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Method for rapidly separating heterotrophic microalgae from natural waters by using combined bacteriostat

A technology for natural waters and microalgae, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of low sterilization efficiency of algae species, lack of pertinence of algae strains, high equipment requirements, etc. The effects of high bacterialization efficiency, clear pertinence, and low equipment requirements

Inactive Publication Date: 2009-12-23
SICHUAN UNIV
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AI Technical Summary

Problems solved by technology

For the screening of heterotrophic algae strains, the main shortcomings of these studies are: (1) mainly rely on the repeated separation and purification of photoautotrophic culture, which has a long cycle and high equipment requirements (generally requires a light culture device); (2) ) The isolated algae strains lack specificity, and most algae strains cannot grow under heterotrophic conditions; (3) The sterilization efficiency of algae species is low, and it is easy to contaminate miscellaneous bacteria after multiple subcultures, especially heterotrophic cultures (4) The current microalgae sterilization method using antibiotics is mainly aimed at bacteria, however, in our study, it was found that for the screening of heterotrophic algae species, overcoming mold contamination is a more difficult problem
[0004] Through domestic patent and literature search, there is no report on the method of directly separating and purifying heterotrophic microalgae from natural waters

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] (1) Preparation of Basal liquid medium (1000mL): KNO 3 1.25g, KH 2 PO 4 1.25g, MgSO 4 1g, A solution 100mL, B solution 10mL. Among them, the formula of liquid A (1000mL): H 3 BO 3 1.142g, CaCl 2 2H 2 O 1.11g, ZnSO 4 ·7H 2 O 0.882g, MnCl 2 4H 2 O 0.142g, CuSO 4 ·5H 2 O 0.157g, MoO 3 0.071g, Co(NO 3 ) 2 ·6H 2 O 0.049g, EDTA 4g; B liquid formula (100mL): FeSO 4 ·7H 2 O 0.498g, EDTA 1g. The pH of the medium was adjusted to 6.1 with 5% NaOH. Add 50 mL of prepared Basal medium into a 500 mL Erlenmeyer flask, seal the bottle mouth with a silica gel stopper, sterilize at 121°C for 15 min, and cool down for later use.

[0023] (2) Prepare heterotrophic Basal solid medium: add 1% glucose and 1.5% agar to Basal liquid medium, heat and stir in a water bath to boil, after cooling slightly, adjust the pH of the medium with 5% NaOH To 6.1, put the culture medium with adjusted pH into the Erlenmeyer flask, seal the mouth of the flask with a silica gel stopper,...

Embodiment 2

[0031] (1) Prepare aquatic No. 4 liquid medium (1000mL): (NH 4 ) 2 SO 4 0.2g, calcium superphosphate Ca(H 2 PO 4 )·H 2 O+2(CaSO 4 ·H 2 O) 0.03g, MgSO 4 ·7H 2 O 0.08g, NaHCO 3 0.10g, KCl 0.025g, FeCl 3 (1% solution) 0.15mL, soil leaching solution 0.5mL; adjust the pH to 6.1 with 5% NaOH. Add 50 mL of prepared Basal medium into a 500 mL Erlenmeyer flask, seal the bottle mouth with a silica gel stopper, sterilize at 121°C for 15 min, and cool down for later use.

[0032] (2) Preparation of Heterotrophic Aquatic No. 4 Solid Medium: Add 1% glucose and 1.5% agar to the No. 4 Aquatic Liquid Medium, heat and stir in a water bath to boil, after cooling slightly, use 5% NaOH to culture Adjust the pH of the base to 6.1, put the adjusted pH medium into the Erlenmeyer flask, seal the mouth of the flask with a silica gel stopper, sterilize at 121°C for 15 minutes, and cool down for later use.

[0033] (3) Bacteriostat plate preparation: Heterotrophic Shuisheng No. 4 solid medi...

Embodiment 3

[0039] (1) Preparation of Basal liquid medium (1000mL): KNO3 1.25g, KH 2 PO 4 1.25g, MgSO 4 1g, A solution 100mL, B solution 10mL. Among them, the formula of liquid A (1000mL): H 3 BO 3 1.142g, CaCl 2 2H 2 O 1.11g, ZnSO 4 ·7H 2 O 0.882g, MnCl 2 4H 2 O 0.142g, CuSO 4 ·5H 2 O 0.157g, MoO 3 0.071g, Co(NO 3 ) 2 ·6H 2 O 0.049g, EDTA 4g; B liquid formula (100mL): FeSO 4 ·7H 2 O 0.498g, EDTA 1g. The pH of the medium was adjusted to 6.1 with 5% NaOH. Add 50mL of the prepared Basal medium into a 500mL Erlenmeyer flask, seal the mouth of the bottle with a silica gel stopper, sterilize at 121°C for 15min, and cool down for later use.

[0040] (2) Prepare heterotrophic Basal solid medium: add 1% glucose and 2% agar to Basal liquid medium, heat and stir in a water bath to boil, after cooling slightly, adjust the pH of the medium with 5% NaOH To 6.1, put the medium with adjusted pH into the Erlenmeyer flask, seal the mouth of the flask with a silica gel stopper, ste...

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Abstract

The invention provides a method for rapidly separating purified heterotrophic microalgae from natural water areas by using a combined bacteriostat. The invention is characterized in that the method comprises the steps as follows: a) preparing microalgae autotrophic liquid nutrient medium; b) preparing microalgae heterotrophic solid medium (adding 1% of glucose and 1.5-2% of agar into the microalgae autotrophic liquid nutrient medium); c) preparing a medium plate containing the combined bacteriostat (20-60mu g / mL of ampicillin and 200-600mu g / mL of Natamycin); d) coating natural water sample or propagated and cultured water sample on the medium plate containing the combined bacteriostat, and then culturing in a constant-temperature incubator at the temperature of 25-35 DEG C; e) after colorful algae grows on the plate, using an inoculating loop to pick the colorful algae to the blank medium plate containing the combined bacteriostat, lining and then culturing the medium plate with the algae in the constant-temperature incubator at the temperature of 25-35 DEG C; and f) repeating the step e) for 3-5 times, and then obtaining the purified heterotrophic microalgae. The method is simple, convenient, rapid and reliable in operation and also can separate and purify the heterotrophic microalgae from the natural waters within a shorter time.

Description

technical field [0001] The invention relates to a method for rapidly separating heterotrophic microalgae from natural waters by using a combined bacteriostatic agent, and belongs to the field of microalgae biotechnology. Background technique [0002] Microalgae are rich in protein, fat, EPA, DHA and other functional active substances, which have important economic value. The development and utilization of microalgae has become a new way for humans to obtain food, medicine, and biofuels. Microalgae belong to lower plants and mainly grow by converting inorganic carbon sources through photosynthesis. The traditional open photoautotrophic method has problems such as light limitation, easy pollution and large footprint, so the production efficiency is not high. Studies have shown that some unicellular microalgae (such as Chlorella) can use organic carbon sources for heterotrophic growth in the absence of light, and their biomass and production efficiency have been greatly impro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/12C12R1/89
Inventor 吴正云张文学曾娟
Owner SICHUAN UNIV
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