CN110819577A - Blue algae culture method with desert soil moisturizing function - Google Patents
Blue algae culture method with desert soil moisturizing function Download PDFInfo
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Abstract
The invention relates to a method for culturing blue algae with a desert soil moisturizing function. The method comprises the following steps: firstly, separating a moisturizing algae culture; secondly, inoculating single moisturizing algae filaments on a solid plate for repeated culture until 5-7 generations of algae are inoculated to obtain algae, and thirdly, inoculating the algae into a liquid culture medium for large-scale open culture for 7-21 days. The invention firstly cultures the moisturizing algae, improves the cell concentration, obtains the moisturizing algae culture solution with high cell concentration, then separates, then purifies to obtain high-yield target algae species, and finally carries out large-scale industrial culture, thus ensuring successful separation of target algae strains.
Description
Technical Field
The invention relates to a method for culturing microbial algae, in particular to a method for culturing blue algae with a desert soil moisturizing function.
Background
Blue-green Algae (Blue-green Algae) is an Algae plant; prokaryotes capable of photosynthetic oxygen evolution. Single cell individuals or populations, or filaments that are arranged in strings of cells that make up algal filaments (cell divisions), unbranched, pseudobranched, or true branched. Nucleated, non-nucleated membranes; the chromatoplasm area mainly comprises thylakoids and related structures thereof, phycobilisomes, glycogen particles and the like, has photosynthetic pigments such as chlorophyll a, phycobilin, carotene, carotenoid and the like, but has no chlorophyll membrane and does not form chloroplasts; has a cell wall. Blue algae have existed on earth for about 30 hundred million years and are the earliest photosynthetic oxygen-releasing organisms. The blue algae has great adaptability and wide distribution. Fresh and sea water, moist and arid soils or rocks, trunks and leaves, hot springs, ice and snow, and even in bittern ponds, rock cracks; some can also live in deep soil. The tacrolimus arid desert moisturizing algae are used as the extreme environment blue algae, and the separated blue algae are used as the extreme environment blue algae, so that the unique physiological and biochemical characteristics which are not possessed by other blue algae are inevitably accumulated in the evolution of tens of thousands of years. The tacrolimus dry desert moisturizing algae cells have extremely strong water binding capacity and can secrete abundant polysaccharide components. However, at present, a mature and complete technological method for separating and culturing the moisturizing algae does not exist, the separation principle of the traditional 10-fold dilution method is to culture the moisturizing algae after separation, but in the method, the biomass of the target algae in a desert soil sample is very low, the successful separation of the target algae and the growth of the target algae cannot be guaranteed, and meanwhile, a large-scale culture method related to the moisturizing algae is not reported at present.
Disclosure of Invention
In view of the problems in the prior art, the invention provides a method for culturing blue algae with a desert soil moisturizing function, which adopts the technical scheme that the method comprises the following steps:
step.1 isolation of moisturizing algae: weighing a desert soil sample, adding BG11 liquid culture medium into the desert soil sample according to the condition that 300ml to 500ml of BG11 liquid culture medium is added into each 100g to 200g of desert soil sample, using a shaking table for 5min to 10min under the condition that the rotation speed is 80rpm to 100rpm to fully mix the sample and the culture medium uniformly, statically culturing the mixture for 7 days to 21 days under the conditions that the temperature is 27 ℃ to 30 ℃ and the illumination is 3000Lux to 3500Lux, coating each 300 mu L to 500 mu L of supernatant on 25ml to 30ml of TAP solid culture medium after the supernatant of the culture turns green, sucking the supernatant under the aseptic condition, coating the supernatant on a culture dish containing BG11 solid culture medium, and culturing for 7 days to 21 days under the conditions that the temperature is 27 ℃ to 30 ℃ and the illumination is 3000Lux to 3500Lux to obtain a moisturizing algae culture;
step.2, purification: selecting single moisturizing algae filaments from each single moisturizing algae filament inoculated on 25-35 ml of BG11 solid culture medium, inoculating the selected single moisturizing algae filaments on a solid plate containing BG11 solid culture medium by a scribing method, culturing for 7-21 days at the temperature of 27-30 ℃ under the illumination of 3000-3500 Lux to obtain a primary moisturizing algae body, selecting single moisturizing algae filaments from the primary moisturizing algae body by the sterilized inoculating ring, inoculating the single moisturizing algae filaments on the solid plate containing a new BG11 solid culture medium, culturing for 7-21 days at the temperature of 27-30 ℃ under the illumination of 3000-3500 Lux to obtain a secondary moisturizing algae body, repeating the step until 5-7 generations are inoculated to obtain a pure moisturizing algae body;
step.3, large-scale culture: selecting single algae filaments from pure moisturizing algae according to the inoculation of each single algae filament on 250ml to 1000ml BG11 liquid culture medium, inoculating the single algae filaments into BG11 liquid culture medium, and carrying out open culture for 7 days to 21 days under the conditions that the temperature is 27 ℃ to 30 ℃ and the illumination is 3000Lux to 3500Lux to prepare the moisturizing algae mother seed suspension with the optical density (OD560) of the algae liquid concentration reaching 1.0. (1) Primary selection: according to the method, a moisturizing algae mother seed suspension with the volume of 1-3% is inoculated into 100-500L BG11 liquid culture medium, the moisturizing algae mother seed suspension is inoculated into a new BG11 liquid culture medium, the optical density (OD560) of the inoculation concentration is preferably 0.10, the continuous stirring and the recarburization are carried out by an electromagnetic air pump or the direct recarburization is carried out by a carbon dioxide compressed gas cylinder, the large-scale open culture is carried out for 7-21 days under the conditions that the temperature is 25-31 ℃ and the illumination is 2000-4000 Lux, and then when the optical density (OD560) of the algae liquid concentration reaches 1.0, the coarse and strong algae filaments with good suspension are selected by 300-mesh silk cloth for checking. (2) Checking: and (3) inoculating the primarily selected algae filaments into a fresh BG11 liquid culture medium again according to the steps, performing large-scale open culture for 7 to 21 days under the conditions that the temperature is between 25 and 31 ℃ and the illumination is between 2000 and 4000Lux, checking the algae filaments by using a 200-mesh silk cloth sieve after the optical density value (OD560) of the algae liquid concentration reaches 1.0, and repeating the steps for 3 to 5 times. (3) Seed expansion: when the average daily temperature is more than 20 ℃, the checked algae seeds are inoculated into a primary culture pond containing 1000L to 5000L BG11 liquid culture medium, and the open culture is carried out for 7 days to 21 days under the conditions that the optical density (OD560) of the inoculated concentration is 0.10, the temperature is 25 ℃ to 31 ℃, and the illumination is 2000Lux to 4000 Lux. Inoculating the algae liquid to a second-stage culture pond containing 5000L-50000L BG11 liquid culture medium when the optical density value (OD560) of the algae liquid concentration reaches more than 1.0, gradually expanding the algae liquid to a third-stage culture pond containing 50000L-200000L BG11 liquid culture medium, and carrying out open culture for 7-21 days under the conditions that the temperature is 25-31 ℃ and the illumination is 2000-4000 Lux.
Further, the BG11 liquid culture medium comprises 1.5g NaNO3，0.04g K2HPO3.3H2O，0.075gMgSO4.7H2O，0.036g CaCl2.2H2O, 0.006g citric acid, 0.006g ferric ammonium citrate, 0.001g EDTA2，0.02g Na2CO31ml of trace elements is dissolved in water to prepare a 1L solution to obtain BG11 liquid culture medium, and the pH value of the BG11 liquid culture medium ranges from 7.0 to 7.2.
A preparation method of BG11 solid culture medium for blue algae culture comprises mixing 1.5g NaNO3，0.04g K2HPO3.3H2O，0.075g MgSO4.7H2O，0.036g CaCl2.2H2O, 0.006g citric acid, 0.006g ferric ammonium citrate, 0.001g EDTA2，0.02g Na2CO31ml of trace elements are dissolved in water to prepare 1L of solution, agar powder accounting for 1.8 to 2 percent of the total volume of the solution is added into the solution, the pH range of the solution is adjusted to 7.0 to 7.2, the solution is subjected to moist heat sterilization at 121 ℃, 0.11MPa and 20min, and then is cooled and solidified at 40 ℃ to be in a solid state, namely the BG11 solid culture medium is obtained.
The invention has the beneficial effects that: the method comprises the steps of culturing desert algae special for desertification control, improving the cell concentration in a desert soil culture solution, separating the desert algae special for desertification control after obtaining the desert soil culture solution with high biomass of target algae seeds, purifying to obtain high-yield target algae seeds, and finally performing large-scale culture, so that the aim of successfully separating the moisturizing algae strains can be ensured. The culture method adopted by the invention is optimized, and the desert algae special for desertification control can be cultured in a large scale under the condition of ensuring the yield of the desert algae special for desertification control. .
Drawings
FIG. 1 is a schematic diagram of cyanobacteria cultured by the culture method of the present invention;
Detailed Description
The invention will be further described with reference to the following drawings and specific examples:
a method for culturing blue algae with a desert soil moisturizing function adopts the technical scheme that the method comprises the following steps:
step.1 isolation of moisturizing algae: weighing a desert soil sample, adding BG11 liquid culture medium into the desert soil sample according to the condition that 300ml of BG11 liquid culture medium is added into each 100g of desert soil sample, fully and uniformly mixing the sample and the culture medium by using a shaking table for 10min under the condition that the rotating speed is 100rpm, statically culturing the mixture for 7 days under the conditions that the temperature is 30 ℃ and the illumination is 3000Lux, coating each 300 microliter of supernatant on 25ml of TAP solid culture medium after the supernatant of the culture turns green, sucking the supernatant under the aseptic condition, coating the supernatant on a culture dish containing BG11 solid culture medium, and culturing for 7 days under the conditions that the temperature is 27-30 ℃ and the illumination is 3000Lux to obtain a moisturizing algae culture;
step.2, purification: selecting single moisturizing algae filaments from a moisturizing algae culture species by using a sterilized inoculating loop according to the inoculation of each single moisturizing algae filament on 25ml of BG11 solid culture medium, inoculating the single moisturizing algae filaments on a solid plate containing BG11 solid culture medium by a scribing method, culturing for 7 days under the conditions of 30 ℃ and 3000Lux to obtain a primary moisturizing algae body, selecting single moisturizing algae filaments from the primary moisturizing algae body by using the sterilized inoculating loop, inoculating the single moisturizing algae filaments on the solid plate containing a new BG11 solid culture medium, culturing for 7 days under the conditions of 30 ℃ and 3000Lux to obtain a secondary moisturizing algae body, repeating the steps until 5 generations of inoculation are carried out, and finally obtaining a pure moisturizing algae body;
step.3, large-scale culture: according to the method, each single algae filament is inoculated on 250ml BG11 liquid culture medium, the single algae filament is selected from pure moisturizing algae, inoculated in BG11 liquid culture medium, and subjected to open culture for 7 days under the conditions that the temperature is 30 ℃ and the illumination is 3000Lux to prepare the moisturizing algae mother-seed suspension with the optical density (OD560) of the algae liquid concentration reaching 1.0. (1) Primary selection: according to the method, a moisturizing algae mother seed suspension is inoculated into a new BG11 liquid culture medium according to the condition that 1% of moisturizing algae mother seed suspension is inoculated into every 100L of BG11 liquid culture medium, the optical density (OD560) of the inoculation concentration is preferably 0.10, the continuous stirring and the recarburization are carried out by an electromagnetic air pump or the direct recarburization is carried out by a carbon dioxide compressed gas cylinder, and after the large-scale open culture is carried out for 7 days under the conditions that the temperature is 31 ℃ and the illumination is 2000Lux, when the optical density (OD560) of the algae liquid concentration reaches 1.0, the fine selection of the algae filaments with good thick suspension property is carried out by 300-mesh silk cloth. (2) Checking: and when the primarily selected algae filaments are inoculated in a fresh BG11 liquid culture medium again according to the above steps, large-scale open culture is carried out for 7 days under the conditions that the temperature is 31 ℃ and the illumination is 2000Lux, after the optical density value (OD560) of the algae liquid concentration reaches 1.0, checking is carried out by using a 200-mesh silk cloth sieve, and the steps are repeated for 3 times. (3) Seed expansion: when the average daily temperature reached 20 ℃ or higher, the checked algal species were inoculated into a primary culture tank containing 1000L of BG11 liquid medium, and were subjected to open culture for 7 days under conditions of an optical density (OD560) of 0.10, a temperature of 31 ℃ and an illumination of 2000Lux after inoculation. When the optical density value (OD560) of the algae liquid concentration reaches 1.0 or more, inoculating the algae liquid into a secondary culture pond containing 5000L BG11 liquid culture medium, gradually expanding the algae liquid concentration to a tertiary culture pond containing 50000L BG11 liquid culture medium, and carrying out open culture for 7 days under the conditions that the temperature is 31 ℃ and the illumination is 2000 Lux.
Further, the BG11 liquid culture medium comprises 1.5g NaNO3，0.04g K2HPO3.3H2O，0.075gMgSO4.7H2O，0.036g CaCl2.2H2O, 0.006g citric acid, 0.006g ferric ammonium citrate, 0.001g EDTA2，0.02g Na2CO31ml of trace elements is dissolved in water to prepare 1L of solution, so that BG11 liquid culture medium is obtained, and the pH range of the BG11 liquid culture medium is 7.0.
A preparation method of BG11 solid culture medium for blue algae culture comprises mixing 1.5g NaNO3，0.04g K2HPO3.3H2O，0.075g MgSO4.7H2O，0.036g CaCl2.2H2O, 0.006g lemonCitric acid, 0.006g ferric ammonium citrate, 0.001g EDTA2，0.02g Na2CO31ml of trace elements are dissolved in water to prepare 1L of solution, agar powder accounting for 1.8 percent of the total volume of the solution is added into the solution, the pH range of the solution is adjusted to 7.0, the solution is subjected to moist heat sterilization at 121 ℃, 0.11MPa and 20min, and then is cooled and solidified at 40 ℃ to be in a solid state, namely the BG11 solid culture medium is obtained.
Example 2
A method for culturing blue algae with a desert soil moisturizing function adopts the technical scheme that the method comprises the following steps:
step.1 isolation of moisturizing algae: weighing a desert soil sample, adding BG11 liquid culture medium into the desert soil sample according to the condition that 500ml of BG11 liquid culture medium is added into every 200g of desert soil sample, fully and uniformly mixing the sample and the culture medium by using a shaking table for 5min under the condition that the rotating speed is 80rpm, statically culturing the mixture for 21 days under the conditions that the temperature is 27 ℃ and the illumination is 3500Lux, coating 30ml of TAP solid culture medium with supernatant liquid of every 500 mu L of the supernatant liquid after the supernatant liquid of the culture turns green, sucking the supernatant liquid under the aseptic condition, coating the supernatant liquid in a culture dish containing BG11 solid culture medium, and culturing for 21 days under the conditions that the temperature is 27 ℃ and the illumination is 3500Lux to obtain a moisturizing algae culture;
step.2, purification: selecting single moisturizing algae filaments from a sterilized inoculating loop on 35ml of BG11 solid culture medium, inoculating the single moisturizing algae filaments on a solid plate containing BG11 solid culture medium by a scribing method, culturing for 21 days at the temperature of 27 ℃ and under the illumination of 3500Lux to obtain a primary moisturizing algae, selecting single moisturizing algae filaments from the primary moisturizing algae by using the sterilized inoculating loop, inoculating the single moisturizing algae filaments on the solid plate containing a new BG11 solid culture medium, culturing for 21 days at the temperature of 27 ℃ and under the illumination of 3500Lux to obtain a secondary moisturizing algae, repeating the step until 7 generations are inoculated, and finally obtaining pure moisturizing algae;
step.3, large-scale culture: according to the method, each single algae filament is inoculated on 1000ml BG11 liquid culture medium, the single algae filament is selected from pure moisturizing algae, inoculated in BG11 liquid culture medium, and subjected to open culture for 21 days under the conditions that the temperature is 27 ℃ and the illumination is 3500Lux to prepare the moisturizing algae mother-seed suspension with the optical density (OD560) of the algae liquid concentration reaching 1.0. (1) Primary selection: according to the method, a moisturizing algae mother seed suspension is inoculated into a new BG11 liquid culture medium according to the condition that 3% of the moisturizing algae mother seed suspension is inoculated into every 500L of BG11 liquid culture medium, the optical density (OD560) of the inoculation concentration is preferably 0.10, the continuous stirring and the recarburization are carried out by an electromagnetic air pump or the direct recarburization is carried out by a carbon dioxide compressed gas cylinder, and after the large-scale open culture is carried out for 21 days under the conditions that the temperature is 25 ℃ and the illumination is 4000Lux, when the optical density (OD560) of the algae liquid concentration reaches 1.0, the selection of the algae filaments with good thick suspension property is carried out by 300-mesh silk cloth. (2) Checking: and when the primarily selected algae filaments are inoculated in a fresh BG11 liquid culture medium again according to the above steps, large-scale open culture is carried out for 21 days under the conditions that the temperature is 25 ℃ and the illumination is 4000Lux, after the optical density value (OD560) of the algae liquid concentration reaches 1.0, checking is carried out by using a 200-mesh silk cloth sieve, and the steps are repeated for 5 times. (3) Seed expansion: when the average daily temperature reached 20 ℃ or higher, the checked algal species were inoculated into a primary culture tank containing 5000L BG11 liquid medium, and were subjected to open culture for 21 days under conditions of an optical density (OD560) of 0.10, a temperature of 25 ℃ and light irradiation of 4000Lux after inoculation. Inoculating the strain into a second culture pond containing 50000L BG11 liquid culture medium when the optical density value (OD560) of the strain liquid concentration reaches 1.0 or more, gradually expanding to a third culture pond containing 200000L BG11 liquid culture medium, and performing open culture at 25 deg.C under illumination of 4000Lux for 21 days.
Further, the BG11 liquid culture medium comprises 1.5g NaNO3，0.04g K2HPO3.3H2O，0.075gMgSO4.7H2O，0.036g CaCl2.2H2O, 0.006g citric acid, 0.006g ferric ammonium citrate, 0.001g EDTA2，0.02g Na2CO31ml of trace elements is dissolved in water to prepare a 1L solution to obtain BG11 liquid culture medium, and the pH range of the BG11 liquid culture medium is 7.2.
A preparation method of BG11 solid culture medium for blue algae culture comprises mixing 1.5g NaNO3，0.04g K2HPO3.3H2O，0.075g MgSO4.7H2O，0.036g CaCl2.2H2O, 0.006g citric acid, 0.006g ferric ammonium citrate, 0.001g EDTA2，0.02g Na2CO31ml of trace elements are dissolved in water to prepare 1L of solution, agar powder accounting for 1.8 to 2 percent of the total volume of the solution is added into the solution, the pH range of the solution is adjusted to 7.0 to 7.2, the solution is subjected to moist heat sterilization at 121 ℃, 0.11MPa and 20min, and then is cooled and solidified at 40 ℃ to be in a solid state, namely the BG11 solid culture medium is obtained.
Example 3
A method for culturing blue algae with a desert soil moisturizing function adopts the technical scheme that the method comprises the following steps:
step.1 isolation of moisturizing algae: weighing a desert soil sample, adding BG11 liquid culture medium into the desert soil sample according to the condition that 400ml of BG11 liquid culture medium is added into each 150g of desert soil sample, fully and uniformly mixing the sample and the culture medium by using a shaking table for 8min under the condition that the rotation speed is 90rpm, statically culturing the mixture for 15 days under the conditions that the temperature is 28 ℃ and the illumination is 3200Lux, coating each 400 mu L of supernatant on 28ml of TAP solid culture medium after the supernatant of the culture turns green, sucking the supernatant under the aseptic condition, coating the supernatant on a culture dish containing BG11 solid culture medium, and culturing for 15 days under the conditions that the temperature is 28 ℃ and the illumination is 3200Lux to obtain a moisturizing algae culture;
step.2, purification: selecting single moisturizing algae filaments from a sterilized inoculating loop on 30ml BG11 solid culture medium, inoculating the single moisturizing algae filaments on a solid plate containing BG11 solid culture medium by a scribing method, culturing for 15 days at 28 ℃ under the condition of illumination of 3200Lux to obtain a primary moisturizing algae, selecting single moisturizing algae filaments from the primary moisturizing algae by using the sterilized inoculating loop, inoculating the single moisturizing algae filaments on the solid plate containing a new BG11 solid culture medium, culturing for 15 days at 28 ℃ under the condition of illumination of 3200Lux to obtain a secondary moisturizing algae, repeating the step until 6 generations are inoculated to finally obtain pure moisturizing algae;
step.3, large-scale culture: according to the method, each single algae filament is inoculated on 500ml BG11 liquid culture medium, the single algae filament is selected from pure moisturizing algae, inoculated in BG11 liquid culture medium, and subjected to open culture for 15 days under the conditions of 28 ℃ and 3200Lux illumination to prepare the moisturizing algae mother-seed suspension with the optical density (OD560) of the algae liquid concentration reaching 1.0. (1) Primary selection: according to the method, a moisturizing algae mother seed suspension is inoculated into a new BG11 liquid culture medium according to the condition that 2% of the moisturizing algae mother seed suspension is inoculated into every 300L of BG11 liquid culture medium, the optical density (OD560) of the inoculation concentration is preferably 0.10, the continuous stirring and the recarburization are carried out by an electromagnetic air pump or the direct recarburization is carried out by a carbon dioxide compressed gas cylinder, and after the large-scale open culture is carried out for 15 days under the conditions that the temperature is 30 ℃ and the illumination is 3000Lux, the selection of strong algae filaments with good suspension property is carried out by 300-mesh silk cloth when the optical density (OD560) of the algae liquid concentration reaches 1.0. (2) Checking: and when the primarily selected algae filaments are inoculated in a fresh BG11 liquid culture medium again according to the above steps, large-scale open culture is carried out for 15 days under the conditions that the temperature is 30 ℃ and the illumination is 3000Lux, after the optical density value (OD560) of the algae liquid concentration reaches 1.0, checking is carried out by using a 200-mesh silk cloth sieve, and the steps are repeated for 4 times. (3) Seed expansion: when the average daily temperature reached 20 ℃ or higher, the checked algal species were inoculated into a primary culture tank containing 3000L BG11 liquid medium, and were subjected to open culture for 15 days under conditions of an optical density (OD560) of 0.10, a temperature of 30 ℃ and an illumination of 3000Lux at the concentration after inoculation. When the optical density value (OD560) of the algal solution concentration reached 1.0 or more, the algal solution was inoculated into a secondary culture tank containing 30000L BG11 liquid medium, and gradually expanded into a tertiary culture tank containing 120000L BG11 liquid medium, and the open culture was carried out for 15 days at 30 ℃ under 3000Lux illumination.
Further, the BG11 liquid culture medium comprises 1.5g NaNO3，0.04g K2HPO3.3H2O，0.075gMgSO4.7H2O，0.036g CaCl2.2H2O, 0.006g citric acid, 0.006g ferric ammonium citrate, 0.001g EDTA2，0.02g Na2CO31ml of trace elements is dissolved in water to prepare a 1L solution to obtain BG11 liquid culture medium, and the pH range of the BG11 liquid culture medium is 7.1.
A preparation method of BG11 solid culture medium for blue algae culture comprises mixing 1.5g NaNO3，0.04g K2HPO3.3H2O，0.075g MgSO4.7H2O，0.036g CaCl2.2H2O, 0.006g citric acid, 0.006g ferric ammonium citrate, 0.001g EDTA2，0.02g Na2CO31ml of trace elements are dissolved in water to prepare 1L of solution, agar powder accounting for 1.9 percent of the total volume of the solution is added into the solution, the pH range of the solution is adjusted to 7.1, and the solution is subjected to moist heat sterilization at 121 ℃, 0.11MPa and 20min, then is cooled and solidified at 40 ℃ to be in a solid state, namely the BG11 solid culture medium is obtained.
The invention relates to a culture method of filamentous algae (blue algae), which is a large-scale culture method of moisturizing algae. The method comprises the following steps: firstly, separating a moisturizing algae culture; secondly, inoculating single moisturizing algae filaments on a solid plate for repeated culture until 5-7 generations of algae are inoculated to obtain algae, and thirdly, inoculating the algae into a liquid culture medium for large-scale open culture for 7-21 days. The invention firstly cultures the moisturizing algae, improves the cell concentration, obtains the moisturizing algae culture solution with high cell concentration, then separates, then purifies to obtain high-yield target algae species, and finally carries out large-scale industrial culture, thus ensuring successful separation of target algae strains. FIG. 1 shows blue algae cultured by the method.
The raw materials and the mixture ratio in the BG11 liquid culture medium are disclosed in the application number: 201810151943.2, to the extent that they are already known in the art, parts not described in detail herein are known in the art.
Although the present invention has been described in detail with reference to the specific embodiments, the present invention is not limited to the above embodiments, and various changes and modifications without inventive changes may be made within the knowledge of those skilled in the art without departing from the spirit of the present invention.
Claims (3)
1. A method for culturing blue algae with a desert soil moisturizing function is characterized by comprising the following steps: the method comprises the following steps:
step.1 isolation of moisturizing algae: weighing a desert soil sample, adding BG11 liquid culture medium into the desert soil sample according to the condition that 300ml to 500ml of BG11 liquid culture medium is added into each 100g to 200g of desert soil sample, using a shaking table for 5min to 10min under the condition that the rotation speed is 80rpm to 100rpm to fully mix the sample and the culture medium uniformly, statically culturing the mixture for 7 days to 21 days under the conditions that the temperature is 27 ℃ to 30 ℃ and the illumination is 3000Lux to 3500Lux, coating each 300 mu L to 500 mu L of supernatant on 25ml to 30ml of TAP solid culture medium after the supernatant of the culture turns green, sucking the supernatant under the aseptic condition, coating the supernatant on a culture dish containing BG11 solid culture medium, and culturing for 7 days to 21 days under the conditions that the temperature is 27 ℃ to 30 ℃ and the illumination is 3000Lux to 3500Lux to obtain a moisturizing algae culture;
step.1 purification: selecting single moisturizing algae filaments from each single moisturizing algae filament inoculated on 25-35 ml of BG11 solid culture medium, inoculating the selected single moisturizing algae filaments on a solid plate containing BG11 solid culture medium by a scribing method, culturing for 7-21 days at the temperature of 27-30 ℃ under the illumination of 3000-3500 Lux to obtain a primary moisturizing algae body, selecting single moisturizing algae filaments from the primary moisturizing algae body by the sterilized inoculating ring, inoculating the single moisturizing algae filaments on the solid plate containing a new BG11 solid culture medium, culturing for 7-21 days at the temperature of 27-30 ℃ under the illumination of 3000-3500 Lux to obtain a secondary moisturizing algae body, repeating the step until 5-7 generations are inoculated to obtain a pure moisturizing algae body;
step.3, large-scale culture: selecting single algae filaments from pure moisturizing algae according to the inoculation of each single algae filament on 250ml to 1000ml BG11 liquid culture medium, inoculating the single algae filaments into BG11 liquid culture medium, and carrying out open culture for 7 days to 21 days under the conditions that the temperature is 27 ℃ to 30 ℃ and the illumination is 3000Lux to 3500Lux to prepare the moisturizing algae mother seed suspension with the optical density (OD560) of the algae liquid concentration reaching 1.0. (1) Primary selection: according to the method, a moisturizing algae mother seed suspension with the volume of 1-3% is inoculated into 100-500L BG11 liquid culture medium, the moisturizing algae mother seed suspension is inoculated into a new BG11 liquid culture medium, the optical density (OD560) of the inoculation concentration is preferably 0.10, the continuous stirring and the recarburization are carried out by an electromagnetic air pump or the direct recarburization is carried out by a carbon dioxide compressed gas cylinder, the large-scale open culture is carried out for 7-21 days under the conditions that the temperature is 25-31 ℃ and the illumination is 2000-4000 Lux, and then when the optical density (OD560) of the algae liquid concentration reaches 1.0, the coarse and strong algae filaments with good suspension are selected by 300-mesh silk cloth for checking. (2) Checking: and (3) inoculating the primarily selected algae filaments into a fresh BG11 liquid culture medium again according to the steps, performing large-scale open culture for 7 to 21 days under the conditions that the temperature is between 25 and 31 ℃ and the illumination is between 2000 and 4000Lux, checking the algae filaments by using a 200-mesh silk cloth sieve after the optical density value (OD560) of the algae liquid concentration reaches 1.0, and repeating the steps for 3 to 5 times. (3) Seed expansion: when the average daily temperature is more than 20 ℃, the checked algae seeds are inoculated into a primary culture pond containing 1000L to 5000L BG11 liquid culture medium, and the open culture is carried out for 7 days to 21 days under the conditions that the optical density (OD560) of the inoculated concentration is 0.10, the temperature is 25 ℃ to 31 ℃, and the illumination is 2000Lux to 4000 Lux. Inoculating the algae liquid to a second-stage culture pond containing 5000L-50000L BG11 liquid culture medium when the optical density value (OD560) of the algae liquid concentration reaches more than 1.0, gradually expanding the algae liquid to a third-stage culture pond containing 50000L-200000L BG11 liquid culture medium, and carrying out open culture for 7-21 days under the conditions that the temperature is 25-31 ℃ and the illumination is 2000-4000 Lux.
2. The method for culturing the blue algae with the desert soil moisturizing function as claimed in claim 1, wherein the method comprises the following steps: the BG11 liquid culture medium comprises 1.5g NaNO3，0.04g K2HPO3.3H2O，0.075g MgSO4.7H2O，0.036gCaCl2.2H2O, 0.006g citric acid, 0.006g ferric ammonium citrate, 0.001g EDTA2，0.02g Na2CO31ml of trace elements is dissolved in water to prepare a 1L solution to obtain BG11 liquid culture medium, and the pH value of the BG11 liquid culture medium ranges from 7.0 to 7.2.
3. A preparation method of BG11 solid culture medium for blue algae culture is characterized in that: 1.5g NaNO3，0.04gK2HPO3.3H2O，0.075g MgSO4.7H2O，0.036g CaCl2.2H2O, 0.006g citric acid, 0.006g ferric ammonium citrate, 0.001g EDTA2，0.02g Na2CO31ml of trace elements are dissolved in water to prepare 1L of solution, agar powder accounting for 1.8 to 2 percent of the total volume of the solution is added into the solution, the pH range of the solution is adjusted to 7.0 to 7.2, the solution is subjected to moist heat sterilization at 121 ℃, 0.11MPa and 20min, and then is cooled and solidified at 40 ℃ to be in a solid state, namely the BG11 solid culture medium is obtained.
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| CN1734021A (en) * | 2005-08-18 | 2006-02-15 | 中国科学院新疆生态与地理研究所 | The new method that terrestrial nitrogen-fixing blue alga fixes the sand |
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