CN109929886A - A method of utilizing walnut shell extract culture single needle algae generation diesel oil - Google Patents
A method of utilizing walnut shell extract culture single needle algae generation diesel oil Download PDFInfo
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- CN109929886A CN109929886A CN201910202514.8A CN201910202514A CN109929886A CN 109929886 A CN109929886 A CN 109929886A CN 201910202514 A CN201910202514 A CN 201910202514A CN 109929886 A CN109929886 A CN 109929886A
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- 241000195493 Cryptophyta Species 0.000 title claims abstract description 134
- 235000009496 Juglans regia Nutrition 0.000 title claims abstract description 50
- 235000020234 walnut Nutrition 0.000 title claims abstract description 50
- 239000000284 extract Substances 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 30
- 239000002283 diesel fuel Substances 0.000 title claims abstract description 14
- 240000007049 Juglans regia Species 0.000 title 1
- 241000758789 Juglans Species 0.000 claims abstract description 49
- 239000002028 Biomass Substances 0.000 claims abstract description 44
- 239000001963 growth medium Substances 0.000 claims abstract description 39
- 239000007788 liquid Substances 0.000 claims abstract description 33
- 239000004519 grease Substances 0.000 claims abstract description 32
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 24
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 21
- 239000007640 basal medium Substances 0.000 claims abstract description 18
- 239000008103 glucose Substances 0.000 claims abstract description 17
- 239000003225 biodiesel Substances 0.000 claims abstract description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 14
- WCYAALZQFZMMOM-UHFFFAOYSA-N methanol;sulfuric acid Chemical compound OC.OS(O)(=O)=O WCYAALZQFZMMOM-UHFFFAOYSA-N 0.000 claims abstract description 9
- 238000005286 illumination Methods 0.000 claims abstract description 8
- 239000003960 organic solvent Substances 0.000 claims abstract description 8
- 238000005886 esterification reaction Methods 0.000 claims abstract description 7
- 238000000926 separation method Methods 0.000 claims abstract description 3
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 12
- 239000002054 inoculum Substances 0.000 claims description 8
- 150000007524 organic acids Chemical class 0.000 claims description 6
- GZCGUPFRVQAUEE-VANKVMQKSA-N aldehydo-L-glucose Chemical compound OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)C=O GZCGUPFRVQAUEE-VANKVMQKSA-N 0.000 claims description 2
- 150000002632 lipids Chemical class 0.000 abstract description 16
- 238000005516 engineering process Methods 0.000 abstract 1
- 230000032050 esterification Effects 0.000 abstract 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 22
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 12
- 239000000843 powder Substances 0.000 description 12
- 238000002835 absorbance Methods 0.000 description 11
- 230000000052 comparative effect Effects 0.000 description 9
- 238000000605 extraction Methods 0.000 description 7
- 230000005526 G1 to G0 transition Effects 0.000 description 6
- 239000006004 Quartz sand Substances 0.000 description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- 239000012153 distilled water Substances 0.000 description 6
- 238000004108 freeze drying Methods 0.000 description 6
- 239000003921 oil Substances 0.000 description 6
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 4
- 239000005864 Sulphur Substances 0.000 description 4
- 239000006481 glucose medium Substances 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 239000002803 fossil fuel Substances 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 229920005610 lignin Polymers 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000002699 waste 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
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 238000010521 absorption reaction Methods 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
- 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
- 239000000446 fuel Substances 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 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
- 239000005416 organic matter Substances 0.000 description 1
- 150000007965 phenolic acids Chemical class 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 235000019871 vegetable fat Nutrition 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
Classifications
-
- 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
Abstract
The present invention relates to a kind of methods using walnut shell extract culture single needle algae generation diesel oil, belong to biodiesel technology field.Water is added in walnut shell and boils 4h with the mass ratio (g:g) of the liquid in system and walnut shell for (4 ~ 32) by the present invention: 1, it is separated by solid-liquid separation, liquid is as basic culture medium, glucose is added in basal medium, it uses sodium hydroxide solution to adjust the pH value of culture medium as 6.8 ~ 7.0, then is sterilized to obtain single needle algae culture medium；Single needle algae is seeded in single needle algae culture medium, 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 of single needle algae under condition of culture is measured, after single needle algae grows 60 ~ 80h, extracts the grease in single needle frustule using organic solvent；Sulfuric acid-methanol is added to the progress anti-3 ~ 3.5h of esterification in grease and obtains biodiesel.Glucose is added using walnut shell extract as basic culture medium in the method for the present invention, carries out the mixotrophic cultivation of single needle algae, improves the lipid-producing of single needle algae.
Description
Technical field
The present invention relates to a kind of methods using walnut shell extract culture single needle algae generation diesel oil, belong to biodiesel
Technical 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.
Single needle algae as production biodiesel raw material have be easy to cultivate, growth cycle is short, fat content is higher, growth
Condition is simple, nontoxicity, is not take up the advantages that arable land.But due to the limitation of the prior art cause the lipid-producing of single needle algae compared with
Low, how preferably to improve single needle algae lipid-producing becomes biodiesel development problem encountered.
Walnut nutriment rich in, in China, plantation is extensive.As generation during walnut product processing
Waste, many kinds of substance such as lignin, phenolic acid class, flavonoids, glycoside are contained in walnut shell.Content of lignin in walnut shell
It is relatively high, it can be used as the source of wood materials.But because substance extraction process is complicated in walnut shell, walnut shell is big at present
More directly burnings are directly abandoned with sewage conduct, and resource is wasted.
In the prior art, the method incubation time using walnut shell extract culture single needle algae Lipid-producing is long, grease yield
It is low.
Summary of the invention
The present invention is long for the method incubation time in the prior art using walnut shell extract culture single needle algae Lipid-producing
And the deficiency that grease yield is low, a kind of method using walnut shell extract culture single needle algae generation diesel oil is provided, the present invention
Glucose is added using walnut shell extract as basic culture medium in method, carries out the mixotrophic cultivation of single needle algae, improves single needle algae
Lipid-producing.
The mixotrophic cultivation mode that the present invention uses, the mixotrophic cultivation of microalgae include that the absorption of the sorption enhanced of luminous energy, CO2 is consolidated
Fixed, assimilation of organic matter etc..The growth of mixotrophic cultivation group single needle algae is with respect to autotrophy culture group, and the speed of growth is in lag phase and index
Growth period does not change significantly, and growth of the mixotrophic cultivation group frustule in the relative growth decline phase maintains original life substantially
Long speed, after glucose is added in the present invention, the consumption supplemented with its carbon source slows down the decline of the frustule speed of growth.
A method of utilizing walnut shell extract culture single needle algae generation diesel oil, 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 (4 ~ 32): 1, Gu
Glucose is added as basic culture medium in liquid separation, liquid in basal medium, adjusts culture medium using sodium hydroxide solution
PH value be 6.8 ~ 7.0, then sterilized to obtain single needle algae culture medium；
(2) single needle algae is seeded in the single needle algae culture medium of step (1), then being placed in temperature is 24 ~ 26 DEG C, intensity of illumination is
It is cultivated under the conditions of 6000-7000 lux；
(3) biomass of single needle algae is mentioned after single needle algae grows 60 ~ 80h using organic solvent under determination step (2) condition of culture
Take the grease in single needle frustule；
(4) sulfuric acid-methanol is added to progress 3 ~ 3.5h of esterification reaction of organic acid in the grease of step (3) and obtains biodiesel；
The solid-to-liquid ratio g:L of step (1) glucose and basal medium is (1 ~ 4): 1.
Single needle algae is single needle algae in rapid (2)MonoraphidiumSp.QLZ-3, the inoculum concentration of single needle algae are 0.4g/
L。
The biomass concentration measuring method of single needle algae in the step (2) are as follows: according to frustule and absorbance A750Value is one
It is linear in fixed range, obtain single needle algaeMonoraphidiumSp. biomass and suction of the QLZ-3 at 750 nm
The relationship of luminosity is as follows:
Measure the A of algae solution750Value, brings formula into, obtains single needle algae biomass；
Grease extraction in step (3) of the present invention in single needle frustule are as follows: will culture to stationary phase (culture 60 ~
It is that 3500 r/min are centrifuged 6min, then are washed with distilled water 2 times that single needle algae 80h), which is placed in centrifugation rate, and being placed in temperature is -80
Freeze-drying obtains dry algae powder under the conditions of DEG C, and the quartz sand for adding 2 times of dry algae powder quality is ground, and then uses chloroform/methanol
(2:1,v/v) extract frustule in grease, repeat extract 3 times, combined extract, then by extracting solution be placed in temperature be 40 DEG C
Under the conditions of dry weighing；
The mass concentration of sulfuric acid is 3% in sulfuric acid-methanol in the step (4)；
Beneficial effects of the present invention:
(1) present invention improves single needle algae lipid-producing using walnut shell extract and shortens single needle algae incubation time, not only realizes
The resource utilization of waste, can also greatly improve single needle algae lipid-producing, and method is simple and easy；
(2) the method for the present invention is 0.4 g/L, the culture that the period is three days in initial inoculum in the cultivation cycle of single needle algae
In the process, solid-liquid ratio 1:8, when glucose content is 2 g/L, the biomass highest of oil-producing microalgae reaches 0.91 g/L, biology
Volume production rate is up to 301.46 mg/ (Ld), is 1.24 times of control group 2；Lipid-producing is up to 150.78 simultaneously
Mg/ (Ld) improves 30.74 % compared with control group 2；
(3) walnut shell residue of the present invention can be used as the utilization of adsorption column, active carbon, increase the utilization efficiency of walnut shell.
Detailed description of the invention
Fig. 1 is the single needle algae growing state of comparative example and embodiment 1 ~ 4；
The case where Fig. 2 is the single needle algae oil-producing of comparative example and embodiment 1 ~ 4.
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: a method of utilizing walnut shell extract culture single needle algae Lipid-producing, 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 8:1, solid-liquid divides
From, liquid as basic culture medium, sodium hydroxide solution is used to adjust the pH value of culture medium as 6.82, then sterilized to obtain list
Needle algae culture medium；
(2) single needle algae is seeded in the single needle algae culture medium of step (1), then being placed in temperature is 25 DEG C, intensity of illumination 6800
It is cultivated under the conditions of lux；Wherein single needle algae is single needle algaeMonoraphidiumSp.QLZ-3, the inoculum concentration of single needle algae are 0.4g/L；
(3) under determination step (2) condition of culture single needle algae biomass, after single needle algae grows 72h, utilize organic solvent to extract single
Grease in needle frustule；
The biomass concentration measuring method of single needle algae are as follows: according to frustule and absorbance A750It is worth linear in a certain range
Relationship obtains single needle algaeMonoraphidiumSp. the relationship of biomass and absorbance of the QLZ-3 at 750 nm is as follows:
Measure the A of algae solution750Value, brings formula into, obtains single needle algae biomass；
Grease extraction in single needle frustule are as follows: it is 3500 r/ that the single needle algae of culture to stationary phase, which is placed in centrifugation rate,
Min is centrifuged 5 min, then is washed with distilled water 2 times, is placed in freeze-drying under the conditions of temperature is -80 DEG C and obtains dry algae powder, adds 2 times
The quartz sand of dry algae powder quality is ground, then using chloroform/methanol (2:1,v/v) extract frustule in grease, repeat
It extracts 3 times, 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, glucose, single needle algae are added without in basal medium
Cultivation cycle be 72h, biomass reaches 0.73 g/L, and fat content reaches 47.08%；The Biomass yield and oil of single needle algae
Rouge yield is respectively 222.00 mgL-1· d-1With 104.44 mgL-1 ·d-1(see Table 1).
Embodiment 1: a method of utilizing walnut shell extract culture single needle algae generation diesel oil, 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 8:1, solid-liquid divides
From glucose is added as basic culture medium in liquid in basal medium, and the pH of culture medium is adjusted using sodium hydroxide solution
Value is 6.82, then is sterilized to obtain single needle algae culture medium；Wherein the solid-to-liquid ratio g:L of glucose and basal medium is 1:1；
(2) single needle algae is seeded in the single needle algae culture medium of step (1), then being placed in temperature is 25 DEG C, intensity of illumination 6800
It is cultivated under the conditions of lux；Wherein single needle algae is single needle algaeMonoraphidiumSp.QLZ-3, the inoculum concentration of single needle algae are 0.4g/L；
(3) under determination step (2) condition of culture single needle algae biomass, after single needle algae grows 72h, utilize organic solvent to extract single
Grease in needle frustule；
The biomass concentration measuring method of single needle algae are as follows: according to frustule and absorbance A750It is worth linear in a certain range
Relationship obtains single needle algaeMonoraphidiumSp. the relationship of biomass and absorbance of the QLZ-3 at 750 nm is as follows:
Measure the A of algae solution750Value, brings formula into, obtains single needle algae biomass；
Grease extraction in single needle frustule are as follows: it is 3500 r/ that the single needle algae of culture to stationary phase, which is placed in centrifugation rate,
Min is centrifuged 5 min, then is washed with distilled water 2 times, is placed in freeze-drying under the conditions of temperature is -80 DEG C and obtains dry algae powder, adds 2 times
The quartz sand of dry algae powder quality is ground, then using chloroform/methanol (2:1,v/v) extract frustule in grease, repeat
It extracts 3 times, 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, glucose is added in basal medium, single needle algae
Cultivation cycle is 72h, and biomass reaches 0.88 g/L, and fat content reaches 49.93%；The Biomass yield and grease of single needle algae
Yield is respectively 243.38 mgL-1· d-1With 115.33 mgL-1 ·d-1, Biomass yield improves compared with comparative example
9.63%, lipid-producing improves 10.43%(and is shown in Table 1)；
(4) sulfuric acid-methanol is added to progress esterification reaction of organic acid 3.2h in the grease of step (3) and obtains biodiesel, wherein sulphur
The mass concentration of sulfuric acid is 3% in acid-methanol.
Embodiment 2: a method of utilizing walnut shell extract culture single needle algae generation diesel oil, 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 8:1, solid-liquid divides
From glucose is added as basic culture medium in liquid in basal medium, and the pH of culture medium is adjusted using sodium hydroxide solution
Value is 7.00, then is sterilized to obtain single needle algae culture medium；Wherein the solid-to-liquid ratio g:L of glucose and basal medium is 2:1；
(2) single needle algae is seeded in the single needle algae culture medium of step (1), then being placed in temperature is 24 DEG C, intensity of illumination 6900
It is cultivated under the conditions of lux；Wherein single needle algae is single needle algaeMonoraphidiumSp.QLZ-3, the inoculum concentration of single needle algae are 0.4g/L；
(3) under determination step (2) condition of culture single needle algae biomass, after single needle algae grows 72h, utilize organic solvent to extract single
Grease in needle frustule；
The biomass concentration measuring method of single needle algae are as follows: according to frustule and absorbance A750It is worth linear in a certain range
Relationship obtains single needle algaeMonoraphidiumSp. the relationship of biomass and absorbance of the QLZ-3 at 750 nm is as follows:
Measure the A of algae solution750Value, brings formula into, obtains single needle algae biomass；
Grease extraction in single needle frustule are as follows: it is 3500 r/ that the single needle algae of culture to stationary phase, which is placed in centrifugation rate,
Min is centrifuged 5 min, then is washed with distilled water 2 times, is placed in freeze-drying under the conditions of temperature is -80 DEG C and obtains dry algae powder, adds 2 times
The quartz sand of dry algae powder quality is ground, then using chloroform/methanol (2:1,v/v) extract frustule in grease, repeat
It extracts 3 times, 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, glucose is added in basal medium, single needle algae
Cultivation cycle is 72h, and biomass reaches 0.90 g/L, and fat content reaches 50.05%；The Biomass yield and grease of single needle algae
Yield is respectively 301.46 mgL-1· d-1With 150.78 mgL-1 ·d-1, Biomass yield improves compared with comparative example
35.79%, lipid-producing improves 44.37%(and is shown in Table 1)；
(4) sulfuric acid-methanol is added to progress esterification reaction of organic acid 3.1h in the grease of step (3) and obtains biodiesel, wherein sulphur
The mass concentration of sulfuric acid is 3% in acid-methanol.
Embodiment 3: a method of utilizing walnut shell extract culture single needle algae generation diesel oil, 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 24:1, solid-liquid divides
From glucose is added as basic culture medium in liquid in basal medium, and the pH of culture medium is adjusted using sodium hydroxide solution
Value is 6.96, then is sterilized to obtain single needle algae culture medium；Wherein the solid-to-liquid ratio g:L of glucose and basal medium is 3:1；
(2) single needle algae is seeded in the single needle algae culture medium of step (1), then being placed in temperature is 26 DEG C, intensity of illumination 6800
It is cultivated under the conditions of lux；Wherein single needle algae is single needle algaeMonoraphidiumSp.QLZ-3, the inoculum concentration of single needle algae are 0.4g/L；
(3) under determination step (2) condition of culture single needle algae biomass, after single needle algae grows 78h, utilize organic solvent to extract single
Grease in needle frustule；
The biomass concentration measuring method of single needle algae are as follows: according to frustule and absorbance A750It is worth linear in a certain range
Relationship obtains single needle algaeMonoraphidiumSp. the relationship of biomass and absorbance of the QLZ-3 at 750 nm is as follows:
Measure the A of algae solution750Value, brings formula into, obtains single needle algae biomass；
Grease extraction in single needle frustule are as follows: it is 3500 r/ that the single needle algae of culture to stationary phase, which is placed in centrifugation rate,
Min is centrifuged 5 min, then is washed with distilled water 2 times, is placed in freeze-drying under the conditions of temperature is -80 DEG C and obtains dry algae powder, adds 2 times
The quartz sand of dry algae powder quality is ground, then using chloroform/methanol (2:1,v/v) extract frustule in grease, repeat
It extracts 3 times, 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, glucose is added in basal medium, single needle algae
Cultivation cycle is 78h, and biomass reaches 0.80 g/L, and fat content reaches 50.00%；The Biomass yield and grease of single needle algae
Yield is respectively 265.82 mgL-1· d-1With 133.02 mgL-1 ·d-1, Biomass yield improves compared with comparative example
19.74%, lipid-producing improves 27.36%(and is shown in Table 1)；
(4) sulfuric acid-methanol is added to progress esterification reaction of organic acid 3.5h in the grease of step (3) and obtains biodiesel, wherein sulphur
The mass concentration of sulfuric acid is 3% in acid-methanol.
Embodiment 4: a method of utilizing walnut shell extract culture single needle algae generation diesel oil, 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 8:1, solid-liquid divides
From glucose is added as basic culture medium in liquid in basal medium, and the pH of culture medium is adjusted using sodium hydroxide solution
Value is 6.92, then is sterilized to obtain single needle algae culture medium；Wherein the solid-to-liquid ratio g:L of glucose and basal medium is 4:1；
(2) single needle algae is seeded in the single needle algae culture medium of step (1), then being placed in temperature is 26 DEG C, intensity of illumination 6800
It is cultivated under the conditions of lux；Wherein single needle algae is single needle algaeMonoraphidiumSp.QLZ-3, the inoculum concentration of single needle algae are 0.4g/L；
(3) under determination step (2) condition of culture single needle algae biomass, after single needle algae grows 72h, utilize organic solvent to extract single
Grease in needle frustule；
The biomass concentration measuring method of single needle algae are as follows: according to frustule and absorbance A750It is worth linear in a certain range
Relationship obtains single needle algaeMonoraphidiumSp. the relationship of biomass and absorbance of the QLZ-3 at 750 nm is as follows:
Measure the A of algae solution750Value, brings formula into, obtains single needle algae biomass；
Grease extraction in single needle frustule are as follows: it is 3500 r/ that the single needle algae of culture to stationary phase, which is placed in centrifugation rate,
Min is centrifuged 5 min, then is washed with distilled water 2 times, is placed in freeze-drying under the conditions of temperature is -80 DEG C and obtains dry algae powder, adds 2 times
The quartz sand of dry algae powder quality is ground, then using chloroform/methanol (2:1,v/v) extract frustule in grease, repeat
It extracts 3 times, combined extract, then extracting solution is placed under the conditions of temperature is 40 DEG C to dry and is weighed；
The different condition of culture of table 1 are grown to single needle algae and the influence of oil and fat accumulation
As known from Table 1, when the present embodiment is using walnut shell extract as basic culture medium, grape is added in basal medium
The cultivation cycle of sugar, single needle algae is 72h, and biomass reaches 0.76 g/L, and fat content reaches 50.12%；The biomass of single needle algae
Yield and lipid-producing are respectively 254.50mgL-1· d-1With 127.51 mgL-1 ·d-1, the biomass compared with comparative example
Yield improves 14.64%, and lipid-producing improves 22.09%；
(4) sulfuric acid-methanol is added to progress esterification reaction of organic acid 3.5h in the grease of step (3) and obtains biodiesel；Wherein sulphur
The mass concentration of sulfuric acid is 3% in acid-methanol；
As known from Table 1, when single needle algaeMonoraphidiumSp. it when QLZ-3 culture is in walnut shell extract, is initially connecing
Kind amount is 0.4 g/L, and the period is solid-liquid ratio 1:8 in the incubation of 72h, when glucose content is 2 g/L, oil-producing microalgae
Biomass highest, reach 0.91 g/L, Biomass yield is up to 301.46 mg/ (Ld), be comparative example 1.24
Times；Lipid-producing is up to 150.78 mg/ (Ld) simultaneously, improves 30.74 % compared with comparative example, illustrates to work as solid-liquid ratio
For 1:8, when glucose additional amount is 2g/L, the growth and oil-producing situation highest of microalgae.
Claims (4)
1. a kind of method using walnut shell extract culture single needle algae generation diesel oil, 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 (4 ~ 32): 1, Gu
Glucose is added as basic culture medium in liquid separation, liquid in basal medium, adjusts culture medium using sodium hydroxide solution
PH value be 6.8 ~ 7.0, then sterilized to obtain single needle algae culture medium；
(2) single needle algae is seeded in the single needle algae culture medium of step (1), then being placed in temperature is 24 ~ 26 DEG C, intensity of illumination is
It is cultivated under the conditions of 6000-7000 lux；
(3) biomass of single needle algae is mentioned after single needle algae grows 60 ~ 80h using organic solvent under determination step (2) condition of culture
Take the grease in single needle frustule；
(4) sulfuric acid-methanol is added to progress 3 ~ 3.5h of esterification reaction of organic acid in the grease of step (3) and obtains biodiesel.
2. utilizing the method for walnut shell extract culture single needle algae generation diesel oil according to claim 1, it is characterised in that:
The solid-to-liquid ratio g:L of step (1) glucose and basal medium is (1 ~ 4): 1.
3. utilizing the method for walnut shell extract culture single needle algae generation diesel oil according to claim 1, it is characterised in that:
Single needle algae is single needle algae in step (2)MonoraphidiumSp.QLZ-3, the inoculum concentration of single needle algae are 0.4g/L.
4. utilizing the method for walnut shell extract culture single needle algae generation diesel oil according to claim 1, it is characterised in that:
The mass concentration of sulfuric acid is 3% in sulfuric acid-methanol in step (4).
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