CN104593265B - One plant height oil-producing scenedesmus and its screening, cultural method and application - Google Patents

One plant height oil-producing scenedesmus and its screening, cultural method and application Download PDF

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CN104593265B
CN104593265B CN201510047493.9A CN201510047493A CN104593265B CN 104593265 B CN104593265 B CN 104593265B CN 201510047493 A CN201510047493 A CN 201510047493A CN 104593265 B CN104593265 B CN 104593265B
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scenedesmus
sdec
algae
sanitary sewage
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裴海燕
韩琳
张硕
宋明明
韩飞
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Abstract

The invention discloses a plant height oil-producing scenedesmus and its screening and its cultural method and application.The algae obtained from Polluted water area separation is identified through the analysis of 18S rDNA sequence and microscopic morphology, determines that the algae is Scenedesmus (Scenedesmus sp.) member, Serial No. KJ413176.Algae strain is named as scenedesmus SDEC-12 (Scenedesmus bijuga SDEC-12), is preserved in China typical culture collection center, deposit number CCTCC NO:M 2014497 in October, 2014.Using the sanitary sewage manually prepared as substitutive medium, the grease yield and lipid-producing of scenedesmus SDEC-12 is all significantly higher than more common BG-11 culture medium, and show that the scenedesmus SDEC-12 through sanitary sewage culture has the characteristics that therefore better oxidation stability and low temperature flow are more suitable for the raw material as biodiesel according to the fatty acid methyl ester component analysis of extraction.The nitrogen and phosphorous nutrient absorbed in sanitary sewage while scenedesmus SDEC-12 is grown realizes being effectively treated for sanitary sewage, therefore has both economy and environmental benefit using sanitary sewage as substitutive medium.

Description

One plant height oil-producing scenedesmus and its screening, cultural method and application
Technical field
The invention belongs to technical field of microalga biology, in particular to a plant height oil-producing scenedesmus and its screening, cultural method and Using.
Background technique
With the fast development of economy and society, consumption and need of the mankind for fossil fuels such as coal, petroleum, natural gases The amount of asking is increasing, however such fossil fuel is all non-renewable.According to countries in the world proven oil reserves in 2009 Investigation result is shown: world oil total amount total 1342.2 × 109Bucket, available 40 years, however the petroleum total amount of China is only Have 16 × 109Bucket, world rankings 13, therefore it is extremely urgent to find reproducible substitution new energy.In recent years microalgae oil-producing at For the research hotspot of various countries, microalgae has that environmental suitability is strong, cell proliferation is fast, growth as the raw material of production biodiesel Period is short, photosynthetic efficiency is high, carbon sequestration capacity is strong, is not take up the advantages that agriculture plantation.However, due to the grease of part microalgae Content is not high, this two large problems at high cost of microdisk electrode constrain microalgae oil-producing and commercially produce on a large scale.Therefore, it screens Oil-rich microalgae improves grease yield, reduces the toxigenic capacity of microalgae, and the biomass yield and lipid-producing for improving microalgae, which become, grinds The hot spot and focus studied carefully.
Early in the renewable energy laboratory of the U.S. in 1978 initiate water biological species project in from ConUS and summer prestige Isolated more than 3000 plants of microalgaes in each water body on smooth island, and then it is screened out from it the microalgae with high yield oil property.It is close Domestic some scholars separation screening oil-rich microalgae from local waters over year.Microalgae is many kinds of, widely distributed, survival ability By force, good basis is provided for screening algae strain.
Research shows that therefore, researcher proposes containing nutriments such as nitrogen phosphorus required for algal grown in sanitary sewage This concept of micro-algae culture medium culture microalgae oil-producing is substituted with sanitary sewage.Yang of TaiWan, China et al. has screened local sea Foreign microalgae handles sanitary sewage, and the grease finally obtained contains the docosahexaenoic acid (Docosahexaenoic of higher concentration Acid, DHA) it is polybasic unsaturated fatty acid (C there are six double bond22H32O2), it is a kind of Ω -3 essential fatty acid, is commonly called as The important element that " docosapentaenoic acid " is nervous system cell growth and is maintained.Tsinghua University Hu Hongying et al. is also lived using second level The scenedesmus (Scenedesmus bijuga sp.LX1) that sewage culture is sieved, research shows that the scenedesmus has preferable oil and fat accumulation Characteristic and nitrogen phosphorus ligands ability.However and not all microalgae can be grown in sanitary sewage, sieved from local water body Microalgae has better environmental suitability and high yield grease performance.The microalgae sieved can absorb the battalion such as nitrogen phosphorus in sanitary sewage It supports the simultaneously synthesizing Self substances of substance and promotes oil and fat accumulation, the content for not only reducing dirty water pollutant enables sewage to reach Mark discharge, greatly reduces the cost of microdisk electrode；And improve grease yield.Therefore sanitary sewage culture microalgae oil-producing this Concept had not only had environmental benefit but also had had economic benefit.
Summary of the invention
The present invention reduces microdisk electrode cost as foothold and provides a kind of high yield oil fence algae to improve microalgae grease yield Screening and its cultural method and application are screened environment and adapted to by flat band method and 96 well plate methods from Jinan City locality pond, lake Property strong oil-rich microalgae, algae is cultivated using sanitary sewage and obtains high biomass and grease yield with this, realizes waste training Support the organic unity of energy microalgae and energy microalgae processing waste water.
To solve the above problems, the present invention is achieved by the following scheme:
One plant height oil-producing scenedesmus SDEC-12, which is characterized in that be Scenedesmus bijuga SDEC-12, preservation Number is CCTCC NO:M 2014497, and the deposit date is on October 19th, 2014, depositary institution was Chinese Typical Representative culture guarantor Hiding center.This plant of scenedesmus SDEC-12 can in the sanitary sewage of human configuration fast-growth and efficient accumulation grease.
Above-mentioned high yield oil fence algae SDEC-12, nucleotide sequence include nucleosides shown in SEQ ID No.KJ413176 Acid sequence.
Above-mentioned high yield oil fence algae SDEC-12 culture medium is sanitary sewage, initial water quality parameter (mg/L) are as follows: CODcr: 340-400,NH4+- N:10-15, TN:70-80, TP:5-7, between 8.1-8.7, the algae cell density after inoculation is pH value 0.35~0.1g/L.
The application of above-mentioned high yield oil fence algae SDEC-12 culture medium, the culture medium culture high yield oil fence algae SDEC- 12, obtain high yield oil fence algae SDEC-12 algae powder.The fat content 27.5% of the high yield oil fence algae SDEC-12 algae powder, grease produce Rate is 7.88mg/L/d.
The present invention also provides a kind of cultural methods for cultivating above-mentioned high yield oil fence algae SDEC-12, include the following steps:
1) scenedesmus SDEC-12 enrichment culture in BG-11 culture medium, BG-11 composition and dosage are NaNO31500mg·L-1, K2HPO422.5~84.3mgL-1, MgSO4·7H2O 75mg·L-1, CaCl2·2H2O 36mg·L-1, citric acid 6mg L-1, ferric citrate 6mgL-1, EDTANa21mg·L-1, Na2CO320mg·L-1, H3BO32.86mg·L-1, MnCl2·4H2O 1.86mg·L-1, ZnSO4·7H2O 0.22mg·L-1, Na2MoO4·2H2O 0.39mg·L-1, CuSO4·5H2O 0.08mg·L-1, Co (NO3)2·6H2O 0.05mg·L-1, initial pH value is between 6.9-7.5；
2) the scenedesmus SDEC-12 after enrichment culture is inoculated in the sewage manually prepared, is placed in the light incubator Culture, condition of culture is illuminance: 4000lux, temperature: 25 ± 1 DEG C, full exposure；Each ingredient in the sewage manually prepared It is soluble starch: 0.1g/L, urea: 0.075g/L, (NH with dosage4)2SO4: 0.15g/L, K2HPO4 .2H2O:0.04g/L, MgSO4 .7H2O:0.075g/L, NaHCO3: 0.30g/L and 6mL liquid microelement；The liquid microelement includes 0.75g/L Na2EDTA, 97.0mg/L FeCl3 .6H2O, 41.0mg/L MnCl2 .4H2O, 5.0mg/L ZnCl2 .6H2O, 2.0mg/L CoCl2 .6H2O, 4.0mg/L Na2MoO4 .2H2O；
3) until scenedesmus SDEC-12 is harvested by centrifugation after growing to stationary phase, the algal gel after harvest be freeze-dried to get The dry algae powder arrived.
Purposes of the above-mentioned high yield oil fence algae SDEC-12 in processing sewage and/or exhaust gas.
Further, the processing is purification.
Further, the sewage is industrial wastewater or city domestic sewage, and the exhaust gas is industrial waste gas.
Purposes of the above-mentioned high yield oil fence algae SDEC-12 in preparation biodiesel and/or grease.
Beneficial effects of the present invention
The present invention sieves to obtain one plant of advantage rich oil scenedesmus SDEC-12 (Scenedesmus in local polluted water body Bijuga SDEC-12), this plant of algae can well adapt to the microenvironment in sanitary sewage and be passed through using the nutrient in sewage Scale evaluation continuously obtains grease-contained biomass.Scenedesmus SDEC-12 of the invention is in the sanitary sewage manually prepared Middle growth is rapidly and acquisition is higher than the biomass in BG-11 culture medium, has relatively good application prospect.In addition grid of the invention Algae SDEC-12 (Scenedesmus bijuga SDEC-12) oil and fat accumulation characteristic with higher, fat content 27.5% Higher than its fat content 18.67% in BG-11 culture medium, lipid-producing is 7.88mg/L/d ratio BG-11 (1.46mg/L/ D) 5 times in are more.
Detailed description of the invention
The position in phylogenetic relationship tree Fig. 1: scenedesmus SDEC-12.
Fig. 2: scenedesmus SDEC-12 micromorphology photo
Wherein, (a) is light micrograph (× 1000), (b) is transmission electron micrograph (× 30000).C, Ye Lv Body；L, grease；N, nucleus；S, amylum body；V, vacuole；W, cell wall.
Growth curve in Fig. 3: the scenedesmus SDEC-12 sanitary sewage prepared in BG-11 culture medium and manually.
Grease yield and grease in Fig. 4: the scenedesmus SDEC-12 sanitary sewage prepared in BG-11 culture medium and manually produce Rate.
Fig. 5: scenedesmus SDEC-12 to nitrogen phosphorus ligands situation in the sanitary sewage manually prepared.
Specific embodiment
The present invention will be further explained by way of examples below, but does not therefore limit the present invention to the reality It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, is conventionally selected with condition It selects.
The separation of 1 scenedesmus SDEC-12 of embodiment is identified:
One, sample acquires
On December 11st, 2012 reflects Xue Hu (contaminated lake) acquisition water sample from Shandong University Building.
Two, scenedesmus SDEC-12 is isolated and purified
Purification procedures: being added BG-11 culture medium and expand culture after the water sample centrifugal concentrating fetched, and about 7 days It is counted under the microscope by blood cell plate counting method afterwards, algae solution density is then diluted to by about 4000/mL according to count number.? On ultra-clean experimental bench, configured good semisolid culturemedium (BG-11 culture solution+0.5-0.6% agar) inverted plate (has been put Set overnight, no varied bacteria growing), the algae solution after dilution is sprayed on the plate marked respectively using laryngeal sprayer, so After cover, be placed in illumination box and cultivate more than ten days, carry out microscope inspection to grow algae strain bacterium colony on culture medium.Pure algae strain It is grafted directly in the conical flask equipped with 100mL BG-11 culture solution and by sterilizing with the oese sterilized, uses gauze And sterile film sealing, it sets and cultivates in the light incubator.Impure algae strain is further isolated and purified using improved 96 orifice plate: being filled Point shaking culture medium, as to be as far as possible separated into frustule group at condensate unicellular for frustule.Hemocytometer is used under the microscope Number plate is counted and (is counted again after need to diluting when concentration is big), according to counted algae cell density, culture solution is diluted to every 50 μ L and is contained 0.9 frustule unit.After mixing well, 50 μ L culture solutions are successively drawn with liquid-transfering gun in 96 well culture plate holes, Mei Gepei It supports in hole and is placed with fresh BG-11 culture solution (200 μ L) in advance, capping is sealed culture plate with sealed membrane, prevents from evaporating. Cultivation plate hole is cultivated in illumination box, after 6~12h, is examined under inverted microscope and marks slender hilum.To When the slender hilum inner cell of label increases to 500 or more or color occurs, switching can be carried out and expand culture.Select unicellular life Its culture is transferred in the culture bottle of the fresh culture equipped with 200mL with liquid-transfering gun and is expanded culture by long hole.It obtains The pure algae strain obtained is inoculated into sewage to investigate whether be resistant to sanitary sewage, finally obtains one plant of pure algae strain by screening, is named as SDEC-12。
Three, the identification of scenedesmus SDEC-12
1. the Morphological Identification of scenedesmus SDEC-12
The algae of scenedesmus SDEC-12 falls morphological feature: after cultivating on culture medium flat plate, green algae can be observed and fall, in circle It is shape, neat in edge, smooth, glossy.
The morphological feature of scenedesmus SDEC-12: frustule shows green under an optical microscope, and coenobium is flat, by 2-4 Unicellular composition, it is stingless.Group's cell straight line arranged side by side forms a line；Cell is oval or long oval, form meet scenedesmus Belong to feature.Cell colony length and width depend on growth period, and group's width of 2 cells is 9-13 μm, a length of 10-18 μm.Transmission electricity Sem observation contains an individual chloroplaset the results show that each frustule is interior, includes an apparent albumen in chloroplaset Core.It can find that many amylum bodies not of uniform size and drip of grease illustrate that algae strain can be used as Lipid-producing or polysaccharide in cell The raw material of class compound.Ultrastructure such as Fig. 2；
2, the molecular biology identification of scenedesmus SDEC-12
The frustule of logarithmic growth phase is collected by centrifugation, extracts genome DNA using kit, 18S rDNA is carried out to it Amplification.PCR amplification system is 50 μ L, including 25 μ L of Taq PCR Master Mix,
1 μ L of DNA sample, 2 μ L of upstream primer (10 μM), 2 20 μ L of μ L, ddH2O of downstream primer (10 μM).PCR amplification program Are as follows: 94 DEG C of initial denaturation 1min；94 DEG C of denaturation 1min, 55 DEG C of renaturation 1min, 72 DEG C of extension 1.5min repeat 30 circulations；72℃ Extend 10min.
Upstream and downstream primer is respectively as follows: CTGGTTGATCCTGCCAG (18S-F) and TTGATCCTTCTGCAGGTTCA (18S- R)。
Pcr amplification product transfers to Sangon Biotech (Shanghai) Co., Ltd. to be sequenced.Pcr amplification product is 18S The partial sequence of rDNA, length are 967bp sequencing result as shown in SEQ ID No.KJ413176.
According to sequencing result, utilize what is found in the related softwares such as Clustalx1.83, Mega 6 and GenBank database Homologous sequence is compared, and uses NJ (neighbor-joining) method phylogenetic tree construction (see Fig. 1).Scenedesmus The Query coverage value of SDEC-12 and Scenedesmus abundans (X73995.1) reach 99%, Max ident value It is 99%.Scenedesmus SDEC-12 evolutionary degree and Scenedesmus were closest before this simultaneously for phylogenetic tree.It reflects in conjunction with microscopic morphology It is fixed, tentatively assert that it, to scenedesmus, is named as Scenedesmus bijuga SDEC-12 to be double.
Growth characteristics of the 2 scenedesmus SDEC-12 of embodiment in the sanitary sewage of human configuration
Scenedesmus SDEC-12 is compared in BG-11 culture medium and the growth characteristics in the sewage manually prepared.
(1) culture medium used in is BG-11 and the sanitary sewage manually prepared
BG-11 culture medium and for example preceding statement of the sanitary sewage ingredient of human configuration, two kinds of culture mediums pass through high pressure sterilization.
(2) growing state of the scenedesmus SDEC-12 in two kinds of culture mediums
Scenedesmus SDEC-12 is by expanding culture with Initial seeding density as 106A/mL is inoculated in BG-11 culture medium respectively In (control group) and the sanitary sewage manually prepared (experimental group), it is subsequently placed in illumination box and cultivates, cultivation temperature 25 ± 1 DEG C, full exposure, intensity of illumination 4000lux.The daily water quality indicator that carries out includes ammonia nitrogen, total nitrogen, the monitoring of total phosphorus and pH value. Incubation pH value first increases, and the four or five day when can reach 11 and then be gradually reduced, pH value measurement display scenedesmus SDEC-12 tolerance High ph-values can survive in slight alkali environment.
The result shows that: scenedesmus SDEC-12 is shown in Fig. 3 in the growth curve of two kinds of culture mediums.Scenedesmus SDEC-12 is trained in BG-11 Growth cycle in nutrient solution is longer, maximum biomass concentration 0.29gL-1, and the growth week in the sanitary sewage of human configuration Phase, shorter only 9 days maximum biomass concentration was 0.45gL-1.Therefore, scenedesmus SDEC-12 is in the sanitary sewage manually prepared With better growth characteristics.
The oil-producing characteristic of 3 scenedesmus SDEC-12 of embodiment
One, the acquisition of scenedesmus SDEC-12 algae powder
According to the condition of embodiment 2 and culture medium culture scenedesmus SDEC-12, until algae, which grows to stationary phase, carries out centrifugation receipts It obtains, the algal gel of harvest obtains dry algae powder as processing in freeze-dryer.
Two, the growth of scenedesmus SDEC-12 and grease yield determine
1. scenedesmus SDEC-12 biomass estimation
By the monitoring daily to frustule biomass concentration, after frustule growth phase enters stationary phase to algae solution into Row is harvested by centrifugation.The volume of record harvest algae solution, contains algae solution with 50mL centrifuge tube, is put into large capacity refrigerated centrifuge and carries out Centrifugation, removal supernatant retain bed mud, obtain algae powder in 60 DEG C of low temperature dryings.Biomass calculates such as equation (1):
In formula: m-algae dry weight, g；
V-algae solution volume, L.
2. the measurement of scenedesmus SDEC-12 fat content
0.1g dry algae powder is weighed in 50mL centrifuge tube, 10mL chloroform/methanol (2:1) solution is added, uses Ultrasonic Cell Disruptor Broken 10min, then 4000r/min is centrifugated 10min, retains organic phase, organic phase is transferred in 60mL separatory funnel, Entire extraction process is repeated twice.According to organic phase volume of separation, 0.9% sodium chloride solution (1:5) is added, is sufficiently shaken up 1min, and stratification 15min, recycling lower layer's grease extract solution and measure its volume, take 5mL recycling solution in 10mL glass In test tube, with being dried with nitrogen, glass tube is placed in 80 DEG C of baking ovens and is dried to weight (about 30 minutes).Equation calculation grease contains Amount:
In formula: LW-is based on the fat content under dry weight, g/g；
m1- algae dried bean noodles weight, g；
m0- 10mL glass tube dry weight, g；
m2- 10mL glass tube the dry weight with grease, g；
The volume of the low phase grease of V-, mL.
As a result as shown in figure 4, acquired results of the present invention are grease of the scenedesmus SDEC-12 in the sanitary sewage manually prepared Yield is up to 27.5%, and much higher than acquisition grease yield 18.7% in BG-11 culture medium, and lipid-producing (7.88mg/L/d) is compared 5 times of BG-11 (1.46mg/L/d) are more.Illustrate that sanitary sewage is more conducive to the oil of scenedesmus SDEC-12 as substitutive medium Rouge accumulates
3. scenedesmus SDEC-12 Fatty Acid Methyl Esters are analyzed
It extracts fatty acid methyl ester method: taking 0.1g algae powder in centrifuge tube, it is molten that the NaOH methanol that 2mL concentration is 5% is added Liquid or methanolic solution place it in thermostat water bath 60 DEG C after water-bath 1 hour, cooling.Add into the solution after reaction Enter n-hexane 1mL, after standing 30min, extract upper solution 100uL and be placed in clean reagent bottle, then by 25uL internal standard compound (2mg/L, methyl margarate) is added in reagent bottle, to be measured.
Fatty acid methyl ester is measured using gas chromatograph-mass spectrometer (GC-MS), using full scan mode, scanning range 50- 300amu.Parameter setting is as follows, chromatographic column: VF-23ms, using the method for temperature programming: 150 DEG C of reservation 1min, then with 1 ° C/min is warming up to 165 DEG C；Injector temperature: 220 DEG C.
Fatty acid component of the 1 scenedesmus SDEC-12 of table in BG-11 and sanitary sewage
SFA=saturated fatty acid (14:0,16:0)；MUFA=monounsaturated fatty acids (16:1,18:1)；PUFA=is mostly not Saturated fatty acid (18:2,18:3)
The result shows that (such as table 1) scenedesmus SDEC-12 does not have essentially comprising C16:0, C16:1 and C18:1 fatty acid Other complicated ingredients, and C16 and C18 system content is up to 92%, wherein C16:0 is main constituent.C16:0 is living Content is up to 60% after cultivating in sewage, higher than its content in BG-11.Furthermore content of the C18:1 in sanitary sewage It is significantly higher than its content in BG-11, it was reported that monounsaturated fatty acids (MUFA) includes that C16:0 and C18:0 has preferably Oxidation stability and low temperature flow, therefore be more suitable for doing biodiesel.It follows that being made with the sanitary sewage manually prepared Do not have an impact oil quality for the substitutive medium of scenedesmus SDEC-12, is more suitable for the raw material as biodiesel on the contrary.
Removal situation of the 4 scenedesmus SDEC-12 of embodiment for nitrogen phosphorus in sanitary sewage
For detecting its water quality indicator situation of change daily in the incubation of scenedesmus SDEC-12, to investigate it to nitrogen phosphorus Removal rate.
1. monitoring method: daily synchronization takes algae solution 20mL to be centrifuged 10min under the revolving speed of 4000r/min, after centrifugation Supernatant be respectively intended to after 0.45 μm of membrane filtration measurement concentration of nitrogen and phosphorus.The detection of ammonia nitrogen uses Nessler's reagent photometer (HJ 535-2009), determination of total nitrogen content is using alkaline chitinase resolution ultraviolet spectrophotometry (HJ636-2012), the survey of total phosphorus Surely ammonium molybdate spectrophotometric method (GB 11893-89) is used.
2. monitoring result: daily concentration of nitrogen and phosphorus is as shown in Figure 5, the results showed that scenedesmus SDEC-12 for phosphorus removal compared with Fastly, the 4th day when total phosphorus concentration less than 1mg/L, the removal rate of phosphorus is close to 100%.Removal of the scenedesmus SDEC-12 for ammonia nitrogen Rate is up to 62%, and the removal rate of total nitrogen only has 27%, illustrates the main ammonia nitrogen that absorbs of scenedesmus SDEC-12 growth as nitrogen source.
Above-mentioned, although the foregoing specific embodiments of the present invention is described with reference to the accompanying drawings, not protects model to the present invention The limitation enclosed, those skilled in the art should understand that, based on the technical solutions of the present invention, those skilled in the art are not Need to make the creative labor the various modifications or changes that can be made still within protection scope of the present invention.

Claims (6)

1. a plant height oil-producing scenedesmus SDEC-12, which is characterized in that its deposit number is CCTCC NO:M2014497, preservation date For on October 19th, 2014, depositary institution was China typical culture collection center.
2. high yield oil fence algae SDEC-12 according to claim 1, nucleotide sequence includes shown in SEQ ID No.1 Nucleotide sequence.
3. purposes of the high yield oil fence algae SDEC-12 described in claim 1 in processing sewage.
4. purposes according to claim 3, which is characterized in that the processing is purification.
5. purposes according to claim 3 or 4, which is characterized in that the sewage is industrial wastewater or city domestic sewage.
6. purposes of the high yield oil fence algae SDEC-12 described in claim 1 in preparation biodiesel and/or grease.
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CN105002089A (en) * 2015-06-30 2015-10-28 上海交通大学 Culture system for microalgae energy efficiency optimization and method thereof
CN107177505B (en) * 2016-03-11 2020-11-10 中国石油化工股份有限公司 Scenedesmus as well as culture method and application thereof
CN107140741B (en) * 2017-05-18 2020-02-11 西北农林科技大学 Method for removing salt in brackish water and producing oil by using scenedesmus obliquus
CN108359609B (en) * 2018-03-15 2020-12-15 石家庄君乐宝乳业有限公司 High-survivability chlorella CV-6, and screening method and application thereof
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CN113214996B (en) * 2021-05-18 2023-03-24 武汉理工大学 Scenedesmus as well as culture method and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130164322A1 (en) * 2011-12-22 2013-06-27 Department Of Veterans Affairs Cultured extremophilic algae species native to new mexico
CN103923838A (en) * 2014-04-28 2014-07-16 黑龙江省科学院大庆分院 Scendesmus quadricauda with high oil content and construction method thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130164322A1 (en) * 2011-12-22 2013-06-27 Department Of Veterans Affairs Cultured extremophilic algae species native to new mexico
CN103923838A (en) * 2014-04-28 2014-07-16 黑龙江省科学院大庆分院 Scendesmus quadricauda with high oil content and construction method thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Identification and characterization of a freshwater microalga Scenedesmus SDEC-8 for nutrient removal and biodiesel production;第129-135页;《Bioresource Technology》;20140403;第162卷;第129-135页，尤其是第130页左栏2.1和2.2 *
Lin Han等.Nutrient removal and lipid accumulation properties of newly isolated microalgal strains.《Bioresource Technology》.2014,第165卷 *
Nutrient removal and lipid accumulation properties of newly isolated microalgal strains;Lin Han等;《Bioresource Technology》;20140831;第165卷;第38-41页，尤其是摘要，第2（39）页第2.2节、第3.1节，第3（40）页第3.5节第1段、图1、表1、右边栏4-6行、第3.5节第1段、第4节，第4（41）页表2 *
栅藻的优化培养及脂质提取方法研究;岳龙等;《核农学报》;20121231;第26卷(第6期);第0894-0899页 *

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