CN107345208B - Low-temperature growth green algae and application thereof in removing nitrogen and phosphorus in sewage - Google Patents
Low-temperature growth green algae and application thereof in removing nitrogen and phosphorus in sewage Download PDFInfo
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Abstract
The invention discloses a green alga which is identified to belong to Chlorophyta (Chlorophyta), Chlorococcales (Chlorophyceae), Scenedesmaceae (Scenedesmaceae), Tetradesmus (Tetradesmus), and has an alga strain name WU4, and is preserved in the China center for type culture collection with the preservation number: CCTCC M2017335, the preservation date is: 6 months and 14 days 2017. The novel green algae can be used for removing nitrogen and phosphorus in sewage, and has an obvious effect. Compared with the prior art, the novel green algae provided by the invention is applied to removing nitrogen and phosphorus in culture sewage under a low-temperature condition, and has the advantages of simple application method, high nitrogen and phosphorus removal rate and obvious effect.
Description
Technical Field
The invention relates to a novel low-temperature growth algae plant and the biotechnology field of treating organic pollutants in the environment by using the same, in particular to a biological degradation treatment of high-concentration nitrogen and phosphorus organic pollutants in breeding wastewater such as pig manure sewage and the like.
Background
In recent years, under the strong support of agricultural departments, the large-scale cultivation in China develops rapidly, the increasing demand and supply of consumers to meat food are effectively ensured, and huge industrial economic benefits are brought; however, a large amount of sewage generated by the large-scale cultivation accumulation is discharged randomly, so that the ecological environment is polluted greatly, epidemic disease transmission and spread are easily caused, and the health of human beings and the harmony and stability of the society are seriously threatened. The pig manure sewage belongs to high-concentration organic sewage, has high nitrogen and phosphorus contents, can induce water eutrophication when being directly discharged, causes serious pollution to water and soil ecological environment, and is a key point of the supervision of the national environmental protection department and is also a difficult point of prevention and control. How to effectively reduce the content of high-concentration nitrogen and phosphorus in the pig manure sewage to enable the content to reach the discharge standard related to the state is not only related to the safety of ecological environment, but also is an important restriction factor for the large-scale sustainable health development of the pig breeding industry, and has become a hot subject to be urgently solved by the multidisciplinary cross-exploration research.
The green algae is a photoautotrophic organism, has the characteristics of high photosynthetic efficiency, short growth period, quick propagation, strong adaptability, simple culture and the like, and needs to absorb nutrient substances such as nitrogen, phosphorus and the like in the environment in the growth process to synthesize complex macromolecular organic matters in the body, thereby reducing the content of organic substances such as nitrogen, phosphorus and the like in the water body and achieving the effect of purifying the water body.
However, the prior green algae applied to the degradation of nitrogen and phosphorus in sewage have some defects, for example, the green algae in the prior art can degrade nitrogen and phosphorus in sewage in a high-temperature environment at normal temperature or even in summer, and the green algae are hindered from growing and have low capability of degrading nitrogen and phosphorus in a low-temperature environment.
Technical scheme
The purpose of the invention is as follows:
aiming at the technical problems, the invention provides novel green algae which has obvious consumption and degradation functions on nitrogen and phosphorus organic matters in pig manure sewage through indoor experiments, and the algae and the application technology thereof have important scientific research values and huge application potentials.
The technical scheme is as follows:
in order to achieve the above objects, the present invention discloses a green alga identified as belonging to the phylum Chlorophyta (Chlorophyta), the class Chlorococcales (Chlorophyceae), the order Chlorococcales (Chlorococcales), the family Scenedesmaceae (Scenedesmaceae), the Tetradesmus curvatus (Tetradesmus acuminatus) in the genus Tetradesmus, the algal strain name WU4, namely the taxonomic name: tetradesmus acuminatus WU4, deposited at the China center for type culture Collection, accession number: china, wuhan university, zip code: 430072, with the preservation number: CCTCC M2017335, the preservation date is: 6 months and 14 days 2017.
The 18SrDNA nucleotide of the green algae is shown as SEQ ID NO. 1.
The culture conditions of the green algae are as follows: the culture temperature is 15 +/-2 ℃, and the culture medium is BG11 culture medium.
The green algae strain is obtained by sampling from a domestic sewage water body with high nitrogen and phosphorus content, and culturing, separating and screening.
The culture separation screening adopts a dilution coating line marking method: coating and streaking are a classic method of obtaining pure cultures of microorganisms. Diluting the algae liquid in a proper proportion, filtering by adopting a filter membrane with the aperture of 0.45 mu m, repeatedly washing the filtered algae liquid by combining a sterile liquid culture medium, and simultaneously adopting the plate culture and single colony picking technology to obtain the sterile algae strains.
The invention also provides application of the green algae in removing nitrogen in sewage, wherein the application temperature is 6-15 ℃, and the sewage is breeding wastewater containing high-concentration nitrogen and phosphorus organic pollutants, such as pig manure sewage and the like.
The invention also provides application of the green algae in removing phosphorus in sewage, wherein the application temperature is 6-15 ℃, and the sewage is breeding wastewater containing high-concentration nitrogen and phosphorus organic pollutants, such as pig manure sewage and the like.
The green algae in the prior art can degrade nitrogen and phosphorus in sewage at normal temperature and even in high-temperature environment in summer, and the green algae are hindered from growing and have low capability of degrading nitrogen and phosphorus in low-temperature environment. Therefore, the low-temperature green algae screened by the method can well grow in a low-temperature environment, can remove the nitrogen and phosphorus content in the pig manure sewage under a low-temperature condition, and has an obvious effect.
Technical effects
Compared with the prior art, the novel green algae of the invention has the following technical advantages:
the novel green algae provided by the invention is applied to removing nitrogen and phosphorus in pig manure sewage under a low-temperature condition, and has the advantages of simple application method, high nitrogen and phosphorus removal rate and obvious effect.
Drawings
FIG. 1 is a standard curve of total phosphorus in the total phosphorus measurement method;
FIG. 2 shows the removal rate of total phosphorus in pig manure wastewater by the WU4 algal strain of the invention
FIG. 3 is a standard curve of total nitrogen in the total nitrogen measurement method;
FIG. 4 shows the total nitrogen removal rate of the WU4 algal strain in swine waste water.
Detailed Description
The invention is further described with reference to specific examples.
The reagents and media formulations referred to in the examples:
(1) BG11 culture medium formula:
the components are as follows: 1L of deionized water contains 30g of NaNO3、0.8g K2HPO4、1.5g MgSO4·7H2O、2.86g H3BO3、1.81g MnCl2·4H2O、0.22g ZnSO4·7H2O、0.39g Na2MoO·2H2O、0.08g CuSO4·2H2O、0.05g Co(NO3)2·6H2O、1.36g CaCl2、2g Na2CO30.3g ferric ammonium citrate, 0.3g citric acid, 0.05g EDTANA2。
(2)(1+1)H2SO4
(3) Molybdate solution: 13g of ammonium molybdate (NH) are dissolved4)6MO7O24·4H2O in 100ml deionized water. 0.35g of antimony potassium tartrate KSbC was dissolved4H4O7·1/2H2O in 100ml deionized water. The ammonium molybdate solution was slowly added to 300ml (1+1) H with constant stirring2SO4And adding antimony potassium tartrate solution and mixing uniformly. Brown glass bottle, 4 ℃ storage.
(4) 10% ascorbic acid solution: 10g of ascorbic acid was dissolved and the volume was adjusted to 100ml with deionized water. Brown glass bottle, 4 ℃ storage.
(5)5％K2S2O8: dissolution 5g K2S2O8And the volume is adjusted to 100ml by deionized water.
(6) Phosphate stock solution: weighing 0.2197gKH2PO45ml of (1+1) H are added2SO4And the volume is adjusted to 1000ml by deionized water.
(7) Phosphate standard solution: 10ml of phosphate stock solution was taken in a 250ml volumetric flask and diluted to the mark with deionized water. The preparation is carried out immediately.
(8) Alkaline potassium persulfate solution: weighing 40g K2S2O815g of NaOH, the volume is adjusted to 1000ml by deionized water, and the mixture is stored in a polyethylene bottle.
(9) (1+9) hydrochloric acid
(10) Standard potassium nitrate stock solution: weighing 0.7218gKNO3And the volume is adjusted to 1000ml by deionized water. Adding 2ml of trichloromethane as a protective agent, and storing at 4 ℃.
(11) Standard potassium nitrate use solution: diluting the stock solution with deionized water by 10 times.
The method comprises the following specific operation steps:
(1) WU4 is separated from sewage water body
Dilution coating and scribing method: coating and streaking are a basic classical method for obtaining pure culture in microorganisms, and the method comprises the following specific steps: collecting the algae from the domestic sewage water body with high nitrogen and phosphorus content by using a plankton net, and putting the collected algae into a sample bottle filled with a culture medium. Sometimes impurity is a lot of from the water sample of field collection, so need carry out preliminary filtration processing to the water sample of gathering. Because the density of algae cells in the sample is lower, the failure of separation culture is easily caused, in order to avoid the failure of separation culture of directly collecting the sample, the sample subjected to filtration treatment can be subjected to enrichment pretreatment, and a proper amount of BG11 culture medium is added into the sample to accelerate the growth of the target algae species, so that the separation culture of the target algae species is facilitated after the algae cells in the sample reach a certain density. And (3) carrying out gradient dilution on the algae liquid sample subjected to enrichment culture to be separated by using a sterilized BG11 culture medium, and then carrying out algae seed separation by a plate coating separation method. Adding agar with the content of 1% into the culture medium, performing high-pressure steam sterilization, and after the sterilization is finished, quickly pouring the culture medium into a sterile culture dish when the temperature of the culture solution is cooled to about 50 ℃, thus preparing the solid culture medium for later use. Uniformly coating the diluted algae solution on the surface of a sterilized culture medium by using a coating rod, then placing the culture medium in a low-temperature illumination incubator for culture, and selecting the best growing monoclonal algae species to complete the separation of new algae strains. Then inoculating the strain into a sterile BG11 culture medium for enrichment growth.
Collecting 50ml of the above concentrated algae solution, centrifuging at 5000r/min for 8 min. Removing supernatant, adding liquid nitrogen, grinding, and placing in a clean centrifuge tube. Add 400. mu.l of PlantZol and mix well with shaking to suspend the sample completely. Add 7.5. mu.l RNaseA to the lysate and mix well. Incubate at 55 ℃ for 15 minutes. Adding phenol-chloroform-isoamyl alcohol (25:24:1) with the same volume, shaking and mixing evenly, and centrifuging at 12000rpm for 5 min. Carefully pipette the upper aqueous phase into a clean centrifuge tube, add an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1), and mix well by inversion. Centrifuge at 12000rpm for 5min, remove supernatant. Add 500. mu.l 70% ethanol, vortex for 5 seconds, centrifuge at 12000rpm for 5min, and remove supernatant. Centrifuging again for 1-2min, and sucking off residual liquid. Air-drying the DNA precipitate, adding 50. mu.l of TE, incubating at 65 ℃ for 10min to dissolve the DNA, flicking for several times to aid dissolution, and storing at 4 ℃.
The primer sequence for amplifying the 18SrDNA of the eukaryotic green algae is as follows:
upstream 5 "-ACCTGGTTGATCCTGCCAGTAG-3" (SEQ ID NO.2)
Downstream 5 "-ACCTTGTTACGACTTCTCCTTCCTCC-3" (SEQ ID NO.3)
And (3) PCR reaction conditions: denaturation at 95 deg.C for 3 min; 30sec at 95 ℃, 45sec at 55 ℃, 1min at 72 ℃ and 32 cycles; finally, extension is carried out for 10min at 72 ℃. The amplification product was sequenced by Nanjing Ongzhike Biometrics Ltd.
The 18SrDNA nucleotide sequence of the strain (WU4) is shown in SEQ ID NO.1, and is identified by observing the form under a microscope and comparing the nucleotide sequence in NCBI databases, wherein the strain belongs to Chlorophyta (Chlorophyta), Chlorococcales (Chlorophyceae), Chlorococcales (Chlorococcales), Scenedesmaceae (Scenedesmaceae), Tetradesmus (Tetradesmus) and has the strain name WU4, namely the strain is classified and named as WU 4: tetradesmus acuminatus WU4, deposited at the China center for type culture Collection, accession number: china, wuhan university, zip code: 430072, with the preservation number: CCTCC M2017335, the preservation date is: 6 months and 14 days 2017.
(2) Culturing the separated algae in an algae culture room of university of Nanjing Master at 15 + -2 deg.C with BG11 culture solution.
(3) The pig farm breeding sewage is obtained from a pig farm of Nanjing institute of agricultural science. The pig manure wastewater was filtered through a 0.45 μm filter membrane, and the filtered pig manure wastewater was used as an experimental material.
Water quality condition of experimental pig farm breeding sewage
(4) The experiment is carried out in 2017, month 2, and the temperature is controlled to be 6-15 ℃. Taking 40ml of the filtered pig manure wastewater into a 100ml conical flask, adding 5ml of WU4 algae liquid into the conical flask, and carrying out three parallels. For WU4 to better adapt to the environment of pig manure wastewater, multiple times of transfer are carried out.
TABLE 1 growth Density of the first inoculation of WU4 (A750nm)
TABLE 2 growth Density of the second transfer of WU4 (A750nm)
TABLE 3 growth Density of WU4 third transfer (A750nm)
(5) And measuring the content of nitrogen and phosphorus in the pig manure wastewater removed by using the third transferred algae strain.
(6) The method for measuring the total phosphorus in the pig manure wastewater in the experiment is an ammonium molybdate spectrophotometry.
① A, taking 750 ml colorimetric tubes with plugs, adding 0, 0.5, 1, 3, 5, 10 and 15ml phosphate standard solution into the colorimetric tubes respectively, using deionized water to fix the volume to 25ml, then adding 4ml 5% potassium persulfate solution into the colorimetric tubes, wrapping a piece of gauze at the tube openings after plugging, and fastening the gauze by using a rope.
② placing the cuvette in a large beaker, and subjecting to high pressureHeating in a sterilizing pot at a temperature of 1.1Kg/cm230min at 120 ℃. And taking out the colorimetric tube and cooling when the pressure pointer in the sterilizing pot is reduced to zero. Then diluted to 50ml of the line.
③ adding 1ml ascorbic acid solution into the digestion solution obtained in step ②, mixing, adding 2ml molybdate solution after 30s, standing for 15min at room temperature after mixing, measuring absorbance at 700nm, deducting the absorbance of blank test, and drawing a working curve (as shown in figure 1) with the corresponding phosphorus content.
④ taking 2ml of water sample, centrifuging for 5000r/min, 3min, taking 1ml of supernatant, placing the supernatant in a 50ml colorimetric tube with a plug, fixing the volume to 25ml with deionized water, adding 4ml of 5% potassium persulfate solution, covering a piece of gauze on the tube opening after plugging, and fastening the gauze with a rope.
⑤ synchronization step ②.
⑥ since the water sample contains color, two volumes of (1+1) H are added to the digestion solution2SO4And one volume of 10% ascorbic acid solution, the mixture being prepared on the day. After mixing well, standing at room temperature for 15min, and measuring the absorbance at 700 nm. After subtracting the absorbance of the blank test, the phosphorus content is found from the standard curve.
TABLE 4 efficiency (%)% of WU4 algal strains in removing total phosphorus in pig manure wastewater
The percentage of phosphorus in the pig manure wastewater removed by the WU4 strain is shown in the accompanying figure 2.
(7) The method for measuring the total nitrogen of the pig manure wastewater in the experiment is potassium persulfate oxidation ultraviolet spectrophotometry.
① A25 ml colorimetric tube with a plug was taken, 0, 0.5, 1, 2, 3, 5, 7, 8ml potassium nitrate standard solution was added to the colorimetric tube, and the colorimetric tube was diluted with deionized water to 10ml mark.
② adding 5ml of alkaline potassium persulfate into the cuvette with a plug, covering the opening with a piece of gauze and fastening the gauze with a rope after the plug is added, putting the cuvette into a big beaker, and putting the beaker into an autoclaveHeating at 1.1Kg/cm230min at 120 ℃. When the pressure pointer in the sterilizing pot is reduced to zero, the colorimetric tube is taken out and cooled to room temperature.
③ (1+9) hydrochloric acid 1ml is added, then diluted with deionized water to 25ml mark line, mixed and left for 10min, the absorbance (A ═ A220-2 xA 275) is measured at 220nm and 275nm respectively with 10mm quartz cuvette, the corrected absorbance is used to draw a standard curve (as shown in figure 3).
④ taking 2ml of water sample, centrifuging for 5000r/min, 3min, taking 1ml of supernatant into a 25ml colorimetric tube with a plug, fixing the volume to 10ml with deionized water, drawing steps ② - ③ according to a standard curve, finding out the total nitrogen amount of response on the calibration curve according to the calibration absorbance, and calculating the total nitrogen content by using the following formula.
Total nitrogen (mg/L) ═ m/v
In the formula: m is the nitrogen content (μ g) found on a standard curve;
v is the volume of the sampled water (ml)
TABLE 5 efficiency (%)% of the strain WU4 for removing total nitrogen in swine waste water
The percentage of nitrogen in pig manure wastewater removed by the strain WU4 is shown in FIG. 4.
The technical solutions of the present invention are described in detail in the above embodiments, it should be understood that the above embodiments are only specific examples of the present invention, and are not intended to limit the present invention, and any modifications or improvements made within the scope of the principles of the present invention should be included in the scope of the present invention.
SEQUENCE LISTING
<110> university of Nanjing university
<120> a low-temperature growing green alga and application thereof in removing nitrogen and phosphorus in sewage
<130>2017.06
<160>3
<170>PatentIn version 3.3
<210>1
<211>1668
<212>DNA
<213> Tetradesmus
<400>1
aactgcttat actgtgaaac tgcgaatggc tcattaaatc agttatagtt tatttggtgg 60
taccttacta ctcggataac cgtagtaatt ctagggctaa tacgtgcgta aatcccgact 120
tctggaaggg acgtatatat tagataaaag gccgaccgag ctttgctcga cccgcggtga 180
accatgatat cttcacgaag cgcatggcct tgagccggcg ctgttccatt caaatttctg 240
ccctatcaac tttcgatggt aggatagagg cctaccatgg tggtaacggg tgacggagga 300
ttagggttcg attccggaga gggagcctga gaaacggcta ccacatccaa ggaaggcagc 360
aggcgcgcaa attacccaat cctgatacgg ggaggtagtg acaataaata acaataccgg 420
gcatttcatg tctggtaatt ggaatgagta caatctaaat cccttaacga ggatccattg 480
gagggcaagt ctggtgccag cagccgcggt aattccagct ccaatagcgt atatttaagt 540
tgttgcagtt aaaaagctcg tagttggatt tcgggtgggt tctagcggtc cgcctatggt 600
gagtactgct atggccttcc tttctgtcgg ggacgggctt ctgggcttca ctgtccggga 660
ctcggagtcg acgtggttac tttgagtaaa ttagagtgtt caaagcaggc ttacgccaga 720
atactttagc atggaataac acgataggac tctggcctat cttgttggtc tgtaggaccg 780
gagtaatgat taagagggac agtcgggggc attcgtattt cattgtcaga ggtgaaattc 840
ttggatttat gaaagacgaa ctactgcgaa agcatttgcc aaggatgttt tcattaatca 900
agaacgaaag ttgggggctc gaagacgatt agataccgtc gtagtctcaa ccataaacga 960
tgccgactag ggattggcga atgttttttt aatgacttcg ccagcacctt atgagaaatc 1020
aaagtttttg ggttccgggg ggagtatggt cgcaaggctg aaacttaaag gaattgacgg 1080
aagggcacca ccaggcgtgg agcctgcggc ttaatttgac tcaacacggg aaaacttacc 1140
aggtccagac atagtgagga ttgacagatt gagagctctt tcttgattct atgggtggtg 1200
gtgcatggcc gttcttagtt ggtgggttgc cttgtcaggt tgattccggt aacgaacgag 1260
acctcagcct gctaaatagt ctcagttgct ttttgcagct ggctgacttc ttagagggac 1320
tattggcgtt tagtcaatgg aagtatgagg caataacagg tctgtgatgc ccttagatgt 1380
tctgggccgc acgcgcgcta cactgatgca ttcaacaagc ctatccttga ccgaagggtc 1440
tgggtaatct ttgaaactgc atcgtgatgg ggatagatta ttgcaattat tagtcttcaa 1500
cgaggaatgc ctagtaagcg caagtcatca gcttgcgttg attacgtccc tgccctttgt 1560
acacaccgcc cgtcgctcct accgattggg tgtgctggtg aagtgttcgg attggcagct 1620
tagggtggca acacctcagg tctgccgaga agttcataaa ccctccca 1668
<210>2
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<213> Artificial sequence
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acctggttga tcctgccagt ag 22
<210>3
<211>26
<212>DNA
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accttgttac gacttctcct tcctcc 26
Claims (4)
1. A green alga characterized by being identified as belonging to the phylum Chlorophyta (Chlorophyta), the class Chlorophyceae (Chlorophyceae), the order Chlorococcales (Chlorococcales), the family scenedemiaceae (Scenedesmaceae), the genus Tetradesmus (Tetradesmus), the strain name WU4, deposited at the chinese collection of type cultures under the deposit number: CCTCCM2017335, the preservation date is: 6 months and 14 days 2017.
2. The green alga of claim 1 wherein the 18SrDNA nucleotide of said green alga is as set forth in SEQ ID No. 1.
3. The green alga of claim 1 wherein said green alga is cultured under the following conditions: the culture temperature is 15 +/-2 ℃, and the culture medium is BG11 culture medium.
4. The green alga of claim 1 wherein said green alga strain is obtained by sampling from a body of domestic sewage containing high contents of nitrogen and phosphorus, culturing, separating and screening.
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| CN104388315A (en) * | 2014-11-07 | 2015-03-04 | 山东大学 | Scnedesmus quadricauda for efficiently treating typical domestic sewage, and culture method and application thereof |
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