CN109355321A - A method of microalgae grease yield is improved using walnut shell extract - Google Patents
A method of microalgae grease yield is improved using walnut shell extract Download PDFInfo
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- CN109355321A CN109355321A CN201811323562.4A CN201811323562A CN109355321A CN 109355321 A CN109355321 A CN 109355321A CN 201811323562 A CN201811323562 A CN 201811323562A CN 109355321 A CN109355321 A CN 109355321A
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- 239000000284 extract Substances 0.000 title claims abstract description 53
- 235000009496 Juglans regia Nutrition 0.000 title claims abstract description 45
- 235000020234 walnut Nutrition 0.000 title claims abstract description 45
- 239000004519 grease Substances 0.000 title claims abstract description 44
- 238000000034 method Methods 0.000 title claims abstract description 36
- 240000007049 Juglans regia Species 0.000 title 1
- 239000002028 Biomass Substances 0.000 claims abstract description 45
- 241000758789 Juglans Species 0.000 claims abstract description 44
- 239000001963 growth medium Substances 0.000 claims abstract description 43
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 24
- 239000007788 liquid Substances 0.000 claims abstract description 24
- 239000008246 gaseous mixture Substances 0.000 claims abstract description 19
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 16
- 230000005526 G1 to G0 transition Effects 0.000 claims abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 15
- 230000005791 algae growth Effects 0.000 claims abstract description 10
- 238000005286 illumination Methods 0.000 claims abstract description 9
- 239000003960 organic solvent Substances 0.000 claims abstract description 9
- 239000007640 basal medium Substances 0.000 claims abstract description 8
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 8
- 238000000926 separation method Methods 0.000 claims abstract description 7
- 241000195493 Cryptophyta Species 0.000 claims description 27
- 239000007789 gas Substances 0.000 claims description 9
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Inorganic materials [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 9
- 239000002609 medium Substances 0.000 claims description 7
- 239000003225 biodiesel Substances 0.000 abstract description 7
- 238000005516 engineering process Methods 0.000 abstract description 2
- 239000002699 waste material Substances 0.000 abstract description 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 21
- 150000002632 lipids Chemical class 0.000 description 18
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 14
- 238000002835 absorbance Methods 0.000 description 14
- 230000000052 comparative effect Effects 0.000 description 9
- 239000006004 Quartz sand Substances 0.000 description 7
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 7
- 239000012153 distilled water Substances 0.000 description 7
- 238000000605 extraction Methods 0.000 description 7
- 230000001954 sterilising effect Effects 0.000 description 7
- 238000005119 centrifugation Methods 0.000 description 6
- 238000004108 freeze drying Methods 0.000 description 6
- 239000002054 inoculum Substances 0.000 description 6
- 239000002803 fossil fuel Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000011149 active material Substances 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 239000000446 fuel Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 229920005610 lignin Polymers 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 235000019737 Animal fat Nutrition 0.000 description 1
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 240000005809 Prunus persica Species 0.000 description 1
- 235000006040 Prunus persica var persica Nutrition 0.000 description 1
- 208000007107 Stomach Ulcer Diseases 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012075 bio-oil Substances 0.000 description 1
- 208000020670 canker sore Diseases 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 150000002148 esters Chemical group 0.000 description 1
- 235000019197 fats Nutrition 0.000 description 1
- 229930003935 flavonoid Natural products 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- 201000005917 gastric ulcer Diseases 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 150000007965 phenolic acids Chemical class 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 235000019871 vegetable fat Nutrition 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
-
- 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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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention relates to a kind of methods for improving microalgae grease yield using walnut shell extract, belong to biodiesel technology field.Water is added in walnut shell and boils 4 h with the mass ratio of the walnut shell in system and liquid for (9.5 ~ 10.5) by the present invention: 1, it is separated by solid-liquid separation, NaNO is added as basic culture medium in liquid in basal medium3Into culture medium, nitrogen content is 1.1 ~ 1.2 g/L, uses sodium hydroxide solution to adjust the pH value of culture medium as 6.8 ~ 7.0, then sterilized to obtain micro-algae culture medium；Microalgae is seeded in micro-algae culture medium, CO is passed through2The gaseous mixture of air, then being placed in temperature is 24 ~ 26 DEG C, intensity of illumination is cultivated under the conditions of being 6000-7000 lux；The biomass for measuring microalgae under condition of culture extracts the grease in microalgae cell using organic solvent after micro algae growth reaches stationary phase.The method of the present invention not only realizes the resource utilization of walnut shell waste, can also greatly improve microalgae grease yield.
Description
Technical field
The present invention relates to a kind of methods for improving microalgae grease yield using walnut shell extract, belong to biodiesel technology
Field.
Background technique
In recent years, due to the extensive use of fossil fuel, lead to the exhaustion of fossil fuel, biodiesel is as a kind of substitution
The environmentally friendly renewable energy of fossil fuel, has obtained extensive concern.Biodiesel refers to vegetable fat, animal fat, micro-
Reproducible biomass fuel is made by ester exchange process for raw material in bio-oil, biodiesel etc..Compared with common diesel,
It has renewable, degradable, CO2The features such as discharge amount is low, Cetane number is high.
Microalgae as production biodiesel raw material have be easy to cultivate, growth cycle is short, fat content is higher, growth item
Part is simple, nontoxicity, is not take up the advantages that arable land.But since the limitation of the prior art causes the lipid-producing of microalgae lower,
How preferably to improve microalgae grease yield becomes biodiesel development problem encountered.
Walnut shell is the rigid shell after walnut kernel-taking, can be used for treating canker sore, gastric ulcer, modern studies have found that core
Peach shell contains the various actives substance such as phenolic acid class, flavonoids, glycoside, lignin, and the lignin of high-content can be used to process life
Wood materials are produced, but is mainly used as fuel at present or directly abandons, not only cause the significant wastage of resource in this way, and pollute ring
Border.The utility value of active material is very big in walnut shell, but develops and uses at present seldom.
Summary of the invention
The present invention utilizes the deficiency of method for microalgae grease low yield in the prior art and walnut shell active material, mentions
For a kind of method for improving microalgae grease yield using walnut shell extract, the method for the present invention is using walnut shell extract as base
Basal culture medium carries out the culture of microalgae, improves the lipid-producing of microalgae.
A method of microalgae grease yield, specific steps are improved using walnut shell extract are as follows:
(1) water is added in walnut shell and boils 4h with the mass ratio of the liquid in system and walnut shell for (9.5 ~ 10.5):
1, it is separated by solid-liquid separation, NaNO is added as basic culture medium in liquid in basal medium3, adjusted and trained using sodium hydroxide solution
The pH value for supporting base is 6.8 ~ 7.0, then is sterilized to obtain micro-algae culture medium；
(2) microalgae is seeded in the micro-algae culture medium of step (1), is passed through CO2The gaseous mixture of air, then be placed in temperature be 24 ~
26 DEG C, intensity of illumination is cultivated under the conditions of being 6000-7000 lux；
(3) biomass of microalgae utilizes organic solvent after micro algae growth reaches stationary phase under determination step (2) condition of culture
Extract the grease in microalgae cell；
Step (1) sterilizing methods can be 20 ± 1 min that sterilize under the conditions of temperature is 121 ± 1 DEG C；
Step (1) NaNO3The content of middle nitrogen in the medium is 1.1 ~ 1.2 g/L；
Microalgae is single needle algae in the step (2)MonoraphidiumSp. QLZ-3(is commercially available)；
Microalgae initial inoculum is 0.375 ~ 0.425 g/L in the step (2)；
CO in the step (2)2CO in the gaseous mixture of air2Volume fraction be 0 ~ 16%, CO2The gas of the gaseous mixture of air
Flow is 0.475 ~ 0.525 L/min；
The biomass concentration measuring method of microalgae in the step (3) are as follows: according to frustule and absorbance A750Value is in certain model
It encloses interior linear, obtains single needle algaeMonoraphidiumSp. the pass of dry weight and absorbance of the QLZ-3 at 750 nm
It is as follows:
Measure the A of algae solution750Value, brings formula into, obtains micro algae biomass；
Grease extraction in step (3) of the present invention in microalgae cell are as follows: will culture to stationary phase microalgae be placed in from
Heart rate is that 3500 r/min are centrifuged 5 min, then is washed with distilled water 2 times, is placed under the conditions of temperature is -80 DEG C to be lyophilized and obtains
Dry microalgae, the quartz sand for adding 2 times of dry microalgae quality are ground, then using chloroform/methanol (2:1,v/v) extract algae it is thin
Grease intracellular repeats to extract 3 times, combined extract, then extracting solution is placed under the conditions of temperature is 40 DEG C to dry and is weighed.
Beneficial effects of the present invention:
(1) present invention improves microalgae grease yield using walnut shell extract, not only realizes the resource utilization of waste, also
It can reply and improve microalgae grease yield, and method is simple and easy；
(2) the method for the present invention is in the cultivation cycle of microalgae, when being passed through CO2Volume fraction is 12 %, Biomass yield and grease
Yield reaches maximum value, and Biomass yield is 197.48 mg/Ld and lipid-producing is 97.59 mg/Ld, Biomass yield
It is 1.45 times of comparative example, lipid-producing is 1.71 times of comparative example.
Specific embodiment
Invention is further described in detail With reference to embodiment, but protection scope of the present invention and unlimited
In the content.
Comparative example: using BG-11 culture medium as microalgae (single needle algaeMonoraphidiumSp. QLZ-3) culture medium, adopt
It is 6.8 with the pH value that dilute hydrochloric acid adjusts culture medium, being subsequently placed in temperature is 121 DEG C of 20 min of sterilizing, by microalgae (single needle algaeMonoraphidiumSp. QLZ-3) in the micro-algae culture medium that is seeded to, it is passed through air, then being placed in temperature is 24 DEG C, illumination is strong
Degree is cultivated under the conditions of being 6000 lux；Daily timing sampling measures the life of microalgae under condition of culture using ultraviolet specrophotometer
Object amount extracts the grease in microalgae cell using organic solvent after micro algae growth reaches stationary phase；Wherein microalgae initial inoculation
Amount is 0.4 g/L, and air velocity is 0.5 L/min；
The biomass concentration measuring method of microalgae are as follows: according to frustule and absorbance A750It is worth linear pass in a certain range
System, obtains single needle algaeMonoraphidiumSp. the relationship of dry weight and absorbance of the QLZ-3 at 750 nm is as follows:
Measure the A of algae solution750Value, brings formula into, obtains micro algae biomass；
Grease extraction in microalgae cell are as follows: will culture to stationary phase microalgae be placed in centrifugation rate be 3500 r/min from
5 min of the heart, then be washed with distilled water 2 times is placed in freeze-drying under the conditions of temperature is -80 DEG C and obtains dry microalgae, add 2 times do it is micro-
The quartz sand of algae quality is ground, then using chloroform/methanol (2:1,v/v) extract frustule in grease, repeat extract 3
It is secondary, combined extract, then extracting solution is placed under the conditions of temperature is 40 DEG C to dry and is weighed；
Using BG-11 as microalgae (single needle algaeMonoraphidiumSp. QLZ-3) culture medium when, single needle algaeMonoraphidiumSp. the cultivation cycle of QLZ-3 is 17 days, and biomass reaches 2.13 g/L, and fat content is 44.37 %；
Biomass yield and lipid-producing are respectively 135.32 mgL-1· d-1With 57.13 mgL-1 ·d-1(see Table 1).
Embodiment 1: a method of microalgae grease yield, specific steps are improved using walnut shell extract are as follows:
(1) water is added in walnut shell and boils 4 h with the mass ratio of the liquid in system and walnut shell for 10:1, solid-liquid divides
From NaNO is added as basic culture medium in liquid in basal medium3, the pH of culture medium is adjusted using sodium hydroxide solution
Value is 6.8, then is sterilized to obtain micro-algae culture medium；Wherein sterilizing methods are to sterilize 20 under the conditions of temperature is 121 ± 1 DEG C
min；NaNO3The content of middle nitrogen in the medium is 1.16 g/L；
(2) by microalgae, (microalgae is single needle algaeMonoraphidiumSp. QLZ-3) it is seeded to the micro-algae culture medium of step (1)
In, it is passed through air, then being placed in temperature is 24 DEG C, intensity of illumination is cultivated under the conditions of being 6000 lux；Wherein microalgae initial inoculum
For 0.375 g/L；The gas flow of air is 0.5 L/min；
(3) biomass of microalgae utilizes organic solvent after micro algae growth reaches stationary phase under determination step (2) condition of culture
Extract the grease in microalgae cell；
The biomass concentration measuring method of microalgae are as follows: according to frustule and absorbance A750It is worth linear pass in a certain range
System, obtains single needle algaeMonoraphidiumSp. the relationship of dry weight and absorbance of the QLZ-3 at 750 nm is as follows:
Measure the A of algae solution750Value, brings formula into, obtains micro algae biomass；
Grease extraction in microalgae cell are as follows: will culture to stationary phase microalgae be placed in centrifugation rate be 3500 r/min from
5 min of the heart, then be washed with distilled water 2 times is placed in freeze-drying under the conditions of temperature is -80 DEG C and obtains dry microalgae, add 2 times do it is micro-
The quartz sand of algae quality is ground, then using chloroform/methanol (2:1,v/v) extract frustule in grease, repeat extract 3
It is secondary, combined extract, then extracting solution is placed under the conditions of temperature is 40 DEG C to dry and is weighed；
When the present embodiment is using walnut shell extract as basic culture medium, under conditions of being only passed through air, the culture week of microalgae
Phase is 6 days, and biomass reaches 0.89 g/L, and fat content reaches 41.13 %；The Biomass yield and lipid-producing of microalgae are distinguished
For 148.56 mgL-1· d-1With 62.30 mgL-1 ·d-1, Biomass yield improves 9.78 % compared with comparative example,
Lipid-producing improves 9.06 %(and is shown in Table 1).
Embodiment 2: a method of microalgae grease yield, specific steps are improved using walnut shell extract are as follows:
(1) in walnut shell be added water and boil 4 h with the mass ratio of the liquid in system and walnut shell be 10: 1, solid-liquid
NaNO is added as basic culture medium in separation, liquid in basal medium3, culture medium is adjusted using sodium hydroxide solution
PH value is 6.9, then is sterilized to obtain micro-algae culture medium；Wherein sterilizing methods are to sterilize 20 under the conditions of temperature is 121 ± 1 DEG C
min；NaNO3The content of middle nitrogen in the medium is 1.1 g/L；
(2) by microalgae, (microalgae is single needle algaeMonoraphidiumSp. QLZ-3) it is seeded to the micro-algae culture medium of step (1)
In, it is passed through CO2The gaseous mixture of air, then being placed in temperature is 25 DEG C, intensity of illumination is cultivated under the conditions of being 6500 lux；It is wherein micro-
Algae initial inoculum is 0.4 g/L；CO2CO in the gaseous mixture of air2Volume fraction be 4 %, CO2The gaseous mixture of air
Gas flow is 0.475 L/min；
(3) biomass of microalgae utilizes organic solvent after micro algae growth reaches stationary phase under determination step (2) condition of culture
Extract the grease in microalgae cell；
The biomass concentration measuring method of microalgae are as follows: according to frustule and absorbance A750It is worth linear pass in a certain range
System, obtains single needle algaeMonoraphidiumSp. the relationship of dry weight and absorbance of the QLZ-3 at 750 nm is as follows:
Measure the A of algae solution750Value, brings formula into, obtains micro algae biomass；
Grease extraction in microalgae cell are as follows: will culture to stationary phase microalgae be placed in centrifugation rate be 3500 r/min from
5 min of the heart, then be washed with distilled water 2 times is placed in freeze-drying under the conditions of temperature is -80 DEG C and obtains dry microalgae, add 2 times do it is micro-
The quartz sand of algae quality is ground, then using chloroform/methanol (2:1,v/v) extract frustule in grease, repeat extract 3
It is secondary, combined extract, then extracting solution is placed under the conditions of temperature is 40 DEG C to dry and is weighed；
It is 4 % CO being passed through containing volume fraction when the present embodiment is using walnut shell extract as basic culture medium2CO2It is empty
When oxygen mixture, the cultivation cycle of microalgae is 6 days, and the total biomass of microalgae reaches 0.98 g/L, and fat content reaches 44.85
%；The Biomass yield and lipid-producing of microalgae are respectively 161.78 mgL-1· d-1With 72.54 mgL-1 ·d-1；With it is right
Ratio improves 19.55 % compared to Biomass yield, and lipid-producing improves 27 %(and is shown in Table 1).
Embodiment 3: a method of microalgae grease yield, specific steps are improved using walnut shell extract are as follows:
(1) in walnut shell be added water and boil 4 h with the mass ratio of the liquid in system and walnut shell be 9.5: 1, solid-liquid
NaNO is added as basic culture medium in separation, liquid in basal medium3, culture medium is adjusted using sodium hydroxide solution
PH value is 7.0, then is sterilized to obtain micro-algae culture medium；Wherein sterilizing methods are to sterilize 20 under the conditions of temperature is 121 ± 1 DEG C
min；NaNO3The content of middle nitrogen in the medium is 1.15 g/L；
(2) by microalgae, (microalgae is single needle algaeMonoraphidiumSp. QLZ-3) it is seeded to the micro-algae culture medium of step (1)
In, it is passed through CO2The gaseous mixture of air, then being placed in temperature is 26 DEG C, intensity of illumination is cultivated under the conditions of being 7000 lux；It is wherein micro-
Algae initial inoculum is 0.425 g/L；CO2CO in the gaseous mixture of air2Volume fraction be 8 %, CO2The gaseous mixture of air
Gas flow be 0.5 L/min；
(3) biomass of microalgae utilizes organic solvent after micro algae growth reaches stationary phase under determination step (2) condition of culture
Extract the grease in microalgae cell；
The biomass concentration measuring method of microalgae are as follows: according to frustule and absorbance A750It is worth linear pass in a certain range
System, obtains single needle algaeMonoraphidiumSp. the relationship of dry weight and absorbance of the QLZ-3 at 750 nm is as follows:
Measure the A of algae solution750Value, brings formula into, obtains micro algae biomass；
Grease extraction in microalgae cell are as follows: will culture to stationary phase microalgae be placed in centrifugation rate be 3500 r/min from
5 min of the heart, then be washed with distilled water 2 times is placed in freeze-drying under the conditions of temperature is -80 DEG C and obtains dry microalgae, add 2 times do it is micro-
The quartz sand of algae quality is ground, then using chloroform/methanol (2:1,v/v) extract frustule in grease, repeat extract 3
It is secondary, combined extract, then extracting solution is placed under the conditions of temperature is 40 DEG C to dry and is weighed；
It is 4 % CO being passed through containing volume fraction when the present embodiment is using walnut shell extract as basic culture medium2CO2It is empty
When oxygen mixture, the cultivation cycle of microalgae is 6 days, and biomass reaches 1.10 g/L, and fat content is 47.90 %.Microalgae
Biomass yield and lipid-producing are respectively 184.11 mgL-1· d-1With 88.21 mgL-1 ·d-1；Compared with comparative example
Biomass yield improves 36.06 %, and lipid-producing improves 27 %(and is shown in Table 1).
Embodiment 4: a method of microalgae grease yield, specific steps are improved using walnut shell extract are as follows:
(1) in walnut shell be added water and boil 4 h with the mass ratio of the liquid in system and walnut shell be 10: 1, solid-liquid
NaNO is added as basic culture medium in separation, liquid in basal medium3, culture medium is adjusted using sodium hydroxide solution
PH value is 6.8, then is sterilized to obtain micro-algae culture medium；Wherein sterilizing methods are to sterilize 20 under the conditions of temperature is 121 ± 1 DEG C
min；NaNO3The content of middle nitrogen in the medium is 1.2 g/L；
(2) by microalgae, (microalgae is single needle algaeMonoraphidiumSp. QLZ-3) it is seeded to the micro-algae culture medium of step (1)
In, it is passed through CO2The gaseous mixture of air, then being placed in temperature is 24 DEG C, intensity of illumination is cultivated under the conditions of being 6000 lux；It is wherein micro-
Algae initial inoculum is 0.4 g/L；CO2CO in the gaseous mixture of air2Volume fraction be 12 %, CO2The gaseous mixture of air
Gas flow is 0.525 L/min；
(3) biomass of microalgae utilizes organic solvent after micro algae growth reaches stationary phase under determination step (2) condition of culture
Extract the grease in microalgae cell；
The biomass concentration measuring method of microalgae are as follows: according to frustule and absorbance A750It is worth linear pass in a certain range
System, obtains single needle algaeMonoraphidiumSp. the relationship of dry weight and absorbance of the QLZ-3 at 750 nm is as follows:
Measure the A of algae solution750Value, brings formula into, obtains micro algae biomass；
Grease extraction in microalgae cell are as follows: will culture to stationary phase microalgae be placed in centrifugation rate be 3500 r/min from
5 min of the heart, then be washed with distilled water 2 times is placed in freeze-drying under the conditions of temperature is -80 DEG C and obtains dry microalgae, add 2 times do it is micro-
The quartz sand of algae quality is ground, then using chloroform/methanol (2:1,v/v) extract frustule in grease, repeat extract 3
It is secondary, combined extract, then extracting solution is placed under the conditions of temperature is 40 DEG C to dry and is weighed；
It is 4% CO being passed through containing volume fraction when the present embodiment is using walnut shell extract as basic culture medium2CO2Air
When mixed gas, the cultivation cycle of microalgae is 6 days, and biomass reaches 1.18 g/L, and fat content is 49.54 %.The life of microalgae
Object amount yield and lipid-producing are respectively 196.85 mgL-1· d-1With 97.52 mgL-1 ·d-1；It is given birth to compared with comparative example
Object amount yield improves 45.47 %, and lipid-producing improves 70.73 %(and is shown in Table 1).
Embodiment 5: a method of microalgae grease yield, specific steps are improved using walnut shell extract are as follows:
(1) water is added in walnut shell and boils 4 h with the mass ratio of the liquid in system and walnut shell for 10.5: 1, Gu
NaNO is added as basic culture medium in liquid separation, liquid in basal medium3, culture medium is adjusted using sodium hydroxide solution
PH value be 6.9, then sterilized to obtain micro-algae culture medium；Wherein sterilizing methods are to sterilize under the conditions of temperature is 121 ± 1 DEG C
20 min；NaNO3The content of middle nitrogen in the medium is 1.16 g/L；
(2) by microalgae, (microalgae is single needle algaeMonoraphidiumSp. QLZ-3) it is seeded to the micro-algae culture medium of step (1)
In, it is passed through CO2The gaseous mixture of air, then being placed in temperature is 26 DEG C, intensity of illumination is cultivated under the conditions of being 7000lux；It is wherein micro-
Algae initial inoculum is 0.410 g/L；CO2CO in the gaseous mixture of air2Volume fraction be 16 %, CO2The gaseous mixture of air
Gas flow be 0.5 L/min；
(3) biomass of microalgae utilizes organic solvent after micro algae growth reaches stationary phase under determination step (2) condition of culture
Extract the grease in microalgae cell；
The biomass concentration measuring method of microalgae are as follows: according to frustule and absorbance A750It is worth linear pass in a certain range
System, obtains single needle algaeMonoraphidiumSp. the relationship of dry weight and absorbance of the QLZ-3 at 750 nm is as follows:
Measure the A of algae solution750Value, brings formula into, obtains micro algae biomass；
Grease extraction in microalgae cell are as follows: will culture to stationary phase microalgae be placed in centrifugation rate be 3500 r/min from
5 min of the heart, then be washed with distilled water 2 times is placed in freeze-drying under the conditions of temperature is -80 DEG C and obtains dry microalgae, add 2 times do it is micro-
The quartz sand of algae quality is ground, then using chloroform/methanol (2:1,v/v) extract frustule in grease, repeat extract 3
It is secondary, combined extract, then extracting solution is placed under the conditions of temperature is 40 DEG C to dry and is weighed；
It is 4% CO being passed through containing volume fraction when the present embodiment is using walnut shell extract as basic culture medium2CO2Air
When mixed gas, the cultivation cycle of microalgae is 6 days, and biomass reaches 1.05g/L, and fat content is 43.08 %.The biology of microalgae
Volume production rate and lipid-producing are respectively 174.05 mgL-1· d-1With 74.97 mgL-1 ·d-1；It is biological compared with comparative example
Volume production rate improves 28.62 %, and lipid-producing improves 31.25 %(and is shown in Table 1)；
Influence of the different condition of culture of table 1 to micro algae growth and oil and fat accumulation
It as known from Table 1, is basic culture medium with walnut shell extract, external source adds NaNO3(1.1 ~ 1.2 g/L), is passed through simultaneously
CO2(volume fraction is 4 %, 8 %, 12 %, 16 %) culture；When being only passed through air, the total biomass of microalgae reaches 0.89 g/
L, fat content reaches 41.13 %；The Biomass yield and lipid-producing of microalgae are respectively 149.50 and 61.49 mgL-1 ·
d-1；Compared with comparative example, 9.78 % and 9.06 % have been respectively increased in Biomass yield and lipid-producing.
When single needle algaeMonoraphidiumSp. when QLZ-3 culture is in walnut shell extract, CO2 Concentration is 12 %
When, the biomass of microalgae is 1.18 g/L, Biomass yield is 197.48 mgL-1 ·d-1, fat content be 49.54 %, oil
Rouge yield is 97.59 mgL-1 ·d-1Reach maximum value；Biomass yield and lipid-producing mention respectively compared with comparative example
High 45.47 % and 70.73 %.
Claims (4)
1. a kind of method for improving microalgae grease yield using walnut shell extract, which is characterized in that specific steps are as follows:
(1) water is added in walnut shell and boils 4h with the mass ratio of the liquid in system and walnut shell for (9.5 ~ 10.5):
1, it is separated by solid-liquid separation, NaNO is added as basic culture medium in liquid in basal medium3, adjusted and trained using sodium hydroxide solution
The pH value for supporting base is 6.8 ~ 7.0, then is sterilized to obtain micro-algae culture medium；
(2) microalgae is seeded in the micro-algae culture medium of step (1), is passed through CO2The gaseous mixture of air, then be placed in temperature be 24 ~
26 DEG C, intensity of illumination is cultivated under the conditions of being 6000-7000 lux；
(3) biomass of microalgae utilizes organic solvent after micro algae growth reaches stationary phase under determination step (2) condition of culture
Extract the grease in microalgae cell.
2. the method for improving microalgae grease yield using walnut shell extract according to claim 1, it is characterised in that: step
(1) NaNO3The content of middle nitrogen in the medium is 1.1 ~ 1.2 g/L.
3. the method for improving microalgae grease yield using walnut shell extract according to claim 1, it is characterised in that: step
(2) microalgae is single needle algae inMonoraphidium sp. QLZ-3。
4. the method for improving microalgae grease yield using walnut shell extract according to claim 1, it is characterised in that: step
(2) CO in2CO in the gaseous mixture of air2Volume fraction be 0 ~ 16 %, CO2The gas flow of the gaseous mixture of air is 0.475
~5.25 L/min。
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| CN109929886A (en) * | 2019-03-18 | 2019-06-25 | 昆明理工大学 | A method of utilizing walnut shell extract culture single needle algae generation diesel oil |
| CN110218652A (en) * | 2019-05-27 | 2019-09-10 | 昆明理工大学 | A method of promoting micro algae growth and oil and fat accumulation in BG-11 culture medium |
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