CN105985989A - Method for producing biodiesel from chlorella - Google Patents

Method for producing biodiesel from chlorella Download PDF

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Publication number
CN105985989A
CN105985989A CN201510050746.8A CN201510050746A CN105985989A CN 105985989 A CN105985989 A CN 105985989A CN 201510050746 A CN201510050746 A CN 201510050746A CN 105985989 A CN105985989 A CN 105985989A
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chlorella
producing biodiesel
oils
light
fats
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孙洪磊
李建忠
齐泮仑
何皓
郑蕾
雪晶
张家仁
孙立明
李萌萌
张佳
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China Petroleum and Natural Gas Co Ltd
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China Petroleum and Natural Gas Co Ltd
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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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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses a method for producing biodiesel from chlorella. The method comprises the following steps: (1) seed culture; (2) seed collection; (3) photoautotrophic cultivation; (4) harvesting of microalgae; and (5) oil extraction. The method provided by the invention uses common chlorella and conditions like mild illumination and light quality, sufficient nutrients, a proper nitrogen-phosphorus ratio and proper temperature and can promote rapid growth of chlorella cells, accumulation of oil and stabilization and improvement of oil quality.

Description

The method producing biodiesel with chlorella
Technical field
The present invention relates to microalgae energy technology, produce high-quality biological diesel oil particularly to a kind of with chlorella Method.
Background technology
Microalgae biodiesel, as the succedaneum of petrifaction diesel, just should meet the use requirement of petrifaction diesel, Its physicochemical property i.e. should be close with petrifaction diesel, its fuel characteristic of guarantee.Weigh biodiesel character Index specifically include that density, dynamic viscosity, flash-point, slip journey, cryogenic property, carbon residue, ash, ten Six alkane values, methanol content, fat content, phosphorus content, glycerol content, glycerol content, iodine number and free-fat Acid etc., wherein evaluate the combustibility of biodiesel with Cetane number.Combustibility is that biodiesel is the heaviest One of quality wanted, and Cetane number is mainly affected by raw material.Additionally, in production of biodiesel raw material The kind of unsaturated fatty acid and quantity have been largely fixed the oxidation stability of biodiesel oil product.So And, microalgae is during cultivating, and its content of fatty acid and distribution easily change with environmental condition and become Change.As growth conditions under, the oils and fats of microalgae accumulation is relatively fewer, and under environmental stress conditions, The accumulation of oils and fats then can be doubled and redoubled.Affect a lot of because have of microalgae grease content, such as chemical factor, Physical factor and the different growth phases etc. of microalgae self.At present, both at home and abroad to microalgae biodiesel research, It is mainly concentrated in improving fat content and how being catalytically converted in biodiesel by oils and fats, and at microalgae Cultivation stage, how on the basis of improving fat content, the quality aspect that simultaneously also can improve oils and fats is several Become blank.
Therefore, how to set about from culture environment and the process aspect of microalgae, design is a kind of produces height with chlorella The method of quality biodiesel, becomes those skilled in the art's problem demanding prompt solution.
Summary of the invention
It is an object of the invention to provide a kind of method producing biodiesel with chlorella so that it is can improve The quality of oils and fats.
For achieving the above object, the present invention provides a kind of method producing biodiesel with chlorella, its step Including:
1) aseptic chlorella seed is inoculated in airtight bioreactor it is enlarged being further cultured for；Light source For LED or fluorescent lamp, intensity of illumination is 8-10Klux, and light quality is white light；Seed culture based component is: Sea salt 24-34g/L, CH3COONa 0.3-0.5g/L, NaNO3200-300mg/L, NaH2PO4 50-100mg/L, Na2EDTA 10mg/L, FeSO4·7H2O 2.5-5.0mg/L, vitamin B1 0.006-0.06mg/L, vitamin B2 0.00005-0.0005mg/L；Cultivating initial pH is 6.0-7.5, ventilation Intensity is 0.2-0.7VVM, CO2Concentration is 0-0.5%, and cultivation temperature is 23-26 DEG C, cultivates 3-5 days, Until cell grows into logarithmic (log) phase；
2) to step 1) the chlorella seed of output is collected；
3) to step 2 in Race-way photobioreactor) the chlorella seed collected carries out light autotrophy cultivation； Light source is fluorescent lamp or LED, and light quality is white light or blue light, and intensity of illumination is 8-10Klux；Light autotrophy Medium component is: sea salt 28-34g/L, NaNO3350-550mg/L, NaH2PO430-80mg/L, Na2EDTA 10-30mg/L, FeSO4·7H2O 2.5-5.0mg/L, CuSO4·5H2O 0.01mg/L, ZnSO4·7H2O 0.03-0.06mg/L, CoCl2·6H2O 0.02-0.05mg/L, MnCl2·4H2O 0.2-0.6mg/L, Na2Mo4·2H2O 0.1-0.3mg/L, vitamin B1 0.006-0.06mg/L, vitamin B2 0.00005-0.0005mg/L, biotin 0.00005-0.0005mg/L, wherein the ratio of nitrogen P elements is 15-25 (N:P=15-25:1)；Cultivating initial pH is 6.0-7.5, and ventilation intensity is 0.5-1.0VVM, CO2Dense Degree is 1.0-2.0%, and cultivation temperature is 22-30 DEG C；
4) to step 3) chlorella that produces gathers；
5) destruction step 4) obtained by chlorella cells, carry out oils and fats extraction.
Wherein, in method one better embodiment producing biodiesel with chlorella of the present invention, Yu Bu Rapid 1), in, the triangle shaking flask that will be equipped with chlorella vulgaris is seeded to aseptic bioreactor, is going out Illumination cultivation in the culture medium of bacterium；Bioreactor is bubble type closed reactor, including CO2Gas bomb, Gas mixing device and gas flowmeter.
Wherein, in method one better embodiment producing biodiesel with chlorella of the present invention, Yu Bu Rapid 2), in, centrifugal or pneumatically supported acquisition technique is used to carry out seed collection.
Wherein, in method one better embodiment producing biodiesel with chlorella of the present invention, Yu Bu Rapid 2), in, under being centrifuged as being 4000-12000rmp at rotating speed, room temperature (20-30 DEG C) is centrifuged 1-10min, After obtaining algae mud, by clean water 2-3 time, more under equal conditions, the centrifugal algae mud obtained, then, The most resuspended with a small amount of clear water, it is the seed of follow-up cultivation.
Wherein, in method one better embodiment producing biodiesel with chlorella of the present invention, Yu Bu Rapid 3), in, inoculum density is in 1.0-2.0 × 107Individual/L.
Wherein, in method one better embodiment producing biodiesel with chlorella of the present invention, Yu Bu Rapid 3) in, being received by ready seed equipped with in the bioreactor of culture medium, this reactor is room Interior open system, including CO2Gas bomb, gas mixing device and gas flowmeter.
Wherein, in method one better embodiment producing biodiesel with chlorella of the present invention, Yu Bu Rapid 3), in, after cultivating the time of one week (one week about 6-8 days, preferable 7 days), step 4 is carried out) Gather.
Wherein, in method one better embodiment producing biodiesel with chlorella of the present invention, Yu Bu Rapid 4), in, with centrifugal, chemical flocculation or aerating collecting algae solution, algae mud is obtained.
Wherein, in method one better embodiment producing biodiesel with chlorella of the present invention, Yu Bu Rapid 4), in, chemical flocculation uses iron sulfate, iron chloride, aluminum sulfate, aluminum chloride or chitosan as chemistry Flocculant.
Wherein, in method one better embodiment producing biodiesel with chlorella of the present invention, Yu Bu Rapid 4), in, chemical flocculation is cultured algae solution to be collected in a reservoir, and in algae solution ratio in container Add the chemical floc of doses (as added FeCl in algae solution320-80mg/L), treat flocculation completely, Incline upper aqueous layer, obtain algae mud.
Wherein, in method one better embodiment producing biodiesel with chlorella of the present invention, Yu Bu Rapid 5), in, use enzyme process, ultrasonic method or liquid nitrogen method to carry out chlorella cells and crush.Wherein, liquid nitrogen method and Enzyme process effect is best.
Wherein, in method one better embodiment producing biodiesel with chlorella of the present invention, Yu Bu Rapid 5) in, enzyme process use enzyme include cellulase, center protein enzyme, alkaline protease, pectase, One or several in wall breaking enzyme, Snailase and lipase are (common with cellulase and protease in enzyme process Crushing effect is best).
Wherein, in method one better embodiment producing biodiesel with chlorella of the present invention, Yu Bu Rapid 5) in, using organic extraction to carry out room temperature oils and fats extraction, organic solvent is the chloroform of equal proportion, first Alcohol and water, or the ether of equal proportion, normal hexane and water (i.e., by volume, chloroform: methanol: water=1: 1:1, or ether: normal hexane: water=1:1:1)；Room temperature during described room temperature oils and fats extracts refers to 20-30 ℃。
Wherein, in method one better embodiment producing biodiesel with chlorella of the present invention, this The bright method producing biodiesel with chlorella, also includes:
Step 6) content of the oils and fats extracted is carried out gas chromatography-mass spectrometry analysis；
Step 7) combustibility of the oils and fats extracted is evaluated.
Wherein, in method one better embodiment producing biodiesel with chlorella of the present invention, Yu Bu Rapid 6), in, GC-MS is used to carry out oils and fats analysis: to take oils and fats and be added in 2mL centrifuge tube, then add successively Enter the normal hexane of 1.5mL and the 0.4mol/L potassium hydroxide methanol solution of 0.2mL；Shake up, stand 10 points About clock；Take in the centrifuge tube of supernatant 0.5mL to 1.5mL, be simultaneously introduced the deionized water of 0.3mL, After shaking up, then under the conditions of 4500rmp, centrifugal 3min；Finally, take appropriate upper strata normal hexane layer, use color Composing pure normal hexane to be diluted, and add anhydrous sodium sulfate, pretreatment terminates, and prepares sample introduction；Chromatostrip Part: Thermo Finnigan TRACE DSQ type gas chromatograph-mass spectrometer (GC-MS), uses DB-5MS hair Capillary column (30m × 0.25mm × 0.25 μm), initial column temperature 50 DEG C, it is warmed up to 250 with 20 DEG C/min DEG C, keeping 2min, injector temperature 200 DEG C, carrier gas is helium, flow rate of carrier gas 1ml/min, Splitless injecting samples, Total run time is 12min, and extracting head is desorbed 5min at injection port at 200 DEG C；Mass Spectrometer Method condition: EI source 70eV, ion source temperature 250 DEG C, transmission line temperature 250 DEG C, sweep limits 50～400aum.
Wherein, in method one better embodiment producing biodiesel with chlorella of the present invention, Yu Bu Rapid 7), in, combustibility evaluation is to evaluate with Cetane number (CN).CN value uses Lapuerta et al. (2009) model that and Tong et al. (2010) is previously mentioned is estimated, and assessment models is as follows:
For satisfied fatty acid:
CN=-107.71+31.126n-2.042n2+0.0499n3
For monounsaturated fatty acid:
CN=109-9.292n+0.354n2
For polyunsaturated fatty acid:
CN=-21.157+ (7.965-1.785db+0.235db2)n-0.099n2
For mixing-in fat acid system:
CN=1.068 ∑ (CNimi)-6.747
Wherein, n represents the amount of carbon atom number of fatty acid, and db represents the quantity of double bond.
Additionally, the ratio of satisfied fatty acid and unsaturated fatty acid is also a performance assessment criteria, represent biological bavin Oil oxidation stability.
For solving the problems of the prior art, the present invention provides a kind of and produces high-quality biological diesel oil with chlorella Method, it is with chlorella for the strain that sets out, use two-step fermentation, by regulate different phase temperature Degree and illumination condition produce the frond raw material of the biodiesel with high-load and high-quality, and it has as follows Technical advantage:
1) present invention sets about in terms of the condition of culture and technique two of microalgae, uses relatively mild illumination and light Matter, sufficient nutrition and the condition such as suitable N/P ratio and suitable temperature, promote the quick of microalgae cell Stable and the improvement of growth, the accumulation of oils and fats and oil quality.
2) present invention is by introducing Cetane number, and the ratio of unsaturated fatty acid and satisfied fatty acid etc. characterizes raw The index of thing diesel quality, cultivates the frond cell of high oil quality, the oil content of regulation and control frond.
3) present invention can not only improve the yield of oils and fats, moreover it is possible to effectively improves the quality of oils and fats, final To the fat content of frustule more than 30%, CN value stabilization more than 50, satisfied fatty acid and unsaturation The ratio of fatty acid at 1.5-2.5, this invention effectively have adjusted from the raw material of source biodiesel yield and Quality, meets microalgae biodiesel industrialized application requirement, is that a kind of preferably production high-quality is micro- The method of algae biodiesel.
Describe the present invention below in conjunction with specific embodiment, but not as a limitation of the invention.
Detailed description of the invention
Below in conjunction with detailed description of the invention, present disclosure is described in detail, but cited embodiment is only For making those skilled in the art be better understood from the present invention, and not as limiting to the invention.
For improving the quality of oils and fats, the present invention provides a kind of method producing biodiesel with chlorella, its step Suddenly include:
1) aseptic chlorella seed is inoculated in airtight bioreactor it is enlarged being further cultured for；Light source For LED or fluorescent lamp, intensity of illumination is 8-10Klux, and light quality is white light；Seed culture based component is: Sea salt 24-34g/L, CH3COONa 0.3-0.5g/L, NaNO3200-300mg/L, NaH2PO4 50-100mg/L, Na2EDTA 10mg/L, FeSO4·7H2O 2.5-5.0mg/L, vitamin B1 0.006-0.06mg/L, vitamin B2 0.00005-0.0005mg/L；Cultivating initial pH is 6.0-7.5, ventilation Intensity is 0.2-0.7VVM, CO2Concentration is 0-0.5%, and cultivation temperature is 23-26 DEG C, cultivates 3-5 days, Until cell grows into logarithmic (log) phase；
2) to step 1) the chlorella seed of output is collected；
3) to step 2 in Race-way photobioreactor) the chlorella seed collected carries out light autotrophy cultivation； Light source is fluorescent lamp or LED, and light quality is white light or blue light, and intensity of illumination is 8-10Klux；Light autotrophy Medium component is: sea salt 28-34g/L, NaNO3350-550mg/L, NaH2PO430-80mg/L, Na2EDTA 10-30mg/L, FeSO4·7H2O 2.5-5.0mg/L, CuSO4·5H2O 0.01mg/L, ZnSO4·7H2O 0.03-0.06mg/L, CoCl2·6H2O 0.02-0.05mg/L, MnCl2·4H2O 0.2-0.6mg/L, Na2Mo4·2H2O 0.1-0.3mg/L, vitamin B1 0.006-0.06mg/L, vitamin B2 0.00005-0.0005mg/L, biotin 0.00005-0.0005mg/L, wherein the ratio of nitrogen P elements is 15-25 (N:P=15-25:1)；Cultivating initial pH is 6.0-7.5, and ventilation intensity is 0.5-1.0VVM, CO2Dense Degree is 1.0-2.0%, and cultivation temperature is 22-30 DEG C；
4) to step 3) chlorella that produces gathers；
5) destruction step 4) obtained by chlorella cells, carry out oils and fats extraction.
Wherein, in method one better embodiment producing biodiesel with chlorella of the present invention, Yu Bu Rapid 1), in, the triangle shaking flask that will be equipped with chlorella vulgaris is seeded to aseptic bioreactor, is going out Illumination cultivation in the culture medium of bacterium；Bioreactor is bubble type closed reactor, including CO2Gas bomb, Gas mixing device and gas flowmeter.
Wherein, in method one better embodiment producing biodiesel with chlorella of the present invention, Yu Bu Rapid 2), in, centrifugal or pneumatically supported acquisition technique is used to carry out seed collection.
Wherein, in method one better embodiment producing biodiesel with chlorella of the present invention, Yu Bu Rapid 2), in, under being centrifuged as being 4000-12000rmp at rotating speed, room temperature (20-30 DEG C) is centrifuged 1-10min, After obtaining algae mud, by clean water 2-3 time, more under equal conditions, the centrifugal algae mud obtained, then, The most resuspended with a small amount of clear water, it is the seed of follow-up cultivation.
Wherein, in method one better embodiment producing biodiesel with chlorella of the present invention, Yu Bu Rapid 3), in, inoculum density is in 1.0-2.0 × 107Individual/L.
Wherein, in method one better embodiment producing biodiesel with chlorella of the present invention, Yu Bu Rapid 3) in, being received by ready seed equipped with in the bioreactor of culture medium, this reactor is room Interior open system, including CO2Gas bomb, gas mixing device and gas flowmeter.
Wherein, in method one better embodiment producing biodiesel with chlorella of the present invention, Yu Bu Rapid 3), in, after cultivating the time of about one week (6-8 days, preferable 7 days), carry out step 4) gather.
Wherein, in method one better embodiment producing biodiesel with chlorella of the present invention, Yu Bu Rapid 4), in, with centrifugal, chemical flocculation or aerating collecting algae solution, algae mud is obtained.
Wherein, in method one better embodiment producing biodiesel with chlorella of the present invention, Yu Bu Rapid 4), in, chemical flocculation uses iron sulfate, iron chloride, aluminum sulfate, aluminum chloride or chitosan as chemistry Flocculant.
Wherein, in method one better embodiment producing biodiesel with chlorella of the present invention, Yu Bu Rapid 4), in, chemical flocculation is cultured algae solution to be collected in a reservoir, and in algae solution ratio in container Add the chemical floc of doses (as added FeCl in algae solution320-80mg/L), treat flocculation completely, Incline upper aqueous layer, obtain algae mud.
Wherein, in method one better embodiment producing biodiesel with chlorella of the present invention, Yu Bu Rapid 5), in, use enzyme process, ultrasonic method or liquid nitrogen method to carry out chlorella cells and crush.Wherein, liquid nitrogen method and Enzyme process effect is best.
Wherein, in method one better embodiment producing biodiesel with chlorella of the present invention, Yu Bu Rapid 5) in, enzyme process use enzyme include cellulase, center protein enzyme, alkaline protease, pectase, One or several in wall breaking enzyme, Snailase and lipase are (common with cellulase and protease in enzyme process Crushing effect is best).
Wherein, in method one better embodiment producing biodiesel with chlorella of the present invention, Yu Bu Rapid 5) in, using organic extraction to carry out room temperature oils and fats extraction, organic solvent is the chloroform of equal proportion, first Alcohol and water, or the ether of equal proportion, normal hexane and water (i.e., by volume, chloroform: methanol: water=1: 1:1, or ether: normal hexane: water=1:1:1)；Room temperature during room temperature oils and fats extracts refers to 20-30 DEG C.
Wherein, in method one better embodiment producing biodiesel with chlorella of the present invention, this The bright method producing biodiesel with chlorella, also includes:
Step 6) content of the oils and fats extracted is carried out gas chromatography-mass spectrometry analysis；
Step 7) combustibility of the oils and fats extracted is evaluated.
Wherein, in method one better embodiment producing biodiesel with chlorella of the present invention, Yu Bu Rapid 6), in, GC-MS is used to carry out oils and fats analysis: to take oils and fats and be added in 2mL centrifuge tube, then add successively Enter the normal hexane of 1.5mL and the 0.4mol/L potassium hydroxide methanol solution of 0.2mL；Shake up, stand 10 points About clock；Take in the centrifuge tube of supernatant 0.5mL to 1.5mL, be simultaneously introduced the deionized water of 0.3mL, After shaking up, then under the conditions of 4500rmp, centrifugal 3min；Finally, take appropriate upper strata normal hexane layer, use color Composing pure normal hexane to be diluted, and add anhydrous sodium sulfate, pretreatment terminates, and prepares sample introduction；Chromatostrip Part: Thermo Finnigan TRACE DSQ type gas chromatograph-mass spectrometer (GC-MS), uses DB-5MS hair Capillary column (30m × 0.25mm × 0.25 μm), initial column temperature 50 DEG C, it is warmed up to 250 with 20 DEG C/min DEG C, keeping 2min, injector temperature 200 DEG C, carrier gas is helium, flow rate of carrier gas 1ml/min, Splitless injecting samples, Total run time is 12min, and extracting head is desorbed 5min at injection port at 200 DEG C；Mass Spectrometer Method condition: EI source 70eV, ion source temperature 250 DEG C, transmission line temperature 250 DEG C, sweep limits 50～400aum.
Wherein, in method one better embodiment producing biodiesel with chlorella of the present invention, Yu Bu Rapid 7), in, combustibility evaluation is to evaluate with Cetane number (CN).CN value uses Lapuerta et al. (2009) model that and Tong et al. (2010) is previously mentioned is estimated, and assessment models is as follows:
For satisfied fatty acid:
CN=-107.71+31.126n-2.042n2+0.0499n3
For monounsaturated fatty acid:
CN=109-9.292n+0.354n2
For polyunsaturated fatty acid:
CN=-21.157+ (7.965-1.785db+0.235db2)n-0.099n2
For mixing-in fat acid system:
CN=1.068 ∑ (CNimi)-6.747
Wherein, n represents the amount of carbon atom number of fatty acid, and db represents the quantity of double bond.
Additionally, the ratio of satisfied fatty acid and unsaturated fatty acid is also a performance assessment criteria, represent biological bavin Oil oxidation stability.
Embodiment a
The present embodiment provides a kind of method producing biodiesel with chlorella, and its step includes:
1) aseptic chlorella seed is inoculated in airtight bioreactor it is enlarged being further cultured for, specifically Step is: the triangle shaking flask that will be equipped with chlorella vulgaris is seeded to aseptic bioreactor, bacterium of having gone out Culture medium in illumination cultivation；Bioreactor is bubble type closed reactor, including CO2Gas bomb, Gas mixing device and gas flowmeter；Light source is LED, and intensity of illumination is 8Klux, and light quality is white light；Kind Sub-medium component is: sea salt 24g/L, CH3COONa 0.3g/L, NaNO3200mg/L, NaH2PO4 50mg/L, Na2EDTA 10mg/L, FeSO4·7H2O 2.5mg/L, vitamin B1 0.006mg/L, Vitamin B2 0.00005mg/L；Cultivating initial pH is 6.0, and ventilation intensity is 0.2VVM, CO2Concentration Being 0%, cultivation temperature is 23 DEG C, cultivates about 3 days, grows into logarithmic (log) phase to cell；
2) to step 1) the chlorella seed of output is collected；Concrete operations are to use centrifugal collection skill Art, under rotating speed is 4000rmp, room temperature (20 DEG C) is centrifuged 1min, after obtaining algae mud, uses clean water 2 times, more under equal conditions, the centrifugal algae mud obtained, then, the most resuspended with a small amount of clear water, i.e. Seed for follow-up cultivation；
3) to step 2 in Race-way photobioreactor) the chlorella seed collected carries out light autotrophy cultivation, Concretely comprise the following steps: being received by ready seed equipped with in the bioreactor of culture medium, this reactor is Indoor open formula system, including CO2Gas bomb, gas mixing device and gas flowmeter；Inoculum density is 1.0 ×107Individual/L；Light source is fluorescent lamp, and light quality is white light, and intensity of illumination is 8Klux；Light autotrophy culture medium becomes It is divided into: sea salt 28g/L, NaNO3350mg/L, NaH2PO430mg/L, Na2EDTA 10mg/L, FeSO4·7H2O 2.5mg/L, CuSO4·5H2O 0.01mg/L, ZnSO4·7H2O 0.03mg/L, CoCl2 ·6H2O 0.02mg/L, MnCl2·4H2O 0.2mg/L, Na2Mo4·2H2O 0.1mg/L, vitamin B1 0.006mg/L, vitamin B2 0.00005mg/L, biotin 0.00005mg/L, wherein nitrogen P elements Ratio be 15 (N:P=15:1)；Cultivating initial pH is 6.0, and ventilation intensity is 0.5VVM, CO2Dense Degree is 1.0%, and cultivation temperature is 22 DEG C；
4) after cultivating time of about 6 days, to step 3) chlorella that produces gathers；Particularly as follows: with Chemical flocculation is gathered algae solution, obtains algae mud；Chemical flocculation is cultured algae solution to be collected in a reservoir, and Chemical floc FeCl is added in algae solution320mg/L, treats flocculation completely, and upper aqueous layer of inclining obtains algae Mud；
5) destruction step 4) obtained by chlorella cells, carry out oils and fats extraction；Crush and concretely comprise the following steps: Use enzyme process to carry out chlorella cells to crush；The enzyme that enzyme process uses includes cellulase and center protein enzyme；Oil Fat extracts and concretely comprises the following steps: using organic extraction to carry out room temperature (20 DEG C) oils and fats and extract, organic solvent is The chloroform of equal proportion, first alcohol and water (i.e., by volume, chloroform: methanol: water=1:1:1)；
6) content of the oils and fats extracted is carried out gas chromatography-mass spectrometry analysis；Concrete operations are as follows: take Oils and fats is added in 2mL centrifuge tube, sequentially adds the normal hexane of 1.5mL and the 0.4mol/L hydrogen of 0.2mL Potassium oxide methanol solution；Shake up, stand about 10 minutes；Take the centrifugal of supernatant 0.5mL to 1.5mL Guan Zhong, is simultaneously introduced the deionized water of 0.3mL, after shaking up, then under the conditions of 4500rmp, and centrifugal 3min； Finally, take appropriate upper strata normal hexane layer, be diluted with chromatographically pure normal hexane, and add anhydrous sodium sulfate, Pretreatment terminates, and prepares sample introduction；Chromatographic condition: Thermo Finnigan TRACE DSQ type gas chromatogram- GC-MS, uses DB-5MS capillary column (30m × 0.25mm × 0.25 μm), initial column temperature 50 DEG C, being warmed up to 250 DEG C with 20 DEG C/min, keep 2min, injector temperature 200 DEG C, carrier gas is helium, carries Gas velocity 1ml/min, Splitless injecting samples, total run time is 12min, extracting head at injection port in 200 5min it is desorbed at DEG C；Mass Spectrometer Method condition: EI source 70eV, ion source temperature 250 DEG C, transmission line temperature 250 DEG C, sweep limits 50～400aum；
7) being evaluated the combustibility of the oils and fats extracted, combustibility evaluation is with Cetane number (CN) evaluate；CN value uses Lapuerta et al. (2009) and Tong et al. (2010) to be previously mentioned Model is estimated, and assessment models is as follows:
For satisfied fatty acid:
CN=-107.71+31.126n-2.042n2+0.0499n3
For monounsaturated fatty acid:
CN=109-9.292n+0.354n2
For polyunsaturated fatty acid:
CN=-21.157+ (7.965-1.785db+0.235db2)n-0.099n2
For mixing-in fat acid system:
CN=1.068 ∑ (CNimi)-6.747
Wherein, n represents the amount of carbon atom number of fatty acid, and db represents the quantity of double bond.
Embodiment b
The present embodiment provides a kind of method producing biodiesel with chlorella, and its step includes:
1) aseptic chlorella seed is inoculated in airtight bioreactor it is enlarged being further cultured for, specifically Step is: the triangle shaking flask that will be equipped with chlorella vulgaris is seeded to aseptic bioreactor, bacterium of having gone out Culture medium in illumination cultivation；Bioreactor is bubble type closed reactor, including CO2Gas bomb, Gas mixing device and gas flowmeter；Light source is fluorescent lamp, and intensity of illumination is 10Klux, and light quality is white light；Kind Sub-medium component is: sea salt 34g/L, CH3COONa 0.5g/L, NaNO3300mg/L, NaH2PO4 100mg/L, Na2EDTA 10mg/L, FeSO4·7H2O 5.0mg/L, vitamin B1 0.06mg/L, Vitamin B2 0.0005mg/L；Cultivating initial pH is 7.5, and ventilation intensity is 0.7VVM, CO2Concentration is 0.5%, cultivation temperature is 26 DEG C, cultivates about 5 days, grows into logarithmic (log) phase to cell；
2) to step 1) the chlorella seed of output is collected；It is specially and uses centrifugal acquisition technique, Under being centrifuged as being 12000rmp at rotating speed, room temperature (30 DEG C) is centrifuged 10min, after obtaining algae mud, uses clear water Clean 3 times, more under equal conditions, the centrifugal algae mud obtained, then, the most resuspended with a small amount of clear water, It is the seed of follow-up cultivation；
3) to step 2 in Race-way photobioreactor) the chlorella seed collected carries out light autotrophy cultivation, Concretely comprise the following steps: being received by ready seed equipped with in the bioreactor of culture medium, this reactor is Indoor open formula system, including CO2Gas bomb, gas mixing device and gas flowmeter；Inoculum density is 2.0 ×107Individual/L；Light source is LED, and light quality is blue light, and intensity of illumination is 10Klux；Light autotrophy culture medium Composition is: sea salt 34g/L, NaNO3550mg/L, NaH2PO480mg/L, Na2EDTA 30mg/L, FeSO4·7H2O 5.0mg/L, CuSO4·5H2O 0.01mg/L, ZnSO4·7H2O 0.06mg/L, CoCl2 ·6H2O 0.05mg/L, MnCl2·4H2O 0.6mg/L, Na2Mo4·2H2O 0.3mg/L, vitamin B1 0.06mg/L, vitamin B2 0.0005mg/L, biotin 0.0005mg/L, the wherein ratio of nitrogen P elements It is 25 (N:P=25:1)；Cultivating initial pH is 7.5, and ventilation intensity is 1.0VVM, CO2Concentration is 2.0%, cultivation temperature is 30 DEG C；
4) after cultivating time of about 8 days, to step 3) chlorella that produces gathers；It is specially to change Learn flocculation to gather algae solution, obtain algae mud；Chemical flocculation is cultured algae solution to be collected in a reservoir, and to Algae solution adds chemical floc FeCl380mg/L, treats flocculation completely, and upper aqueous layer of inclining obtains algae mud；
5) destruction step 4) obtained by chlorella cells, carry out oils and fats extraction；Crush and concretely comprise the following steps: Use enzyme process to carry out chlorella cells to crush；The enzyme that enzyme process uses includes alkaline protease, pectase, breaking cellular wall Enzyme, Snailase and lipase；Oils and fats extracts and concretely comprises the following steps: use organic extraction to carry out room temperature (30 DEG C) Oils and fats extracts, and organic solvent is the ether of equal proportion, normal hexane and water (i.e., by volume, ether: just Hexane: water=1:1:1)；
6) content of the oils and fats extracted is carried out gas chromatography-mass spectrometry analysis；The same embodiment of concrete operations a；
7) being evaluated the combustibility of the oils and fats extracted, combustibility evaluation is with Cetane number (CN) evaluate；Specifically with embodiment a.
Embodiment c
The present embodiment provides a kind of method producing biodiesel with chlorella, and its step includes:
1) aseptic chlorella seed is inoculated in airtight bioreactor it is enlarged being further cultured for, specifically Step is: the triangle shaking flask that will be equipped with chlorella vulgaris is seeded to aseptic bioreactor, bacterium of having gone out Culture medium in illumination cultivation；Bioreactor is bubble type closed reactor, including CO2Gas bomb, Gas mixing device and gas flowmeter；Light source is LED, and intensity of illumination is 9Klux, and light quality is white light；Kind Sub-medium component is: sea salt 28g/L, CH3COONa 0.4g/L, NaNO3240mg/L, NaH2PO4 60mg/L, Na2EDTA 10mg/L, FeSO4·7H2O 3.0mg/L, vitamin B1 0.01mg/L, dimension Raw element B2 0.0001mg/L；Cultivating initial pH is 6.5, and ventilation intensity is 0.4VVM, CO2Concentration is 0.2%, cultivation temperature is 24 DEG C, cultivates 4 days, until cell grows into logarithmic (log) phase；
2) to step 1) the chlorella seed of output is collected；Use pneumatically supported acquisition technique；
3) to step 2 in Race-way photobioreactor) the chlorella seed collected carries out light autotrophy cultivation, Concretely comprise the following steps: being received by ready seed equipped with in the bioreactor of culture medium, this reactor is Indoor open formula system, including CO2Gas bomb, gas mixing device and gas flowmeter；Inoculum density is 1.4 ×107Individual/L；Light source is fluorescent lamp, and light quality is white light, and intensity of illumination is 9Klux；Light autotrophy culture medium becomes It is divided into: sea salt 30g/L, NaNO3400mg/L, NaH2PO440mg/L, Na2EDTA 15mg/L, FeSO4·7H2O 3.0mg/L, CuSO4·5H2O 0.01mg/L, ZnSO4·7H2O 0.04mg/L, CoCl2 ·6H2O 0.03mg/L, MnCl2·4H2O 0.3mg/L, Na2Mo4·2H2O 0.2mg/L, vitamin B1 0.01mg/L, vitamin B2 0.001mg/L, biotin 0.0001mg/L, the wherein ratio of nitrogen P elements It is 18 (N:P=18:1)；Cultivating initial pH is 7.0, and ventilation intensity is 0.7VVM, CO2Concentration is 1.5%, cultivation temperature is 25 DEG C；
4) after cultivating time of about 7 days, to step 3) chlorella that produces gathers；Gather with centrifugal Algae solution, obtains algae mud；
5) destruction step 4) obtained by chlorella cells, carry out oils and fats extraction；Crush and concretely comprise the following steps: Use ultrasonic method to carry out chlorella cells to crush；Oils and fats extracts and concretely comprises the following steps: use organic extraction to carry out Room temperature (22 DEG C) oils and fats extract, organic solvent be the chloroform of equal proportion, first alcohol and water (i.e., by volume, Chloroform: methanol: water=1:1:1)；
6) content of the oils and fats extracted is carried out gas chromatography-mass spectrometry analysis；The same embodiment of concrete operations a；
7) being evaluated the combustibility of the oils and fats extracted, combustibility evaluation is with Cetane number (CN) evaluate；Specifically with embodiment a.
Embodiment d
The present embodiment provides a kind of method producing biodiesel with chlorella, and its step includes:
1) aseptic chlorella seed is inoculated in airtight bioreactor it is enlarged being further cultured for, specifically Step is: the triangle shaking flask that will be equipped with chlorella vulgaris is seeded to aseptic bioreactor, bacterium of having gone out Culture medium in illumination cultivation；Bioreactor is bubble type closed reactor, including CO2Gas bomb, Gas mixing device and gas flowmeter；Light source is fluorescent lamp, and intensity of illumination is 9Klux, and light quality is white light；Kind Sub-medium component is: sea salt 32g/L, CH3COONa 0.4g/L, NaNO3280mg/L, NaH2PO4 80mg/L, Na2EDTA 10mg/L, FeSO4·7H2O 4.0mg/L, vitamin B1 0.03mg/L, dimension Raw element B2 0.0003mg/L；Cultivating initial pH is 7.0, and ventilation intensity is 0.6VVM, CO2Concentration is 0.4%, cultivation temperature is 25 DEG C, cultivates 4 days, until cell grows into logarithmic (log) phase；
2) to step 1) the chlorella seed of output is collected；Use pneumatically supported acquisition technique；
3) to step 2 in Race-way photobioreactor) the chlorella seed collected carries out light autotrophy cultivation, Concretely comprise the following steps: being received by ready seed equipped with in the bioreactor of culture medium, this reactor is Indoor open formula system, including CO2Gas bomb, gas mixing device and gas flowmeter；Inoculum density is 1.8 ×107Individual/L；Light source is LED, and light quality is blue light, and intensity of illumination is 9Klux；Light autotrophy culture medium Composition is: sea salt 32g/L, NaNO3450mg/L, NaH2PO460mg/L, Na2EDTA 25mg/L, FeSO4·7H2O 4.0mg/L, CuSO4·5H2O 0.01mg/L, ZnSO4·7H2O 0.05mg/L, CoCl2 ·6H2O 0.04mg/L, MnCl2·4H2O 0.4mg/L, Na2Mo4·2H2O 0.25mg/L, vitamin B1 0.03mg/L, vitamin B2 0.0003mg/L, biotin 0.0003mg/L, the wherein ratio of nitrogen P elements It is 22 (N:P=22:1)；Cultivating initial pH is 7.2, and ventilation intensity is 0.9VVM, CO2Concentration is 1.8%, cultivation temperature is 28 DEG C；
4) after cultivating time of about 7 days, to step 3) chlorella that produces gathers；With aerating collecting Algae solution, obtains algae mud；
5) destruction step 4) obtained by chlorella cells, carry out oils and fats extraction；Crush and concretely comprise the following steps: Use liquid nitrogen method to carry out chlorella cells to crush；Oils and fats extracts and concretely comprises the following steps: use organic extraction to carry out Room temperature (28 DEG C) oils and fats extract, organic solvent be the ether of equal proportion, normal hexane and water (i.e., by volume Ratio, ether: normal hexane: water=1:1:1)；
6) content of the oils and fats extracted is carried out gas chromatography-mass spectrometry analysis；The same embodiment of concrete operations a；
7) being evaluated the combustibility of the oils and fats extracted, combustibility evaluation is with Cetane number (CN) evaluate；Specifically with embodiment a.
For solving the problems of the prior art, the present invention provides a kind of and produces high-quality biological diesel oil with chlorella Method, it is with chlorella for the strain that sets out, use two-step fermentation, by regulate different phase temperature Degree and illumination condition produce the frond raw material of the biodiesel with high-load and high-quality, and it has as follows Technical advantage:
1) present invention sets about in terms of the condition of culture and technique two of microalgae, uses relatively mild illumination and light Matter, sufficient nutrition and the condition such as suitable N/P ratio and suitable temperature, promote the quick of microalgae cell Stable and the improvement of growth, the accumulation of oils and fats and oil quality.
2) present invention is by introducing Cetane number, and the ratio of unsaturated fatty acid and satisfied fatty acid etc. characterizes raw The index of thing diesel quality, cultivates the frond cell of high oil quality, the oil content of regulation and control frond.
3) present invention can not only improve the yield of oils and fats, moreover it is possible to effectively improves the quality of oils and fats, final To the fat content of frustule more than 30%, CN value stabilization more than 50, satisfied fatty acid and unsaturation The ratio of fatty acid at 1.5-2.5, this invention effectively have adjusted from the raw material of source biodiesel yield and Quality, meets microalgae biodiesel industrialized application requirement, is that a kind of preferably production high-quality is micro- The method of algae biodiesel.
Below in conjunction with embodiment and experimental result data thereof, the present invention is described in detail, to clearly show that it Technique effect.
It addition, all use deionized water conduct when the solvent of used medium is not explicitly labeled in the inventive method Solvent.PH regulator mode in the inventive method: adjust with the sulphuric acid of 1mol/L and the sodium hydroxide of 2mol/L Joint pH.
Embodiment 1:
In the present invention, algae kind is chlorella, and its light autotrophy medium component should be as follows:
Sea salt 28-34g/L, NaNO3350-550mg/L, NaH2PO4 30-80mg/L, Na2EDTA 10-30mg/L, FeSO4·7H2O 2.5-5.0mg/L, CuSO4·5H2O 0.01mg/L, ZnSO4·7H2O 0.03-0.06mg/L, CoCl2·6H2O 0.02-0.05mg/L, MnCl2·4H2O 0.2-0.6mg/L, Na2Mo4 ·2H2O 0.1-0.3mg/L, Vitamin B1 0.006-0.06mg/L, Vitamin B2 0.00005-0.0005mg/L, biotin 0.00005-0.0005mg/L, wherein the ratio of nitrogen P elements is 15-25 Most preferably；
Specific in the present embodiment 1, each step is with embodiment a, difference:
Light autotrophy condition of culture: light quality is white light, intensity of illumination is 9Klux, and initial pH is 6.5, ventilation Intensity is 0.6VVM, CO2Concentration is 1.5%, and cultivation temperature is 25 DEG C.
Final experimental result, the final fat content of chlorella that embodiment 1 is cultivated reaches 33%, and CN value is 51.0。
Embodiment 2:
In the present invention, algae kind is chlorella, and its light autotrophy medium component should be as follows:
Sea salt 28-34g/L, NaNO3350-550mg/L, NaH2PO4 30-80mg/L, Na2EDTA 10-30mg/L, FeSO4·7H2O 2.5-5.0mg/L, CuSO4·5H2O 0.01mg/L, ZnSO4·7H2O 0.03-0.06mg/L, CoCl2·6H2O 0.02-0.05mg/L, MnCl2·4H2O 0.2-0.6mg/L, Na2Mo4 ·2H2O 0.1-0.3mg/L, Vitamin B1 0.006-0.06mg/L, Vitamin B2 0.00005-0.0005mg/L, biotin 0.00005-0.0005mg/L, wherein the ratio of nitrogen P elements is 15-25 Most preferably；
Specific in the present embodiment 2, each step is with embodiment a, difference:
Light autotrophy condition of culture: light quality is blue light, intensity of illumination is 9Klux, and initial pH is 6.5, ventilation Intensity is 0.6VVM, CO2Concentration is 1.5%, and cultivation temperature is 25 DEG C.
Final experimental result, the final fat content of chlorella that embodiment 2 is cultivated reaches 35%, and CN value is 52.5。
Embodiment 3:
In the present invention, algae kind is chlorella, and its light autotrophy medium component should be as follows:
Sea salt 28-34g/L, NaNO3350-550mg/L, NaH2PO4 30-80mg/L, Na2EDTA 10-30mg/L, FeSO4·7H2O 2.5-5.0mg/L, CuSO4·5H2O 0.01mg/L, ZnSO4·7H2O 0.03-0.06mg/L, CoCl2·6H2O 0.02-0.05mg/L, MnCl2·4H2O 0.2-0.6mg/L, Na2Mo4 ·2H2O 0.1-0.3mg/L, Vitamin B1 0.006-0.06mg/L, Vitamin B2 0.00005-0.0005mg/L, biotin 0.00005-0.0005mg/L, wherein the ratio of nitrogen P elements is 15-25 Most preferably；
Specific in the present embodiment 3, each step is with embodiment a, difference:
Light autotrophy condition of culture: light quality is white light, intensity of illumination is 10Klux, and initial pH is 7.0, ventilation Intensity is 1.0VVM, CO2Concentration is 2.0%, and cultivation temperature is 24 DEG C.
Final experimental result, the final fat content of chlorella that embodiment 3 is cultivated reaches 40%, and CN value is 54.0。
Certainly, the present invention also can have other various embodiments, without departing substantially from present invention spirit and the feelings of essence thereof Under condition, those of ordinary skill in the art can make various corresponding change and deformation according to the present invention, but this A little corresponding changes and deformation all should belong to the protection domain of the claims in the present invention.

Claims (10)

1. the method producing biodiesel with chlorella, it is characterised in that its step includes:
1) aseptic chlorella seed is inoculated in airtight bioreactor it is enlarged being further cultured for；Light Source is LED or fluorescent lamp, and intensity of illumination is 8-10Klux, and light quality is white light；Seed culture based component For: sea salt 24-34g/L, CH3COONa 0.3-0.5g/L, NaNO3200-300mg/L, NaH2PO4 50-100mg/L, Na2EDTA 10mg/L, FeSO4·7H2O 2.5-5.0mg/L, vitamin B1 0.006-0.06mg/L, vitamin B2 0.00005-0.0005mg/L；Cultivating initial pH is 6.0-7.5, logical Gas intensity is 0.2-0.7VVM, CO2Concentration is 0-0.5%, and cultivation temperature is 23-26 DEG C, cultivates 3-5 days, Until cell grows into logarithmic (log) phase；
2) to step 1) the chlorella seed of output is collected；
3) to step 2 in Race-way photobioreactor) the chlorella seed collected carries out light autotrophy training Support；Light source is fluorescent lamp or LED, and light quality is white light or blue light, and intensity of illumination is 8-10Klux；Light Autotrophy medium component is: sea salt 28-34g/L, NaNO3350-550mg/L, NaH2PO430-80mg/L, Na2EDTA 10-30mg/L, FeSO4·7H2O 2.5-5.0mg/L, CuSO4·5H2O 0.01mg/L, ZnSO4·7H2O 0.03-0.06mg/L, CoCl2·6H2O 0.02-0.05mg/L, MnCl2·4H2O 0.2-0.6mg/L, Na2Mo4·2H2O 0.1-0.3mg/L, vitaminB10 .006-0.06mg/L, dimension is raw Element B20.00005-0.0005mg/L, biotin 0.00005-0.0005mg/L, wherein the ratio of nitrogen P elements is 15-25；Cultivating initial pH is 6.0-7.5, and ventilation intensity is 0.5-1.0VVM, CO2Concentration is 1.0-2.0%, Cultivation temperature is 22-30 DEG C；
4) to step 3) chlorella that produces gathers；
5) destruction step 4) obtained by chlorella cells, carry out oils and fats extraction.
The method producing biodiesel with chlorella the most according to claim 1, it is characterised in that In step 2) in, use centrifugal or pneumatically supported acquisition technique to carry out seed collection.
The method producing biodiesel with chlorella the most according to claim 1, it is characterised in that In step 3) in, inoculum density is in 1.0-2.0 × 107Individual/L.
The method producing biodiesel with chlorella the most according to claim 1, it is characterised in that In step 4) in, with centrifugal, chemical flocculation or aerating collecting algae solution, obtain algae mud.
The method producing biodiesel with chlorella the most according to claim 4, it is characterised in that In step 4) in, chemical flocculation uses iron sulfate, iron chloride, aluminum sulfate, aluminum chloride or chitosan conduct Chemical floc.
The method producing biodiesel with chlorella the most according to claim 1, it is characterised in that In step 5) in, use enzyme process, ultrasonic method or liquid nitrogen method to carry out chlorella cells and crush.
The method producing biodiesel with chlorella the most according to claim 6, it is characterised in that In step 5) in, the enzyme that enzyme process uses includes cellulase, center protein enzyme, alkaline protease, pectin One or several in enzyme, wall breaking enzyme, Snailase and lipase.
The method producing biodiesel with chlorella the most according to claim 1, it is characterised in that In step 5) in, use organic extraction carry out room temperature oils and fats extraction, organic solvent be equal proportion chloroform, First alcohol and water, or the ether of equal proportion, normal hexane and water.
The method producing biodiesel with chlorella the most according to claim 1, it is characterised in that Also include:
Step 6) content of the oils and fats extracted is carried out gas chromatography-mass spectrometry analysis；
Step 7) combustibility of the oils and fats extracted is evaluated.
The method producing biodiesel with chlorella the most according to claim 9, it is characterised in that In step 7) in, combustibility evaluation is to evaluate with Cetane number.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106520559A (en) * 2016-12-28 2017-03-22 海南绿藻世界生物科技有限公司 High-efficiency light autotrophic culture method for chlorella
CN109337940A (en) * 2018-11-28 2019-02-15 北京大学 The method for being catalyzed wet chlorella preparation biodiesel using two step enzyme methods
CN110756115A (en) * 2019-10-18 2020-02-07 中国石油大学(北京) Sulfate type double-hydrocarbon-chain surfactant and preparation method and application thereof
CN114958931A (en) * 2022-04-21 2022-08-30 暨南大学 Method for increasing yield of xanthomonas oil and palmitoleic acid

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CN103451101A (en) * 2012-05-28 2013-12-18 中国石油天然气股份有限公司 Production method of high-quality microalgae biodiesel

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CN103451101A (en) * 2012-05-28 2013-12-18 中国石油天然气股份有限公司 Production method of high-quality microalgae biodiesel

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106520559A (en) * 2016-12-28 2017-03-22 海南绿藻世界生物科技有限公司 High-efficiency light autotrophic culture method for chlorella
CN106520559B (en) * 2016-12-28 2019-05-31 海南绿藻世界生物科技有限公司 A kind of chlorella high efficiency light autotrophy cultural method
CN109337940A (en) * 2018-11-28 2019-02-15 北京大学 The method for being catalyzed wet chlorella preparation biodiesel using two step enzyme methods
CN110756115A (en) * 2019-10-18 2020-02-07 中国石油大学(北京) Sulfate type double-hydrocarbon-chain surfactant and preparation method and application thereof
CN114958931A (en) * 2022-04-21 2022-08-30 暨南大学 Method for increasing yield of xanthomonas oil and palmitoleic acid

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