CN113717854A - Oil-producing chlorella ZM-5 and application thereof - Google Patents
Oil-producing chlorella ZM-5 and application thereof Download PDFInfo
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Abstract
An oil-producing chlorella ZM-5 and application thereof, relating to oil-producing chlorella. It aims to solve the problem that the purification effect of the existing algae species on the pig raising wastewater is not good. It has been preserved in China center for type culture Collection with the preservation numbers: CCTCC No. M20211015, preservation time 2021, 8 months and 10 days, preservation address is Wuhan university in Wuhan City, which is Chlorella (Chlorella sorokiniana) ZM-5. The invention adopts the PRECI SCS single-cell sorter to carry out single-cell sorting on the specific microalgae, has simple method, realizes the separation of single algae cells, does not damage the cell structure, and ensures the subsequent expanded culture and research of the single cells. The chlorella ZM-5 has high-efficiency COD, TN and TP purification capacity on the pig raising wastewater, is suitable for large-scale popularization and use, and has great significance on the pollution treatment and reclamation of the wastewater. The chlorella ZM-5 is applied to purifying the pig-raising wastewater.
Description
Technical Field
The invention belongs to the field of environmental microbiology, and particularly relates to an oil-producing chlorella ZM-5 and application thereof.
Background
The microalgae has the advantages of small size, large specific surface area and the like, so that the microalgae can efficiently obtain nutrient substances from the external environment. The microalgae not only can absorb and decompose nutrient substances, but also can utilize the nutrient substances to synthesize biomass with high economic value such as grease, protein, carbohydrate and the like. Based on the physiological characteristics of the microalgae, people can utilize the microalgae to treat the pollution of the pig raising wastewater and realize the resource utilization of the wastewater. Because the efficiency of purifying the pig raising wastewater by the microalgae depends on the growth of the microalgae, the screening of the algae species with high-efficiency purification effect on the pig raising wastewater has great research significance in the aspect of sewage treatment.
Today, the most widely used and traditional cell sorting method is the plate sorting method. Because three-area line drawing operation needs to be carried out for many times, the risk of contamination is greatly improved. And for some complex environment samples, the traditional sorting method is not suitable and has great limitation. For example, algae strains such as oscillatoria and the like are widely distributed in the artificial wetland due to the high organic matter content of the water body, and the separation of chlorella from such complicated samples by using the traditional separation method is extremely difficult. In addition, the sorting result of the traditional cell sorting method has great randomness, and particularly, for samples with abundant microalgae species, the target microalgae are difficult to sort, so that the alternative sorting mode is urgently selected. Currently, the separation and screening of cells using flow cytometric fluorescence sorting (FACS) is partially studied. Wherein, the cells or the cell-like particles are the detection and analysis objects of FACS, and the accuracy of the final result is closely related to the preparation of the sample. However, in the process of dissociating the tissue into single cells, FACS may damage the cell structure, making the single cells unable to be cultured subsequently or difficult to culture.
Disclosure of Invention
The invention aims to provide oil-producing chlorella ZM-5 and application thereof, and solves the problem that the existing algae strain has poor purification effect on pig raising wastewater.
An oil-producing chlorella ZM-5 strain which is preserved in China center for type culture collection with the preservation number as follows: CCTCC No. M20211015, preservation time is 8 months and 10 days in 2021, preservation address is Wuhan university in Wuhan, China, which is Chlorella (Chlorella sorokiniana) ZM-5.
The application of the oil-producing chlorella ZM-5 is as follows: it is applied to purifying and treating pig-raising wastewater.
The chlorella ZM-5 of the invention has the morphological characteristics of the strain: single cell, regular spherical, light green and smooth surface.
The chlorella ZM-5 of the invention has the physical and chemical properties of the strain: the microalgae is oil-producing microalgae, and the product of the microalgae comprises 40.29% of oil, 22.04% of polysaccharide and 37.67% of protein; the chlorella ZM-5 can efficiently degrade the pig-raising wastewater.
The chlorella ZM-5 of the invention has the following molecular biological identification results of the strain: the 18SrRNA sequence GenBank registration number is MZ557824, the similarity with the Chlorella sorokiniana sequence reaches 99.47 percent, and the affinity is nearest; chlorella ZM-5 is a new kind of microalgae.
The invention adopts the PRECI SCS single cell sorter to carry out single cell sorting on the specific microalgae, the single cell sorting method is simple, the process is easy to control, the separation of single algae cells is realized, and the cell structure is not damaged; the PRECI SCS single-cell sorter is based on a Laser-Induced Forward Transfer (LIFT) technology, has the characteristic of visualization of microscopic imaging, can realize the separation of single algae cells, ensures the purity and the sorting accuracy of single-cell microalgae, and prevents the pollution of mixed bacteria; the principle is that the pulsed laser interacts with a thin layer material on a transparent glass slide to deform the thin layer material, so that single cells attached to the thin layer material are pushed into a receiving container below the thin layer material, and the marking-free and non-contact separation of the single cells in a sample is realized; because the laser and the cells to be separated have no direct interaction, the algae can not be damaged in the sorting process; meanwhile, the separation process does not depend on labels such as fluorescence, and the influence of exogenous labels on the cell activity is avoided. Therefore, the activity and the function of the sorted algae in the in-situ state can be truly reflected, and the subsequent expanded culture of single cells and the subsequent research are ensured.
The method quickly selects oil-producing chlorella ZM-5 from the artificial wetland water body near a certain pig farm around Harbin of Heilongjiang province in China, has high-efficiency COD, TN and TP purification capacity on the pig raising wastewater, inoculates the oil-producing chlorella ZM-5 into the pig raising wastewater preparation water, and detects that the degradation rates of the chlorella ZM-5 on the COD, TN and TP of the pig raising wastewater are 86.66%, 88.75% and 84.59% respectively; therefore, the single strain of oil-producing chlorella ZM-5 is adopted to purify the pig-raising wastewater, so that a good treatment effect can be achieved, the environmental pollution is reduced, and the cost is saved.
The oil-producing chlorella ZM-5 is suitable for purifying pig-raising wastewater.
Drawings
FIG. 1 is a schematic representation of visual imaging (part A) and single cell sorting (part B) of Chlorella ZM-5 of the present invention;
FIG. 2 is a plate morphology (part A) and a 100X optical microscope morphology (part B) of Chlorella vulgaris ZM-5 according to the present invention;
FIG. 3 is a phylogenetic tree diagram of Chlorella ZM-5 of the present invention;
FIG. 4 is a graph of the growth of Chlorella ZM-5 of the present invention;
FIG. 5 is a diagram showing the physicochemical property profile of Chlorella ZM-5 of the present invention;
FIG. 6 is a bar graph showing the degradation of COD, TN and TP in swine wastewater by Chlorella ZM-5 of the present invention, wherein a represents COD, b represents TP and c represents TN.
Detailed Description
The first embodiment is as follows: the oil-producing chlorella ZM-5 strain of the embodiment is preserved in China center for type culture Collection with the preservation number as follows: CCTCC No. M20211015, preservation time is 8 months and 10 days in 2021, preservation address is Wuhan university in Wuhan, China, which is Chlorella (Chlorella sorokiniana) ZM-5.
The oil-producing chlorella ZM-5 strain has the following sorting and culturing processes:
firstly, enabling a PRECI SCS single-cell sorter to reach a sterile state; taking an artificial wetland water body near a certain pig farm around Harbin of Heilongjiang province in China as a sample, shaking uniformly, placing 5 mu l of the artificial wetland water body on a sorting chip, shooting a microimaging picture of the sample before sorting by adopting a PRECI SCS single cell sorter, then positioning and sorting single cells of target microalgae according to the form of the microalgae in the microimaging, collecting the single cells of the microalgae, and shooting a microimaging picture after sorting;
secondly, transferring the collected microalgae single cells into a sterilized cell culture tube containing a BG-11 liquid culture medium containing 3g/L glucose, placing the cell culture tube into a shaking incubator for culture to obtain turbid light green culture solution, transferring the turbid light green culture solution into a sterilized aseptic conical flask containing the BG-11 liquid culture medium containing 3g/L glucose, placing the aseptic conical flask into the shaking incubator for amplification culture, transferring the algae solution successfully subjected to amplification culture into a BG-11 solid culture medium for flat dilution and coating, and screening single algae colonies;
wherein the BG-11 liquid medium components: NaNO3 1.5g/L，K2HPO4·3H2O 0.04g/L，MgSO4·7H2O 0.075g/L，CaCl2·2H2O 0.036g/L，C6H8O70.006g/L，C6H8FeNO7 0.006g/L，EDTA 0.001g/L，Na2CO3 0.02g/L，H3BO3 0.00286g/L，MnCl2·H2O 0.00181g/L，ZnSO4·7H2O 0.000222g/L， CuSO4·5H2O 0.000079g/L，Na2MoO4·2H2O 0.00039g/L，Co(NO3)2·6H2O0.000049 g/L and the balance of distilled water;
the culture conditions are as follows: the culture medium is illuminated for 24 hours every day, the illumination intensity is 6000lux, the culture temperature is 30 +/-2 ℃, the oscillation speed is 180r/min, and BG-11 liquid culture medium is supplemented every four days.
In the first step of the present embodiment, the microscopic imaging image of the sample before sorting and the microscopic imaging image after sorting, which are taken by the PRECI SCS single cell sorter, are respectively shown in fig. 1 a and B, which are the visualization imaging and single cell sorting of the microalgae single cell.
In the second step of the present embodiment, the obtained single algae colonies are screened, and the single algae colonies are picked and observed under an optical microscope for microscopic morphology: morphological characteristics of single algae colonies are observed by naked eyes, the algae colonies are green, regular and round, and the surfaces of the algae colonies are smooth; the algae colony morphology is single cell, regular spherical and light green when observed under a 100X optical microscope, and is preliminarily identified as chlorella (figure 2).
In the second step of the embodiment, the obtained single algal colony is screened, after the third-generation purification, the single algal colony in the BG-11 solid culture medium is picked, and the 18S rRNA sequencing technology is adopted to identify the algal species:
(1) extracting microalgae DNA by using a bacterial genome DNA extraction kit (Tiangen Biochemical technology Co., Ltd.);
(2) amplifying 18S rRNA genes from microalgae total DNA by using Polymerase Chain Reaction (PCR), and amplifying target alga-like 18S sequences by using 18S rRNA universal primers 18SF (5'-CCTGGTTGATCCTGCCAG-3') and 18SR (5'-TTGATCCTTCTGCAGGTTCA-3'); the PCR product was sequenced by Beijing Liuhe Hua Dagen science and technology Co., Ltd (Chinese Dalian) (see SEQ ID No: 3); using the BLAST program to compare the sequences to sequences available in the NCBI database;
(3) the PCR reaction system is as follows: 1ng of template DNA, 15 mu L of Taq enzyme mixed solution, 1 mu L of primer 18SF and 1 mu L of primer 18SR, and adding sterile deionized water to 30 mu L;
PCR amplification conditions: pre-denaturation at 96 ℃ for 5min, denaturation at 96 ℃ for 20S, annealing at 56 ℃ for 20S, extension at 72 ℃ for 20S, circulation for 35 times, and extension at 72 ℃ for 10 min.
Amplifying a target alga-like 18S sequence by adopting 18S rRNA universal primers 18SF (5'-CCTGGTTGATCCTGCCAG-3') and 18SR (5'-TTGATCCTTCTGCAGGTTCA-3'), and sequencing by utilizing a high-throughput sequencing technology to perform molecular identification; and finally, judging the species level taxonomy information of the target algae sample by NCBI database comparison based on a molecular biology means.
The identification results show (see fig. 3): the 18SrRNA sequence GenBank registration number is MZ557824, the similarity with the Chlorella sorokiniana sequence reaches 99.47 percent, and the affinity is nearest; is named as Chlorella (Chlorella sorokiniana) ZM-5.
Test 1: growth of Chlorella ZM-5: inoculating chlorella ZM-5 into the pig-raising wastewater, and sampling once every 2 h; measuring absorbance at a wavelength of 600nm by using a spectrophotometer, drawing a growth curve (see fig. 4), and according to the growth curve, displaying that the life cycle of chlorella ZM-5 in pig wastewater (COD is 400mg/L) is 40h, and entering logarithmic growth phase for 22h after 4h inoculation; after 26h, a stationary phase was entered, lasting 8 h.
Test 2: physical and chemical properties of chlorella ZM-5:
(1) and (3) oil and fat determination: sucking 20ml of algae liquid which is successfully expanded and cultured into a centrifuge tube by using a pipette; centrifuging at 8000r/min for 10min, and removing supernatant; repeatedly washing with deionized water for 3 times, and collecting algae sample; adding 2ml chloroform and 4ml methanol into the algae sample, mixing uniformly, and then carrying out ultrasonic crushing for 10min (ultrasonic conditions: power 400w, working time 5s, and pause time 2 s); centrifuging at the rotating speed of 4500r/min for 10min, collecting the supernatant in a new 10mL centrifuge tube, standing for layering with 2mL of 1% NaCl solution; repeating the above operation for the rest algae sample until the color of the algae sample turns white; weighing a blank test tube, recording the weight as m1, sucking the lower layer liquid after standing into the weighed test tube, and evaporating to dryness in a water bath at 65 ℃ until the weight is constant; weighing the test tube after drying to dryness, and recording the weight as m 2; the oil content is m2-m 1.
(2) And (3) polysaccharide determination: freeze-drying the microalgae cells to prepare algae powder; weighing 0.1g of algae powder, adding 4% NaOH according to a ratio of 1:25, and uniformly mixing; performing ultrasonic extraction at 80w for 20min, performing ice bath for 10min, and repeating extraction for 3 times; carrying out water bath in a water bath kettle at 80 ℃ for 2h, cooling, centrifuging at 4800r/min for 10min, and taking supernatant; washing with deionized water for 2 times, and mixing the supernatants; adding anhydrous ethanol with three times of volume into the supernatant, uniformly mixing, and standing in a refrigerator at 4 ℃ overnight; after freezing and centrifuging, taking the precipitate, and adding 3% of TCA into the precipitate until the precipitate is not dissolved any more. Filtering with 0.45um filter membrane, adding anhydrous alcohol with three times volume again to precipitate supernatant; centrifuging at 4 deg.C, collecting precipitate, and freeze drying to obtain crude polysaccharide powder. Dissolving the crude polysaccharide powder with deionized water; 1mL of the anthrone-sulfuric acid solution was added to the test tube, and 4mL of the anthrone-sulfuric acid solution was added. Mixing, boiling in water bath for 10min, and measuring light absorption value at 620nm after ice bath.
(3) Protein yield determination: microalgae cells were diluted with 1 × PBS and subjected to the procedure of a Bradford method protein quantitative determination kit (chinese bioengineering, ltd.).
The measurement results show (see figure 5), that the oil accounts for 40.29%, the polysaccharide accounts for 22.04%, and the protein accounts for 37.67% in the chlorella ZM-5 product; therefore, the chlorella ZM-5 is an oleaginous microalgae.
The second embodiment is as follows: the application of the oil-producing chlorella ZM-5 of the embodiment is as follows: it is applied to purifying and treating pig-raising wastewater.
The third concrete implementation mode: the second difference between this embodiment and the second embodiment is that the application process is as follows: centrifuging the algae solution successfully cultured in an enlarged way for 10min at 8000r/min, then discarding the supernatant, inoculating the supernatant into the pig-raising wastewater and water, and culturing for 40h at 30 ℃ at 400r/min to finish the culture; the inoculation amount of the chlorella ZM-5 is 0.01g/ml for water distribution of pig-raising wastewater; the pig raising wastewater comprises the following components (g/L): 0.0620464g/L NaCl, CaCl2·2H2O 0.234872g/L， MgSO4 0.151662g/L，NH4Cl 1.019244g/L，K2HPO4 0.05239g/L，NaNO3 0.16014g/L， C6H12O60.3768 g/L; the water quality index (mg/L) of the pig raising wastewater is as follows: COD is 400 mg/L; TN 9.315 mg/L; TP 293.147 mg/L. Other steps and parameters are the same as those in the first embodiment.
Test 3: the chlorella ZM-5 degrades pig-raising wastewater:
centrifuging the algae solution successfully cultured in an amplification way for 10min at 8000r/min, discarding the supernatant, inoculating the algae solution into the pig-raising wastewater water (with the inoculation amount of 0.01 g/ml), and sampling once every 4 h. Respectively adopting a potassium dichromate-sulfuric acid method to determine the COD content of the pig-raising wastewater; measuring the TP content of the pig raising wastewater by adopting a molybdenum-antimony anti-spectrophotometry method; and (3) measuring the TN content of the pig-raising wastewater by adopting an alkaline potassium persulfate method.
The assay results show (see fig. 6): the degradation rates of chlorella ZM-5 on COD, TN and TP of the pig-raising wastewater are calculated to be 86.66%, 88.75% and 84.59% respectively.
Sequence listing
<110> university of northeast agriculture, Harbin university of industry, Changchun Chang Guangxi Bioscience Instrument Co., Ltd
<120> oil-producing chlorella ZM-5 and application thereof
<160> 3
<210> 1
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<212> DNA
<223> Artificial sequence
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<223> nucleotide sequence of primer 18SF
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cctggttgat cctgccag 18
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<223> Artificial sequence
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<223> nucleotide sequence of primer 18SR
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ttgatccttc tgcaggttca 20
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<223> Chlorella sorokiniana
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attagccatg catgtctaag tataaactgc tttatactgt gaaactgcga atggctcatt 60
aaatcagtta tagtttattt gatggtacct actactcgga tacccgtagt aaatctagag 120
ctaatacgtg cgtaaatccc gacttctgga agggacgtat ttattagata aaaggccgac 180
cgggctctgc ccgactcgcg gtgaatcatg ataacttcac gaatcgcatg gccttgtgcc 240
ggcgatgttt cattcaaatt tctgccctat caactttcga tggtaggata gaggcctacc 300
atggtggtaa cgggtgacgg aggattaggg ttcgattccg gagagggagc ctgagaaacg 360
gctaccacat ccaaggaagg cagcaggcgc gcaaattacc caatcctgac acagggaggt 420
agtgacaata aataacaata ctgggccttt tcaggtctgg taattggaat gagtacaatc 480
taaacccctt aacgaggatc aattggaggg caagtctggt gccagcagcc gcggtaattc 540
cagctccaat agcgtatatt taagttgctg cagttaaaaa gctcgtagtt ggatttcggg 600
tggggcctgc cggtccgccg tttcggtgtg cactggcagg gcccaccttg ttgccgggga 660
cgggctcctg ggcttaattg tccgggactc ggagtcggcg ctgttacttt gagtaaatta 720
gagtgttcaa gcaggcctac gctctgaata cattagcatg gaataacacg ataggactct 780
ggcctatcct gttggtctgt aggaccggag taatgattaa gagggacagt cgggggcatt 840
cgtatttcat tgtcagaggt gaaattcttg gatttatgaa agacgaacta ctgcgaaagc 900
atttgccaag gatgttttca ttaatcaaga acgaaagttg ggggctcgaa gacgattaga 960
taccgtccta gtctcaacca taaacgatgc cgactaggga tcggcggatg tttcttcgat 1020
gactccgccg gcaccttatg agaaatcaaa gtttttgggt tccgggggga gtatggtcgc 1080
aaggctgaaa cttaaaggaa ttgacggaag ggcaccacca ggcgtggagc ctgcggctta 1140
atttgactca acacgggaaa acttaccagg tccagacata gtgaggattg acagattgag 1200
agctctttct tgattctatg ggtggtggtg catggccgtt cttagttggt gggttgcctt 1260
gtcaggttga ttccggtaac gaacgagacc tcagcctgct aaatagtcac ggttggttct 1320
ccagccggcg gacttcttag agggactatt ggcgactagc caatggaagc atgaggcaat 1380
aacaggtctg tgatgccctt agatgttctg ggccgcacgc gcgctacact gatgcattca 1440
acgagcctag ccttgaccga gaggtccggg taatctttga aactgcatcg tgatggggat 1500
agattattgc aattattaat cttcaacgag gaatgcctag taagcgcaat tcatcagatt 1560
gcgttgatta cgtccctgcc ctttgtacac accgcccgtc gctcctaccg attgggtgtg 1620
ctggtgaagt gttcggattg gcgaccgggg gcggtctccg ctctcggccg ccgagaagtt 1680
cattaaaccc tcccacctag aggaagagaa gt 1712
Claims (3)
1. An oil-producing chlorella ZM-5 is characterized in that the oil-producing chlorella ZM-5 is preserved in China center for type culture collection with the preservation number as follows: CCTCC No. M20211015, preservation time is 8 months and 10 days in 2021, preservation address is Wuhan university in Wuhan City, which is Chlorella (Chlorella sorokiniana) ZM-5.
2. The use of the oil-producing chlorella ZM-5 as defined in claim 1, wherein the oil-producing chlorella ZM-5 is used for purifying pig wastewater.
3. The use of an oleaginous chlorella ZM-5 according to claim 2, characterized in that the application process is as follows: centrifuging the algae solution successfully cultured for 10min at 8000r/min, discarding supernatant, inoculating into pig wastewater, and culturing at 400rMin, and culturing for 40h at 30 ℃ to finish; the inoculation amount of the chlorella ZM-5 is 0.01g/ml for water distribution of pig-raising wastewater; the pig raising wastewater comprises the following components (g/L): 0.0620464g/L NaCl, CaCl2·2H2O 0.234872g/L，MgSO40.151662g/L，NH4Cl 1.019244g/L，K2HPO4 0.05239g/L，NaNO3 0.16014g/L，C6H12O60.3768 g/L; the water quality index (mg/L) of the pig raising wastewater is as follows: COD is 400 mg/L; TN 9.315 mg/L; TP 293.147 mg/L.
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