CN103468578A - Chlorella strain for synchronously treating municipal sewage and implementing grease accumulation - Google Patents
Chlorella strain for synchronously treating municipal sewage and implementing grease accumulation Download PDFInfo
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- CN103468578A CN103468578A CN2013104757800A CN201310475780A CN103468578A CN 103468578 A CN103468578 A CN 103468578A CN 2013104757800 A CN2013104757800 A CN 2013104757800A CN 201310475780 A CN201310475780 A CN 201310475780A CN 103468578 A CN103468578 A CN 103468578A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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Abstract
The invention relates to Chlorella, particularly a Chlorella strain for synchronously treating municipal sewage and implementing grease accumulation. The invention aims to solve the problems of poor stress tolerance, low alga cell grease content, poor decontamination capacity, difficulty in degrading carbon source substances in sewage, and requirement of adding exogenous nutrient substances for growth in sewage in the coupling process of a microalga biodiesel production system and a municipal sewage treatment system. The Chlorella sp. PF2 is collected at China General Microbiological Culture Collection Center, the collection address is Beichenxi Road No.1 Yard 3, Chaoyang District, Beijing City, the collection date is July 30th, 2013, and the collection number is CGMCC No.8014. The Chlorella sp. PF2 has strong anti-pollution capacity, and can grow and reproduce in common nonsterile municipal sewage by using carbon, nitrogen, phosphorus and other pollutants as nutrient substances while accumulating grease.
Description
Technical field
The present invention relates to a kind of chlorella.
Background technology
In the face of increasingly serious fossil energy and the challenge of environmental pollution, utilize micro-algae production biofuel more and more to come into one's own, this is because micro-algae biofuel is renewable energy source, has an energy conversion efficiency high, the advantage that microalgae grease content is high and rate of propagation is fast.In addition, micro-algae production of biodiesel process does not increase greenhouse gas emission, is conducive to environment protection.But the high production cost of micro-algae biofuel has limited its large-scale production and has been widely used, the nutritive substances such as carbon nitrogen phosphorus source that wherein micro algae growth needs are the important component parts of micro-algae production of biodiesel cost.Contain abundant carbon nitrogen and phosphorus pollutants in municipal wastewater, a large amount of discharges of these materials have caused white elephant to environment.Therefore, if micro-algae biodiesel manufacturing system and the coupling of municipal wastewater treatment system can be become the pollutents such as carbon nitrogen phosphorus in sewage to the nutrition of micro-algae production of biodiesel, can reduce the production cost of micro-algae biofuel, can realize waste water reclaiming, environmental contamination reduction again.Run in sewage disposal and micro-algae biodiesel manufacturing system coupling process that algae kind resistance is poor, fat content is low, the problem of the ability of depolluting, substrate utilization ability.Therefore, obtain adaptive capacity to environment strong, can in sewage, stablize high-speed rapid growth, fat content higher, depollute the algae kind effective for realizing that sewage disposal resource utilization and the commercialization of micro-algae production of biodiesel are significant.
Summary of the invention
The present invention be to solve that the algae kind resistance run in micro-algae biodiesel manufacturing system and municipal wastewater treatment system coupling process is poor, frustule fat content is low, the carbon source material in the ability of depolluting, hard degradation sewage and the problem that growth needs to add the exogenous nutrition material in sewage, the chlorella that a strain synchronous processing municipal wastewater is provided and has realized oil and fat accumulation.
The present invention's one strain synchronous processing municipal wastewater and the chlorella of realizing oil and fat accumulation, it is chlorella (Chlorella sp.) PF2, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, the preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date is on July 30th, 2013, and preserving number is CGMCC No.8014.
Chlorella of the present invention (Chlorella sp.) PF2, unicellular, spherical, the children is elliposoidal often, cell walls is thin.The long diameter 4-12 of cell μ m.The cell green, the chromatoplast cup-shaped, account for the cell major part, pyrenoids of tool.
The chlorella that the present invention separates a strain synchronous processing municipal wastewater and realizes oil and fat accumulation from wetland park lake, outbeach, West, the Sun Island, Chinese Harbin City, Heilongjiang Province, the phylogenetic tree that small subunit ribosome gene order 18S rDNA based on the nucleus coding builds, algae kind PF-2 and Chlorella (Chlorella) sibship is close, is positioned on same evolutionary branching.Through order-checking, sample 18S rDNA clip size to be identified is 1121bp.Data in this sequence and NCBI Genebank database are carried out to sequence homology and compare, find that the sibship of this algae strain and Chlorella (Chlorella) and Micractinium pusillum (Micratinium) is nearest.According to microscopic examination and 18srRNA molecular sequences analysis-by-synthesis, judge that micro-algae PF2 is a kind of chlorella (Chlorella sp.), be under the jurisdiction of Chlorophyta (Chlorophyta), Chlorophyceae (Chlorophyceae), Chlorococcale (Chlorococcales), chlorella section (Chlorellaceae), Chlorella (Chlorella), final decision PF2 is a kind of chlorella (Chlorella sp.).
Chlorella of the present invention (Chlorella sp.) PF2, have a contamination resistance strong, can in unsterilised general municipal effluent, utilize the pollutents such as its carbon, nitrogen, phosphorus for the nutritive substance growth and breeding and can accumulate the advantage of grease.Municipal wastewater is after Chlorella sp.PF-2 processes, the pollutants removal rates such as COD, nitrogen, phosphorus can reach respectively 80%, 65%, more than 97%, COD, nitrogen, tp removal rate best result Bie Keda 90%, 90%, 100%, reach " urban wastewater treatment firm pollutant emission standard " (GB8978-2002).Simultaneously, this chlorella can change into bio-oil by the carbon source pollutent in sewage, and the fat content of frustule reaches more than 20%, has realized the recycling treatment of municipal wastewater.
The present invention relates to chlorella (Chlorella sp.) PF2, it is under the jurisdiction of Chlorophyta (Chlorophyta), Chlorophyceae (Chlorophyceae), Chlorococcale (Chlorococcales), chlorella section (Chlorellaceae), Chlorella (Chlorella).Be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, the preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and preservation date is on July 30th, 2013, and preserving number is CGMCC No.8014.
The accompanying drawing explanation
Fig. 1 is that chlorella PF2 cellular form of the present invention is amplified the Photomicrograph of 400 times;
Fig. 2 is that chlorella PF2 cellular form of the present invention is amplified the Photomicrograph of 400 times;
Fig. 3 is the maximum brief tree gene constructed based on chlorella PF218S rDNA of the present invention.
Embodiment
Embodiment one: present embodiment one strain synchronous processing municipal wastewater and the chlorella of realizing oil and fat accumulation, it is chlorella (Chlorella sp.) PF2, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, the preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date is on July 30th, 2013, and preserving number is CGMCC No.8014.
The chlorella of present embodiment (Chlorella sp.) PF2, unicellular, spherical, the children is elliposoidal often, cell walls is thin.The long diameter 4-12 of cell μ m.The cell green, the chromatoplast cup-shaped, account for the cell major part, has a pyrenoids (as depicted in figs. 1 and 2).
The chlorella of present embodiment (Chlorella sp.) PF2, have a contamination resistance strong, can in unsterilised general municipal effluent, utilize the pollutents such as its carbon, nitrogen, phosphorus for the nutritive substance growth and breeding and can accumulate the advantage of grease.Municipal wastewater is after Chlorella sp.PF2 processes, the pollutants removal rates such as COD, nitrogen, phosphorus can reach respectively 80%, 65%, more than 97%, COD, nitrogen, tp removal rate best result Bie Keda 90%, 90%, 100%, reach " urban wastewater treatment firm pollutant emission standard " (GB8978-2002).Simultaneously, this chlorella can change into bio-oil by the carbon source pollutent in sewage, and the fat content of frustule reaches more than 20%, has realized the recycling treatment of municipal wastewater.
Present embodiment relates to chlorella (Chlorella sp) PF2, and it is under the jurisdiction of Chlorophyta (Chlorophyta), Chlorophyceae (Chlorophyceae), Chlorococcale (Chlorococcales), chlorella section (Chlorellaceae), Chlorella (Chlorella).Be deposited in Chinese common micro-organisms culture presevation administrative center, the preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and preservation date is on August 7th, 2013, and preserving number is CGMCC No.8014.
Embodiment two: the chlorella of present embodiment (Chlorella sp.) PF2 screens and obtains from wetland park lake, outbeach, West, the Sun Island, Harbin, Heilungkiang.Screening is carried out according to the following steps:
(1) carefully gather the planktonic microalgae in water with aseptic beaker, the water sample of collection is preserved with the clear-glass bottle that aseptic culture medium is housed, and vial is put into the vacuum flask that ice cube is housed, and then brings laboratory into and carries out separation and purification in 24 hours.
(2) getting appropriate algae source adds in the triangular flask that fills sterilizing BG11 substratum, add a small amount of granulated glass sphere simultaneously, be placed in concussion 24h left and right on shaking table, static 30min left and right after taking out, draw middle level liquid 50mL, add in the triangular flask that fills the aseptic BG11 substratum in 150mL left and right, be placed in the constant temperature illumination box and cultivate, every 4h rocks once, once shakes 2min.
(3) get the enrichment algae liquid of 1mL, with sterilized water, dilute successively 10
4, 10
5, 10
6doubly, then draw 0.5ml, splash in the aseptic flat board that contains solid BG11 substratum and be coated with, be placed in the constant temperature illumination box and cultivate.Wait to grow obvious visible algae and fall behind, fall with transfering loop picking list algae, access aseptic flat board and carry out three rides, being placed in the illumination constant incubator cultivates, wait to grow obvious visible algae and fall, access in the triangular flask of aseptic orienting enriching cultivation, be placed in illumination box and cultivate.Repeat above-mentioned three rides-liquid nutrient medium-tri-ride step 3-6 time, the think pure strain consistent with cellular form under microscope until the algae on flat board falls, after further confirming with 18S rDNA, just can obtain single pure strain.To the algae kind obtained, it is cultivated in sewage, measure its grease productive rate, algae density and pollutant removal situation, repeatedly measure until stable, select the best algae kind of grease productive rate as purpose algae kind.
The chlorella PF-2 that screening is obtained carries out Molecular Identification, carries out according to the following steps:
One, adopt CTAB (Cetyltrimethylammonium bromide) method to extract the algae genome, and, with reference to " Molecular systematics ", concrete grammar is as follows:
Get 10ml algae liquid, the centrifugal 10min of 8000rpm, add 100 μ L TE, the vibration suspension cell, add again 800 μ L lysates and 0.8 μ L beta-mercaptoethanol, fully vibration mixes rapidly, be placed in 65 ℃ of temperature and bathe 30min, put upside down back and forth therebetween for several times, , add again 700 μ L chloroforms, fully mix, the centrifugal 5min of 12000rpm, after centrifugal, supernatant is transferred in new centrifuge tube, add 700uL binding buffer liquid, fully mix, join in centrifugal adsorbing column and go again, place 1min, the centrifugal 1min of 12000rpm, outwell the waste liquid in collection tube, add again 700 μ L lavation buffer solutions, the centrifugal 1min of 12000rpm, repeated washing 3 times, the last centrifugal 2min of 12000rpm, fully remove lavation buffer solution, take out centrifugal adsorbing column, it is inserted in to a clean 1.5mL centrifuge tube, and add 50 μ L elution buffers, after placing 1min, in the centrifugal 1min of 12000rpm, take out centrifugal adsorbing column, get 10 μ L electrophoresis.
Two, sequence amplification and mensuration
Primer sequence for pcr amplification:
18SF：：5′-GTCAGAGGTGAAATTCTTGGATTTA-3′
18SR：5′-AGGGCAGGGACGTAATCAACG-3′
50 μ L18S rDNA PCR reaction systems comprise: 10 * PCR buffer, 0.2mM dNTP, 2.5mM Mg2+, 1U Taq enzyme, template DNA, forward and reverse primer, ddH2O.Response procedures is: 95 ℃ of sex change 5min, and then by 95 ℃ of 30s, 55 ℃ of 30s, 34 circulations of 72 ℃ of 1min programs, last 72 ℃ are extended 10min.The PCR product is electrophoresis in 1.5% agarose gel.After the PCR product is purified, by Hua Da genome company, checked order, through order-checking, sample 18S rDNA clip size to be identified is that 1118bp(is as shown in sequence table Seq ID No:3).Data in this sequence and NCBI Genebank database are carried out to sequence homology and compare, find that the sibship of this algae strain and Chlorella (Chlorella) and Micractinium pusillum (Micratinium) is nearest.
Three, growing tree builds
Utilize the Blast function, resulting sample 18S rDNA sequence and GenBank database are compared.Download the sequence of the upper similar algae of GenBank, utilize Clustal X to compare to gene order, be aided with the manual correction of SEAVIEW, adopt MEGA5 to build morphological development tree (as shown in Figure 3), the phylogeny position of analytic sample, algae kind PF-2 and Chlorella (Chlorella) sibship is close, is positioned on same evolutionary branching.
Chlorella (Chlorella sp.) PF2 of present embodiment is processed to the functional checking of municipal wastewater and oil and fat accumulation:
Test 1, chlorella (Chlorella sp.) PF2 is inoculated in the municipal wastewater of the sterilized that pH is 7.5, COD content is 351mg/L, TN content 33mg/L, TP content 4.0mg/L, initial inoculum is 5*10
5individual/ml makes the nutrient growth of algae kind in utilizing sewage under source of artificial light irradiates, and culture condition is: Light To Dark Ratio 12h:12h, light intensity 2000lux, stir speed (S.S.) 150r/min, aeration speed 200ml/min, 25 ℃ of temperature.Cultivate after 8 days, adopt centrifugal method to collect micro-algae, discharge and process rear water.After processing, COD, the TN of sewage, the clearance of TP are respectively 76.1%, 65.2%, 98.0%, reach " urban wastewater treatment firm pollutant emission standard " (GB8978-2002) secondary discharge standard.The microalgae grease content of collecting is 20.0%.
Test 3, chlorella (Chlorella sp.) PF2 are better than the processing to sterilizing sewage to COD, nitrogen, the phosphorus removal effect of unpasteurized municipal wastewater, but fat content is a little less than sterilizing sewage.Embodiment as: chlorella Chlorella sp.PF2 is inoculated into respectively in the sewage that sterilizing and unsterilised, pH are 7.5, COD content is 310mg/L, TN content 33mg/L, TP content 4.0mg/L, and initial inoculum is 1*10
6individual/ml makes the nutrient growth of algae kind in utilizing sewage under source of artificial light irradiates, and culture condition is: Light To Dark Ratio 12h:12h, light intensity 1800lux, stir speed (S.S.) 150r/min, aeration speed 200ml/min, 25 ℃ of temperature.Cultivate after 8 days, adopt centrifugal method to collect micro-algae, discharge and process rear water.After treatment, the clearance of COD, TN, TP is respectively 75.0%, 61.6%, 96.4% to unpasteurized sewage, and the fat content of micro-algae of collection is 20%; After treatment, the clearance of COD, TN, TP is respectively 65.6%, 47.4%, 94.6% to the sewage of sterilizing, and the fat content of micro-algae of collection is 22%.
Claims (1)
1. a strain synchronous processing municipal wastewater and the chlorella of realizing oil and fat accumulation, it is characterized in that it is chlorella (Chlorella sp.) PF2, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, the preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date is on July 30th, 2013, and preserving number is CGMCC No.8014.
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| CN103966272A (en) * | 2014-05-05 | 2014-08-06 | 北京航空航天大学 | Method for purifying sewage and accumulating grease by using microalgae |
| CN105733951A (en) * | 2014-12-10 | 2016-07-06 | 中国农业大学 | A chlorella strain producing grease and applications thereof |
| CN109576160A (en) * | 2017-09-29 | 2019-04-05 | 武汉藻优生物科技有限公司 | A kind of energy removes chlorella W3 and its application of the heavy metal in high content of beary metal water body |
| CN109971647A (en) * | 2017-12-28 | 2019-07-05 | 深圳华大生命科学研究院 | Chlorella and application thereof and preparation method |
| CN110157622A (en) * | 2019-05-15 | 2019-08-23 | 中国辐射防护研究院 | A kind of screening technique of the chlorella algae strain for production of biodiesel |
| CN110387332A (en) * | 2019-07-09 | 2019-10-29 | 天津大学 | It is a kind of to be accumulated using artificial municipal wastewater culture chlorella and extract the research method of protein |
| CN112457992A (en) * | 2020-09-21 | 2021-03-09 | 中国科学院烟台海岸带研究所 | Chlorella mutant strain, culture method thereof and application thereof in livestock and poultry breeding wastewater treatment |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105733951A (en) * | 2014-12-10 | 2016-07-06 | 中国农业大学 | A chlorella strain producing grease and applications thereof |
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| CN109576160A (en) * | 2017-09-29 | 2019-04-05 | 武汉藻优生物科技有限公司 | A kind of energy removes chlorella W3 and its application of the heavy metal in high content of beary metal water body |
| CN109576160B (en) * | 2017-09-29 | 2021-08-31 | 武汉藻优生物科技有限公司 | Chlorella W3 capable of removing heavy metals in water with high heavy metal content and application thereof |
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| CN110157622A (en) * | 2019-05-15 | 2019-08-23 | 中国辐射防护研究院 | A kind of screening technique of the chlorella algae strain for production of biodiesel |
| CN110387332A (en) * | 2019-07-09 | 2019-10-29 | 天津大学 | It is a kind of to be accumulated using artificial municipal wastewater culture chlorella and extract the research method of protein |
| CN112457992A (en) * | 2020-09-21 | 2021-03-09 | 中国科学院烟台海岸带研究所 | Chlorella mutant strain, culture method thereof and application thereof in livestock and poultry breeding wastewater treatment |
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