CN108587917A - Chlorella pyrenoidosa cell utilizes the method that manioc waste is that primary raw material prepares biodiesel - Google Patents
Chlorella pyrenoidosa cell utilizes the method that manioc waste is that primary raw material prepares biodiesel Download PDFInfo
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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
- C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- 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
- C12P7/6436—Fatty acid esters
- C12P7/6445—Glycerides
- C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
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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
- C12P7/6436—Fatty acid esters
- C12P7/649—Biodiesel, i.e. fatty acid alkyl esters
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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
Abstract
The invention belongs to bioenergy technical fields, disclose chlorella pyrenoidosa cell using the method that manioc waste is primary raw material preparation biodiesel comprising following steps：Step 1）Microorganism is mixed, step 2）Extract grease, step 3）Prepare biodiesel.The method of the present invention reduces cost, and grease yield improves.Biodiesel production rate and quality prepared by the present invention is high, can be as the desirable material of substitute of diesel fuel.
Description
Technical field
The invention belongs to biological field of new energy technologies, and in particular to chlorella pyrenoidosa cell is main using manioc waste
The method that raw material prepares biodiesel.
Background technology
Biodiesel is a kind of long chain fatty acids Ester, is by short-chain alcohols substance (methanol or ethyl alcohol) and certain
A little fatty oil substances product obtained by the reaction.The most common production method of biodiesel is ester-interchange method, i.e., in vegetable fat
A certain amount of methanol is added, is heated to certain temperature.Reaction generates fatty acid methyl ester under catalyst (acid or alkali) effect, and divides
Separate out the process of byproduct glycerine.As the clean biometric fuel of alternative petrifaction diesel, the performance of biodiesel with it is existing
It is substantially suitable with petrifaction diesel, and there is better performance, including：Biological degradability is high, good environmental protection, has preferable low
Warm engine start function and lubricating function, lightning is high, has a safety feature, has renewable performance, there is vast potential for future development.
The production method of biodiesel includes mainly Physical and chemical method at present, and the most commonly used is chemical method.Chemistry
Method is that animal and plant fat is carried out chemical conversion, changes its molecular structure, fundamentally changes its mobility and viscosity, thus is made
Biodiesel obtained has the dynamic characteristics and combustion characteristics similar to petrifaction diesel.The source of grease is the weight studied at present
Point, main source include：Plant origin, animal origin and alga-derived.Plant origin is to utilize rapeseed, soybean, peanut
And the grease that various oil crops are extracted is raw material；Animal origin is to utilize the animal tallows such as lard, butter and sheep oil
Or used edible oil；Alga-derived, microalgae can synthesize a large amount of greases in growth course, and microalgae grease belongs to Unicell Oils and Fats,
Its key component is glycerine and aliphatic acid, be by microalgae under certain conditions, utilize carbohydrate, hydrocarbon and general
Logical grease synthesizes, mainly as biofilm components, metabolin and energy source as carbon source in frond.Since plant comes
Source, animal origin, raw material life cycle is long, and total resources is insufficient, and economic benefit is low and it provides agricultural product price, arable land
The influence in source, grain security constrains the development of biodiesel.Relative to traditional plant origin and animal origin biology bavin
Oily raw material, microalgae have it is widely distributed, growth cycle is short, biomass is big, strong environmental adaptability, fat content height, not with grain
It strives ground, strive the huge advantages such as grain with people, pursued energetically by scientific research personnel, it is considered to be solve current biodiesel raw material
One of insufficient important channel.Currently, alga-derived grease is the hot spot of research.It is micro- as biodiesel raw material of new generation
Algae possesses many advantages.Algal kind is various, is distributed widely in fresh water and seawater.The identified microalgae in the whole world has tens of thousands of
Kind, and its quantity is also being continuously increased.Relative to traditional oil crops, microalgae is big with biomass, growth cycle is short.It is micro-
The growth rate of algae is significantly larger than terrestrial crop, and general microalgae can be doubled in its interior biomass for 24 hours, in exponential phase of growth
The biomass doubling time is generally 3-5h.The ingredient of microalgae oil is similar to vegetable oil, is the substitute of vegetable oil, can directly use
The prior art produces biodiesel.Under regular culture conditions, the oil content of general microalgae is all up 20%~50%, part microalgae
Cultivation in sea water can also be used, the extreme environments such as desert, punja, half-dried nonirrigated farmland is resistant to, is not take up arable land, therefore will not be to grain
The production of crop constitutes a threat to.Microalgae can absorb and using a large amount of C02 and nitride given off in industrial and agricultural production or from useless
Nitrogen, phosphorus etc. are obtained in water, are conducive to improve environment.
The biochemical composition of microalgae can be adjusted by the change of environmental condition, to improve oil content.Zhejiang
Regional algae species are abundant, have the natural advantage for carrying out algae production biodiesel research.It is produced currently with algae
The problem of diesel oil industrialization maximum is how to reduce toxigenic capacity and raising oil productivity.Due to the grease of variety classes microalgae
Content and growth ability have differences, and need to screen microalgae type before carrying out microdisk electrode.The microalgae bacterium of selection
Strain must have high productivity and high fat content, have stronger stain resistance, and can adapt to the variation of environment.Most often
The microalgae seen has chlorella, Du Shi algaes, micro- quasi- ball algae, purple ball algae, scenedesmus etc., because of itself higher fat content and growth rate
It is commonly used for the production of microalgae biodiesel.But also there are many researchers by changing the growing environment of microalgae or utilizing gene
Engineering improves the fat content and growth rate of microalgae.
Chlorella pyrenoidosa can be accumulated as a kind of renewable energy source biomass under the condition of culture of light autotrophy and heterotrophism
Tired grease can efficiently use solar energy and have function to carry out fast-growth and accumulation grease, and the grease of chlorella pyrenoidosa can be with
As the renewable sources of energy for making biodiesel.But there is slow-growing, biologies for current chlorella pyrenoidosa pilot scale culture
Measure low, the low problem of fat content is unfavorable for large-scale culture.CN106754383A discloses chlorella and oleaginous yeast is common
The method to improve grease yield is cultivated, this method is by the two while to be inoculated into culture medium, is mixed microbial dry powder
Total lipid content can reach 40.55%, and total fatty acids yield reaches 175.64mg/l/d, and the microalgae noticeably greater than individually cultivated is thin
Born of the same parents are twice or more of the yeast cells individually cultivated.But there are still fat content is to be improved, fermentation costs it is higher with
And the defect of period length." Wang Bujiang etc., TanJin Agricultural College journal, shadow of the fermentation condition to chlorella pyrenoidosa fat content
Ring ", the influence to its biomass and fat content such as carbon source, nitrogen source, carbon-nitrogen ratio, pH, temperature in culture medium is had studied, is used
Glucose is primary raw material, and the frond content in zymotic fluid reaches 5.6g/L, and fat content can reach 1.46g/L.Manioc waste is
Cassava extracts the by-product after starch, and leading indicator includes crude fibre, coarse ash, moisture, and nutritional cost is relatively low, is typically used as raising
Material is discarded.There is a large amount of starch factory in In Hangzhou Region of Zhe Jiang Province, will produce many manioc wastes when processing starch with cassava, such as
It is also the technical issues that need to address that, which to manioc waste efficiently use,.Patented technology " pyrenoids bead before applicant
Frustule utilizes the method that manioc waste is that primary raw material prepares grease " by mixed culture, it uses manioc waste for primary raw material, carries
High grease yield, reduces fermentation costs；On this basis, applicant continues to study, by oil and fat preparation at biological bavin
Oil.
Invention content
It is low and the defects of toxigenic capacity is high present invention aim to address prior art algae generation diesel oil efficiency, it provides
Chlorella pyrenoidosa cell utilizes the method that manioc waste is that primary raw material prepares biodiesel.
The present invention is achieved by the following technical solution：
Chlorella pyrenoidosa cell utilizes the method that manioc waste is that primary raw material prepares biodiesel comprising following steps：Step
Rapid 1）Microorganism is mixed, step 2）Extract grease, step 3）Prepare biodiesel.
Specifically, the step 1）Microorganism is mixed, and includes the following steps：By the pyrenoids in exponential phase
Bead algae solution and trichoderma reesei seed liquor are inoculated into the culture medium containing manioc waste, and temperature is 28 DEG C, 24 hours continuous lights,
Intensity is 6000-8000Lux, and rotating speed 100rpm, fermentation time is 2-3 days, then accesses aspergillus niger seed liquor, continues to cultivate
3-4 days, after culture, after centrifugation, washing and freeze-drying, obtain microorganism powder；The composition of the culture medium is such as
Under：Manioc waste 50-80g/L, sodium nitrate 1-2g/L, potassium dihydrogen phosphate 0.5-1g/L, sodium chloride 0.1-0.2g/L, seven water sulphur
Sour magnesium 100-200mg/L, calcium chloride 50-70mg/L, ferric citrate 20-30mg/L, white vitriol 10-15mg/L.
Specifically, the step 2）Grease is extracted, is included the following steps：Microorganism powder is handled using impulse electric field,
Then powder is added in chloroform methanol mixed solution, additive amount is 1g powder：2ml chloroform methanol mixed solutions, ultrasound carry
It takes, is then centrifuged for, collect chloroform phase, be placed in nitrogen and dry up, and be dried in vacuo, obtain grease.
Specifically, the step 3）Biodiesel is prepared, is included the following steps：
By grease, lipase and methanol according to 5ml：1g：The volume ratio of 6ml is added in reactor, catalysis reaction 12 hours,
Then addition accounts for the methanol of grease two volumes, continues catalysis reaction 20h, terminates reaction, and catalysis reaction is 45 DEG C in temperature control
It is carried out in shaking table, shaking speed 100rpm；The mixture of reaction system is centrifuged 10 minutes with 12000rpm, by upper phase
Part removes, stratification；Take out lower layer's liquid phase after stratification, as biodiesel crude product, again with 12000rpm from
The heart 10 minutes, take out upper phase, the ultra-pure water of 2 times of 50 DEG C of volumes be added, fully agitation is uniform, again with 12000rpm from
The heart 10 minutes, the upper organic phase obtained after stratification, separation take out upper organic phase to get biodiesel.
Preferably, the trichoderma reesei seed liquor is prepared according to following technique：Trichoderma reesei streak inoculation is trained in PDA
It supports and is cultivated on base, obtain single bacterium colony；Picking single bacterium colony is inoculated into primary-seed medium and is cultivated, and then carries out secondary seed
Medium culture obtains trichoderma reesei seed liquor；The primary-seed medium and secondary seed medium are the training of PDA liquid
Support base.
Preferably, the chlorella pyrenoidosa liquid is prepared according to following technique：Picking chlorella pyrenoidosa, kind is to containing
Have in the container of growth medium, intensity of illumination 6000lux, 28 DEG C of cultures are shaken every day container 2-3 times, to be grown to logarithm
Growth period obtains chlorella pyrenoidosa liquid；The group of the growth medium is divided into：Glucose 10g/L, ammonium chloride 2g/L, nitric acid
Sodium 1g/L, potassium dihydrogen phosphate 0.5g/L, sodium chloride 0.1g/L, epsom salt 100mg/L, calcium chloride 30mg/L, lemon
Sour iron ammonium 20mg/L, white vitriol 10mg/L, manganese sulfate 10mg/L.
Preferably, the aspergillus niger seed liquor is prepared according to following technique：Aspergillus niger streak inoculation is trained on inclined-plane
It supports and is cultivated on base, obtain single bacterium colony；Picking single bacterium colony is inoculated into primary-seed medium and is cultivated, and then carries out secondary seed
Medium culture obtains aspergillus niger seed liquor；The slant medium group is divided into：Potato 150g/L, sucrose 20g/L, agar
15g/L；The component of the primary-seed medium and secondary seed medium is：Corn flour 50g/L, sucrose 10g/L, sulfuric acid
Ammonium 5g/L, potassium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate 1g/L.
Preferably, the impulse electric field, which is handled, is：Electric field strength 20kV/cm, pulse width 4us, processing time 200us.
Preferably, the ultrasonic extraction is：Extracting temperature is 60-65 DEG C, ultrasonic power 100-200W, and extraction time is
60-90min。
Preferably, the chloroform methanol mixed solution is 2 according to volume ratio by chloroform and methanol:1 is made.
Compared with prior art, the advantageous effect that the present invention obtains includes but is not limited to mainly several aspects：
The increment and grease yield of algae are not fully positively correlated ratio, by adjusting extraneous factor so that increment is in
In reasonable range and grease yield maximizes, and is technological difficulties all the time.Trichoderma reesei is generated using fermentation cassava slag
Reduced sugar, reduced sugar can promote the growth rate of algae, increase biomass, and after algae reaches certain growth amount, inoculation is black
Aspergillus, aspergillus niger can utilize nitrogen source and partial reduction sugar faster, to generate competition with algae so that algae limits in nitrogen
System and nutrient are deprived under stress conditions, and higher fat content is obtained by nitrogen limitation or nutrition limitation.And aspergillus niger is also
The inorganic carbon source of a large amount of carbon dioxide can be generated, to promote the grease yield of algae.Trichoderma reesei and aspergillus niger also can
Generate oil substances, and used manioc waste as primary raw material in culture of the present invention, it is cheap, reduce enterprise at
This.The oxygen that microalgae photosynthesis discharges in mixed culture can be utilized by somatic cells, so that co-culture system is in
One equilibrium state.The highfield of suitable treatments time can have an impact intracellular polar molecule, be generated on cell membrane
Concussion, occurs expendable destruction so that permeability of cell membrane enhances, and may finally accelerate the exchange of the outer grease of intracellular
Efficiency.When ul-trasonic irradiation is in liquid reaction system, due to the cavitation of ultrasonic wave, a large amount of small gas are had in liquid
Bubble is formed, and the generation of these bubbles and vanishes very rapid, and reaction system will produce local high temperature and high pressure, Ke Yida
To the effect for destroying cell wall, the touch opportunity of solvent and intracellular organic matter can be increased.The present invention is cooperateed with super using impulse electric field
It is an efficient method that sound wave, which extracts grease, combines the advantage of the two, not only shortens the time of reaction, can also be reduced anti-
It should be able to consume.It is high using the biodiesel production rate of oil and fat preparation of the present invention, C16 and C18 fatty acid methyl esters in biodiesel ingredient
Percentage composition reaches 95% or more, can be as the desirable material of substitute of diesel fuel.
Description of the drawings
Fig. 1：Influence of the different impulse electric field times to grease yield；
Fig. 2：The influence of ultrasonic power and ultrasonic time to grease yield.
Specific implementation mode
Those skilled in the art can use for reference present disclosure, be suitably modified technological parameter realization.In particular, it should be pointed out that
All similar substitutions and modifications are apparent to those skilled in the art, they are considered as being included in this hair
It is bright.The product and method of the present invention is described by preferred embodiment, and related personnel can obviously not depart from this hair
Product as described herein and method are modified or are suitably changed and combined in bright content, spirit and scope, to realize and answer
Use the technology of the present invention.For a further understanding of the present invention, the following describes the present invention in detail with reference to examples.
Embodiment 1
Chlorella pyrenoidosa cell utilizes the method that manioc waste is that primary raw material prepares grease comprising following steps：
Trichoderma reesei streak inoculation is cultivated in PDA culture medium, obtains single bacterium colony；Picking single bacterium colony is inoculated into first order seed training
Foster base is cultivated, and secondary seed medium culture is then carried out, and obtains trichoderma reesei seed liquor；The primary-seed medium
It is PDA liquid medium with secondary seed medium；
Picking chlorella pyrenoidosa is planted into the container containing growth medium, intensity of illumination 6000lux, 28 DEG C of cultures, daily
Shake container 2-3 times, it is to be grown to exponential phase, obtain chlorella pyrenoidosa liquid；The group of the growth medium is divided into：Portugal
Grape sugar 10g/L, ammonium chloride 2g/L, sodium nitrate 1g/L, potassium dihydrogen phosphate 0.5g/L, sodium chloride 0.1g/L, epsom salt
100mg/L, calcium chloride 30mg/L, ferric citrate 20mg/L, white vitriol 10mg/L, manganese sulfate 10mg/L；
Aspergillus niger streak inoculation is cultivated on slant medium, obtains single bacterium colony；Picking single bacterium colony is inoculated into first order seed training
Foster base is cultivated, and secondary seed medium culture is then carried out, and obtains aspergillus niger seed liquor；The slant medium component
For：Potato 150g/L, sucrose 20g/L, agar 15g/L；The component of the primary-seed medium and secondary seed medium
It is：Corn flour 50g/L, sucrose 10g/L, ammonium sulfate 5g/L, potassium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate 1g/L.
By in exponential phase chlorella pyrenoidosa liquid and trichoderma reesei seed liquor be inoculated into containing the anti-of culture medium
The inoculum density of Ying Chizhong, chlorella pyrenoidosa and trichoderma reesei is respectively 1 × 106A/ml and 1 × 107Cfu/ml, temperature are
28 DEG C, 24 hours continuous lights, intensity 6000Lux, rotating speed 100rpm, fermentation time is 3 days, then accesses aspergillus niger kind
Sub- liquid, inoculum density are 5 × 107Cfu/ml continues culture 3 days, after culture, after centrifugation, washing and freeze-drying,
Obtain powder；The concrete composition of the culture medium is as follows：Manioc waste 50g/L, sodium nitrate 1g/L, potassium dihydrogen phosphate 0.5g/
L, sodium chloride 0.1g/L, epsom salt 100mg/L, calcium chloride 50mg/L, ferric citrate 20mg/L, white vitriol
10mg/L。
Powder is handled using impulse electric field, electric field strength 20kV/cm, pulse width 4us, processing time 200us, then
Powder is added to chloroform methanol mixed solution（The volume ratio of chloroform and methanol is 2:1）In, additive amount is 1g powder：2ml chlorine
Imitative methanol mixed solution, ultrasonic extraction, Extracting temperature are 60 DEG C, ultrasonic power 200W, extraction time 60min, then from
The heart collects chloroform phase, is placed in nitrogen and dries up, and be dried in vacuo, obtain grease；
Grease, lipase（10000 U/g）And methanol is according to 5ml：1g：The volume ratio of 6ml is added in reactor, catalysis reaction
12 hours, then addition accounted for the methanol of grease two volumes, continued catalysis reaction 20h, terminated reaction, and catalysis reaction is in temperature control
It is carried out in 45 DEG C of shaking table, shaking speed 100rpm；The mixture of reaction system is centrifuged 10 minutes, 12000rpm, it will be upper
Layer liquid phase part removes, stratification；Lower layer's liquid phase after stratification, as biodiesel crude product are taken out, centrifuges 10 again
Minute, 12000rpm takes out upper phase, and the ultra-pure water of 2 times of 50 DEG C of volumes is added, and fully agitation is uniform, centrifuges 10 points again
Clock, 12000rpm, the upper organic phase obtained after stratification, separation take out upper organic phase to get biodiesel.
Embodiment 2
Chlorella pyrenoidosa cell utilizes the method that manioc waste is that primary raw material prepares grease comprising following steps：
Trichoderma reesei streak inoculation is cultivated in PDA culture medium, obtains single bacterium colony；Picking single bacterium colony is inoculated into first order seed training
Foster base is cultivated, and secondary seed medium culture is then carried out, and obtains trichoderma reesei seed liquor；The primary-seed medium
It is PDA liquid medium with secondary seed medium；
Picking chlorella pyrenoidosa is planted into the container containing growth medium, intensity of illumination 6000lux, 28 DEG C of cultures, daily
Shake container 2-3 times, it is to be grown to exponential phase, obtain chlorella pyrenoidosa liquid；The group of the growth medium is divided into：Portugal
Grape sugar 10g/L, ammonium chloride 2g/L, sodium nitrate 1g/L, potassium dihydrogen phosphate 0.5g/L, sodium chloride 0.1g/L, epsom salt
100mg/L, calcium chloride 30mg/L, ferric citrate 20mg/L, white vitriol 10mg/L, manganese sulfate 10mg/L；
Aspergillus niger streak inoculation is cultivated on slant medium, obtains single bacterium colony；Picking single bacterium colony is inoculated into first order seed training
Foster base is cultivated, and secondary seed medium culture is then carried out, and obtains aspergillus niger seed liquor；The slant medium component
For：Potato 150g/L, sucrose 20g/L, agar 15g/L；The component of the primary-seed medium and secondary seed medium
It is：Corn flour 50g/L, sucrose 10g/L, ammonium sulfate 5g/L, potassium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate 1g/L.
By in exponential phase chlorella pyrenoidosa liquid and trichoderma reesei seed liquor be inoculated into containing the anti-of culture medium
The inoculum density of Ying Chizhong, chlorella pyrenoidosa and trichoderma reesei is respectively 1 × 106A/ml and 1 × 107Cfu/ml, temperature are
28 DEG C, 24 hours continuous lights, intensity 7000Lux, rotating speed 100rpm, fermentation time is 3 days, then accesses aspergillus niger kind
Sub- liquid, inoculum density are 5 × 107Cfu/ml continues culture 4 days, after culture, after centrifugation, washing and freeze-drying,
Obtain powder；The concrete composition of the culture medium is as follows：Manioc waste 80g/L, sodium nitrate 2g/L, potassium dihydrogen phosphate 1g/L,
Sodium chloride 0.2g/L, epsom salt 200mg/L, calcium chloride 70mg/L, ferric citrate 30mg/L, white vitriol
15mg/L。
Powder is handled using impulse electric field, electric field strength 20kV/cm, pulse width 4us, processing time 200us, then
Powder is added to chloroform methanol mixed solution（The volume ratio of chloroform and methanol is 2:1）In, additive amount is 1g powder：2ml chlorine
Imitative methanol mixed solution, ultrasonic extraction, Extracting temperature are 65 DEG C, ultrasonic power 100W, extraction time 90min, then from
The heart collects chloroform phase, is placed in nitrogen and dries up, and be dried in vacuo, obtain grease；
Grease, lipase（10000 U/g）And methanol is according to 5ml：1g：The volume ratio of 6ml is added in reactor, catalysis reaction
12 hours, then addition accounted for the methanol of grease two volumes, continued catalysis reaction 20h, terminated reaction, and catalysis reaction is in temperature control
It is carried out in 45 DEG C of shaking table, shaking speed 100rpm；The mixture of reaction system is centrifuged 10 minutes, 12000rpm, it will be upper
Layer liquid phase part removes, stratification；Lower layer's liquid phase after stratification, as biodiesel crude product are taken out, centrifuges 10 again
Minute, 12000rpm takes out upper phase, and the ultra-pure water of 2 times of 50 DEG C of volumes is added, and fully agitation is uniform, centrifuges 10 points again
Clock, 12000rpm, the upper organic phase obtained after stratification, separation take out upper organic phase to get biodiesel.
Comparative example 1
Trichoderma reesei and aspergillus niger are not added, remaining is the same as embodiment 1.
Comparative example 2
Aspergillus niger is not added, remaining is the same as embodiment 1.
Comparative example 3
Chlorella pyrenoidosa, trichoderma reesei and aspergillus niger add simultaneously, remaining is the same as embodiment 1.
Embodiment 3
Biomass dry weight content, total lipid content in the embodiment of the present invention and comparative example（Account for the percentage of biomass dry weight）And
Grease yield detects.Specific testing result is shown in Table 1；
Table 1
| Group | Incubation time d | Biomass dry weight content g/L | Total lipid content % | Grease yield g/L |
| Embodiment 1 | 6 | 4.37 | 44.5 | 1.94 |
| Comparative example 1 | 6 | 3.02 | 35.2 | 1.06 |
| Comparative example 2 | 6 | 4.19 | 37.8 | 1.58 |
| Comparative example 3 | 6 | 3.74 | 41.6 | 1.56 |
Conclusion：Strain type is demonstrated by table 1, addition opportunity produces biomass dry weight content, total lipid content and grease
The influence of amount, it is found that 1 substance dry weight content of embodiment, total lipid content and each index of grease yield are above comparative example 1-
3；Comparative example 1 does not add bacterial strain, and only with algae culture, various aspects index is minimum, and grease yield reduces 40% than embodiment 1
Left and right；Comparative example 2 is only with trichoderma reesei, wherein trichoderma reesei generates reduced sugar using fermentation cassava slag, and reduced sugar can promote
Into the growth rate of algae, increase biomass, but compared with Example 1, continue sufficient nutrition although can maintain higher
Microbial quality, but be reduction of fat content；Comparative example 3 while inoculated aspergillus niger cause aspergillus niger transition to compete carbon source,
It causes algae nutrient insufficient, causes algal grown slow；Embodiment 1 is inoculated after algae basically reaches compared with height increament
Aspergillus niger, aspergillus niger speed can be competed so that algae using nitrogen source and partial reduction sugar to be generated with algae faster
Under the stress conditions that nitrogen limitation and nutrient are deprived, higher fat content is obtained by nutrient limitation, and aspergillus niger is also
The inorganic carbon source of a large amount of carbon dioxide can be generated, to promote the grease yield of algae.
Embodiment 4
Influence of the different impulse electric field times to grease yield is respectively set 0,100,200,400 by taking embodiment 1 as an example,
Influences of the 600us to grease yield, as shown in Figure 1, with the increase of processing time, grease yield amplification is apparent, after 200us,
Increase processing time can't bring to grease yield to be significantly affected, therefore selects 200us the most suitable.The suitable treatments time
Highfield can have an impact intracellular polar molecule, and concussion is generated on cell membrane, expendable destruction occurs,
So that permeability of cell membrane enhancing, may finally accelerate the exchange efficiency of the outer grease of intracellular.
Embodiment 5
The influence of ultrasonic power and ultrasonic time to grease yield：
Setting ultrasonic power is 50w, 100w, 200w, 400w, 800w；The ultrasonic extraction time is 15,30,60,90,120min, such as
Shown in Fig. 2, with the increase of ultrasonic power and ultrasonic time, grease yield is stepped up, and final ultrasonic power selects 100-
When 200W, processing time are 60-90min, the extraction effect of grease is best.When ul-trasonic irradiation is in liquid reaction system, by
In having a large amount of small bubble formations in the cavitation of ultrasonic wave, liquid, and the generation of these bubbles and vanish very
Rapidly, reaction system will produce local high temperature and high pressure, can have the function that destroy plant cell wall, can increase solvent
With the touch opportunity of intracellular organic matter.The present invention is an efficient method using impulse electric field synergistic supersonic wave extraction grease, knot
The advantage of the two has been closed, the time of reaction is not only shortened, can also reduce energy consumption of reaction.
Embodiment 6
On the basis of embodiment 4, the present invention also has detected the yield of each group biodiesel（The calculating of biodiesel production rate,
Yield (Y)=（Biodiesel weight/microalgae grease weight）× 100%, the composition of biodiesel is analyzed by GC-MS, specifically
It is shown in Table 2.
Table 2
| Group | The yield % of biodiesel | C16 total contents % | C18 total contents % |
| Embodiment 1 | 81.6 | 39.2 | 57.7 |
| Comparative example 1 | 82.1 | 28.5 | 60.8 |
| Comparative example 2 | 81.3 | 35.6 | 56.4 |
| Comparative example 3 | 80.1 | 31.7 | 58.1 |
As shown in table 2, the yield of each group biodiesel is close, 80% or so.C atoms in diesel oil are relatively closed at 16-18
It is suitable, have preferable circulation, be easy to aoxidize, can ensure fully to burn, in embodiment 1 biodiesel main component C16 and
The percentage composition of C18 accounts for the 96.9% of grease total amount, not the presence of aromatic hydrocarbon, it is ensured that fully burning not will produce charcoal
It is black；The percentage composition of C16 and C18 is apparently higher than comparative example 1-3, similar with conventional diesel, can compare conjunction as conventional diesel
Suitable substitute.
Finally it should be noted that：The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although reference
Invention is explained in detail for previous embodiment, it will be understood by those of ordinary skill in the art that：It still can be right
Technical solution recorded in previous embodiment is modified or equivalent replacement of some of the technical features；And these
Modification or replacement, the spirit and scope for technical solution of the embodiment of the present invention that it does not separate the essence of the corresponding technical solution.
Claims (10)
1. chlorella pyrenoidosa cell utilizes the method that manioc waste is that primary raw material prepares biodiesel comprising following steps：
Step 1）Microorganism is mixed, step 2）Extract grease, step 3）Prepare biodiesel.
2. according to the method described in claim 1, it is characterized in that, the step 1）Microorganism is mixed, including walks as follows
Suddenly：By in exponential phase chlorella pyrenoidosa liquid and trichoderma reesei seed liquor be inoculated into the culture medium containing manioc waste
In, temperature is 28 DEG C, 24 hours continuous lights, intensity 6000-8000Lux, rotating speed 100rpm, and fermentation time is 2-3 days,
Then aspergillus niger seed liquor is accessed, continues culture 3-4 days, after culture, after centrifugation, washing and freeze-drying, obtains
Microorganism powder；The composition of the culture medium is as follows：Manioc waste 50-80g/L, sodium nitrate 1-2g/L, potassium dihydrogen phosphate 0.5-1g/
L, sodium chloride 0.1-0.2g/L, epsom salt 0.1-0.2g/L, calcium chloride 50-70mg/L, ferric citrate 20-30mg/L,
White vitriol 10-15mg/L.
3. according to the method described in claim 2, it is characterized in that, the step 2）Grease is extracted, is included the following steps：It will be micro-
Biological powder is handled using impulse electric field, and then powder is added in chloroform methanol mixed solution, and additive amount is 1g powder：
2ml chloroform methanol mixed solutions, ultrasonic extraction are then centrifuged for, and are collected chloroform phase, are placed in nitrogen and dry up, and be dried in vacuo,
Obtain grease.
4. according to the method described in claim 3, it is characterized in that, the step 3）Biodiesel is prepared, is included the following steps：
By grease, lipase and methanol according to 5ml：1g：The volume ratio of 6ml is added in reactor, catalysis reaction 12 hours,
Then addition accounts for the methanol of grease two volumes, continues catalysis reaction 20h, terminates reaction；By the mixture of reaction system with
12000rpm is centrifuged 10 minutes, is then removed upper phase part, stratification；Lower layer's liquid phase after stratification is taken out,
As biodiesel crude product is centrifuged 10 minutes with 12000rpm again, upper phase is taken out, the ultrapure of 2 times of 50 DEG C of volumes is added
Water, fully agitation are uniform, are centrifuged 10 minutes with 12000rpm again, stratification, and separation takes out upper organic phase to get life
Object diesel oil.
5. according to the method described in claim 2, it is characterized in that, the trichoderma reesei seed liquor prepared according to following technique and
：Trichoderma reesei streak inoculation is cultivated in PDA culture medium, obtains single bacterium colony；Picking single bacterium colony is inoculated into first order seed culture
Base is cultivated, and secondary seed medium culture is then accessed, and obtains trichoderma reesei seed liquor；The primary-seed medium and
Secondary seed medium is PDA liquid medium.
6. according to the method described in claim 2, it is characterized in that, the chlorella pyrenoidosa liquid prepared according to following technique and
：Picking chlorella pyrenoidosa is inoculated into the container containing growth medium, intensity of illumination 6000lux, 28 DEG C of cultures, often
Its shake container 2-3 times, it is to be grown to exponential phase, obtain chlorella pyrenoidosa liquid；The group of the growth medium is divided into：
Glucose 10g/L, ammonium chloride 2g/L, sodium nitrate 1g/L, potassium dihydrogen phosphate 0.5g/L, sodium chloride 0.1g/L, seven water sulfuric acid
Magnesium 0.1g/L, calcium chloride 30mg/L, ferric citrate 20mg/L, white vitriol 10mg/L, manganese sulfate 10mg/L.
7. according to the method described in claim 2, it is characterized in that, the aspergillus niger seed liquor prepared according to following technique and
：Aspergillus niger streak inoculation is cultivated on slant medium, obtains single bacterium colony；Picking single bacterium colony is inoculated into first order seed culture
Base is cultivated, and secondary seed medium culture is then accessed, and obtains aspergillus niger seed liquor；The slant medium group is divided into：
Potato 150g/L, sucrose 20g/L, agar 15g/L；The component of the primary-seed medium and secondary seed medium is equal
For：Corn flour 50g/L, sucrose 10g/L, ammonium sulfate 5g/L, potassium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate 1g/L.
8. according to the method described in claim 3, it is characterized in that, impulse electric field processing is：Electric field strength 20kV/cm,
Pulse width 4us, processing time 200us.
9. according to the method described in claim 3, it is characterized in that, the ultrasonic extraction is：Extracting temperature is 60-65 DEG C, is surpassed
Acoustical power is 100-200W, extraction time 60-90min.
10. according to the method described in claim 3, it is characterized in that, the chloroform methanol mixed solution is pressed by chloroform and methanol
It is 2 according to volume ratio:1 is made.
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