CN103468578B - 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 PDF

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CN103468578B
CN103468578B CN201310475780.0A CN201310475780A CN103468578B CN 103468578 B CN103468578 B CN 103468578B CN 201310475780 A CN201310475780 A CN 201310475780A CN 103468578 B CN103468578 B CN 103468578B
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chlorella
sewage
municipal sewage
grease
collection
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CN103468578A (en
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于艳玲
冯玉杰
李姗姗
张大伟
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Harbin Institute of Technology
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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

One strain synchronous processing municipal wastewater and the chlorella realizing oil and fat accumulation
Technical field
The present invention relates to a kind of chlorella.
Background technology
In the face of the challenge of increasingly serious fossil energy and environmental pollution, micro-algae production biofuel is utilized more and more to come into one's own, this is because microalgae biodiesel is renewable energy source, there is energy conversion efficiency high, the high and advantage that rate of propagation is fast of microalgae grease content.In addition, microalgae biodiesel production process does not increase greenhouse gas emission, is conducive to environment protection.But the high production cost of microalgae biodiesel limits its large-scale production and widely uses, wherein the nutritive substance such as the carbon nitrogen phosphorus source that needs of micro algae growth is the important component part of microalgae biodiesel production cost.Containing abundant carbon nitrogen and phosphorus pollutants in municipal wastewater, a large amount of discharges of these materials cause white elephant to environment.Therefore, if microalgae biodiesel production system can be coupled with municipal sewage system, the pollutents such as the carbon nitrogen phosphorus in sewage be become the nutrition that microalgae biodiesel is produced, the production cost of microalgae biodiesel can be reduced, can waste water reclaiming be realized again, reduce environmental pollution.In sewage disposal and microalgae biodiesel production system coupling process, encounter that algae kind resistance is poor, fat content is low, the problem of ability of depolluting, substrate utilization ability.Therefore, obtain adaptive capacity to environment strong, can stablize in sewage high-speed rapid growth, fat content higher, depollute algae kind effective for realizing sewage disposal resource utilization and the microalgae biodiesel commercialization of production is significant.
Summary of the invention
The present invention will solve that the algae kind resistance run in microalgae biodiesel production system and municipal sewage system coupling process is poor, frustule fat content is low, ability of depolluting, is difficult to the carbon source material in degradation of sewage and in sewage, grows the problem need adding exogenous nutrition material, provides a strain synchronous processing municipal wastewater and realizes the chlorella of oil and fat accumulation.
The present invention one strain synchronous processing municipal wastewater and the chlorella 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, 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, children is elliposoidal often, and cell walls is thin.The long diameter 4-12 μm of cell.Cell is green, and chromatoplast cup-shaped, accounts for cell major part, tool pyrenoids.
The present invention separates a strain synchronous processing municipal wastewater and realizes the chlorella of oil and fat accumulation from wetland park lake, outbeach, West, the Sun Island, Chinese Harbin City, Heilongjiang Province, based on the phylogenetic tree that the small subunit ribosome gene order 18S rDNA of nucleus coding builds, algae kind PF-2 and Chlorella (Chlorella) sibship closely, are 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 sequence homology comparison, finds that the sibship of this algae strain and Chlorella (Chlorella) and Micractinium pusillum (Micratinium) is nearest.Comprehensively analyze according to microscopic examination and 18srRNA molecular sequences, 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 judgement PF2 is a kind of chlorella (Chlorella sp.).
Chlorella of the present invention (Chlorella sp.) PF2, has contamination resistance strong, the pollutents such as its carbon, nitrogen, phosphorus can be utilized for nutritive substance growth and breeding and can accumulate the advantage of grease in unsterilised general municipal effluent.Municipal wastewater is after Chlorella sp.PF-2 process, the pollutants removal rates such as COD, nitrogen, phosphorus can reach 80%, 65%, more than 97% respectively, COD, nitrogen, tp removal rate best result Bie Keda 90%, 90%, 100%, reach " urban wastewater treatment firm pollutant emission standard " (GB8978-2002).Meanwhile, the carbon source contamination transform in sewage can be become bio-oil by this chlorella, and the fat content of frustule reaches more than 20%, achieves 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, 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.
Accompanying drawing explanation
Fig. 1 is the Photomicrograph that chlorella PF2 cellular form of the present invention amplifies 400 times;
Fig. 2 is the Photomicrograph that chlorella PF2 cellular form of the present invention amplifies 400 times;
Fig. 3 is based on the gene constructed maximum brief tree of chlorella PF218S rDNA of the present invention.
Embodiment
Embodiment one: present embodiment one strain synchronous processing municipal wastewater and the chlorella 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, 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 (Chlorella sp.) PF2 of present embodiment, unicellular, spherical, children is elliposoidal often, and cell walls is thin.The long diameter 4-12 μm of cell.Cell is green, and chromatoplast cup-shaped, accounts for cell major part, has a pyrenoids (as depicted in figs. 1 and 2).
Chlorella (Chlorella sp.) PF2 of present embodiment, has contamination resistance strong, the pollutents such as its carbon, nitrogen, phosphorus can be utilized for nutritive substance growth and breeding and can accumulate the advantage of grease in unsterilised general municipal effluent.Municipal wastewater is after Chlorella sp.PF2 process, the pollutants removal rates such as COD, nitrogen, phosphorus can reach 80%, 65%, more than 97% respectively, COD, nitrogen, tp removal rate best result Bie Keda 90%, 90%, 100%, reach " urban wastewater treatment firm pollutant emission standard " (GB8978-2002).Meanwhile, the carbon source contamination transform in sewage can be become bio-oil by this chlorella, and the fat content of frustule reaches more than 20%, achieves 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 China General Microbiological culture presevation administrative center, 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: chlorella (Chlorella sp.) PF2 of present embodiment 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 sterile beaker, the water sample of the collection clear-glass bottle that aseptic culture medium is housed are preserved, and the vacuum flask that ice cube is housed put into by vial, then bring laboratory in 24 hours and carry out separation and purification.
(2) getting appropriate algae source adds in the triangular flask filling sterilizing BG11 substratum, add a small amount of granulated glass sphere simultaneously, be placed on shaking table and shake about 24h, static about 30min after taking out, draw middle level liquid 50mL, add in the triangular flask filling the aseptic BG11 substratum of about 150mL, be placed in constant temperature illumination box and cultivate, every 4h rocks once, once shakes 2min.
(3) get the enrichment algae liquid of 1mL, dilute 10 successively with sterilized water 4, 10 5, 10 6doubly, then draw 0.5ml, instill aseptic being coated with containing in the flat board of solid BG11 substratum, be placed in constant temperature illumination box and cultivate.Wait that growing obvious visible algae falls behind, and falls with transfering loop picking list algae, accesses aseptic flat board and carry out three rides, be placed in illumination constant incubator to cultivate, wait that growing obvious visible algae falls, 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, until the algae on flat board falls and unanimously with the cellular form under microscope thinks pure strain, after confirming with 18S rDNA further, just can obtain single pure strain.To the algae kind obtained, it cultivated in sewage, measure its grease productive rate, algae density and pollutant removal situation, repeatedly measure until stable, the algae kind selecting grease productive rate best is as object algae kind.
Carrying out Molecular Identification to screening the chlorella PF-2 obtained, carrying out according to the following steps:
One, adopt CTAB (Cetyltrimethylammonium bromide) method to extract algal gene group, 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, vibration suspension cell, add 800 μ L lysates and 0.8 μ L beta-mercaptoethanol again, rapid mixing of fully vibrating, be placed in 65 DEG C of temperature bath 30min, put upside down for several times therebetween back and forth, , add 700 μ L chloroforms again, abundant mixing, the centrifugal 5min of 12000rpm, supernatant is transferred in new centrifuge tube after centrifugal, add 700uL binding buffer liquid, fully mix, join in centrifugal adsorbing column again and go, place 1min, the centrifugal 1min of 12000rpm, outwell the waste liquid in collection tube, add 700 μ L lavation buffer solutions again, the centrifugal 1min of 12000rpm, repeated washing 3 times, the centrifugal 2min of last 12000rpm, abundant removing lavation buffer solution, take out centrifugal adsorbing column, be inserted in 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 DEG C of sex change 5min, and then by 95 DEG C of 30s, 55 DEG C of 30s, 34 circulations of 72 DEG C of 1min programs, last 72 DEG C extend 10min.PCR primer is electrophoresis in the agarose gel of 1.5%.Checked order by Hua Da genome company after PCR primer is purified, through order-checking, sample 18S rDNA clip size to be identified is for 1118bp(is as shown in sequence table Seq ID No:3).Data in this sequence and NCBI Genebank database are carried out sequence homology comparison, finds that the sibship of this algae strain and Chlorella (Chlorella) and Micractinium pusillum (Micratinium) is nearest.
Three, grow tree to build
Utilize Blast function, obtained sample 18S rDNA sequence and GenBank database are compared.Download the sequence of similar algae on GenBank, Clustal X is utilized to compare to gene order, be aided with SEAVIEW manual correction, MEGA5 is adopted to build morphological development tree (as shown in Figure 3), the phylogeny position of analytic sample, algae kind PF-2 and Chlorella (Chlorella) sibship closely, are positioned on same evolutionary branching.
The functional authorization processing municipal wastewater and oil and fat accumulation is carried out to chlorella (Chlorella sp.) PF2 of present embodiment:
Test 1, chlorella (Chlorella sp.) PF2 is inoculated into pH is 7.5, COD content is that in the municipal wastewater of the sterilized of 351mg/L, TN content 33mg/L, TP content 4.0mg/L, initial inoculum is 5*10 5individual/ml, under source of artificial light irradiates, make algae kind utilize the nutrient in sewage to grow, culture condition is: Light To Dark Ratio 12h:12h, light intensity 2000lux, stir speed (S.S.) 150r/min, aeration rate 200ml/min, temperature 25 DEG C.Cultivate after 8 days, adopt centrifugal method to collect micro-algae, discharge the rear water of process.After process, the clearance of COD, TN, TP of sewage is respectively 76.1%, 65.2%, 98.0%, reaches " urban wastewater treatment firm pollutant emission standard " (GB8978-2002) secondary discharge standard.The microalgae grease content collected is 20.0%.
Test 2, chlorella (Chlorella sp.) PF2 is inoculated into pH is 7.5, COD content is that in the sewage of 1100mg/L, TN content 35mg/L, TP content 4.0mg/L, initial inoculum is 1*10 6individual/ml, under source of artificial light irradiates, make algae kind utilize the nutrient in sewage to grow, culture condition is: Light To Dark Ratio 12h:12h, light intensity 1800lux, stir speed (S.S.) 150r/min, aeration rate 200ml/min, temperature 25 DEG C.Cultivate after 10 days, adopt centrifugal method to collect micro-algae, discharge the rear water of process.After process, the clearance of COD, TN, TP of sewage is respectively 85.2%, 88.2%, 99.5%, and the fat content of micro-algae of collection is 22.1%.
Test 3, chlorella (Chlorella sp.) PF2 are better than process to sterilizing sewage to the COD of unpasteurized municipal wastewater, nitrogen, phosphorus ligands effect, but fat content is a little less than sterilizing sewage.Embodiment as: chlorella Chlorella sp.PF2 is inoculated into respectively sterilizing and unsterilised, pH be 7.5, COD content is that in the sewage of 310mg/L, TN content 33mg/L, TP content 4.0mg/L, initial inoculum is 1*10 6individual/ml, under source of artificial light irradiates, make algae kind utilize the nutrient in sewage to grow, culture condition is: Light To Dark Ratio 12h:12h, light intensity 1800lux, stir speed (S.S.) 150r/min, aeration rate 200ml/min, temperature 25 DEG C.Cultivate after 8 days, adopt centrifugal method to collect micro-algae, discharge the rear water of process.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 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, 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
CN105733951B (en) * 2014-12-10 2020-02-18 中国农业大学 Chlorella capable of producing grease and application thereof
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
CN109971647B (en) * 2017-12-28 2022-07-05 深圳华大生命科学研究院 Chlorella and its use 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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CN101565675B (en) * 2009-05-13 2011-05-11 清华大学 Chlorella, cultivating method and application thereof in producing biodiesel
CN102093955B (en) * 2010-12-03 2012-06-27 福建漳州鼎能生物科技有限公司 Chlorella strain and application thereof
CN102586116B (en) * 2011-01-14 2013-06-05 江南大学 Common chlorella as well as culturing method and application thereof
CN102746991B (en) * 2012-07-16 2013-07-03 哈尔滨工业大学 Method for carrying out heterotrophic culture on chlorella with acid-producing wastewater
CN103146582A (en) * 2013-03-25 2013-06-12 哈尔滨工业大学 High-throughput screening method of oil-rich microalgae

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