CN113717854B - Chlorella oleaginous ZM-5 and application thereof - Google Patents
Chlorella oleaginous ZM-5 and application thereof Download PDFInfo
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- C02F3/00—Biological treatment of water, waste water, or sewage
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Abstract
An oleaginous chlorella ZM-5 and application thereof relate to oleaginous chlorella. It solves the problem that the purification effect of the existing algae seeds on the pig raising wastewater is poor. It has been deposited in China center for type culture Collection with accession number: CCTCC No. M20211015, the preservation time of 2021 is 8 months and 10 days, the preservation address is university of Wuhan in Wuhan, and the preservation address is Chlorella (Chlorella sorokiniana) ZM-5. The invention adopts the PRECI SCS single cell sorter to sort the single cells of the specific microalgae, has simple method, realizes the separation of single algae cells, does not damage the cell structure, and ensures the subsequent expansion culture and research of single cells. The chlorella ZM-5 has high-efficiency COD, TN, TP purifying capacity on the pig raising wastewater, is suitable for large-scale popularization and application, and has great significance on pollution treatment and recycling of the wastewater. Chlorella ZM-5 is used for purifying pig raising wastewater.
Description
Technical Field
The invention belongs to the field of environmental microbiology, and particularly relates to an oleaginous chlorella ZM-5 and application thereof.
Background
Microalgae have the advantages of small individual, large specific surface area and the like, so that the microalgae can efficiently obtain nutrient substances from the external environment. 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 realize the resource utilization of the waste water while treating the pig raising waste water pollution by using the microalgae. The efficiency of purifying the pig raising wastewater by the microalgae is more dependent on the growth of the microalgae, so that 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.
The most widely used and traditional cell sorting method today is the plate sorting method. As the three-region line drawing operation is needed for a plurality of times, the risk of bacteria infection is greatly improved. Moreover, for some complex environmental samples, the traditional sorting method is not applicable and has great limitation. For example, in artificial wetlands, algae strains such as Oscillatoria are widely distributed due to high organic matter content in water bodies, and the difficulty of separating chlorella from such complex samples by using a traditional separation method is great. In addition, the sorting result of the conventional cell sorting method has great randomness, and particularly for samples with abundant microalgae, the target microalgae are difficult to sort out, so that the selection of alternative sorting modes is urgent. Currently, there are some studies on cell isolation and selection using flow cytometry fluorescence sorting (FACS). Wherein, cells or cell-like particles are the detection analysis object of FACS, and the accuracy of the final result is closely related to the preparation of samples. However, during dissociation of the tissue into single cells, FACS may damage the cell structure, making subsequent single cell culture impossible or difficult.
Disclosure of Invention
The invention aims to provide an oleaginous chlorella ZM-5 and application thereof, which solve the problem that the existing algae species have poor purification effect on pig raising wastewater.
Chlorella oleaginous ZM-5, which has been deposited in China center for type culture Collection, accession number: CCTCC No. M20211015, the preservation time is 2021, 8 months and 10 days, the preservation address is Wuhan university of Chinese, and the preservation address is Chlorella (Chlorella sorokiniana) ZM-5.
The application of the chlorella vulgaris ZM-5 strain for producing oil is as follows: it is applied to purifying and treating pig raising waste water.
The chlorella ZM-5 of the invention has morphological characteristics of the strain: single cells are displayed, the spherical shape is regular, the light green color is realized, and the surface is smooth.
The chlorella ZM-5 has physical and chemical properties of the strain: the microalgae is a microalgae for producing grease, wherein grease accounts for 40.29 percent, polysaccharide accounts for 22.04 percent, and protein accounts for 37.67 percent; chlorella ZM-5 can efficiently degrade pig raising wastewater.
The molecular biological identification result of the chlorella ZM-5 strain disclosed by the invention is as follows: the registration number of the 18SrRNA sequence GenBank is MZ557824, the similarity with Chlorella sorokiniana sequences reaches 99.47%, and the parent relationship is nearest; chlorella ZM-5 is a new type of microalgae.
The invention adopts the PRECI SCS single-cell sorter to sort the specific microalgae single cells, 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 technology (Laser-Induced Forward Transfer, LIFT), has the characteristics of microscopic imaging and visualization, can realize separation of single algae cells, ensures purity of single-cell microalgae and sorting accuracy, and prevents contamination of mixed bacteria; the principle is that pulse laser is used to interact with the thin layer material on the transparent slide to deform the thin layer material, so that single cells attached to the thin layer material are pushed into a receiving container below, and the non-marking and non-contact separation of single cells in a sample is realized; because the laser has no direct interaction with the cells to be separated, algae cannot be damaged in the separation process; meanwhile, the separation process does not depend on fluorescence and other markers, so that the influence of exogenous markers on the activity of cells is avoided. Therefore, the selected algae can truly reflect the activity and the function in the in-situ state, and the subsequent single cell expansion culture and subsequent research are ensured.
According to the invention, the oil-producing chlorella ZM-5 is rapidly separated from a water body of an artificial wetland near a pig farm around Harbin in Heilongjiang province of China, the chlorella ZM-5 has high-efficiency COD, TN, TP purifying capacity on pig wastewater, and is inoculated into pig wastewater water distribution, and the degradation rates of the chlorella ZM-5 on the pig wastewater COD, TN and TP are 86.66%, 88.75% and 84.59% respectively; the invention can achieve good treatment effect by adopting the single algae species of the oil-producing chlorella ZM-5 to carry out the purification treatment of the pig raising wastewater, reduce the environmental pollution, save the cost, ensure that the accumulated grease of the chlorella ZM-5 is an oil-producing microalgae, can be converted into clean energy, namely biodiesel, through the extraction and transfection processes, has better economic value, is suitable for large-scale popularization and application, and has great significance for pollution treatment and recycling of the wastewater.
The chlorella ZM-5 producing oil is suitable for purifying pig raising waste water.
Drawings
FIG. 1 is a schematic diagram of visual imaging (part A) and single cell sorting (part B) of Chlorella ZM-5 according to the present invention;
FIG. 2 is a graph of the morphological characteristics of the plates of Chlorella ZM-5 (section A) and of the 100X optical microscope (section B) according to the present invention;
FIG. 3 is a phylogenetic tree spectrum of Chlorella ZM-5 according to the present invention;
FIG. 4 is a graph showing the growth of Chlorella ZM-5 according to the present invention;
FIG. 5 is a graph showing the physicochemical property profile of Chlorella ZM-5 according to the present invention;
FIG. 6 is a bar graph showing the degradation of COD, TN, 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 chlorella oleaginous ZM-5 strain of the embodiment is preserved in China center for type culture Collection, and the preservation number is as follows: CCTCC No. M20211015, the preservation time is 2021, 8 months and 10 days, the preservation address is Wuhan university of Chinese, and the preservation address is Chlorella (Chlorella sorokiniana) ZM-5.
The separation and culture process of the chlorella oleaginous ZM-5 strain is as follows:
1. enabling the PRECI SCS single cell sorter to achieve a sterile state; taking a constructed wetland water body near a pig farm around Harbin in Heilongjiang province of China as a sample, shaking uniformly, taking 5 μl, placing on a sorting chip, shooting a microscopic imaging image of the sample before sorting by adopting a PRECI SCS single cell sorter, positioning single cells of target microalgae according to algae morphology in microscopic imaging, sorting, collecting the microalgae single cells, and shooting a microscopic imaging image after sorting;
2. transferring the collected microalgae single cells into a cell culture tube of a sterilized BG-11 liquid culture medium containing 3g/L glucose, placing the cell culture tube in a shaking incubator for culture to obtain a turbid light green culture solution, transferring the culture solution into a sterilized sterile conical flask of the BG-11 liquid culture medium containing 3g/L glucose, placing the culture solution in the shaking incubator for expansion culture, transferring the algae solution which is successfully subjected to expansion culture into a BG-11 solid culture medium for plate dilution coating, and screening the single algae fall;
wherein the BG-11 liquid medium comprises the following components: naNO 3 1.5g/L，K 2 HPO 4 ·3H 2 O 0.04g/L，MgSO 4 ·7H 2 O 0.075g/L，CaCl 2 ·2H 2 O 0.036g/L，C 6 H 8 O 7 0.006g/L，C 6 H 8 FeNO 7 0.006g/L，EDTA 0.001g/L，Na 2 CO 3 0.02g/L，H 3 BO 3 0.00286g/L，MnCl 2 ·H 2 O 0.00181g/L，ZnSO 4 ·7H 2 O 0.000222g/L， CuSO 4 ·5H 2 O 0.000079g/L，Na 2 MoO 4 ·2H 2 O 0.00039g/L，Co(NO 3 ) 2 ·6H 2 O0.000049 g/L and the balance of distilled water;
the culture conditions are as follows: the light is irradiated for 24 hours every day, the light intensity is 6000lux, the culture temperature is 30+/-2 ℃, the shaking speed is 180r/min, and the BG-11 liquid culture medium is supplemented every four days.
In the first embodiment, the pre-sorting sample is photographed by a presi SCS single-cell sorter to obtain a microscopic image and a microscopic image after sorting, see parts a and B in fig. 1, respectively, which are visual imaging of microalgae single cells and single-cell sorting.
In the second step of this embodiment, the obtained single algae is selected, and the microscopic morphology is observed under an optical microscope: visually observing morphological characteristics of the single algae, wherein the algae are green regular round and smooth in surface; the algal morphology was observed under a 100X light microscope to appear as single cells, regular spherical, light green, and was initially identified as Chlorella (FIG. 2).
Screening the obtained single algae in the second step of the implementation mode, picking the single algae in the BG-11 solid medium after three generations of purification, and carrying out algae species identification by adopting an 18S rRNA sequencing technology:
(1) Microalgae DNA was extracted using a bacterial genomic DNA extraction kit (tiangen biochemical technologies limited);
(2) Amplifying an 18S rRNA gene from total DNA of microalgae by Polymerase Chain Reaction (PCR), and amplifying a target algae-like 18S sequence by using 18S rRNA universal primers 18SF (5'-CCTGGTTGATCCTGCCAG-3') and 18SR (5'-TTGATCCTTCTGCAGGTTCA-3'); the PCR products were sequenced by Beijing Liuhua Dada Gene technology Co., ltd (Dalian China) (see SEQ ID No: 3); comparing the sequences to sequences available in the NCBI database using the BLAST program;
(3) The PCR reaction system is as follows: 1ng of template DNA, 15 mu L of Taq enzyme mixture, 1 mu L of primer 18SF,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, and cycle for 35 times, extension at 72℃for 10min.
The 18S rRNA universal primers 18SF (5'-CCTGGTTGATCCTGCCAG-3') and 18SR (5'-TTGATCCTTCTGCAGGTTCA-3') are adopted to amplify the 18S sequence of the target algae sample, and then the high-throughput sequencing technology is used for sequencing, so as to carry out molecular identification; finally, judging the species level taxonomy information of the target alga sample based on a molecular biology means by NCBI database comparison.
The identification result shows (see fig. 3): the registration number of the 18SrRNA sequence GenBank is MZ557824, the similarity with Chlorella sorokiniana sequences reaches 99.47%, and the parent relationship is nearest; named chlorella (Chlorella sorokiniana) ZM-5.
Test 1: growth of Chlorella ZM-5: inoculating chlorella ZM-5 into pig raising waste water and water distribution, and sampling every 2 hours; measuring absorbance at 600nm wavelength 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 raising wastewater (COD=400 mg/L) is 40h, inoculating for 4h, and entering into logarithmic growth phase for 22h; after 26 hours, the stable phase is started for 8 hours.
Test 2: physicochemical properties of Chlorella ZM-5:
(1) And (3) oil and fat measurement: sucking 20ml of algae liquid which is successfully cultured by expansion into a centrifuge tube by using a pipetting gun; centrifuging at 8000r/min for 10min, and discarding supernatant; repeatedly cleaning with deionized water for 3 times, and collecting algae samples; adding 2ml chloroform and 4ml methanol into the algae sample, uniformly mixing, and performing ultrasonic crushing for 10min (ultrasonic conditions: power 400w, work 5s and intermittent 2 s); centrifuging for 10min at 4500r/min, collecting 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 algae sample turns white; weighing a blank test tube, marking the weight as m1, sucking the lower liquid after standing in the weighed test tube, and evaporating the lower liquid in a water bath at 65 ℃ until the lower liquid reaches constant weight; weighing the test tube after being evaporated to dryness, and recording the weight of the test tube as m2; the grease content is m2-m1.
(2) Polysaccharide assay: freeze drying microalgae cells to obtain algae powder; weighing 0.1g of algae powder, and adding 4% NaOH according to a ratio of 1:25 to uniformly mix; extracting with 80w ultrasound for 20min, and then ice-bathing for 10min, and repeating the extraction for 3 times; water-bathing for 2h in a water bath kettle at 80 ℃, cooling, centrifuging for 10min at a rotating speed of 4800r/min, and taking supernatant; washing with deionized water for 2 times, and mixing the supernatants; adding three times of absolute ethyl alcohol into the supernatant, uniformly mixing, and standing in a refrigerator at 4 ℃ overnight; after freeze centrifugation, the pellet was taken and 3% TCA was added to the pellet until the pellet was no longer dissolved. Filtering with 0.45um filter membrane, adding three times volume of absolute ethanol again to precipitate supernatant; centrifuging at 4deg.C, collecting precipitate, and lyophilizing to obtain crude polysaccharide powder. Dissolving the crude polysaccharide powder with deionized water; 1mL of the solution was taken in a test tube, and 4mL of anthrone-sulfuric acid solution was added. After mixing, the mixture was boiled for 10min, and after ice bath, the absorbance was measured at 620 nm.
(3) Protein yield determination: microalgae cells were diluted with 1 XPBS and subjected to the procedure of Bradford method protein quantitative detection kit (China biological engineering Co., ltd.).
The measurement result shows (see figure 5), the grease in the chlorella ZM-5 product accounts for 40.29 percent, the polysaccharide accounts for 22.04 percent, and the protein accounts for 37.67 percent; therefore, the chlorella ZM-5 is a microalgae producing grease.
The second embodiment is as follows: the application of the chlorella oleaginous ZM-5 in the embodiment is as follows: it is applied to purifying and treating pig raising waste water.
And a third specific embodiment: this embodiment differs from the second embodiment in the process of the application: centrifuging the algae liquid which is successfully subjected to the expansion culture for 10min at 8000r/min, discarding the supernatant, inoculating into pig raising wastewater and water distribution, and culturing for 40h at 30 ℃ at 400r/min to obtain the finished product; the chlorella ZM-5The inoculation amount of the feed is 0.01g/ml pig raising wastewater for water distribution; the pig raising wastewater contains the following components (g/L): 0.0620464g/L NaCl and CaCl 2 ·2H 2 O 0.234872g/L， MgSO 4 0.151662g/L，NH 4 Cl 1.019244g/L，K 2 HPO 4 0.05239g/L，NaNO 3 0.16014g/L， C 6 H 12 O 6 0.3768g/L; the water quality index (mg/L) of the pig raising wastewater is as follows: COD 400mg/L; TN 9.315mg/L; TP 293.147mg/L. Other steps and parameters are the same as in the first embodiment.
Test 3: chlorella ZM-5 degrades pig raising waste water:
centrifuging the algae liquid which is successfully subjected to the expansion culture for 10min at 8000r/min, discarding the supernatant, inoculating the supernatant into pig raising wastewater water distribution (the inoculation amount is 0.01g/ml of pig raising wastewater water distribution), and sampling every 4 hours. Respectively adopting a potassium dichromate-sulfuric acid method to measure the COD content of the pig raising wastewater; determining TP content of the pig raising wastewater by adopting a molybdenum-antimony anti-spectrophotometry; and determining TN content of the pig raising wastewater by adopting an alkaline potassium persulfate method.
The measurement results showed (see fig. 6): the degradation rates of the chlorella ZM-5 on the pig raising wastewater COD, TN and TP are 86.66%, 88.75% and 84.59% respectively.
Sequence list
<110> university of northeast agriculture, university of Harbin industry, company of Changchun long light English biosciences instruments Co., ltd
<120> Chlorella oleaginous ZM-5 and application thereof
<160> 3
<210> 1
<211> 18
<212> DNA
<223> Artificial sequence
<220>
<223> nucleotide sequence of primer 18SF
<400> 1
cctggttgat cctgccag 18
<210> 2
<211>20
<212> DNA
<223> Artificial sequence
<220>
<223> nucleotide sequence of primer 18SR
<400> 2
ttgatccttc tgcaggttca 20
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<211> 1712
<212> 18S rRNA
<223> Chlorella 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 oleaginous chlorella ZM-5, which is characterized in that it has been deposited in China center for type culture Collection with accession number: CCTCC No. M20211015, the preservation time is 2021, 8 and 10, the preservation address is university of Wuhan in Wuhan, and the classification is named asChlorella sorokiniana。
2. The use of a Chlorella oleaginous ZM-5 strain according to claim 1 for purifying waste water from pig farming.
3. The use of a Chlorella oleaginous ZM-5 strain according to claim 2The method is characterized by comprising the following application processes: centrifuging the algae liquid which is successfully subjected to the expansion culture for 10min at 8000r/min, discarding the supernatant, inoculating into pig raising wastewater and water distribution, and culturing for 40h at 30 ℃ at 400r/min to obtain the finished product; the inoculation amount of the chlorella ZM-5 is 0.01g/ml, and water distribution is carried out on the pig raising wastewater; the pig raising wastewater contains the following components (g/L): naCl 0.0620464g/L, caCl 2 ·2H 2 O 0.234872 g/L，MgSO 4 0.151662 g/L，NH 4 Cl 1.019244 g/L，K 2 HPO 4 0.05239 g/L，NaNO 3 0.16014 g/L，C 6 H 12 O 6 0.3768g/L; the water quality index (mg/L) of the pig raising wastewater is as follows: COD 400mg/L; TN 9.315mg/L; TP 293.147mg/L.
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