻Desmodesmus brasiliensis，2015年10月购于中科院水生所藻种库。
本发明以Desmodesmus brasiliensis作为微藻的例子，但这一微藻并不对本发明进行限定，实验证明，使用其它的微藻也可以用本发明的装置进行培养。
实施例1
一种基于纸质培养装置的微藻培养方法，见图1，图2，包括如下步骤：
1)在电脑上设计培养装置图形，培养装置图形为中间有24个圆形1分布的矩形，圆形的直径为5.0mm；
2)使用喷蜡打印机，按照步骤1)的设计，向定性滤纸上喷蜡打印，打印的蜡是位于矩形除圆形处以外的部分2；
3)将喷蜡打印后的滤纸放在加热盘中，在140℃加热3分钟，使蜡穿透滤纸为疏水区域，圆形处为亲水区域，得纸质培养装置，将滤纸的喷蜡打印的一面称为A面，另一面称为B面；
4)微藻(Desmodesmus brasiliensis)的培养：将第一种琼脂水溶液20μL滴在步骤3)获得的纸质培养装置B面的圆形处；将加入了微藻的第二种琼脂水溶液(在BG-11培养液中培育的微藻液(OD 4.2)与第二种琼脂水溶液按体积比1：4的比例混合)取10μL滴在步骤3)获得的纸质培养装置A面的圆形处；
5)将步骤4)获得的装置B面浸于含有10mL BG-11培养液的90mm玻璃培养皿中，在25℃，光照强度2000lx，光照5天培养，对微藻进行采收(取出24个圆形的纸质培养装置进行干燥)，得到的OD大于8.0，见图3。
第一种琼脂水溶液用下述方法制成：取1.0g琼脂加水至100mL，混匀，加热溶解；
第二种琼脂水溶液用下述方法制成：取0.3g琼脂加水至100mL，混匀，加热溶解。
该微藻密度很高，均附着在琼脂凝胶中，无需离心或絮凝等方式进行微藻采收。在节省采收成本的同时，防止使用絮凝剂对水体造成的污染。同时，由于微藻只存在于凝胶中，因此该培养液可重复使用。
培养装置图形中的每一个圆形相当于传统锥形瓶培养的一个锥形瓶；一个24个圆形的纸质培养装置，就相当于24个锥形瓶。因此本发明的方法可以大大节省培养空间和材料消耗。
实施例2
一种基于纸质培养装置的微藻培养方法，包括如下步骤：
1)在电脑上设计培养装置图形，培养装置图形为中间有6个圆形分布的矩形；圆形的直径为7.8mm；
2)使用喷蜡打印机，按照步骤1)的设计，向定性滤纸上喷蜡打印，打印的蜡是位于矩形除圆形处以外的部分；
3)将喷蜡打印后的滤纸放在加热盘中，在120℃加热5分钟，使蜡穿透滤纸为疏水区域，圆形处为亲水区域，得纸质培养装置，将滤纸的喷蜡打印的一面称为A面，另一面称为B面；
4)微藻(Desmodesmus brasiliensis)的培养：将第一种琼脂水溶液15μL滴在步骤3)获得的纸质培养装置B面的圆形处；将加入了微藻的第二种琼脂水溶液(在BG-11培养液中培育的微藻液(OD 4.2)与第二种琼脂水溶液按体积比1：4的比例混合)取15μL滴在步骤3)获得的纸质培养装置A面的圆形处；
5)将步骤4)获得的装置B面浸于含有10mL BG-11培养液的90mm玻璃培养皿中，在20℃，光照强度2000lx，光照8天培养，对微藻进行采收(取出6个圆形的纸质培养装置进行干燥)，得到的OD大于8.0。
第一种琼脂水溶液用下述方法制成：取0.8g琼脂加水至100mL，混匀，加热溶解；
第二种琼脂水溶液用下述方法制成：取0.1g琼脂加水至100mL，混匀，加热溶解。
实施例3
一种基于纸质培养装置的微藻培养方法，包括如下步骤：
1)在电脑上设计培养装置图形，培养装置图形为中间有96个圆形分布的矩形；圆形的直径为5.0mm；
2)使用喷蜡打印机，按照步骤1)的设计，向定量滤纸上喷蜡打印，打印的蜡是位于矩形除圆形处以外的部分；
3)将喷蜡打印后的滤纸放在加热盘中，在150℃加热0.5分钟，使蜡穿透滤纸为疏水区域，圆形处为亲水区域，得纸质培养装置，将滤纸的喷蜡打印的一面称为A面，另一面称为B面；
4)微藻(Desmodesmus brasiliensis)的培养：将第一种琼脂水溶液15μL滴在步骤3)获得的纸质培养装置B面的圆形处；将加入了微藻的第二种琼脂水溶液(在BG-11培养液中培育的微藻液(OD 4.2)与第二种琼脂水溶液按体积比1：4的比例混合)取15μL滴在步骤3)获得的纸质培养装置A面的圆形处；
5)将步骤4)获得的装置B面浸于含有10mL BG-11培养液的90mm玻璃培养皿中，在35℃，光照强度2000lx，光照4天培养，对微藻进行采收(取出96个圆形的纸质培养装置进行干燥)，得到的OD大于8.0。
第一种琼脂水溶液用下述方法制成：取1.5g琼脂加水至100mL，混匀，加热溶解；
第二种琼脂水溶液用下述方法制成：取0.5g琼脂加水至100mL，混匀，加热溶解。
实施例4
单盐浓度实验
1)、2)、3)同实施例1步骤1)、2)、3)；
4)微藻(Desmodesmus brasiliensis)的培养：将第一种琼脂水溶液20μL滴在步骤3)获得的纸质培养装置B面的圆形处；将在BG-11培养液中培育的微藻液进行离心，除去培养液，将微藻重悬在超纯水中，得到OD 4.0微藻液，将所述微藻液和第二种琼脂水溶液按体积比1：4的比例混合，取10μL滴在步骤3)获得的纸质培养装置A面的圆形处，随后在已凝结的微藻胶体上分别滴加一种盐水溶液，滴加的体积均为4μL，
盐溶液：
Figure PCTCN2017085869-appb-000001
Figure PCTCN2017085869-appb-000002
阳性对照：上述5种盐，各自浓度同上。
阴性对照：水。
5)将步骤4)获得的装置B面浸于含有10mL BG-11超纯水的90mm玻璃培养皿中，在25℃，光照强度2000lx，光照5天培养；对微藻进行采收(取出纸质培养装置进行干燥)，得到的OD大于8.0。
第一种琼脂水溶液用下述方法制成：取1.0g琼脂加水至100mL，混匀，加热溶解；
第二种琼脂水溶液用下述方法制成：取0.3g琼脂加水至100mL，混匀，加热溶解。
微藻培养结果如图4所示，三个主要营养物质，P源、N源和C源对比，加入NaNO3的微藻生长最快，因此N源是该微藻生长的必须营养；加入Na2CO3的微藻在前期生长较快，后期减慢，可能由于C源的消耗较快；单一的P源加入对微藻的生长没有促进作用。两种微量元素Ca和Mg对比，加入CaCl2的微藻，其生长速度慢于阴性对照，可能由于单一Ca离子过多对该种微藻的生长有所抑制，或促进油脂积累；加入MgSO4·7H2O的微藻的生长速度也慢于阴性对照，可能由于Mg的加入导致叶绿素的累积，从而抑制了微藻的生长。阳性对照加入了5种盐，生长速度最快。
实验证明，各实施例微藻和第二种琼脂水溶液的体积比也可以是选1：5、1：6或1：7等等。

Claims (4)

  1. 一种基于纸质培养装置的微藻培养方法，其特征是包括如下步骤：
    1)设计培养装置图形，培养装置图形为中间有圆形(1)分布的矩形；
    2)使用喷蜡打印机，按照步骤1)的设计，向滤纸上喷蜡打印，打印的蜡是位于矩形除圆形处以外的部分(2)；
    3)将喷蜡打印后的滤纸放加热，使蜡穿透滤纸，得纸质培养装置，将滤纸的喷蜡打印的一面称为A面，另一面称为B面；
    4)微藻的培养：将第一种琼脂水溶液滴在步骤3)获得的纸质培养装置B面的圆形处；将加入了微藻的第二种琼脂水溶液滴在步骤3)获得的纸质培养装置A面的圆形处；
    5)将步骤4)获得的装置B面浸于含有BG-11培养液的玻璃培养皿中，培养；
    所述第一种琼脂水溶液用下述方法制成：取0.8-1.5g琼脂加水至100mL，混匀，加热溶解；
    所述第二种琼脂水溶液用下述方法制成：取0.1-0.5g琼脂加水至100mL，混匀，加热溶解。
  2. 根据权利要求1所述的方法，其特征是所述圆形的个数为6-96个。
  3. 根据权利要求1所述的方法，其特征是所述滤纸为定性滤纸或定量滤纸。
  4. 根据权利要求1所述的方法，其特征是步骤(4)培养的条件为：在20-35℃，光照4-8天。
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101978272A (zh) * 2008-03-27 2011-02-16 哈佛学院院长等 基于纸的细胞阵列
CN102373156A (zh) * 2010-08-10 2012-03-14 中国科学院青岛生物能源与过程研究所 一种用于微藻工业化生产的半干固态培养方法
CN103834567A (zh) * 2014-02-26 2014-06-04 新奥科技发展有限公司 一种微藻培养方法
CN104677896A (zh) * 2015-01-31 2015-06-03 太原理工大学 一种用于比色分析的纸基微流控芯片的制备及应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014105581A1 (en) * 2012-12-26 2014-07-03 Konica Minolta Laboratory U.S.A., Inc. Apparatus and method for rapid 3d cell culture analysis using paper stacks

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101978272A (zh) * 2008-03-27 2011-02-16 哈佛学院院长等 基于纸的细胞阵列
CN102373156A (zh) * 2010-08-10 2012-03-14 中国科学院青岛生物能源与过程研究所 一种用于微藻工业化生产的半干固态培养方法
CN103834567A (zh) * 2014-02-26 2014-06-04 新奥科技发展有限公司 一种微藻培养方法
CN104677896A (zh) * 2015-01-31 2015-06-03 太原理工大学 一种用于比色分析的纸基微流控芯片的制备及应用

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