CN107937382B - Preparation method of immobilized microalgae - Google Patents

Preparation method of immobilized microalgae Download PDF

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CN107937382B
CN107937382B CN201711428923.7A CN201711428923A CN107937382B CN 107937382 B CN107937382 B CN 107937382B CN 201711428923 A CN201711428923 A CN 201711428923A CN 107937382 B CN107937382 B CN 107937382B
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microalgae
solution
hours
culture
weight
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CN107937382A (en
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周燕
王建泉
周洁
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Anhui Renwei Environmental Protection Engineering Technology Co ltd
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Anhui Renwei Environmental Protection Engineering Technology Co ltd
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/10Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/04Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres

Abstract

The invention discloses a preparation method of immobilized microalgae, which comprises the following steps: under the aseptic operation condition, inoculating chlorella into a culture medium, and circularly and alternately culturing for 60-75 hours under the light and dark conditions to obtain a microalgae culture solution; adjusting pH of microalgae culture solution to 4.5-6, adding sodium citrate, stirring for 20-30 min, adding calcium chloride, stirring for 40-50 min, and centrifuging to obtain microalgae solution; preparing a carrier solution containing sodium alginate and magnesium chloride, adding the microalgae solution into the carrier solution, uniformly mixing, dripping the microalgae solution into a calcium chloride solution with the mass fraction of 2.6-3.2% by using an injector, fixing the microalgae solution at the temperature of 8-10 ℃ for 2-2.5 hours, taking out the microalgae solution, and desalting the microalgae solution for 1-2 hours by using deionized water. The immobilized microalgae prepared by the method has the advantages of stable treatment effect, capability of effectively preventing secondary pollution and the like.

Description

Preparation method of immobilized microalgae
Technical Field
The invention relates to the field of preparation of microalgae sewage treatment agents, in particular to a preparation method of immobilized microalgae.
Background
With the large-scale development of livestock and poultry breeding industry in China, nearly billions of tons of livestock and poultry breeding manure can be generated every year, and serious threat is caused to limited water resources.
At present, the treatment mode of breeding sewage mainly has two kinds: firstly, the biogas digester is adopted for anaerobic fermentation treatment, but the generated biogas slurry with huge amount still contains high-concentration nutrient salts such as nitrogen and phosphorus, and with the promotion of rural urbanization process, cultivated land for absorbing biogas slurry is gradually insufficient, and the generated biogas slurry is directly discharged into water body, so that the natural water body is seriously eutrophicated; and secondly, biological removal is carried out, wherein biological treatment is to degrade and/or assimilate pollutants in sewage by using microorganisms such as bacteria and fungi to purify the sewage, but the carbon source generally available for the culture sewage after anaerobic treatment is too low to meet the requirement of heterotrophic functional bacteria, so that the treatment effect on nitrate and phosphate is poor, and various modes such as physical filtration and chemical adsorption are required to be combined for use, so that the treatment cost is increased, and the potential risk of secondary pollution is increased.
The microalgae treatment method is a novel sewage treatment mode at present, rich nutrient substances such as nitrogen, phosphorus and the like contained in the livestock and poultry breeding sewage can provide nutrition for the microalgae, and the microalgae treatment sewage can be coupled with the large-scale production of the microalgae at the same time, so that the cyclic utilization of resources is realized.
However, since the microalgae cells are generally smaller than 30 μm, have negative charges and density close to that of water, the microalgae cells are always in a stable suspension state in the water due to the characteristics, and the microalgae cells are greatly lost along with the treated water, which not only can secondarily pollute the treated water, but also can cause difficulty in maintaining the biomass of the microalgae cells in the treated sewage in a large amount (generally, the biomass of the microalgae cells is only 0.2-0.6 g/L). The low culture density results in low removal efficiency, making the treatment effect less stable.
Disclosure of Invention
Based on the technical problems in the background art, the invention provides a preparation method of immobilized microalgae, and the immobilized microalgae prepared by the method has the advantages of stable treatment effect, capability of effectively preventing secondary pollution and the like.
The invention provides a preparation method of immobilized microalgae, which comprises the following steps:
s1, under the aseptic operation condition, the chlorella is processed at 1 × 1061X 10 per mL7Inoculating the inoculum size of each/mL into a culture medium, and circularly and alternately culturing for 60-75 hours in the dark under the conditions that the temperature is 25-27 ℃ and the rotating speed is 110-130 r/min to obtain a microalgae culture solution;
s2, adjusting the pH value of the microalgae culture solution obtained in S1 to 4.5-6, adding sodium citrate, stirring for 20-30 minutes, adding calcium chloride, stirring for 40-50 minutes, and performing centrifugal separation at the revolution of 2000-2200 rpm to obtain a microalgae solution;
s3, preparing a carrier solution containing sodium alginate and magnesium chloride, adding the microalgae solution obtained in S2 into the carrier solution, uniformly mixing, dripping the microalgae solution into a calcium chloride solution with the mass fraction of 2.6-3.2% by using an injector, fixing for 2-2.5 hours at 8-10 ℃, taking out, and desalting for 1-2 hours by using deionized water.
Preferably, the raw materials of the culture medium in S1 comprise, by weight: 1-2 parts of glucose, 4-6 parts of starch sugar, 0.01-0.03 part of zinc sulfate, 0.1-0.3 part of magnesium sulfate, 0.15-0.3 part of ammonium sulfate, 0.2-0.4 part of monopotassium phosphate, 0.1-0.3 part of sodium nitrate, 9-12 parts of sodium chloride and 240-260 parts of deionized water.
Preferably, in the S1 light and dark cycle alternating culture, the culture time in the light environment is 8-10 hours, the light intensity is 3500-3800Lux, the culture time in the dark environment is 11-13 hours, and the light environment and the dark environment are cyclically and alternately carried out.
Preferably, the pH of the microalgae culture solution is adjusted to 5.1-5.4 in S2.
Preferably, the amount of the sodium citrate added into the S2 is 1.0-2.5% of the weight of the microalgae culture solution.
Preferably, the amount of the sodium citrate added into the S2 is 1.5 to 1.7 percent of the weight of the microalgae culture solution.
Preferably, the amount of calcium chloride added in the S2 is 3-5% of the weight of the microalgae culture solution.
Preferably, the amount of the calcium chloride added into the S2 is 3.8-4.3% of the weight of the microalgae culture solution.
Preferably, the carrier solution in S3 contains sodium alginate 2-3 wt% and magnesium chloride 0.6-0.8 wt%.
Preferably, the amount of the carrier solution used in S3 is 2.3 to 2.7 times the weight of the microalgal solution.
The preparation method of the immobilized microalgae mainly comprises three parts of culture, harvesting and microalgae immobilization of the chlorella. Culturing chlorella by adopting a light and dark cycle alternate culture method, preferably selecting light environment and dark environment culture time, and finishing the culture after the light and dark cycle alternate culture is carried out for 60-75 hours. The technicians of the invention find that the growth speed of the chlorella obtained by finishing the culture in the period of 60-75 hours is better than that of the chlorella obtained by finishing the culture in other periods after the chlorella enters the livestock and poultry breeding sewage. The chlorella is collected by adjusting the pH value of the microalgae culture solution and adding sodium citrate and calcium chloride to flocculate the chlorella so as to be separated from the liquid. The immobilization of the chlorella adopts a method for preparing microalgae gel spheres with the diameter of 4-6 mm by adopting a sodium alginate carrier, and magnesium chloride is added into a sodium alginate carrier solution, so that the microalgae gel spheres have the effects of preventing the microalgae gel spheres from being broken and deformed.
The immobilized microalgae prepared by the method of the invention absorbs organic matters, nitrogen, phosphorus and other nutrient substances in the culture sewage in the growth process of the microalgae, synthesizes a self cytoskeleton to purify the livestock and poultry culture sewage, and can remove heavy metals and refractory organic matters. After the microalgae is immobilized by the method, the absorption and enrichment of substances such as nitrogen, phosphorus, heavy metals and the like are improved, and the method has the advantages of high reaction speed, strong poison resistance, stable and reliable operation, less loss of microalgae cells, easy separation of products and the like, and can effectively prevent secondary pollution of treated water.
Detailed Description
The technical solution of the present invention will be described in detail below with reference to specific examples.
Example 1
The invention provides a preparation method of immobilized microalgae, which comprises the following steps:
s1, under the aseptic operation condition, the common chlorella is processed by mixing at 5.4 x 106Inoculating the inoculum size of each/mL into a culture medium, carrying out illumination and dark cycle alternate culture under the conditions that the temperature is 26 ℃ and the rotating speed is 122 r/min, carrying out culture for 9 hours under the illumination environment, wherein the illumination intensity is 3700Lux, the culture time under the dark environment is 11 hours, and carrying out illumination and dark cycle alternate culture for 68 hours to obtain a microalgae culture solution;
s2, adjusting the pH value of the microalgae culture solution obtained in S1 to 5.2, adding 1.58% of sodium citrate based on the weight of the microalgae culture solution, stirring for 25 minutes, adding 4.1% of calcium chloride based on the weight of the microalgae culture solution, stirring for 44 minutes, and performing centrifugal separation at 2100 revolutions per minute to obtain a microalgae culture solution;
s3, preparing a carrier solution containing 2.6 mass percent of sodium alginate and 0.68 mass percent of magnesium chloride, adding the microalgae liquid obtained in the step S2 into the carrier solution, uniformly mixing, dripping the microalgae liquid into 2.9 mass percent of calcium chloride solution by using an injector, fixing for 2.3 hours at 9 ℃, taking out, and desalting for 1.2 hours by using deionized water to obtain the immobilized microalgae gel spheres. Wherein the amount of the carrier solution is 2.6 times of the weight of the microalgae solution.
The raw materials of the culture medium in the S1 comprise the following components in parts by weight: 1.6 parts of glucose, 5.2 parts of starch sugar, 0.02 part of zinc sulfate, 0.19 part of magnesium sulfate, 0.23 part of ammonium sulfate, 0.28 part of monopotassium phosphate, 0.21 part of sodium nitrate, 11 parts of sodium chloride and 250 parts of deionized water.
Example 2
The invention provides a preparation method of immobilized microalgae, which comprises the following steps:
s1, under the aseptic operation condition, mixing the chlorella vulgaris at the ratio of 1.6 multiplied by 106Inoculating the strain/mL into a culture medium, and carrying out illumination and dark cycle alternate culture under the conditions that the temperature is 25 ℃ and the rotating speed is 128 r/min, wherein the culture is carried out for 10 hours under the illumination environment, the illumination intensity is 3600Lux, the culture time is 12 hours under the dark environment, and the microalgae culture solution is obtained after the illumination environment and the dark environment are cyclically and alternately cultured for 73 hours;
s2, adjusting the pH value of the microalgae culture solution obtained in S1 to 5.3, adding 1.67% of sodium citrate based on the weight of the microalgae culture solution, stirring for 22 minutes, adding 3.9% of calcium chloride based on the weight of the microalgae culture solution, stirring for 48 minutes, and centrifuging at a speed of 2200 revolutions per minute to obtain a microalgae culture solution;
s3, preparing a carrier solution containing 2.8 mass percent of sodium alginate and 0.62 mass percent of magnesium chloride, adding the microalgae liquid obtained in the step S2 into the carrier solution, uniformly mixing, dripping the microalgae liquid into 2.7 mass percent of calcium chloride solution by using an injector, fixing the microalgae liquid at 10 ℃ for 2.4 hours, taking out the microalgae liquid, and desalting the microalgae liquid by using deionized water for 1.4 hours to obtain the immobilized microalgae gel spheres. Wherein the amount of the carrier solution is 2.4 times of the weight of the microalgae solution.
Wherein the raw materials of the culture medium in the S1 comprise the following components in parts by weight: 1.9 parts of glucose, 4.3 parts of starch sugar, 0.01 part of zinc sulfate, 0.12 part of magnesium sulfate, 0.17 part of ammonium sulfate, 0.38 part of monopotassium phosphate, 0.27 part of sodium nitrate, 10 parts of sodium chloride and 256 parts of deionized water.
Example 3
The invention provides a preparation method of immobilized microalgae, which comprises the following steps:
s1, under the aseptic operation condition, the common chlorella is mixed with 8.9 multiplied by 106Inoculating to culture medium at 27 deg.C and 113 rpm, culturing under light and dark conditions for 8 hr with light intensity of 3800Lux and dark conditionsThe lower culture time is 13 hours, and microalgae culture solution is obtained after the microalgae culture solution is circularly and alternately cultured for 62 hours in a light environment and a dark environment;
s2, adjusting the pH value of the microalgae culture solution obtained in S1 to 5.1, adding 1.51% of sodium citrate based on the weight of the microalgae culture solution, stirring for 28 minutes, adding 4.2% of calcium chloride based on the weight of the microalgae culture solution, stirring for 41 minutes, and performing centrifugal separation at 2000 revolutions per minute to obtain a microalgae culture solution;
s3, preparing a carrier solution containing 2.3 mass percent of sodium alginate and 0.77 mass percent of magnesium chloride, adding the microalgae solution obtained in the step S2 into the carrier solution, uniformly mixing, dripping the microalgae solution into 3.1 mass percent of calcium chloride solution by using an injector, fixing for 2.1 hours at 8 ℃, taking out, and desalting for 1.8 hours by using deionized water to obtain the immobilized microalgae gel spheres. Wherein the amount of the carrier solution is 2.5 times of the weight of the microalgae solution.
Wherein the raw materials of the culture medium in the S1 comprise the following components in parts by weight: 1.2 parts of glucose, 5.8 parts of starch sugar, 0.03 part of zinc sulfate, 0.27 part of magnesium sulfate, 0.28 part of ammonium sulfate, 0.23 part of monopotassium phosphate, 0.14 part of sodium nitrate, 9 parts of sodium chloride and 241 parts of deionized water.
Example 4
The method for preparing immobilized microalgae according to example 1, wherein the pH of the microalgae culture solution is further adjusted to 4.8 in S2.
Example 5
The method for preparing immobilized microalgae according to example 1, wherein the pH of the microalgae culture solution is further adjusted to 5.9 in S2.
Example 6
The method for preparing immobilized microalgae according to example 1, wherein the pH of the microalgae culture solution is further adjusted to 5.6 in S2.
Example 7
The method for preparing immobilized microalgae in example 1, wherein the amount of sodium citrate added in S2 is 1.2% of the weight of the microalgae culture solution.
Example 8
The method for preparing immobilized microalgae in example 1, wherein the amount of sodium citrate added in S2 is also 2.1% of the weight of the microalgae culture solution.
Example 9
The method for preparing immobilized microalgae in example 1, wherein the amount of calcium chloride added in S2 is 3.3% of the weight of the microalgae culture solution.
Example 10
The method for preparing immobilized microalgae in example 1, wherein the amount of calcium chloride added in S2 is 4.8% of the weight of the microalgae culture solution.
Example 11
The method for preparing immobilized microalgae in example 1, wherein the amount of sodium citrate added in S2 may also be 2.4% of the weight of the microalgae culture solution, and the amount of calcium chloride added may also be 3.6% of the weight of the microalgae culture solution.
Example 12
The method for preparing immobilized microalgae in example 1, wherein the amount of sodium citrate added in S2 may also be 1.3% of the weight of the microalgae culture solution, and the amount of calcium chloride added may also be 4.6% of the weight of the microalgae culture solution.
The chlorella referred to in S1 of the present invention is preferably chlorella vulgaris (A)Chlorella vulgaris) The chlorella inoculated into the culture medium is preferably activated chlorella, and the activation method can be selected from the activation methods known to those skilled in the art, preferably slant activation method. The pH of the microalgae culture solution in the S2 can be adjusted by adding hydrochloric acid, and preferably, the hydrochloric acid with the mass fraction of 30-40% is added.
The methods of inoculation, agitation, centrifugation, and preparation of the culture medium involved in the present invention are all common knowledge in the art, and are not described herein.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Claims (10)

1. The preparation method of the immobilized microalgae is characterized by comprising the following steps:
s1, under the aseptic operation condition, the chlorella is processed at 1 × 1061X 10 per mL7Inoculating the inoculum size of each/mL into a culture medium, and circularly and alternately culturing for 60-75 hours in the dark under the conditions that the temperature is 25-27 ℃ and the rotating speed is 110-130 r/min to obtain a microalgae culture solution;
s2, adjusting the pH value of the microalgae culture solution obtained in S1 to 4.5-6, adding sodium citrate, stirring for 20-30 minutes, adding calcium chloride, stirring for 40-50 minutes, and performing centrifugal separation at the revolution of 2000-2200 rpm to obtain a microalgae solution;
s3, preparing a carrier solution containing sodium alginate and magnesium chloride, adding the microalgae solution obtained in S2 into the carrier solution, uniformly mixing, dripping the microalgae solution into a calcium chloride solution with the mass fraction of 2.6-3.2% by using an injector, fixing for 2-2.5 hours at 8-10 ℃, taking out, and desalting for 1-2 hours by using deionized water.
2. The method for preparing immobilized microalgae according to claim 1, wherein the raw materials of the culture medium in S1 comprise, by weight: 1-2 parts of glucose, 4-6 parts of starch sugar, 0.01-0.03 part of zinc sulfate, 0.1-0.3 part of magnesium sulfate, 0.15-0.3 part of ammonium sulfate, 0.2-0.4 part of monopotassium phosphate, 0.1-0.3 part of sodium nitrate, 9-12 parts of sodium chloride and 240-260 parts of deionized water.
3. The method for preparing immobilized microalgae according to claim 1, wherein in the S1 cultivation under light and dark cycles, the cultivation time under light is 8-10 hours, the light intensity is 3500-3800Lux, the cultivation time under dark is 11-13 hours, and the light and dark cycles are alternately performed.
4. The method of claim 1, wherein the pH of the microalgae culture is adjusted to 5.1-5.4 in S2.
5. The method for preparing immobilized microalgae according to claim 1, wherein the amount of sodium citrate added in S2 is 1.0% -2.5% of the weight of microalgae culture solution.
6. The method for preparing immobilized microalgae according to claim 1, wherein the amount of sodium citrate added in S2 is 1.5-1.7% of the weight of microalgae culture solution.
7. The method for preparing immobilized microalgae according to claim 1, wherein the amount of calcium chloride added in S2 is 3-5% of the weight of microalgae culture solution.
8. The method for preparing immobilized microalgae according to claim 1, wherein the amount of calcium chloride added to S2 is 3.8% -4.3% of the weight of microalgae culture solution.
9. The method for preparing immobilized microalgae according to claim 1, wherein the carrier solution in S3 contains sodium alginate in an amount of 2-3% by mass and magnesium chloride in an amount of 0.6-0.8% by mass.
10. The method of claim 1, wherein the amount of the carrier solution used in S3 is 2.3-2.7 times the weight of the microalgae solution.
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