CN111808754A - Mining area soil microalgae and separation and purification method and application thereof - Google Patents
Mining area soil microalgae and separation and purification method and application thereof Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; 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
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/89—Algae ; Processes using algae
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/32—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae
- C02F3/322—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae use of algae
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- C12N1/12—Unicellular algae; Culture media therefor
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- B—PERFORMING OPERATIONS; TRANSPORTING
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Abstract
The invention provides a mining area soil microalgae and a separation and purification method and application thereof, the mining area soil microalgae is soil microalgae separated and purified from biological crust on the surface of tailings, and the mining area soil microalgae comprises one or more of chlorella, cylindracea, diatom, rhombohedral algae, schizochytrium, dunaliella, scenedesmus, nannochlorococcus, chlamydomonas, platonas and euglena. The mining area soil microalgae is obtained by separating and purifying from biological crust on the surface of tailings, the obtained microalgae is more in variety, and the obtained microalgae has better adsorption and absorption capacity on pollutants such as heavy metal, phosphorus and the like in polluted soil and water, so that the pollutants such as heavy metal, phosphorus and the like in the polluted soil and water can be effectively treated, and the treatment cost of the pollutants such as heavy metal, phosphorus and the like in the polluted soil and water is greatly reduced.
Description
Technical Field
The invention relates to the technical field of microalgae biology, in particular to mining area soil microalgae and a separation and purification method and application thereof.
Background
Soil microalgae, an important component of soil microorganisms, refers to a collection of algae from various groups such as soil surface layers, soil subsurface layers, and stone-growing and water-land-growing algae related to soil formation, succession, and soil composition, and is most widely distributed in woodland, cultivated land, paddy field, and soil with high organic matter content.
Soil microalgae are abundant in species, and in the field of environment, microalgae is used as one of organisms with adsorption capacity, has the advantages of high photosynthetic rate, high propagation speed, strong environmental adaptability and the like, and a great deal of research reports now show that microalgae is used as a biological adsorbent in the aspects of sewage treatment, soil pollution and the like. Some oil produced by microalgae such as chlorella, etc. can be converted into biodiesel (fatty acid methyl ester, etc.) after esterification, and the algae residue can be comprehensively utilized to produce animal feed, organic fertilizer, methane, etc. With the rapid development of the microalgae biotechnology industry, the microalgae biotechnology industry is more and more highly concerned by governments, scientific research institutions and enterprises.
The soil with the same texture has different microalgae group distribution, and the soil microalgae can be divided into three categories of soil-borne algae, stone-borne algae and cave-inhabited algae according to the specific soil living environment of each algae. Among them, the research on the soil-borne algae is the most intensive, and the species diversity of the microalgae in various soil environments such as cultivated land, forest land, grassland, desert and the like has been widely researched. The soil with different properties provides different physicochemical conditions for the growth of algae, so the algae species adapting to the survival are different.
The tailings refer to waste residue slag discharged after mineral separation by mine enterprises, generally form a tailing pond due to long-time accumulation, generally contain high-content heavy metals, the types of the main heavy metals contained in the tailings are different according to different types of mineral deposits, and the common heavy metals contained in the tailings comprise copper, iron, zinc, cadmium, chromium, lead and the like. The biological crust on the surface of the tailings refers to a complex formed by living organisms such as bacteria, fungi, algae, bryophytes and the like and surface tailings on the surface of the tailings. Algae and mosses, which are the earliest pioneer plants to colonize, account for the major part of biological crust, and many studies have shown that, on one hand, algae in crust have high tolerance to heavy metals and still have strong vitality under high heavy metal concentration, and on the other hand, algae in crust have better ability to adsorb heavy metals. However, at present, few researches on the species of microalgae in the biological crust on the surface of the tailings are carried out, and the application direction of the microalgae is also little involved.
Therefore, it is of great significance to obtain microalgae from biological crust on the surface of tailings in a tailings field and research the species and application direction of the microalgae.
Disclosure of Invention
In view of the above, the present invention is directed to provide a mining area soil microalgae, which is obtained by purifying the mining area soil crust with high heavy metal content, has good tolerance to heavy metals and adaptability to extreme environments, and better solves the problems that the existing pollutants such as heavy metals and phosphorus in the polluted soil and water are difficult to treat and the treatment cost is high.
In order to achieve the purpose, the technical scheme of the invention is realized as follows:
mining area soil microalgae, which is soil microalgae separated and purified from biological crust on the surface of tailings, and comprises one or more of chlorella (chlorella sp), cylindracea (cylindracea sp.), diatom (diatom), rhombohedral (Nitzschia sp), schizochytrium (schizochytrium sp.), dunaliella (dunaliella), Scenedesmus (Scenedesmus sp.), nannochlorococcus (nannochlororis sp.), chlamydomonas (chlamydomonas sp.), tetraselmis (Tetraselmissp.), eudoria (Eudorina sp.).
The second purpose of the invention is to provide a separation and purification method of the mining area soil microalgae, which comprises the following steps:
1) shoveling biological crust on the surface of the tailings, kneading, and sieving to obtain a mixture of algae and moss in the crust;
2) placing the algae and moss mixture in a sterile conventional algae liquid culture medium, culturing for 12-48 h in a constant-temperature illumination incubator at the temperature of 22-29 ℃ and the illumination intensity of 8000-12000 lx, and separating to obtain an algae liquid;
3) putting the algae liquid into a first solid culture medium, sealing, and then placing the algae liquid into a constant-temperature illumination incubator with the temperature of 22-29 ℃, the illumination intensity of 8000-12000 lx and the light-dark ratio of 14: 10h for preservation and culture for 3-10 days;
4) after the algae grow out from the first solid culture medium, inoculating the algae into a second solid culture medium in time, sealing, and then placing in a constant-temperature illumination incubator with the temperature of 22-29 ℃, the illumination intensity of 8000-12000 lx and the light-dark ratio of 14: 10h for preservation and culture for 3-10 days;
5) and (3) repeating the algae inoculation process in the step 4) until no foreign bacteria and other organisms appear in the second solid culture medium and only single algae of the mining area soil microalgae exist, transferring the mining area soil microalgae in the second solid culture medium into a liquid culture medium to obtain a culture solution containing the mining area soil microalgae.
The third purpose of the invention is to provide an application of the mining area soil microalgae in the treatment of polluted soil or polluted water, which comprises the following steps:
inoculating the soil microalgae in the mining area into a microalgae culture medium, enriching, amplifying and culturing to a stable growth period, adding into the polluted soil or the polluted water body, and carrying out pollutant treatment on the polluted soil or the polluted water body.
Optionally, the microalgae culture medium is a BG-11 culture medium, and the microalgae culture medium is sterilized at 121 ℃ for 20min, cooled to room temperature, and then inoculated with the soil microalgae in the mining area.
Optionally, the culture temperature of the enrichment amplification culture is 22-29 ℃, the culture time is 20-30 d, and the culture illumination intensity is 8000-12000 lx.
Optionally, purified air is introduced into the enrichment amplification culture process, and the pressure of the purified air is 0.1-0.6 Mpa.
Optionally, the adding amount of the mining area soil microalgae in the contaminated soil is 0.01-0.2 mg/g in dry weight; the adding amount of the mining area soil microalgae in the polluted water body is 0.01-0.2 g/L.
Optionally, the treatment time for treating the pollutants in the polluted soil or the polluted water body is 20-30 d.
Compared with the prior art, the mining area soil microalgae disclosed by the invention has the following advantages:
1. the mining area soil microalgae is obtained by separating and purifying from biological crust on the surface of tailings, the obtained microalgae is more in variety, and the obtained microalgae has better adsorption and absorption capacity on pollutants such as heavy metal, phosphorus and the like in polluted soil and water, so that the pollutants such as heavy metal, phosphorus and the like in the polluted soil and water can be effectively treated, and the treatment cost of the pollutants such as heavy metal, phosphorus and the like in the polluted soil and water is greatly reduced.
2. The invention firstly obtains the microalgae with the capability of treating the polluted soil and the water body of the mining area by separating and purifying the soil microalgae in the mining area, extracts, purifies and cultures the soil microalgae obtained by separation, and expands and cultures the water body rich in heavy metal ions in tailings after determining the species of the microalgae.
3. The microalgae can be recycled, and has high economic value, for example, the microalgae can utilize nutrient substances in the wastewater for the growth and development of cells per se, and can produce biodiesel or be used as animal feed after being recycled; the algae cells have broad-spectrum adsorption effect, and after dehydration, drying and incineration treatment, ash content has potential rare earth recovery value.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate an embodiment of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 is a photograph under a microscope of microalgae according to example 1 of the present invention;
FIG. 2 is a microalgae concentration-time curve for treating phosphorus-containing wastewater using the microalgae of example 1 of the present invention;
FIG. 3 is a graph of phosphorus concentration versus time for treating phosphorus-containing wastewater using microalgae according to example 1 of the present invention.
Detailed Description
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
The present invention will be described in detail below with reference to the drawings and examples.
Example 1
The mining area soil microalgae is soil microalgae separated and purified from biological crust on the surface of tailings, and is separated and purified by the following method:
1) shoveling biological crust on the surface of the tailings, slightly kneading and crushing the tailings, and then sieving the crushed tailings with a 100-mesh sieve to obtain a mixture of algae and moss in the crust, wherein the biological crust on the surface of the tailings is shoveled from the tailings in the region of a minizone in Ezhou city, Hubei province;
2) placing the mixture of the algae and the moss in the step 1) in a sterile conventional algae liquid culture medium, culturing for 48 hours in a constant-temperature illumination incubator with illumination intensity of 10000lx at the temperature of 25 ℃, and separating to obtain algae liquid;
3) putting 1-5 drops of the alga liquid obtained in the step 2) into a first solid culture medium, uniformly covering the surface of the first solid culture medium with the sample liquid, sealing a sealing film, and then preserving and culturing in a constant-temperature illumination incubator with the illumination intensity of 10000lx and the light-dark ratio of 14: 10h at 25 ℃ for 3-10 d;
4) after the algae grow out from the first solid culture medium in the step 3), timely inoculating the grown algae into a second solid culture medium by using an inoculating loop, sealing a sealing film, and then storing and culturing for 3-10 days in a constant-temperature illumination culture box with the illumination intensity of 10000lx and the light-dark ratio of 14: 10h at 25 ℃;
5) and (3) repeating the algae inoculation process in the step 4) until no foreign bacteria and other organisms appear in the second solid culture medium, and transferring the mining area soil microalgae in the second solid culture medium into a liquid culture medium after observing the mining area soil microalgae only containing single algae under a microscope to obtain a culture solution of the usable mining area soil microalgae (single strain algae).
In order to ensure the rigorous effectiveness of the experiment, all the experimental instruments are sterilized in a high-pressure steam sterilization pot for 20min, and the experimental operation process is carried out in an ultra-clean workbench sterilized by ultraviolet light.
The mining area soil microalgae prepared in the embodiment is subjected to morphology test by using a microscope, and the test result is shown in fig. 1.
As can be seen from fig. 1, the microalgae extracted from the tailing soil is spherical microalgae, which belongs to one of chlorella, and it can be inferred from the microalgae cells of a single kind and form, at which time the separation and purification of microalgae are completed. In addition, the microalgae identified by 18sDNA had 99% homology to Didymogens palatina and was named Didymogens palatina XR, Didymogens.
The mining area soil microalgae prepared by the embodiment is used for treating phosphorus-containing wastewater, wherein the phosphorus-containing wastewater is simulated by adopting BG-11 culture media with phosphate (potassium dihydrogen phosphate) concentrations of 45mg/L and 160mg/L respectively, and the specific treatment method comprises the following steps:
placing prepared BG-11 culture medium containing phosphate with different concentrations into a high pressure steam sterilization pot, sterilizing at 121 deg.C for 20min, cooling to room temperature, taking out, and placing in a super clean bench;
according to the concentration of 0.01-0.2 g/L, namely the adding amount of Didymogens.XR microalgae in the BG-11 culture medium containing phosphate with different concentrations is 0.01-0.2 g/L, the Didymogens.XR microalgae is inoculated into the BG-11 culture medium containing phosphate with different concentrations for standby, and is cultured for 20-30 days under the environment of light-dark ratio of 10000lx illumination intensity and 14: 10h at 25 ℃, under the culture condition, the Didymogens.XR microalgae can be rapidly enriched and expanded to a stable growth period, and further the rapid absorption of phosphorus in the phosphorus-containing wastewater is realized.
In this embodiment, each component in the BG-11 medium solution can provide nutrient elements for microalgae, promote healthy growth and rapid propagation of microalgae, and rapidly enrich and expand to a stable growth period, so as to improve the life activity of microalgae, and further improve the efficacy of the nutrient restoration solution, thereby improving the treatment effect on pollutants such as phosphorus. Moreover, the purified air with the pressure of 0.1-0.6 Mpa can be continuously introduced into the algae liquid to promote the growth and the propagation of microalgae cells, and the purified air continuously introduced can stir the algae liquid, so that the microalgae is uniformly distributed in the algae liquid, the uniformity and the consistency of the growth and the propagation of the microalgae are greatly improved, and the treatment effect of the microalgae on pollutants such as heavy metals, phosphorus and the like is improved.
Xr algae concentration and phosphorus concentration were measured every two days in BG-11 medium and the results are shown in fig. 2 and 3.
As can be seen from fig. 2 and fig. 3, the high phosphorus promotes the growth of the didymogen.xr microalgae, and meanwhile, the microalgae almost completely absorbs and utilizes the phosphorus, the microalgae has excellent treatment effect on the phosphorus-containing wastewater, and when the phosphorus concentration is low (45mg/L), the absorption and utilization of the phosphorus reaches 100%, and the phosphorus concentration of the water body reaches the primary standard of pollutant discharge standard of urban sewage treatment plant (GB 18918-; the absorption utilization rate of the high-concentration phosphorus (160mg/L) microalgae on phosphorus in the water body reaches 98%, the phosphorus concentration of the treated water body reaches the secondary standard of pollutant discharge Standard of urban Sewage treatment plant (GB 18918-.
The present invention is not limited to the above preferred embodiments, and any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. The mining area soil microalgae is characterized in that the mining area soil microalgae is soil microalgae separated and purified from biological crust on the surface of tailings, and the mining area soil microalgae comprises one or more of chlorella, cylindracea, diatom, rhombohedral algae, schizochytrium, dunaliella, scenedesmus, nannochlorococcus, chlamydomonas, platymyces and euglena.
2. The method for separating and purifying the soil microalgae in the mining area of claim 1, which is characterized by comprising the following steps:
1) shoveling biological crust on the surface of the tailings, kneading, and sieving to obtain a mixture of algae and moss in the crust;
2) placing the algae and moss mixture in a sterile conventional algae liquid culture medium, culturing for 12-48 h in a constant-temperature illumination incubator at the temperature of 22-29 ℃ and the illumination intensity of 8000-12000 lx, and separating to obtain an algae liquid;
3) putting the algae liquid into a first solid culture medium, sealing, and then placing the algae liquid into a constant-temperature illumination incubator with the temperature of 22-29 ℃, the illumination intensity of 8000-12000 lx and the light-dark ratio of 14: 10h for preservation and culture for 3-10 days;
4) after the algae grow out from the first solid culture medium, inoculating the algae into a second solid culture medium in time, sealing, and then placing in a constant-temperature illumination incubator with the temperature of 22-29 ℃, the illumination intensity of 8000-12000 lx and the light-dark ratio of 14: 10h for preservation and culture for 3-10 days;
5) and (3) repeating the algae inoculation process in the step 4) until no foreign bacteria and other organisms appear in the second solid culture medium and only single algae of the mining area soil microalgae exist, transferring the mining area soil microalgae in the second solid culture medium into a liquid culture medium to obtain a culture solution containing the mining area soil microalgae.
3. The use of the mining area soil microalgae according to claim 1 for treating contaminated soil or contaminated water, comprising the steps of:
inoculating the soil microalgae in the mining area into a microalgae culture medium, enriching, amplifying and culturing to a stable growth period, adding into the polluted soil or the polluted water body, and carrying out pollutant treatment on the polluted soil or the polluted water body.
4. The application of the mining area soil microalgae in the treatment of the polluted soil or the polluted water body according to claim 3, wherein the microalgae culture medium is BG-11 culture medium, and the microalgae culture medium is sterilized at 121 ℃ for 20min, cooled to room temperature, and then inoculated with the mining area soil microalgae.
5. The application of the mining area soil microalgae in treating polluted soil or polluted water according to claim 3, wherein the culture temperature of the enrichment amplification culture is 22-29 ℃, the culture time is 20-30 d, and the culture illumination intensity is 8000-12000 lx.
6. The application of the mining area soil microalgae in treating polluted soil or polluted water according to claim 3, wherein purified air is introduced in the culture process of enrichment and amplification culture, and the pressure of the purified air is 0.1-0.6 MPa.
7. The application of the mining area soil microalgae in the treatment of the polluted soil or the polluted water body according to claim 3, wherein the addition amount of the mining area soil microalgae in the polluted soil is 0.01-0.2 mg/g in dry weight; the adding amount of the mining area soil microalgae in the polluted water body is 0.01-0.2 g/L.
8. The application of the mining area soil microalgae in treating the polluted soil or the polluted water body according to claim 3, wherein the treatment time for treating the pollutants in the polluted soil or the polluted water body is 20-30 d.
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